TY - JOUR ID - 201 TI - Imaging of plant calcium-sensor kinase conformation monitors real time calcium-dependent decoding in planta JO - Plant Cell PY - 2024 SP - 276-296 AU - Liese, A. AU - Eichstädt, B. AU - Lederer, S. AU - Schulz, P. AU - Oehlschläger, J. AU - Matschi, S. AU - Feijó, J. A. AU - Schulze, W. X. AU - Konrad, K. R. AU - Romeis, T. AU - VL - 36 UR - https://doi.org/10.1093/plcell/koad196 DO - 10.1093/plcell/koad196 AB - Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here we developed a genetically encoded FRET (Förster resonance energy transfer)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 265 TI - A conserved microtubule-binding region in Xanthomonas XopL is indispensable for induced plant cell death reactions JO - PLOS Pathog. PY - 2023 SP - e1011263 AU - Ortmann, S. AU - Marx, J. AU - Lampe, C. AU - Handrick, V. AU - Ehnert, T.-M. AU - Zinecker, S. AU - Reimers, M. AU - Bonas, U. AU - Erickson, J. AU - VL - 19 UR - https://doi.org/10.1371/journal.ppat.1011263 DO - 10.1371/journal.ppat.1011263 AB - Pathogenic Xanthomonas bacteria cause disease on more than 400 plant species. These Gram-negative bacteria utilize the type III secretion system to inject type III effector proteins (T3Es) directly into the plant cell cytosol where they can manipulate plant pathways to promote virulence. The host range of a given Xanthomonas species is limited, and T3E repertoires are specialized during interactions with specific plant species. Some effectors, however, are retained across most strains, such as Xanthomonas Outer Protein L (XopL). As an ‘ancestral’ effector, XopL contributes to the virulence of multiple xanthomonads, infecting diverse plant species. XopL homologs harbor a combination of a leucine-rich-repeat (LRR) domain and an XL-box which has E3 ligase activity. Despite similar domain structure there is evidence to suggest that XopL function has diverged, exemplified by the finding that XopLs expressed in plants often display bacterial species-dependent differences in their sub-cellular localization and plant cell death reactions. We found that XopL from X. euvesicatoria (XopLXe) directly associates with plant microtubules (MTs) and causes strong cell death in agroinfection assays in N. benthamiana. Localization of XopLXe homologs from three additional Xanthomonas species, of diverse infection strategy and plant host, revealed that the distantly related X. campestris pv. campestris harbors a XopL (XopLXcc) that fails to localize to MTs and to cause plant cell death. Comparative sequence analyses of MT-binding XopLs and XopLXcc identified a proline-rich-region (PRR)/α-helical region important for MT localization. Functional analyses of XopLXe truncations and amino acid exchanges within the PRR suggest that MT-localized XopL activity is required for plant cell death reactions. This study exemplifies how the study of a T3E within the context of a genus rather than a single species can shed light on how effector localization is linked to biochemical activity. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 259 TI - The secreted PAMP-induced peptide StPIP1_1 activates immune responses in potato JO - Sci. Rep. PY - 2023 SP - 20534 AU - Nietzschmann, L. AU - Smolka, U. AU - Perino, E. H. B. AU - Gorzolka, K. AU - Stamm, G. AU - Marillonnet, S. AU - Bürstenbinder, K. AU - Rosahl, S. AU - VL - 13 UR - https://doi.org/10.1038/s41598-023-47648-x DO - 10.1038/s41598-023-47648-x AB - Treatment of potato plants with the pathogen-associated molecular pattern Pep-13 leads to the activation of more than 1200 genes. One of these, StPIP1_1, encodes a protein of 76 amino acids with sequence homology to PAMP-induced secreted peptides (PIPs) from Arabidopsis thaliana. Expression of StPIP1_1 is also induced in response to infection with Phytophthora infestans, the causal agent of late blight disease. Apoplastic localization of StPIP1_1-mCherry fusion proteins is dependent on the presence of the predicted signal peptide. A synthetic peptide corresponding to the last 13 amino acids of StPIP1_1 elicits the expression of the StPIP1_1 gene itself, as well as that of pathogenesis related genes. The oxidative burst induced by exogenously applied StPIP1_1 peptide in potato leaf disks is dependent on functional StSERK3A/B, suggesting that StPIP1_1 perception occurs via a receptor complex involving the co-receptor StSERK3A/B. Moreover, StPIP1_1 induces expression of FRK1 in Arabidopsis in an RLK7-dependent manner. Expression of an RLK from potato with high sequence homology to AtRLK7 is induced by StPIP1_1, by Pep-13 and in response to infection with P. infestans. These observations are consistent with the hypothesis that, upon secretion, StPIP1_1 acts as an endogenous peptide required for amplification of the defense response. A2 - C1 - Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 239 TI - Distinct metabolite classes in root exudates are indicative for field- or hydroponically-grown cover crops JO - Front. Plant Sci. PY - 2023 SP - 1122285 AU - Heuermann, D. AU - Döll, S. AU - Schweneker, D. AU - Feuerstein, U. AU - Gentsch, N. AU - von Wirén, N. AU - VL - 14 UR - https://doi.org/10.3389/fpls.2023.1122285 DO - 10.3389/fpls.2023.1122285 AB - Introduction: Plants release a large variety of metabolites via their roots to shape physico-chemical soil properties and biological processes in the rhizosphere. While hydroponic growth conditions facilitate accessibility of the root system and recovery of root exudates, the natural soil environment can alter root metabolism and exudate secretion, raising the question to what extent the quantity and composition of root exudates released in hydroponic growth systems reflect those recovered from soil-grown roots. Methods: Using a root washing method, we sampled root exudates from four field-grown cover crop species with wide taxonomic distance, namely white mustard, lacy phacelia, bristle oat, and Egyptian clover. A set of primary metabolites and secondary metabolites were analysed in a targeted and untargeted LC-MS-based approach, respectively, for comparison with exudates obtained from hydroponically cultured plants. Results and discussion: We found that hydroponically cultivated plants released a larger amount of total carbon, but that the recovery of total carbon was not indicative for the diversity of metabolites in root exudates. In the field, root exudates from phacelia and clover contained 2.4 to 3.8 times more secondary metabolites, whereas carbon exudation in hydroponics was 5- to 4-fold higher. The composition of the set of metabolites identified using the untargeted approach was much more distinct among all species and growth conditions than that of quantified primary metabolites. Among secondary metabolite classes, the presence of lipids and lipid-like molecules was highly indicative for field samples, while the release of a large amount of phenylpropanoids, organoheterocyclic compounds or benzenoids was characteristic for clover, mustard or oat, respectively, irrespective of the cultivation condition. However, at the compound level the bulk of released metabolites was specific for cultivation conditions in every species, which implies that hydroponically sampled root exudates poorly reflect the metabolic complexity of root exudates recovered from field-grown plants. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 214 TI - An LRR receptor kinase controls ABC transporter substrate preferences during plant growth-defense decisions JO - Curr. Biol. PY - 2023 SP - 2008-2023 AU - Aryal, B. AU - Xia, J. AU - Hu, Z. AU - Stumpe, M. AU - Tsering, T. AU - Liu, J. AU - Huynh, J. AU - Fukao, Y. AU - Glöckner, N. AU - Huang, H.-Y. AU - Sancho-Andrés, G. AU - Pakula, K. AU - Ziegler, J. AU - Gorzolka, K. AU - Zwiewka, M. AU - Nodzynski, T. AU - Harter, K. AU - Sánchez-Rodríguez, C. AU - Jasiński, M. AU - Rosahl, S. AU - Geisler, M. M. AU - VL - 33 UR - https://doi.org/10.1016/j.cub.2023.04.029 DO - 10.1016/j.cub.2023.04.029 AB - The exporter of the auxin precursor indole-3-butyric acid (IBA), ABCG36/PDR8/PEN3, from the model plant Arabidopsis has recently been proposed to also function in the transport of the phytoalexin camalexin. Based on these bonafide substrates, it has been suggested that ABCG36 functions at the interface between growth and defense. Here, we provide evidence that ABCG36 catalyzes the direct, ATP-dependent export of camalexin across the plasma membrane. We identify the leucine-rich repeat receptor kinase, QIAN SHOU KINASE1 (QSK1), as a functional kinase that physically interacts with and phosphorylates ABCG36. Phosphorylation of ABCG36 by QSK1 unilaterally represses IBA export, allowing camalexin export by ABCG36 conferring pathogen resistance. As a consequence, phospho-dead mutants of ABCG36, as well as qsk1 and abcg36 alleles, are hypersensitive to infection with the root pathogen Fusarium oxysporum, caused by elevated fungal progression. Our findings indicate a direct regulatory circuit between a receptor kinase and an ABC transporter that functions to control transporter substrate preference during plant growth and defense balance decisions. A2 - C1 - Biochemistry of Plant Interactions; Molecular Signal Processing ER - TY - JOUR ID - 211 TI - Changing turn-over rates regulate abundance of tryptophan, GS biosynthesis, IAA transport and photosynthesis proteins in Arabidopsis growth defense transitions JO - BMC Biol. PY - 2023 SP - 249 AU - Abukhalaf, M. AU - Proksch, C. AU - Thieme, D. AU - Ziegler, J. AU - Hoehenwarter, W. AU - VL - 21 UR - https://doi.org/10.1186/s12915-023-01739-3 DO - 10.1186/s12915-023-01739-3 AB - Background Shifts in dynamic equilibria of the abundance of cellular molecules in plant-pathogen interactions need further exploration. We induced PTI in optimally growing Arabidopsis thaliana seedlings for 16 h, returning them to growth conditions for another 16 h. Methods Turn-over and abundance of 99 flg22 responding proteins were measured chronologically using a stable heavy nitrogen isotope partial labeling strategy and targeted liquid chromatography coupled to mass spectrometry (PRM LC–MS). These experiments were complemented by measurements of mRNA and phytohormone levels. Results Changes in synthesis and degradation rate constants (Ks and Kd) regulated tryptophane and glucosinolate, IAA transport, and photosynthesis-associated protein (PAP) homeostasis in growth/PTI transitions independently of mRNA levels. Ks values increased after elicitation while protein and mRNA levels became uncorrelated. mRNA returned to pre-elicitation levels, yet protein abundance remained at PTI levels even 16 h after media exchange, indicating protein levels were robust and unresponsive to transition back to growth. The abundance of 23 PAPs including FERREDOXIN-NADP( +)-OXIDOREDUCTASE (FNR1) decreased 16 h after PAMP exposure, their depletion was nearly abolished in the myc234 mutant. FNR1 Kd increased as mRNA levels decreased early in PTI, its Ks decreased in prolonged PTI. FNR1 Kd was lower in myc234, mRNA levels decreased as in wild type. Conclusions Protein Kd and Ks values change in response to flg22 exposure and constitute an additional layer of protein abundance regulation in growth defense transitions next to changes in mRNA levels. Our results suggest photosystem remodeling in PTI to direct electron flow away from the photosynthetic carbon reaction towards ROS production as an active defense mechanism controlled post-transcriptionally and by MYC2 and homologs. Target proteins accumulated later and PAP and auxin/IAA depletion was repressed in myc234 indicating a positive effect of the transcription factors in the establishment of PTI. A2 - C1 - Biochemistry of Plant Interactions; Molecular Signal Processing ER - TY - JOUR ID - 294 TI - Poly(ADP-ribose)-binding protein RCD1 is a plant PARylation reader regulated by Photoregulatory Protein Kinases JO - Commun. Biol. PY - 2023 SP - 429 AU - Vainonen, J. P. AU - Gossens, R. AU - Krasensky-Wrzaczek, J. AU - De Masi, R. AU - Danciu, I. AU - Puukko, T. AU - Battchikova, N. AU - Jonak, C. AU - Wirthmueller, L. AU - Wrzaczek, M. AU - Shapiguzov, A. AU - Kangasjärvi, J. AU - VL - 6 UR - https://doi.org/10.1038/s42003-023-04794-2 DO - 10.1038/s42003-023-04794-2 AB - Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 276 TI - Effector XopQ-induced stromule formation in Nicotiana benthamiana depends on ETI signaling components ADR1 and NRG1 JO - Plant Physiol. PY - 2023 SP - 161-176 AU - Prautsch, J. AU - Erickson, J. AU - Özyürek, S. AU - Gormanns, R. AU - Franke, L. AU - Lu, Y. AU - Marx, J. AU - Niemeyer, F. AU - Parker, J. E. AU - Stuttmann, J. AU - Schattat, M. H. AU - VL - 191 UR - https://doi.org/10.1093/plphys/kiac481 DO - 10.1093/plphys/kiac481 AB - In Nicotiana benthamiana, the expression of the Xanthomonas effector XANTHOMONAS OUTER PROTEIN Q (XopQ) triggers RECOGNITION OF XOPQ1 (ROQ1)-dependent effector-triggered immunity (ETI) responses accompanied by the accumulation of plastids around the nucleus and the formation of stromules. Both plastid clustering and stromules were proposed to contribute to ETI-related hypersensitive cell death and thereby to plant immunity. Whether these reactions are directly connected to ETI signaling events has not been tested. Here, we utilized transient expression experiments to determine whether XopQ-triggered plastid reactions are a result of XopQ perception by the immune receptor ROQ1 or a consequence of XopQ virulence activity. We found that N. benthamiana mutants lacking ROQ1, ENHANCED DISEASE SUSCEPTIBILITY 1, or the helper NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT IMMUNE RECEPTORS (NLRs) N-REQUIRED GENE 1 (NRG1) and ACTIVATED DISEASE RESISTANCE GENE 1 (ADR1), fail to elicit XopQ-dependent host cell death and stromule formation. Mutants lacking only NRG1 lost XopQ-dependent cell death but retained some stromule induction that was abolished in the nrg1_adr1 double mutant. This analysis aligns XopQ-triggered stromules with the ETI signaling cascade but not to host programmed cell death. Furthermore, data reveal that XopQ-triggered plastid clustering is not strictly linked to stromule formation during ETI. Our data suggest that stromule formation, in contrast to chloroplast perinuclear dynamics, is an integral part of the N. benthamiana ETI response and that both NRG1 and ADR1 hNLRs play a role in this ETI response. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 324 TI - What’s new in protein kinase/phosphatase signalling in the control of plant immunity? JO - Essays in Biochemistry PY - 2022 SP - 621-634 AU - Erickson, J. AU - Weckwerth, P. AU - Romeis, T. AU - Lee, J. AU - VL - 66 UR - https://doi.org/10.1042/EBC20210088 DO - 10.1042/ebc20210088 AB - Plant immunity is crucial to plant health but comes at an expense. For optimal plant growth, tight immune regulation is required to prevent unnecessary rechannelling of valuable resources. Pattern- and effector-triggered immunity (PTI/ETI) represent the two tiers of immunity initiated after sensing microbial patterns at the cell surface or pathogen effectors secreted into plant cells, respectively. Recent evidence of PTI-ETI cross-potentiation suggests a close interplay of signalling pathways and defense responses downstream of perception that is still poorly understood. This review will focus on controls on plant immunity through phosphorylation, a universal and key cellular regulatory mechanism. Rather than a complete overview, we highlight “what’s new in protein kinase/phosphatase signalling” in the immunity field. In addition to phosphoregulation of components in the pattern recognition receptor (PRR) complex, we will cover the actions of the major immunity-relevant intracellular protein kinases/phosphatases in the ‘signal relay’, namely calcium-regulated kinases (e.g. calcium-dependent protein kinases, CDPKs), mitogen-activated protein kinases (MAPKs), and various protein phosphatases. We discuss how these factors define a phosphocode that generates cellular decision-making ‘logic gates’, which contribute to signalling fidelity, amplitude, and duration. To underscore the importance of phosphorylation, we summarize strategies employed by pathogens to subvert plant immune phosphopathways. In view of recent game-changing discoveries of ETI-derived resistosomes organizing into calcium-permeable pores, we speculate on a possible calcium-regulated phosphocode as the mechanistic control of the PTI-ETI continuum. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 354 TI - An epiphany for plant resistance proteins and its impact on calcium‐based immune signalling JO - New Phytol. PY - 2022 SP - 769-772 AU - Lee, J. AU - Romeis, T. AU - VL - 234 UR - https://doi.org/10.1111/nph.18085 DO - 10.1111/nph.18085 AB - A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 346 TI - Endocytosis is a significant contributor to uranium(VI) uptake in tobacco (Nicotiana tabacum) BY-2 cells in phosphate-deficient culture JO - Sci. Total Environ. PY - 2022 SP - 153700 AU - John, W. A. AU - Lückel, B. AU - Matschiavelli, N. AU - Hübner, R. AU - Matschi, S. AU - Hoehenwarter, W. AU - Sachs, S. AU - VL - 823 UR - https://doi.org/10.1016/j.scitotenv.2022.153700 DO - 10.1016/j.scitotenv.2022.153700 AB - Endocytosis of metals in plants is a growing field of study involving metal uptake from the rhizosphere. Uranium, which is naturally and artificially released into the rhizosphere, is known to be taken up by certain species of plant, such as Nicotiana tabacum, and we hypothesize that endocytosis contributes to the uptake of uranium in tobacco. The endocytic uptake of uranium was investigated in tobacco BY-2 cells using an optimized setup of culture in phosphate-deficient medium. A combination of methods in biochemistry, microscopy and spectroscopy, supplemented by proteomics, were used to study the interaction of uranium and the plant cell. We found that under environmentally relevant uranium concentrations, endocytosis remained active and contributed to 14% of the total uranium bioassociation. Proteomics analyses revealed that uranium induced a change in expression of the clathrin heavy chain variant, signifying a shift in the type of endocytosis taking place. However, the rate of endocytosis remained largely unaltered. Electron microscopy and energy-dispersive X-ray spectroscopy showed an adsorption of uranium to cell surfaces and deposition in vacuoles. Our results demonstrate that endocytosis constitutes a considerable proportion of uranium uptake in BY-2 cells, and that endocytosed uranium is likely targeted to the vacuole for sequestration, providing a physiologically safer route for the plant than uranium transported through the cytosol.Graphical abstract A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 344 TI - The terminal enzymatic step in piperine biosynthesis is co‐localized with the product piperine in specialized cells of black pepper (Piper nigrum L.) JO - Plant J. PY - 2022 SP - 731–747 AU - Jäckel, L. AU - Schnabel, A. AU - Stellmach, H. AU - Klauß, U. AU - Matschi, S. AU - Hause, G. AU - Vogt, T. AU - VL - 111 UR - https://doi.org/10.1111/tpj.15847 DO - 10.1111/tpj.15847 AB - Piperine (1-piperoyl piperidine) is responsible for the pungent perception of dried black pepper (Pipernigrum) fruits and essentially contributes to the aromatic properties of this spice in combination with ablend of terpenoids. The final step in piperine biosynthesis involves piperine synthase (PS), which catalyzesthe reaction of piperoyl CoA and piperidine to the biologically active and pungent amide. Nevertheless, experimental data on the cellular localization of piperine and the complete biosynthetic pathway are missing. Not only co-localization of enzymes and products, but also potential transport of piperamides to thesink organs is a possible alternative. This work, which includes purification of the native enzyme, immunolocalization, laser microdissection, fluorescence microscopy, and electron microscopy combinedwith liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), providesexperimental evidence that piperine and PS are co-localized in specialized cells of the black pepper fruit peri-sperm. PS accumulates during early stages of fruit development and its level declines before the fruits arefully mature. The product piperine is co-localized to PS and can be monitored at the cellular level by itsstrong bluish fluorescence. Rising piperine levels during fruit maturation are consistent with the increasingnumbers of fluorescent cells within the perisperm. Signal intensities of individual laser-dissected cells whenmonitored by LC-ESI-MS/MS indicate molar concentrations of this alkaloid. Significant levels of piperineand additional piperamides were also detected in cells distributed in the cortex of black pepper roots. Insummary, the data provide comprehensive experimental evidence of and insights into cell-specific biosyn-thesis and storage of piperidine alkaloids, specific and characteristic for the Piperaceae. By a combination offluorescence microscopy and LC-MS/MS analysis we localized the major piperidine alkaloids to specific cellsof the fruit perisperm and the root cortex. Immunolocalization of native piperine and piperamide synthasesshows that enzymes are co-localized with high concentrations of products in these idioblasts. A2 - C1 - Biochemistry of Plant Interactions; Cell and Metabolic Biology ER - TY - JOUR ID - 395 TI - The superior salinity tolerance of bread wheat cultivar Shanrong No. 3 is unlikely to be caused by elevated Ta-sro1 poly-(ADP-ribose) polymerase activity JO - Plant Cell PY - 2022 SP - 4130–4137 AU - Vogt, S. AU - Feijs, K. AU - Hosch, S. AU - De Masi, R. AU - Lintermann, R. AU - Loll, B. AU - Wirthmueller, L. AU - VL - 34 UR - https://doi.org/10.1093/plcell/koac261 DO - 10.1093/plcell/koac261 AB - A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 408 TI - EDS1 complexes are not required for PRR responses and execute TNL‐ETI from the nucleus in Nicotiana benthamiana JO - New Phytol. PY - 2022 SP - 2249-2264 AU - Zönnchen, J. AU - Gantner, J. AU - Lapin, D. AU - Barthel, K. AU - Eschen‐Lippold, L. AU - Erickson, J. L. AU - Landeo Villanueva, S. AU - Zantop, S. AU - Kretschmer, C. AU - Joosten, M. H. A. J. AU - Parker, J. E. AU - Guerois, R. AU - Stuttmann, J. AU - VL - 236 UR - https://doi.org/10.1111/nph.18511 DO - 10.1111/nph.18511 AB - Heterodimeric complexes incorporating the lipase-li ke proteins EDS1 wi th PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in A. thaliana, bolstering of signaling and resistance mediated by cell-s u r face pattern recognition receptors (PRRs). Increasing evidence points to the activation of EDS1 complexes by small molecule binding. •We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in N. benthamiana. •We do not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, in assays monitoring functions of SlEDS1-NbEDS1 complexes in N. benthamiana, mutations within the SlEDS1 catalytic triad can abolish or enhance TNL immunity. Furthermore, nuclear EDS1 accumulation is sufficient for N. benthamianaTNL (Roq1) immunity.•Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Although Solanaceae EDS1 functionally depends on catalytic triad residues in some contexts, our data do not support binding of a TNL-derived small molecule in the triad environment. Whether and how nuclear EDS1 activity connects to membrane pore-f orming RNLs remains unknown. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 505 TI - Possible role of WRKY transcription factors in regulating immunity in Oryza sativa ssp. indica JO - Physiol. Mol. Plant Pathol. PY - 2021 SP - 101623 AU - Sheikh, A. H. AU - Fraz Hussain, R. M. AU - Tabassum, N. AU - Badmi, R. AU - Marillonnet, S. AU - Scheel, D. AU - Lee, J. AU - Sinha, A. AU - VL - 114 UR - https://doi.org/10.1016/j.pmpp.2021.101623 DO - 10.1016/j.pmpp.2021.101623 AB - Plants have developed a robust transcription machinery to combat potential pathogenic organisms. One of the hallmarks of early immune responses is the activation of the WRKY transcription factors post infection. Specific WRKYs proteins from Arabidopsis are known substrates of MAPK pathway to mediate the flg22 elicited early immunity. In the current study, using the Golden Gate cloning strategy, we aim to clone the entire WRKY transcription factor family from Oryza sativa ssp. indica consisting of more than 100 members and study their MAPK interaction and subsequent role in PTI. Using a reporter LUC assay in protoplasts we investigated the early defense responses in a few interesting OsWRKY candidates. Interestingly, we observed stringent regulation of WRKY expression in cells and their transcriptional expression only under specific stress responses. The phenomenon of gene expression regulation by intron retention (IR) was prevalently observed in rice WRKY transcripts. We could show the role of WRKY8, 24, and 77 in early defense responses. It was observed that WRKY24 enhanced the expression of early defense response marker genes like NHL10 while WRKY8 and WRKY77 supressed their expression. This study highlights the complicated mechanism by which OsWRKYs expression is possibly regulated and the distinctive roles of some individual members in plant immunity. At the same time this study serves as a cautionary warning for plant researchers to be mindful of the intron retention mechanism while cloning OsWRKYs. A2 - C1 - Biochemistry of Plant Interactions; Cell and Metabolic Biology ER - TY - JOUR ID - 444 TI - Lysophosphatidylcholine 17:1 from the Leaf Surface of the Wild Potato Species Solanum bulbocastanum Inhibits Phytophthora infestans JO - J. Agr. Food Chem. PY - 2021 SP - 5607-5617 AU - Gorzolka, K. AU - Perino, E. H. B. AU - Lederer, S. AU - Smolka, U. AU - Rosahl, S. AU - VL - 69 UR - https://doi.org/10.1021/acs.jafc.0c07199 DO - 10.1021/acs.jafc.0c07199 AB - Late blight, caused by the oomycete Phytophthora infestans, is economically the most important foliar disease of potato. To assess the importance of the leaf surface, as the site of the first encounter of pathogen and host, we performed untargeted profiling by liquid chromatography–mass spectrometry of leaf surface metabolites of the susceptible cultivated potato Solanum tuberosum and the resistant wild potato species Solanum bulbocastanum. Hydroxycinnamic acid amides, typical phytoalexins of potato, were abundant on the surface of S. tuberosum, but not on S. bulbocastanum. One of the metabolites accumulating on the surface of the wild potato was identified as lysophosphatidylcholine carrying heptadecenoic acid, LPC17:1. In vitro assays revealed that both spore germination and mycelial growth of P. infestans were efficiently inhibited by LPC17:1, suggesting that leaf surface metabolites from wild potato species could contribute to early defense responses against P. infestans. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 436 TI - Plant immune memory in systemic tissue does not involve changes in rapid calcium signaling JO - Front. Plant Sci. PY - 2021 SP - 798230 AU - Eichstädt, B. AU - Lederer, S. AU - Trempel, F. AU - Jiang, X. AU - Guerra, T. AU - Waadt, R. AU - Lee, J. AU - Liese, A. AU - Romeis, T. AU - VL - 12 UR - https://doi.org/10.3389/fpls.2021.798230 DO - 10.3389/fpls.2021.798230 AB - Upon pathogen recognition, a transient rise in cytoplasmic calcium levels is one of the earliest events in plants and a prerequisite for defense initiation and signal propagation from a local site to systemic plant tissues. However, it is unclear if calcium signaling differs in the context of priming: Do plants exposed to a first pathogen stimulus and have consequently established systemic acquired resistance (SAR) display altered calcium responses to a second pathogen stimulus? Several calcium indicator systems including aequorin, YC3.6 or R-GECO1 have been used to document local calcium responses to the bacterial flg22 peptide but systemic calcium imaging within a single plant remains a technical challenge. Here, we report on an experimental approach to monitor flg22-induced calcium responses in systemic leaves of primed plants. The calcium-dependent protein kinase CPK5 is a key calcium sensor and regulator of the NADPH oxidase RBOHD and plays a role in the systemic calcium-ROS signal propagation. We therefore compared flg22-induced cytoplasmic calcium changes in Arabidopsis wild-type, cpk5 mutant and CPK5-overexpressing plants (exhibiting constitutive priming) by introgressing the calcium indicator R-GECO1-mTurquoise that allows internal normalization through mTurquoise fluorescence. Aequorin-based analyses were included for comparison. Based on the R-GECO1-mTurquoise data, CPK5-OE appears to reinforce an “oscillatory-like” Ca2+ signature in flg22-treated local tissues. However, no change was observed in the flg22-induced calcium response in the systemic tissues of plants that had been pre-challenged by a priming stimulus – neither in wild-type nor in cpk5 or CPK5-OE-lines. These data indicate that the mechanistic manifestation of a plant immune memory in distal plant parts required for enhanced pathogen resistance does not include changes in rapid calcium signaling upstream of CPK5 but rather relies on downstream defense responses. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 427 TI - Exocyst subunit Exo70B2 is linked to immune signaling and autophagy JO - Plant Cell PY - 2021 SP - 404-419 AU - Brillada, C. AU - Teh, O.-K. AU - Ditengou, F. A. AU - Lee, C.-W. AU - Klecker, T. AU - Saeed, B. AU - Furlan, G. AU - Zietz, M. AU - Hause, G. AU - Eschen-Lippold, L. AU - Hoehenwarter, W. AU - Lee, J. AU - Ott, T. AU - Trujillo, M. AU - VL - 33 UR - DO - 10.1093/plcell/koaa022 AB - During the immune response, activation of the secretory pathway is key to mounting an effective response, while gauging its output is important to maintain cellular homeostasis. The Exo70 subunit of the exocyst functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst regulation remains challenging. We show that, in Arabidopsis thaliana, Exo70B2 behaves as a bona fide exocyst subunit. Conversely, treatment with the salicylic acid (SA) defence hormone analog benzothiadiazole (BTH), or the immunogenic peptide flg22, induced Exo70B2 transport into the vacuole. We reveal that Exo70B2 interacts with AUTOPHAGY-RELATED PROTEIN 8 (ATG8) via two ATG8-interacting motives (AIMs) and its transport into the vacuole is dependent on autophagy. In line with its role in immunity, we discovered that Exo70B2 interacted with and was phosphorylated by the kinase MPK3. Mimicking phosphorylation had a dual impact on Exo70B2: first, by inhibiting localization at sites of active secretion, and second, it increased the interaction with ATG8. Phosphonull variants displayed higher effector-triggered immunity (ETI) and were hypersensitive to BTH, which induce secretion and autophagy. Our results suggest a molecular mechanism by which phosphorylation diverts Exo70B2 from the secretory into the autophagy pathway for its degradation, to dampen secretory activity. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 467 TI - Functional requirement of the Arabidopsis importin-α nuclear transport receptor family in autoimmunity mediated by the NLR protein SNC1 JO - Plant J. PY - 2021 SP - 994-1009 AU - Lüdke, D. AU - Roth, C. AU - Kamrad, S. A. AU - Messerschmidt, J. AU - Hartken, D. AU - Apel, J. AU - Hörnich, B. F. AU - Yan, Q. AU - Kusch, S. AU - Klenke, M. AU - Gunkel, A. AU - Wirthmüller, L. AU - Wiermer, M. AU - VL - 105 UR - https://onlinelibrary.wiley.com/doi/full/10.1111/tpj.15082 DO - 10.1111/tpj.15082 AB - Functional requirement of the Arabidopsis importin-a nuclear transport receptor familIMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is one of nine importin-α isoforms in Arabidopsis that recruit nuclear localization signal-containing cargo proteins to the nuclear import machinery. IMP-α3/MOS6 is required genetically for full autoimmunity of the nucleotide-binding leucine-rich repeat immune receptor mutant snc1 (suppressor of npr1-1, constitutive 1) and MOS6 also contributes to basal disease resistance. Here, we investigated the contribution of the other importin-α genes to both types of immune responses, and we analyzed potential interactions of all importin-α isoforms with SNC1. By using reverse-genetic analyses in Arabidopsis and protein-protein interaction assays in Nicotiana benthamiana, we provide evidence that among the nine α-importins in Arabidopsis, IMP-α3/MOS6 is the main nuclear transport receptor of SNC1, and that IMP-α3/MOS6 is required selectively for autoimmunity of snc1 and basal resistance to mildly virulent Pseudomonas syringae in Arabidopsis. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 466 TI - An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca2+ and H+ reveals new insights into ion signaling in plants JO - New Phytol. PY - 2021 SP - 2292-2310 AU - Li, K. AU - Prada, J. AU - Damineli, D. S. C. AU - Liese, A. AU - Romeis, T. AU - Dandekar, T. AU - Feijó, J. A. AU - Hedrich, R. AU - Konrad, K. R. AU - VL - 230 UR - https://doi.org/10.1111/nph.17202 DO - 10.1111/nph.17202 AB - Whereas the role of calcium ions (Ca2+) in plant signaling is well studied, the physiological significance of pH-changes remains largely undefined.Here we developed CapHensor, an optimized dual-reporter for simultaneous Ca2+ and pH ratio-imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio-temporal relationships between membrane voltage, Ca2+- and pH-dynamics revealed interconnections previously not described.In tobacco PTs, we demonstrated Ca2+-dynamics lag behind pH-dynamics during oscillatory growth, and pH correlates more with growth than Ca2+. In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ elevation. Preventing the alkalization blocked GC ABA-responses and even opened stomata in the presence of ABA, disclosing an important pH-dependent GC signaling node. In MCs, a flg22-induced membrane depolarization preceded Ca2+-increases and cytosolic acidification by c. 2 min, suggesting a Ca2+/pH-independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage-, Ca2+- and pH-responses.We propose close interrelation in Ca2+- and pH-signaling that is cell type- and stimulus-specific and the pH having crucial roles in regulating PT growth and stomata movement. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 493 TI - A phosphoinositide 5-phosphatase from Solanum tuberosum is activated by PAMP-treatment and may antagonize phosphatidylinositol 4,5-bisphosphate at Phytophthora infestans infection sites JO - New Phytol. PY - 2021 SP - 469-487 AU - Rausche, J. AU - Stenzel, I. AU - Stauder, R. AU - Fratini, M. AU - Trujillo, M. AU - Heilmann, I. AU - Rosahl, S. AU - VL - 229 UR - DO - 10.1111/nph.16853 AB - Potato (Solanum tuberosum) plants susceptible to late blight disease caused by the oomycete Phytophthora infestans display enhanced resistance upon infiltration with the pathogen-associated molecular pattern (PAMP), Pep-13. Here, we characterize a potato gene similar to Arabidopsis 5-phosphatases which was identified in transcript arrays performed to identify Pep-13 regulated genes, and termed StIPP.Recombinant StIPP protein specifically dephosphorylated the D5-position of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in vitro. Other phosphoinositides or soluble inositolpolyphosphates were not converted.When transiently expressed in tobacco (Nicotiana tabacum) pollen tubes, a StIPP-YFP fusion localized to the subapical plasma membrane and antagonized PtdIns(4,5)P2-dependent effects on cell morphology, indicating in vivo functionality. Phytophthora infestans-infection of N. benthamiana leaf epidermis cells resulted in relocalization of StIPP-GFP from the plasma membrane to the extra-haustorial membrane (EHM). Colocalizion with the effector protein RFP-AvrBlb2 at infection sites is consistent with a role of StIPP in the plant–oomycete interaction. Correlation analysis of fluorescence distributions of StIPP-GFP and biosensors for PtdIns(4,5)P2 or phosphatidylinositol 4-phosphate (PtdIns4P) indicate StIPP activity predominantly at the EHM.In Arabidopsis protoplasts, expression of StIPP resulted in the stabilization of the PAMP receptor, FLAGELLIN-SENSITIVE 2, indicating that StIPP may act as a PAMP-induced and localized antagonist of PtdIns(4,5)P2-dependent processes during plant immunity. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 502 TI - Improving plant drought tolerance and growth under water limitation through combinatorial engineering of signaling networks JO - Plant Biotechnol. J. PY - 2021 SP - 74–86 AU - Schulz, P. AU - Piepenburg, K. AU - Lintermann, R. AU - Herde, M. AU - Schöttler, M. A. AU - Schmidt, L. K. AU - Ruf, S. AU - Kudla, J. AU - Romeis, T. AU - Bock, R. AU - VL - 19 UR - DO - 10.1111/pbi.13441 AB - Agriculture is by far the biggest water consumer on our planet, accounting for 70 percent of all freshwater withdrawals. Climate change and a growing world population increase pressure on agriculture to use water more efficiently (‘more crop per drop’). Water‐use efficiency (WUE) and drought tolerance of crops are complex traits that are determined by many physiological processes whose interplay is not well understood. Here we describe a combinatorial engineering approach to optimize signaling networks involved in the control of stress tolerance. Screening a large population of combinatorially transformed plant lines, we identified a combination of calcium‐dependent protein kinase genes that confers enhanced drought stress tolerance and improved growth under water‐limiting conditions. Targeted introduction of this gene combination into plants increased plant survival under drought and enhanced growth under water‐limited conditions. Our work provides an efficient strategy for engineering complex signaling networks to improve plant performance under adverse environmental conditions, which does not depend on prior understanding of network function. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 540 TI - Calcium-Dependent Protein Kinase CPK1 Controls Cell Death by In Vivo Phosphorylation of Senescence Master Regulator ORE1 JO - Plant Cell PY - 2020 SP - 1610-1625 AU - Durian, G. AU - Sedaghatmehr, M. AU - Matallana-Ramirez, L. P. AU - Schilling, S. M. AU - Schaepe, S. AU - Guerra, T. AU - Herde, M. AU - Witte, C.-P. AU - Mueller-Roeber, B. AU - Schulze, W. X. AU - Balazadeh, S. AU - Romeis, T. AU - VL - 32 UR - DO - 10.1105/tpc.19.00810 AB - Calcium-regulated protein kinases are key components of are key components of intracellular signaling in plants that mediate rapid stress-induced responses to changes in the environment. To identify in vivo phosphorylation substrates of CALCIUM-DEPENDENT PROTEIN KINASE1 (CPK1), we analyzed the conditional expression of constitutively active CPK1 in conjunction with in vivo phosphoproteomics. We identified Arabidopsis thaliana ORESARA1 (ORE1), the developmental master regulator of senescence, as a direct CPK1 phosphorylation substrate. CPK1 phosphorylates ORE1 at a hotspot within an intrinsically disordered region. This augments transcriptional activation by ORE1 of its downstream target gene BIFUNCTIONAL NUCLEASE1 (BFN1). Plants that overexpress ORE1, but not an ORE1 variant lacking the CPK1 phosphorylation hotspot, promote early senescence. Furthermore, ORE1 is required for enhanced cell death induced by CPK1 signaling. Our data validate the use of conditional expression of an active enzyme combined with phosphoproteomics to decipher specific kinase target proteins of low abundance, of transient phosphorylation, or in yet undescribed biological contexts. Here, we have identified that senescence is not just under molecular surveillance manifested by stringent gene regulatory control over ORE1. In addition, the decision to die is superimposed by an additional layer of control towards ORE1 via its post-translational modification linked to the calcium-regulatory network through CPK1. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 538 TI - Root exudate composition of grass and forb species in natural grasslands JO - Sci. Rep. PY - 2020 SP - 10691 AU - Dietz, S. AU - Herz, K. AU - Gorzolka, K. AU - Jandt, U. AU - Bruelheide, H. AU - Scheel, D. AU - VL - 10 UR - DO - 10.1038/s41598-019-54309-5 AB - Plants exude a diverse cocktail of metabolites into the soil as response to exogenous and endogenous factors. So far, root exudates have mainly been studied under artificial conditions due to methodological difficulties. In this study, each five perennial grass and forb species were investigated for polar and semi-polar metabolites in exudates under field conditions. Metabolite collection and untargeted profiling approaches combined with a novel classification method allowed the designation of 182 metabolites. The composition of exuded polar metabolites depended mainly on the local environment, especially soil conditions, whereas the pattern of semi-polar metabolites was primarily affected by the species identity. The profiles of both polar and semi-polar metabolites differed between growth forms, with grass species being generally more similar to each other and more responsive to the abiotic environment than forb species. This study demonstrated the feasibility of investigating exudates under field conditions and to identify the driving factors of exudate composition. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 530 TI - Reshaping of the Arabidopsis thaliana Proteome Landscape and Co-regulation of Proteins in Development and Immunity JO - Mol. Plant PY - 2020 SP - 1709-1732 AU - Bassal, M. AU - Abukhalaf, M. AU - Majovsky, P. AU - Thieme, D. AU - Herr, T. AU - Ayash, M. AU - Tabassum, N. AU - Al Shweiki, M. R. AU - Proksch, C. AU - Hmedat, A. AU - Ziegler, J. AU - Lee, J. AU - Neumann, S. AU - Hoehenwarter, W. AU - VL - 13 UR - DO - 10.1016/j.molp.2020.09.024 AB - Proteome remodeling is a fundamental adaptive response, and proteins in complexes and functionally related proteins are often co-expressed. Using a deep sampling strategy we define core proteomes of Arabidopsis thaliana tissues with around 10 000 proteins per tissue, and absolutely quantify (copy numbers per cell) nearly 16 000 proteins throughout the plant lifecycle. A proteome-wide survey of global post-translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue- and age-specific roles of entire signaling modules regulating transcription in photosynthesis, seed development, and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of cysteine-rich receptor-like kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were found to be co-expressed in a tissue- and age-specific manner, indicating functional promiscuity in the assembly of these less-studied protein complexes in Arabidopsis. Furthermore, we characterized detailed proteome remodeling in basal immunity by treating Arabidopsis seeldings with flg22. Through simultaneously monitoring phytohormone and transcript changes upon flg22 treatment, we obtained strong evidence of suppression of jasmonate (JA) and JA-isoleucine (JA-Ile) levels by deconjugation and hydroxylation by IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2), respectively, under the control of JASMONATE INSENSITIVE 1 (MYC2), suggesting an unrecognized role of a new JA regulatory switch in pattern-triggered immunity. Taken together, the datasets generated in this study present extensive coverage of the Arabidopsis proteome in various biological scenarios, providing a rich resource available to the whole plant science community. A2 - C1 - Biochemistry of Plant Interactions; Molecular Signal Processing ER - TY - JOUR ID - 554 TI - Phosphorylation of the CAMTA3 Transcription Factor Triggers Its Destabilization and Nuclear Export JO - Plant Physiol. PY - 2020 SP - 1056-1071 AU - Jiang, X. AU - Hoehenwarter, W. AU - Scheel, D. AU - Lee, J. AU - VL - 184 UR - https://academic.oup.com/plphys/article/184/2/1056/6117871 DO - 10.1104/pp.20.00795 AB - The Arabidopsis (Arabidopsis thaliana) calmodulin-binding transcription activator3 (CAMTA3) is a repressor of immunity-related genes but an activator of cold-induced or general stress-responsive genes in plants. Post-transcriptional or posttranslational mechanisms have been proposed to control CAMTA3 functions in different stress responses. Here, we show that treatment with the bacterial flg22 elicitor induces CAMTA3 phosphorylation, which is accompanied by its destabilization and nuclear export. Two flg22-responsive mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, directly phosphorylate CAMTA3, with the phospho-sites contributing to CAMTA3 degradation and suppression of downstream target gene expression. However, the flg22-induced nuclear export and phospho-mobility shift can still be observed for the CAMTA3 phospho-null variant of the MAPK-modified sites, suggesting additional flg22-responsive kinases might be involved. Taken together, we propose that flg22-induced CAMTA3 depletion facilitates de-repression of downstream defense target genes, which involves phosphorylation, increased protein turnover, and nucleo-cytoplasmic trafficking. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 548 TI - Calcium‐dependent protein kinase 5 links calcium‐signaling with N‐Hydroxy‐L‐pipecolic acid‐ and SARD1‐dependent immune memory in systemic acquired resistance JO - New Phytol. PY - 2020 SP - 310-325 AU - Guerra, T. AU - Schilling, S. AU - Hake, K. AU - Gorzolka, K. AU - Sylvester, F.-P. AU - Conrads, B. AU - Westermann, B. AU - Romeis, T. AU - VL - 225 UR - DO - 10.1111/nph.16147 AB - Systemic acquired resistance (SAR) prepares infected plants for faster and stronger defense activation upon subsequent attacks. SAR requires an information relay from primary infection to distal tissue and the initiation and maintenance of a self‐maintaining phytohormone salicylic acid (SA)‐defense loop.In spatial and temporal resolution, we show that calcium‐dependent protein kinase CPK5 contributes to immunity and SAR. In local basal resistance, CPK5 functions upstream of SA synthesis, perception, and signaling. In systemic tissue, CPK5 signaling leads to accumulation of SAR‐inducing metabolite N‐hydroxy‐L‐pipecolic acid (NHP) and SAR marker genes, including Systemic Acquired Resistance Deficient 1 (SARD1)Plants of increased CPK5, but not CPK6, signaling display an ‘enhanced SAR’ phenotype towards a secondary bacterial infection. In the sard1‐1 background, CPK5‐mediated basal resistance is still mounted, but NHP concentration is reduced and enhanced SAR is lost.The biochemical analysis estimated CPK5 half maximal kinase activity for calcium, K50 [Ca2+], to be c. 100 nM, close to the cytoplasmic resting level. This low threshold uniquely qualifies CPK5 to decode subtle changes in calcium, a prerequisite to signal relay and onset and maintenance of priming at later time points in distal tissue. Our data explain why CPK5 functions as a hub in basal and systemic plant immunity. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER - TY - JOUR ID - 547 TI - N-hydroxypipecolic acid: a general and conserved activator of systemic plant immunity JO - J. Exp. Bot. PY - 2020 SP - 6193-6196 AU - Guerra, T. AU - Romeis, T. AU - VL - 71 UR - DO - 10.1093/jxb/eraa345 AB - Long-lasting and broad-spectrum disease resistance throughout plants is an ever-important objective in basic and applied plant and crop research. While the recent identification of N-hydroxpipecolic acid (NHP) and its central role in systemic plant immunity in the model Arabidopsis thaliana provides a conceptual framework toward this goal, Schnake et al. (2020) quantify levels of NHP and its direct precursor in six mono- and dicotyledonous plant species subsequent to attacks by their natural pathogens, thereby implicating (phloem-mobile) NHP as a general and conserved activator of disease resistance. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 614 TI - Sp(l)icing up PepR signalling JO - Nat. Plants PY - 2020 SP - 912-913 AU - Wirthmueller, L. AU - Romeis, T. AU - VL - 6 UR - DO - 10.1038/s41477-020-0708-1 AB - Alternative splicing provides a fundamental and ubiquitous mechanism of gene regulation. Stimuli-induced retention of introns introduces novel proteoforms with altered signalling output: full-length CPK28 blocks immune signalling, while a truncated variant, lacking calcium responsiveness, promotes it. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 601 TI - Phosphorylation‐dependent control of an RNA granule‐localized protein that fine‐tunes defence gene expression at a post‐transcriptional level JO - Plant J. PY - 2020 SP - 1023-1039 AU - Tabassum, N. AU - Eschen-Lippold, L. AU - Athmer, B. AU - Baruah, M. AU - Brode, M. AU - Maldonado-Bonilla, L. D. AU - Hoehenwarter, W. AU - Hause, G. AU - Scheel, D. AU - Lee, J. AU - VL - 101 UR - DO - 10.1111/tpj.14573 AB - Mitogen‐activated protein kinase (MAPK) cascades are key signalling modules of plant defence responses to pathogen‐associated molecular patterns (PAMPs, e.g. bacterial flg22 peptide). The Tandem Zinc Finger Protein 9 (TZF9) is an RNA‐binding protein that is phosphorylated by two PAMP‐responsive MAPKs, MPK3 and MPK6. We mapped the major phosphosites in TZF9 and showed their importance for controlling in vitro RNA‐binding activity, in vivo flg22‐induced rapid disappearance of TZF9‐labelled processing body‐like structures and TZF9 protein turnover. Microarray analysis showed a strong discordance between transcriptome (total mRNA) and translatome (polysome‐associated mRNA) in the tzf9 mutant, with more mRNAs associated to ribosomes in the absence of TZF9. This suggests that TZF9 may sequester and inhibit translation of subsets of mRNAs. Fittingly, TZF9 physically interacts with poly(A)‐binding protein 2 (PAB2), a hallmark constituent of stress granules – a site for stress‐induced translational stalling/arrest. TZF9 even promotes stress granule assembly in the absence of stress. Hence, MAPKs may control defence gene expression post‐transcriptionally through release from translation arrest within TZF9‐PAB2‐containing RNA granules or perturbing PAB2 functions in translation control (e.g. in the mRNA closed‐loop model of translation). A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions; Cell and Metabolic Biology ER - TY - JOUR ID - 610 TI - The Arabidopsis exocyst subunits EXO70B1 and EXO70B2 regulate FLS2 homeostasis at the plasma membrane JO - New Phytol. PY - 2020 SP - 529-544 AU - Wang, W. AU - Liu, N. AU - Gao, C. AU - Cai, H. AU - Romeis, T. AU - Tang, D. AU - VL - 227 UR - DO - 10.1111/nph.16515 AB - The plasma membrane (PM)‐localized receptor kinase FLAGELLIN SENSING 2 (FLS2) recognizes bacterial flagellin or its immunogenic epitope flg22, and initiates microbe‐associated molecular pattern‐triggered immunity, which inhibits infection by bacterial pathogens. The localization, abundance and activity of FLS2 are under dynamic control.Here, we demonstrate that Arabidopsis thaliana EXO70B1, a subunit of the exocyst complex, plays a critical role in FLS2 signaling that is independent of the truncated Toll/interleukin‐1 receptor‐nucleotide binding sequence protein TIR‐NBS2 (TN2). In the exo70B1‐3 mutant, the abundance of FLS2 protein at the PM is diminished, consistent with the impaired flg22 response of this mutant. EXO70B1‐GFP plants showed increased FLS2 accumulation at the PM and therefore enhanced FLS2 signaling.The EXO70B1‐mediated trafficking of FLS2 to the PM is partially independent of the PENETRATION 1 (PEN1)‐containing secretory pathway. In addition, EXO70B1 interacts with EXO70B2, a close homolog of EXO70B1, and both proteins associate with FLS2 and contribute to the accumulation of FLS2 at the PM.Taken together, our data suggest that the exocyst complex subunits EXO70B1 and EXO70B2 regulate the trafficking of FLS2 to the PM, which represents a new layer of regulation of FLS2 function in plant immunity. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 604 TI - A mutation in Asparagine‐Linked Glycosylation 12 (ALG12) leads to receptor misglycosylation and attenuated responses to multiple microbial elicitors JO - FEBS Lett. PY - 2020 SP - 2440-2451 AU - Trempel, F. AU - Eschen‐Lippold, L. AU - Bauer, N. AU - Ranf, S. AU - Westphal, L. AU - Scheel, D. AU - Lee, J. AU - VL - 594 UR - DO - 10.1002/1873-3468.13850 AB - Changes in cellular calcium levels are one of the earliest signalling events in plants exposed to pathogens or other exogenous factors. In a genetic screen, we identified an Arabidopsis thaliana ‘changed calcium elevation 1 ’ (cce1 ) mutant with attenuated calcium response to the bacterial flagellin flg22 peptide and several other elicitors. Whole genome re‐sequencing revealed a mutation in ALG12 (Asparagine‐Linked Glycosylation 12 ) that encodes the mannosyltransferase responsible for adding the eighth mannose residue in an α‐1,6 linkage to the dolichol‐PP‐oligosaccharide N ‐glycosylation glycan tree precursors. While properly targeted to the plasma membrane, misglycosylation of several receptors in the cce1 background suggests that N ‐glycosylation is required for proper functioning of client proteins. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 744 TI - pH effects on plant calcium fluxes: lessons from acidification-mediated calcium elevation induced by the γ-glutamyl-leucine dipeptide identified from Phytophthora infestans JO - Sci. Rep. PY - 2019 SP - 4733 AU - Westphal, L. AU - Strehmel, N. AU - Eschen-Lippold, L. AU - Bauer, N. AU - Westermann, B. AU - Rosahl, S. AU - Scheel, D. AU - Lee, J. AU - VL - 9 UR - DO - 10.1038/s41598-019-41276-0 AB - Cytosolic Ca2+ ([Ca2+]cyt) elevation is an early signaling response upon exposure to pathogen-derived molecules (so-called microbe-associated molecular patterns, MAMPs) and has been successfully used as a quantitative read-out in genetic screens to identify MAMP receptors or their associated components. Here, we isolated and identified by mass spectrometry the dipeptide γ-Glu-Leu as a component of a Phytophthora infestans mycelium extract that induces [Ca2+]cyt elevation. Treatment of Arabidopsis seedlings with synthetic γ-Glu-Leu revealed stimulatory effects on defense signaling, including a weak enhancement of the expression of some MAMP-inducible genes or affecting the refractory period to a second MAMP elicitation. However, γ-Glu-Leu is not a classical MAMP since pH adjustment abolished these activities and importantly, the observed effects of γ-Glu-Leu could be recapitulated by mimicking extracellular acidification. Thus, although γ-Glu-Leu can act as a direct agonist of calcium sensing receptors in animal systems, the Ca2+-mobilizing activity in plants reported here is due to acidification. Low pH also shapes the Ca2+ signature of well-studied MAMPs (e.g. flg22) or excitatory amino acids such as glutamate. Overall, this work serves as a cautionary reminder that in defense signaling studies where Ca2+ flux measurements are concerned, it is important to monitor and consider the effects of pH. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER - TY - JOUR ID - 664 TI - Differential N-end Rule Degradation of RIN4/NOI Fragments Generated by the AvrRpt2 Effector Protease JO - Plant Physiol. PY - 2019 SP - 2272-2289 AU - Goslin, K. AU - Eschen-Lippold, L. AU - Naumann, C. AU - Linster, E. AU - Sorel, M. AU - Klecker, M. AU - de Marchi, R. AU - Kind, A. AU - Wirtz, M. AU - Lee, J. AU - Rajjou, L. AU - Graciet, E. AU - VL - 180 UR - DO - 10.1104/pp.19.00251 AB - In plants, the protein RPM1-INTERACTING PROTEIN4 (RIN4) is a central regulator of both pattern-triggered immunity and effector-triggered immunity. RIN4 is targeted by several effectors, including the Pseudomonas syringae protease effector AvrRpt2. Cleavage of RIN4 by AvrRpt2 generates potentially unstable RIN4 fragments, whose degradation leads to the activation of the resistance protein RESISTANT TO P. SYRINGAE2. Hence, identifying the determinants of RIN4 degradation is key to understanding RESISTANT TO P. SYRINGAE2–mediated effector-triggered immunity, as well as virulence functions of AvrRpt2. In addition to RIN4, AvrRpt2 cleaves host proteins from the nitrate-induced (NOI) domain family. Although cleavage of NOI domain proteins by AvrRpt2 may contribute to pattern-triggered immunity regulation, the (in)stability of these proteolytic fragments and the determinants regulating their stability remain unexamined. Notably, a common feature of RIN4, and of many NOI domain protein fragments generated by AvrRpt2 cleavage, is the exposure of a new N-terminal residue that is destabilizing according to the N-end rule. Using antibodies raised against endogenous RIN4, we show that the destabilization of AvrRpt2-cleaved RIN4 fragments is independent of the N-end rule pathway (recently renamed the N-degron pathway). By contrast, several NOI domain protein fragments are genuine substrates of the N-degron pathway. The discovery of this set of substrates considerably expands the number of known proteins targeted for degradation by this ubiquitin-dependent pathway in plants. These results advance our current understanding of the role of AvrRpt2 in promoting bacterial virulence. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 698 TI - Early Pep-13-induced immune responses are SERK3A/B-dependent in potato JO - Sci. Rep. PY - 2019 SP - 18380 AU - Nietzschmann, L. AU - Gorzolka, K. AU - Smolka, U. AU - Matern, A. AU - Eschen-Lippold, L. AU - Scheel, D. AU - Rosahl, S. AU - VL - 9 UR - DO - 10.1038/s41598-019-54944-y AB - Potato plants treated with the pathogen-associated molecular pattern Pep-13 mount salicylic acid- and jasmonic acid-dependent defense responses, leading to enhanced resistance against Phytophthora infestans, the causal agent of late blight disease. Recognition of Pep-13 is assumed to occur by binding to a yet unknown plasma membrane-localized receptor kinase. The potato genes annotated to encode the co-receptor BAK1, StSERK3A and StSERK3B, are activated in response to Pep-13 treatment. Transgenic RNAi-potato plants with reduced expression of both SERK3A and SERK3B were generated. In response to Pep-13 treatment, the formation of reactive oxygen species and MAP kinase activation, observed in wild type plants, is highly reduced in StSERK3A/B-RNAi plants, suggesting that StSERK3A/B are required for perception of Pep-13 in potato. In contrast, defense gene expression is induced by Pep-13 in both control and StSERK3A/B-depleted plants. Altered morphology of StSERK3A/B-RNAi plants correlates with major shifts in metabolism, as determined by untargeted metabolite profiling. Enhanced levels of hydroxycinnamic acid amides, typical phytoalexins of potato, in StSERK3A/B-RNAi plants are accompanied by significantly decreased levels of flavonoids and steroidal glycoalkaloids. Thus, altered metabolism in StSERK3A/B-RNAi plants correlates with the ability of StSERK3A/B-depleted plants to mount defense, despite highly decreased early immune responses. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 691 TI - A PAMP‐triggered MAPK cascade inhibits phosphatidylinositol 4,5‐bisphosphate production by PIP5K6 in Arabidopsis thaliana JO - New Phytol. PY - 2019 SP - 833-847 AU - Menzel, W. AU - Stenzel, I. AU - Helbig, L. AU - Krishnamoorthy, P. AU - Neumann, S. AU - Eschen-Lippold, L. AU - Heilmann, M. AU - Lee, J. AU - Heilmann, I. AU - VL - 224 UR - DO - 10.1111/nph.16069 AB - The phosphoinositide kinase PIP5K6 has recently been identified as a target for the mitogen‐activated protein kinase (MAPK) MPK6. Phosphorylation of PIP5K6 inhibited the production of phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2), impacting membrane trafficking and cell expansion in pollen tubes. Here, we analyzed whether MPK6 regulated PIP5K6 in vegetative Arabidopsis cells in response to the pathogen‐associated molecular pattern (PAMP) flg22.Promoter‐β‐glucuronidase analyses and quantitative real‐time reverse transcription polymerase chain reaction data show PIP5K6 expressed throughout Arabidopsis tissues. Upon flg22 treatment of transgenic protoplasts, the PIP5K6 protein was phosphorylated, and this modification was reduced for a PIP5K6 variant lacking MPK6‐targeted residues, or in protoplasts from mpk6 mutants.Upon flg22 treatment of Arabidopsis plants, phosphoinositide levels mildly decreased and a fluorescent reporter for PtdIns(4,5)P2 displayed reduced plasma membrane association, contrasting with phosphoinositide increases reported for abiotic stress responses. Flg22 treatment and chemical induction of the upstream MAPK kinase, MKK5, decreased phosphatidylinositol 4‐phosphate 5‐kinase activity in mesophyll protoplasts, indicating that the flg22‐activated MAPK cascade limited PtdIns(4,5)P2 production. PIP5K6 expression or PIP5K6 protein abundance changed only marginally upon flg22 treatment, consistent with post‐translational control of PIP5K6 activity. PtdIns(4,5)P2‐dependent endocytosis of FM 4‐64, PIN2 and the NADPH‐oxidase RbohD were reduced upon flg22 treatment or MKK5 induction. Reduced RbohD‐endocytosis was correlated with enhanced ROS production.We conclude that MPK6‐mediated phosphorylation of PIP5K6 limits the production of a functional PtdIns(4,5)P2 pool upon PAMP perception. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 688 TI - A substrate of the ABC transporter PEN3 stimulates bacterial flagellin (flg22)-induced callose deposition in Arabidopsis thaliana JO - J. Biol. Chem. PY - 2019 SP - 6857-6870 AU - Matern, A. AU - Böttcher, C. AU - Eschen-Lippold, L. AU - Westermann, B. AU - Smolka, U. AU - Döll, S. AU - Trempel, F. AU - Aryal, B. AU - Scheel, D. AU - Geisler, M. AU - Rosahl, S. AU - VL - 294 UR - DO - 10.1074/jbc.RA119.007676 AB - Nonhost resistance of Arabidopsis thaliana against Phytophthora infestans, a filamentous eukaryotic microbe and the causal agent of potato late blight, is based on a multilayered defense system. Arabidopsis thaliana controls pathogen entry through the penetration-resistance genes PEN2 and PEN3, encoding an atypical myrosinase and an ABC transporter, respectively, required for synthesis and export of unknown indole compounds. To identify pathogen-elicited leaf surface metabolites and further unravel nonhost resistance in Arabidopsis, we performed untargeted metabolite profiling by incubating a P. infestans zoospore suspension on leaves of WT or pen3 mutant Arabidopsis plants. Among the plant-secreted metabolites, 4-methoxyindol-3-yl-methanol and S-(4-methoxy-indol-3-yl-methyl) cysteine were detected in spore suspensions recollected from WT plants, but at reduced levels from the pen3 mutant plants. In both whole-cell and microsome-based assays, 4-methoxyindol-3-yl-methanol was transported in a PEN3-dependent manner, suggesting that this compound is a PEN3 substrate. The syntheses of both compounds were dependent on functional PEN2 and phytochelatin synthase 1. None of these compounds inhibited mycelial growth of P. infestans in vitro. Of note, exogenous application of 4-methoxyindol-3-yl methanol slightly elevated cytosolic Ca2+ levels and enhanced callose deposition in hydathodes of seedlings treated with a bacterial pathogen-associated molecular pattern (PAMP), flagellin (flg22). Loss of flg22-induced callose deposition in leaves of pen3 seedlings was partially reverted by the addition of 4-methoxyindol-3-yl methanol. In conclusion, we have identified a specific indole compound that is a substrate for PEN3 and contributes to the plant defense response against microbial pathogens. A2 - C1 - Biochemistry of Plant Interactions; Bioorganic Chemistry ER - TY - JOUR ID - 848 TI - Two Strategies of Pseudomonas syringae to Avoid Recognition of the HopQ1 Effector in Nicotiana Species JO - Front. Plant Sci. PY - 2018 SP - 978 AU - Zembek, P. AU - Danilecka, A. AU - Hoser, R. AU - Eschen-Lippold, L. AU - Benicka, M. AU - Grech-Baran, M. AU - Rymaszewski, W. AU - Barymow-Filoniuk, I. AU - Morgiewicz, K. AU - Kwiatkowski, J. AU - Piechocki, M. AU - Poznanski, J. AU - Lee, J. AU - Hennig, J. AU - Krzymowska, M. AU - VL - 9 UR - DO - 10.3389/fpls.2018.00978 AB - Pseudomonas syringae employs a battery of type three secretion effectors to subvert plant immune responses. In turn, plants have developed receptors that recognize some of the bacterial effectors. Two strain-specific HopQ1 effector variants (for Hrp outer protein Q) from the pathovars phaseolicola 1448A (Pph) and tomato DC3000 (Pto) showed considerable differences in their ability to evoke disease symptoms in Nicotiana benthamiana. Surprisingly, the variants differ by only six amino acids located mostly in the N-terminal disordered region of HopQ1. We found that the presence of serine 87 and leucine 91 renders PtoHopQ1 susceptible to N-terminal processing by plant proteases. Substitutions at these two positions did not strongly affect PtoHopQ1 virulence properties in a susceptible host but they reduced bacterial growth and accelerated onset of cell death in a resistant host, suggesting that N-terminal mutations rendered PtoHopQ1 susceptible to processing in planta and, thus, represent a mechanism of recognition avoidance. Furthermore, we found that co-expression of HopR1, another effector encoded within the same gene cluster masks HopQ1 recognition in a strain-dependent manner. Together, these data suggest that HopQ1 is under high host-pathogen co-evolutionary selection pressure and P. syringae may have evolved differential effector processing or masking as two independent strategies to evade HopQ1 recognition, thus revealing another level of complexity in plant – microbe interactions. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 846 TI - Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to RADICAL-INDUCED CELL DEATH1 JO - New Phytol. PY - 2018 SP - 232-248 AU - Wirthmueller, L. AU - Asai, S. AU - Rallapalli, G. AU - Sklenar, J. AU - Fabro, G. AU - Kim, D. S. AU - Lintermann, R. AU - Jaspers, P. AU - Wrzaczek, M. AU - Kangasjärvi, J. AU - MacLean, D. AU - Menke, F. L. H. AU - Banfield, M. J. AU - Jones, J. D. G. AU - VL - 220 UR - DO - 10.1111/nph.15277 AB - The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL‐INDUCED CELL DEATH1 (RCD1).We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes.We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)‐induced defense genes and alters plant growth responses to light. HaRxL106‐mediated suppression of immunity is abolished in RCD1 loss‐of‐function mutants. We report that RCD1‐type proteins are phosphorylated, and we identified Mut9‐like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1‐interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA‐induced defense marker gene expression compared with wild‐type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling.Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 757 TI - Nuclear Import of Arabidopsis Poly(ADP-Ribose) Polymerase 2 Is Mediated by Importin-α and a Nuclear Localization Sequence Located Between the Predicted SAP Domains JO - Front. Plant Sci. PY - 2018 SP - 1581 AU - Chen, C. AU - Masi, R. D. AU - Lintermann, R. AU - Wirthmueller, L. AU - VL - 9 UR - DO - 10.3389/fpls.2018.01581 AB - Proteins of the Poly(ADP-Ribose) Polymerase (PARP) family modify target proteins by covalent attachment of ADP-ribose moieties onto amino acid side chains. In Arabidopsis, PARP proteins contribute to repair of DNA lesions and modulate plant responses to various abiotic and biotic stressors. Arabidopsis PARP1 and PARP2 are nuclear proteins and given that their molecular weights exceed the diffusion limit of nuclear pore complexes, an active import mechanism into the nucleus is likely. Here we use confocal microscopy of fluorescent protein-tagged Arabidopsis PARP2 and PARP2 deletion constructs in combination with site-directed mutagenesis to identify a nuclear localization sequence in PARP2 that is required for nuclear import. We report that in co-immunoprecipitation assays PARP2 interacts with several isoforms of the importin-α group of nuclear transport adapters and that PARP2 binding to IMPORTIN-α2 is mediated by the identified nuclear localization sequence. Our results demonstrate that PARP2 is a cargo protein of the canonical importin-α/β nuclear import pathway. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 826 TI - YODA MAP3K kinase regulates plant immune responses conferring broad-spectrum disease resistance JO - New Phytol. PY - 2018 SP - 661-680 AU - Sopeña-Torres, S. AU - Jordá, L. AU - Sánchez-Rodríguez, C. AU - Miedes, E. AU - Escudero, V. AU - Swami, S. AU - López, G. AU - Piślewska-Bednarek, M. AU - Lassowskat, I. AU - Lee, J. AU - Gu, Y. AU - Haigis, S. AU - Alexander, D. AU - Pattathil, S. AU - Muñoz-Barrios, A. AU - Bednarek, P. AU - Somerville, S. AU - Schulze-Lefert, P. AU - Hahn, M. G. AU - Scheel, D. AU - Molina, A. AU - VL - 218 UR - DO - 10.1111/nph.15007 AB - Mitogen‐activated protein kinases (MAPKs) cascades play essential roles in plants by transducing developmental cues and environmental signals into cellular responses. Among the latter are microbe‐associated molecular patterns perceived by pattern recognition receptors (PRRs), which trigger immunity.We found that YODA (YDA) – a MAPK kinase kinase regulating several Arabidopsis developmental processes, like stomatal patterning – also modulates immune responses. Resistance to pathogens is compromised in yda alleles, whereas plants expressing the constitutively active YDA (CA‐YDA) protein show broad‐spectrum resistance to fungi, bacteria, and oomycetes with different colonization modes. YDA functions in the same pathway as ERECTA (ER) Receptor‐Like Kinase, regulating both immunity and stomatal patterning.ER‐YDA‐mediated immune responses act in parallel to canonical disease resistance pathways regulated by phytohormones and PRRs. CA‐YDA plants exhibit altered cell‐wall integrity and constitutively express defense‐associated genes, including some encoding putative small secreted peptides and PRRs whose impairment resulted in enhanced susceptibility phenotypes. CA‐YDA plants show strong reprogramming of their phosphoproteome, which contains protein targets distinct from described MAPKs substrates.Our results suggest that, in addition to stomata development, the ER‐YDA pathway regulates an immune surveillance system conferring broad‐spectrum disease resistance that is distinct from the canonical pathways mediated by described PRRs and defense Hormones. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 937 TI - Stress-Related Mitogen-Activated Protein Kinases Stimulate the Accumulation of Small Molecules and Proteins in Arabidopsis thaliana Root Exudates JO - Front. Plant Sci. PY - 2017 SP - 1292 AU - Strehmel, N. AU - Hoehenwarter, W. AU - Mönchgesang, S. AU - Majovsky, P. AU - Krüger, S. AU - Scheel, D. AU - Lee, J. AU - VL - 8 UR - DO - 10.3389/fpls.2017.01292 AB - A delicate balance in cellular signaling is required for plants to respond to microorganisms or to changes in their environment. Mitogen-activated protein kinase (MAPK) cascades are one of the signaling modules that mediate transduction of extracellular microbial signals into appropriate cellular responses. Here, we employ a transgenic system that simulates activation of two pathogen/stress-responsive MAPKs to study release of metabolites and proteins into root exudates. The premise is based on our previous proteomics study that suggests upregulation of secretory processes in this transgenic system. An advantage of this experimental set-up is the direct focus on MAPK-regulated processes without the confounding complications of other signaling pathways activated by exposure to microbes or microbial molecules. Using non-targeted metabolomics and proteomics studies, we show that MAPK activation can indeed drive the appearance of dipeptides, defense-related metabolites and proteins in root apoplastic fluid. However, the relative levels of other compounds in the exudates were decreased. This points to a bidirectional control of metabolite and protein release into the apoplast. The putative roles for some of the identified apoplastic metabolites and proteins are discussed with respect to possible antimicrobial/defense or allelopathic properties. Overall, our findings demonstrate that sustained activation of MAPKs alters the composition of apoplastic root metabolites and proteins, presumably to influence the plant-microbe interactions in the rhizosphere. The reported metabolomics and proteomics data are available via Metabolights (Identifier: MTBLS441) and ProteomeXchange (Identifier: PXD006328), respectively. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 915 TI - A novel family of proline/serine-rich proteins, which are phospho-targets of stress-related mitogen-activated protein kinases, differentially regulates growth and pathogen defense in Arabidopsis thaliana JO - Plant Mol. Biol. PY - 2017 SP - 123-140 AU - Palm-Forster, M. A. T. AU - Eschen-Lippold, L. AU - Uhrig, J. AU - Scheel, D. AU - Lee, J. AU - VL - 95 UR - DO - 10.1007/s11103-017-0641-5 AB - The molecular actions of mitogen-activated protein kinases (MAPKs) are ultimately accomplished by the substrate proteins where phosphorylation affects their molecular properties and function(s), but knowledge regarding plant MAPK substrates is currently still fragmentary. Here, we uncovered a previously uncharacterized protein family consisting of three proline/serine-rich proteins (PRPs) that are substrates of stress-related MAPKs. We demonstrated the importance of a MAPK docking domain necessary for protein–protein interaction with MAPKs and consequently also for phosphorylation. The main phosphorylated site was mapped to a residue conserved between all three proteins, which when mutated to a non-phosphorylatable form, differentially affected their protein stability. Together with their distinct gene expression patterns, this differential accumulation of the three proteins upon phosphorylation probably contributes to their distinct function(s). Transgenic over-expression of PRP, the founding member, led to plants with enhanced resistance to Pseudomonas syringae pv. tomato DC3000. Older plants of the over-expressing lines have curly leaves and were generally smaller in stature. This growth phenotype was lost in plants expressing the phosphosite variant, suggesting a phosphorylation-dependent effect. Thus, this novel family of PRPs may be involved in MAPK regulation of plant development and / or pathogen resistance responses. As datamining associates PRP expression profiles with hypoxia or oxidative stress and PRP-overexpressing plants have elevated levels of reactive oxygen species, PRP may connect MAPK and oxidative stress signaling. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 860 TI - A 1-phytase type III effector interferes with plant hormone signaling JO - Nat. Commun. PY - 2017 SP - 2159 AU - Blüher, D. AU - Laha, D. AU - Thieme, S. AU - Hofer, A. AU - Eschen-Lippold, L. AU - Masch, A. AU - Balcke, G. AU - Pavlovic, I. AU - Nagel, O. AU - Schonsky, A. AU - Hinkelmann, R. AU - Wörner, J. AU - Parvin, N. AU - Greiner, R. AU - Weber, S. AU - Tissier, A. AU - Schutkowski, M. AU - Lee, J. AU - Jessen, H. AU - Schaaf, G. AU - Bonas, U. AU - VL - 8 UR - DO - 10.1038/s41467-017-02195-8 AB - Most Gram-negative phytopathogenic bacteria inject type III effector (T3E) proteins into plant cells to manipulate signaling pathways to the pathogen’s benefit. In resistant plants, specialized immune receptors recognize single T3Es or their biochemical activities, thus halting pathogen ingress. However, molecular function and mode of recognition for most T3Es remains elusive. Here, we show that the Xanthomonas T3E XopH possesses phytase activity, i.e., dephosphorylates phytate (myo-inositol-hexakisphosphate, InsP6), the major phosphate storage compound in plants, which is also involved in pathogen defense. A combination of biochemical approaches, including a new NMR-based method to discriminate inositol polyphosphate enantiomers, identifies XopH as a naturally occurring 1-phytase that dephosphorylates InsP6 at C1. Infection of Nicotiana benthamiana and pepper by Xanthomonas results in a XopH-dependent conversion of InsP6 to InsP5. 1-phytase activity is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene, and for induction of jasmonate- and ethylene-responsive genes in N. benthamiana. A2 - C1 - Cell and Metabolic Biology; Biochemistry of Plant Interactions ER - TY - JOUR ID - 896 TI - Potato plants with genetically engineered tropane alkaloid precursors JO - Planta PY - 2017 SP - 355-365 AU - Küster, N. AU - Rosahl, S. AU - Dräger, B. AU - VL - 245 UR - DO - 10.1007/s00425-016-2610-7 AB - Main conclusionSolanum tuberosum tropinone reductase I reduced tropinone in vivo. Suppression of tropinone reductase II strongly reduced calystegines in sprouts. Overexpression of putrescine N -methyltransferase did not alter calystegine accumulation.Calystegines are hydroxylated alkaloids formed by the tropane alkaloid pathway. They accumulate in potato (Solanum tuberosum L., Solanaceae) roots and sprouting tubers. Calystegines inhibit various glycosidases in vitro due to their sugar-mimic structure, but functions of calystegines in plants are not understood. Enzymes participating in or competing with calystegine biosynthesis, including putrescine N-methyltransferase (PMT) and tropinone reductases (TRI and TRII), were altered in their activity in potato plants by RNA interference (RNAi) and by overexpression. The genetically altered potato plants were investigated for the accumulation of calystegines and for intermediates of their biosynthesis. An increase in N-methylputrescine provided by DsPMT expression was not sufficient to increase calystegine accumulation. Overexpression and gene knockdown of StTRI proved that S. tuberosum TRI is a functional tropinone reductase in vivo, but no influence on calystegine accumulation was observed. When StTRII expression was suppressed by RNAi, calystegine formation was severely compromised in the transformed plants. Under phytochamber and green house conditions, the StTRII RNAi plants did not show phenotypic alterations. Further investigation of calystegines function in potato plants under natural conditions is enabled by the calystegine deprived StTRII RNAi plants. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 882 TI - MAPKs Influence Pollen Tube Growth by Controlling the Formation of Phosphatidylinositol 4,5-Bisphosphate in an Apical Plasma Membrane Domain JO - Plant Cell PY - 2017 SP - 3030-3050 AU - Hempel, F. AU - Stenzel, I. AU - Heilmann, M. AU - Krishnamoorthy, P. AU - Menzel, W. AU - Golbik, R. AU - Helm, S. AU - Dobritzsch, D. AU - Baginsky, S. AU - Lee, J. AU - Hoehenwarter, W. AU - Heilmann, I. AU - VL - 29 UR - DO - 10.1105/tpc.17.00543 AB - An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P2 by phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P2 reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing to directional growth. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 877 TI - Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response JO - Plant Cell PY - 2017 SP - 726-745 AU - Furlan, G. AU - Nakagami, H. AU - Eschen-Lippold, L. AU - Jiang, X. AU - Majovsky, P. AU - Kowarschik, K. AU - Hoehenwarter, W. AU - Lee, J. AU - Trujillo, M. AU - VL - 29 UR - DO - 10.1105/tpc.16.00654 AB - Crosstalk between posttranslational modifications, such as ubiquitination and phosphorylation, play key roles in controlling the duration and intensity of signaling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here, we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signaling. PUB22’s stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and, thus, autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent to most single unit E3s in vitro, can function as a self-regulatory mechanism in vivo. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 978 TI - Bacterial AvrRpt2-Like Cysteine Proteases Block Activation of the Arabidopsis Mitogen-Activated Protein Kinases, MPK4 and MPK11 JO - Plant Physiol. PY - 2016 SP - 2223-2238 AU - Eschen-Lippold, L. AU - Jiang, X. AU - Elmore, J. M. AU - Mackey, D. AU - Shan, L. AU - Coaker, G. AU - Scheel, D. AU - Lee, J. AU - VL - 171 UR - DO - 10.1104/pp.16.00336 AB - To establish infection, pathogens deliver effectors into host cells to target immune signaling components, including elements of mitogen-activated protein kinase (MPK) cascades. The virulence function of AvrRpt2, one of the first identified Pseudomonas syringae effectors, involves cleavage of the plant defense regulator, RPM1-INTERACTING PROTEIN4 (RIN4), and interference with plant auxin signaling. We show now that AvrRpt2 specifically suppresses the flagellin-induced phosphorylation of Arabidopsis (Arabidopsis thaliana) MPK4 and MPK11 but not MPK3 or MPK6. This inhibition requires the proteolytic activity of AvrRpt2, is associated with reduced expression of some plant defense genes, and correlates with enhanced pathogen infection in AvrRpt2-expressing transgenic plants. Diverse AvrRpt2-like homologs can be found in some phytopathogens, plant-associated and soil bacteria. Employing these putative bacterial AvrRpt2 homologs and inactive AvrRpt2 variants, we can uncouple the inhibition of MPK4/MPK11 activation from the cleavage of RIN4 and related members from the so-called nitrate-induced family as well as from auxin signaling. Thus, this selective suppression of specific mitogen-activated protein kinases is independent of the previously known AvrRpt2 targets and potentially represents a novel virulence function of AvrRpt2. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 974 TI - MATE Transporter-Dependent Export of Hydroxycinnamic Acid Amides JO - Plant Cell PY - 2016 SP - 583-596 AU - Dobritzsch, M. AU - Lübken, T. AU - Eschen-Lippold, L. AU - Gorzolka, K. AU - Blum, E. AU - Matern, A. AU - Marillonnet, S. AU - Böttcher, C. AU - Dräger, B. AU - Rosahl, S. AU - VL - 28 UR - DO - 10.1105/tpc.15.00706 AB - The ability of Arabidopsis thaliana to successfully prevent colonization by Phytophthora infestans, the causal agent of late blight disease of potato (Solanum tuberosum), depends on multilayered defense responses. To address the role of surface-localized secondary metabolites for entry control, droplets of a P. infestans zoospore suspension, incubated on Arabidopsis leaves, were subjected to untargeted metabolite profiling. The hydroxycinnamic acid amide coumaroylagmatine was among the metabolites secreted into the inoculum. In vitro assays revealed an inhibitory activity of coumaroylagmatine on P. infestans spore germination. Mutant analyses suggested a requirement of the p-coumaroyl-CoA:agmatine N4-p-coumaroyl transferase ACT for the biosynthesis and of the MATE transporter DTX18 for the extracellular accumulation of coumaroylagmatine. The host plant potato is not able to efficiently secrete coumaroylagmatine. This inability is overcome in transgenic potato plants expressing the two Arabidopsis genes ACT and DTX18. These plants secrete agmatine and putrescine conjugates to high levels, indicating that DTX18 is a hydroxycinnamic acid amide transporter with a distinct specificity. The export of hydroxycinnamic acid amides correlates with a decreased ability of P. infestans spores to germinate, suggesting a contribution of secreted antimicrobial compounds to pathogen defense at the leaf surface. A2 - C1 - Cell and Metabolic Biology; Biochemistry of Plant Interactions ER - TY - JOUR ID - 970 TI - The functional divergence between SPA1 and SPA2 in Arabidopsis photomorphogenesis maps primarily to the respective N-terminal kinase-like domain JO - BMC Plant Biol. PY - 2016 SP - 165 AU - Chen, S. AU - Wirthmueller, L. AU - Stauber, J. AU - Lory, N. AU - Holtkotte, X. AU - Leson, L. AU - Schenkel, C. AU - Ahmad, M. AU - Hoecker, U. AU - VL - 16 UR - DO - 10.1186/s12870-016-0854-9 AB - BackgroundPlants have evolved complex mechanisms to adapt growth and development to the light environment. The COP1/SPA complex is a key repressor of photomorphogenesis in dark-grown Arabidopsis plants and acts as an E3 ubiquitin ligase to ubiquitinate transcription factors involved in the light response. In the light, COP1/SPA activity is inhibited by photoreceptors, thereby allowing accumulation of these transcription factors and a subsequent light response. Previous results have shown that the four members of the SPA family exhibit partially divergent functions. In particular, SPA1 and SPA2 strongly differ in their responsiveness to light, while they have indistinguishable activities in darkness. The much higher light-responsiveness of SPA2 is partially explained by the much stronger light-induced degradation of SPA2 when compared to SPA1. Here, we have conducted SPA1/SPA2 domain swap experiments to identify the protein domain(s) responsible for the functional divergence between SPA1 and SPA2.ResultsWe have individually swapped the three domains between SPA1 and SPA2 - the N-terminal kinase-like domain, the coiled-coil domain and the WD-repeat domain - and expressed them in spa mutant Arabidopsis plants. The phenotypes of transgenic seedlings show that the respective N-terminal kinase-like domain is primarily responsible for the respective light-responsiveness of SPA1 and SPA2. Furthermore, the most divergent part of the N-terminal domain was sufficient to confer a SPA1- or SPA2-like activity to the respective SPA protein. The stronger light-induced degradation of SPA2 when compared to SPA1 was also primarily conferred by the SPA2 N-terminal domain. At last, the different affinities of SPA1 and SPA2 for cryptochrome 2 are defined by the N-terminal domain of the respective SPA protein. In contrast, both SPA1 and SPA2 similarly interacted with COP1 in light-grown seedlings.ConclusionsOur results show that the distinct activities and protein stabilities of SPA1 and SPA2 in light-grown seedlings are primarily encoded by their N-terminal kinase-like domains. Similarly, the different affinities of SPA1 and SPA2 for cry2 are explained by their respective N-terminal domain. Hence, after a duplication event during evolution, the N-terminal domains of SPA1 and SPA2 underwent subfunctionalization, possibly to allow optimal adaptation of growth and development to a changing light environment. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1043 TI - Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana JO - Front. Plant Sci. PY - 2016 SP - 61 AU - Sheikh, A. H. AU - Eschen-Lippold, L. AU - Pecher, P. AU - Hoehenwarter, W. AU - Sinha, A. K. AU - Scheel, D. AU - Lee, J. AU - VL - 7 UR - DO - 10.3389/fpls.2016.00061 AB - Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1033 TI - Challenges in the identification of microbe-associated molecular patterns in plant and animal innate immunity: a case study with bacterial lipopolysaccharide JO - Mol. Plant Pathol. PY - 2016 SP - 1165-1169 AU - Ranf, S. AU - Scheel, D. AU - Lee, J. AU - VL - 17 UR - DO - 10.1111/mpp.12452 AB - Immunity against pathogen infection depends on a host's ability to sense invading pathogens and to rapidly trigger defence reactions that block pathogen proliferation. Both plants and animals detect conserved structural motifs of microbe‐specific compounds, so‐called microbe‐associated molecular patterns (MAMPs), through germline‐encoded immune sensors, which are accordingly termed pattern recognition receptors (PRRs) (Akira et al., 2006; Boller and Felix, 2009). Activated PRRs initiate signal transduction and trigger innate immune responses. MAMPs are generally derived from elements essential for microbial fitness and are conserved across species, thus enabling the host to detect a range of potential pathogens. In mammals, innate immune sensing of MAMPs is not only crucial for basal immune responses but is also tightly connected with and required for a subsequent adaptive, antibody‐mediated immunity (Akira et al., 2006; Janeway and Medzhitov, 2002). Plants, lacking an adaptive immune system, have apparently evolved a greater capacity to detect a broader repertoire of MAMPs. Different plant species possess distinct sets of highly specific PRRs, but the downstream signalling pathways are rather conserved and converge on common signalling steps. This allows the transfer of PRRs, even to different plant families, whilst maintaining their functionality and specificity (Zipfel, 2014). This also enables researchers to use well‐studied, genetically amenable model systems for the identification of MAMPs and their respective PRRs. Several examples of interfamily PRR transfer have demonstrated that the introduction of novel PRRs into plant species can confer relevant levels of resistance to otherwise susceptible plants (e.g. Afroz et al., 2011; Hao et al., 2015; Lacombe et al., 2010; Mendes et al., 2010; Schoonbeek et al., 2015; Tripathi et al., 2014). Hence, MAMP sensing by PRRs has great potential for the engineering of disease resistance in crop plants. In recent years, it has therefore become a major task to identify and isolate MAMPs from a range of microorganisms, and their respective PRRs, to study their role in innate immunity and their application potential. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1029 TI - Comparative genomics to explore phylogenetic relationship, cryptic sexual potential and host specificity of Rhynchosporium species on grasses JO - BMC Genomics PY - 2016 SP - 953 AU - Penselin, D. AU - Münsterkötter, M. AU - Kirsten, S. AU - Felder, M. AU - Taudien, S. AU - Platzer, M. AU - Ashelford, K. AU - Paskiewicz, K. H. AU - Harrison, R. J. AU - Hughes, D. J. AU - Wolf, T. AU - Shelest, E. AU - Graap, J. AU - Hoffmann, J. AU - Wenzel, C. AU - Wöltje, N. AU - King, K. M. AU - Fitt, B. D. L. AU - Güldener, U. AU - Avrova, A. AU - Knogge, W. AU - VL - 17 UR - DO - 10.1186/s12864-016-3299-5 AB - BackgroundThe Rhynchosporium species complex consists of hemibiotrophic fungal pathogens specialized to different sweet grass species including the cereal crops barley and rye. A sexual stage has not been described, but several lines of evidence suggest the occurrence of sexual reproduction. Therefore, a comparative genomics approach was carried out to disclose the evolutionary relationship of the species and to identify genes demonstrating the potential for a sexual cycle. Furthermore, due to the evolutionary very young age of the five species currently known, this genus appears to be well-suited to address the question at the molecular level of how pathogenic fungi adapt to their hosts.ResultsThe genomes of the different Rhynchosporium species were sequenced, assembled and annotated using ab initio gene predictors trained on several fungal genomes as well as on Rhynchosporium expressed sequence tags. Structures of the rDNA regions and genome-wide single nucleotide polymorphisms provided a hypothesis for intra-genus evolution. Homology screening detected core meiotic genes along with most genes crucial for sexual recombination in ascomycete fungi. In addition, a large number of cell wall-degrading enzymes that is characteristic for hemibiotrophic and necrotrophic fungi infecting monocotyledonous hosts were found. Furthermore, the Rhynchosporium genomes carry a repertoire of genes coding for polyketide synthases and non-ribosomal peptide synthetases. Several of these genes are missing from the genome of the closest sequenced relative, the poplar pathogen Marssonina brunnea, and are possibly involved in adaptation to the grass hosts. Most importantly, six species-specific genes coding for protein effectors were identified in R. commune. Their deletion yielded mutants that grew more vigorously in planta than the wild type.ConclusionBoth cryptic sexuality and secondary metabolites may have contributed to host adaptation. Most importantly, however, the growth-retarding activity of the species-specific effectors suggests that host adaptation of R. commune aims at extending the biotrophic stage at the expense of the necrotrophic stage of pathogenesis. Like other apoplastic fungi Rhynchosporium colonizes the intercellular matrix of host leaves relatively slowly without causing symptoms, reminiscent of the development of endophytic fungi. Rhynchosporium may therefore become an object for studying the mutualism-parasitism transition. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1016 TI - Plant-to-Plant Variability in Root Metabolite Profiles of 19 Arabidopsis thaliana Accessions Is Substance-Class-Dependent JO - Int. J. Mol. Sci. PY - 2016 SP - 1565 AU - Mönchgesang, S. AU - Strehmel, N. AU - Trutschel, D. AU - Westphal, L. AU - Neumann, S. AU - Scheel, D. AU - VL - 17 UR - DO - 10.3390/ijms17091565 AB - Natural variation of secondary metabolism between different accessions of Arabidopsis thaliana (A. thaliana) has been studied extensively. In this study, we extended the natural variation approach by including biological variability (plant-to-plant variability) and analysed root metabolic patterns as well as their variability between plants and naturally occurring accessions. To screen 19 accessions of A. thaliana, comprehensive non-targeted metabolite profiling of single plant root extracts was performed using ultra performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS). Linear mixed models were applied to dissect the total observed variance. All metabolic profiles pointed towards a larger plant-to-plant variability than natural variation between accessions and variance of experimental batches. Ratios of plant-to-plant to total variability were high and distinct for certain secondary metabolites. None of the investigated accessions displayed a specifically high or low biological variability for these substance classes. This study provides recommendations for future natural variation analyses of glucosinolates, flavonoids, and phenylpropanoids and also reference data for additional substance classes. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1015 TI - Natural variation of root exudates in Arabidopsis thaliana-linking metabolomic and genomic data JO - Sci. Rep. PY - 2016 SP - 29033 AU - Mönchgesang, S. AU - Strehmel, N. AU - Schmidt, S. AU - Westphal, L. AU - Taruttis, F. AU - Müller, E. AU - Herklotz, S. AU - Neumann, S. AU - Scheel, D. AU - VL - 6 UR - DO - 10.1038/srep29033 AB - Many metabolomics studies focus on aboveground parts of the plant, while metabolism within roots and the chemical composition of the rhizosphere, as influenced by exudation, are not deeply investigated. In this study, we analysed exudate metabolic patterns of Arabidopsis thaliana and their variation in genetically diverse accessions. For this project, we used the 19 parental accessions of the Arabidopsis MAGIC collection. Plants were grown in a hydroponic system, their exudates were harvested before bolting and subjected to UPLC/ESI-QTOF-MS analysis. Metabolite profiles were analysed together with the genome sequence information. Our study uncovered distinct metabolite profiles for root exudates of the 19 accessions. Hierarchical clustering revealed similarities in the exudate metabolite profiles, which were partly reflected by the genetic distances. An association of metabolite absence with nonsense mutations was detected for the biosynthetic pathways of an indolic glucosinolate hydrolysis product, a hydroxycinnamic acid amine and a flavonoid triglycoside. Consequently, a direct link between metabolic phenotype and genotype was detected without using segregating populations. Moreover, genomics can help to identify biosynthetic enzymes in metabolomics experiments. Our study elucidates the chemical composition of the rhizosphere and its natural variation in A. thaliana, which is important for the attraction and shaping of microbial communities. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1048 TI - Altered glycosylation of exported proteins, including surface immune receptors, compromises calcium and downstream signaling responses to microbe-associated molecular patterns in Arabidopsis thaliana JO - BMC Plant Biol. PY - 2016 SP - 31 AU - Trempel, F. AU - Kajiura, H. AU - Ranf, S. AU - Grimmer, J. AU - Westphal, L. AU - Zipfel, C. AU - Scheel, D. AU - Fujiyama, K. AU - Lee, J. AU - VL - 16 UR - DO - 10.1186/s12870-016-0718-3 AB - BackgroundCalcium, as a second messenger, transduces extracellular signals into cellular reactions. A rise in cytosolic calcium concentration is one of the first plant responses after exposure to microbe-associated molecular patterns (MAMPs). We reported previously the isolation of Arabidopsis thaliana mutants with a “changed calcium elevation” (cce) response to flg22, a 22-amino-acid MAMP derived from bacterial flagellin.ResultsHere, we characterized the cce2 mutant and its weaker allelic mutant, cce3. Besides flg22, the mutants respond with a reduced calcium elevation to several other MAMPs and a plant endogenous peptide that is proteolytically processed from pre-pro-proteins during wounding. Downstream defense-related events such flg22-induced mitogen-activated protein kinase activation, accumulation of reactive oxygen species and growth arrest are also attenuated in cce2/cce3. By genetic mapping, next-generation sequencing and allelism assay, CCE2/CCE3 was identified to be ALG3 (Asparagine-linked glycosylation 3). This encodes the α-1,3-mannosyltransferase responsible for the first step of core oligosaccharide Glc3Man9GlcNAc2 glycan assembly on the endoplasmic reticulum (ER) luminal side. Complementation assays and glycan analysis in yeast alg3 mutant confirmed the reduced enzymatic function of the proteins encoded by the cce2/cce3 alleles – leading to accumulation of M5ER, the immature five mannose-containing oligosaccharide structure found in the ER. Proper protein glycosylation is required for ER/Golgi processing and trafficking of membrane proteins to the plasma membrane. Endoglycosidase H-insensitivity of flg22 receptor, FLS2, in the cce2/cce3 mutants suggests altered glycan structures in the receptor.ConclusionProper glycosylation of MAMP receptors (or other exported proteins) is required for optimal responses to MAMPs and is important for immune signaling of host plants. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 990 TI - Nucleoporin-Regulated MAP Kinase Signaling in Immunity to a Necrotrophic Fungal Pathogen JO - Plant Physiol. PY - 2016 SP - 1293-1305 AU - Genenncher, B. AU - Wirthmueller, L. AU - Roth, C. AU - Klenke, M. AU - Ma, L. AU - Sharon, A. AU - Wiermer, M. AU - VL - 172 UR - DO - 10.1104/pp.16.00832 AB - Pathogen-responsive mitogen-activated protein kinase (MAPK or MPK) cascades relay signals from activated immune receptors across the nuclear envelope to intranuclear targets. However, in plants, little is known about the spatial control of MAPK signaling. Here, we report that the Arabidopsis (Arabidopsis thaliana) nuclear pore complex protein Nup88/MOS7 is essential for immunity to the necrotrophic fungus Botrytis cinerea. The mos7-1 mutation, causing a four-amino acid deletion, compromises B. cinerea-induced activation of the key immunoregulatory MAPKs MPK3/MPK6 and reduces MPK3 protein levels posttranscriptionally. Furthermore, MOS7 contributes to retaining a sufficient MPK3 abundance in the nucleus, which is required for full immunity to B. cinerea. Finally, we present a structural model of MOS7 and show that the mos7-1 mutation compromises interactions with Nup98a/b, two phenylalanine-glycine repeat nucleoporins implicated in maintaining the selective nuclear pore complex permeability barrier. Together, our analysis uncovered MOS7 and Nup98 as novel components of plant immunity toward a necrotrophic pathogen and provides mechanistic insights into how these nucleoporins coordinate nucleocytoplasmic transport to mount a robust immune response. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 979 TI - Teaching an old dog new tricks: Suppressing activation of specific mitogen-activated kinases as a potential virulence function of the bacterial AvrRpt2 effector protein JO - Plant Signal Behav. PY - 2016 SP - e1257456 AU - Eschen-Lippold, L. AU - Scheel, D. AU - Lee, J. AU - VL - 11 UR - DO - 10.1080/15592324.2016.1257456 AB - AvrRpt2 is one of the first Pseudomonas syringae effector proteins demonstrated to be delivered into host cells. It suppresses plant immunity by modulating auxin signaling and cleavage of the membrane-localized defense regulator RIN4. We recently uncovered a novel potential virulence function of AvrRpt2, where it specifically blocked activation of mitogen-activated protein kinases, MPK4 and MPK11, but not of MPK3 and MPK6. Putative AvrRpt2 homologs from different phytopathogens and plant-associated bacteria showed distinct activities with respect to MPK4/11 activation suppression and RIN4 cleavage. Apart from differences in sequence similarity, 3 of the analyzed homologs were apparently “truncated.” To examine the role of the AvrRpt2 N-terminus, we modeled the structures of these AvrRpt2 homologs and performed deletion and domain swap experiments. Our results strengthen the finding that RIN4 cleavage is irrelevant for the ability to suppress defense-related MPK4/11 activation and indicate that full protease activity or cleavage specificity is affected by the N-terminus. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1166 TI - Probing formation of cargo/importin-α transport complexes in plant cells using a pathogen effector JO - Plant J. PY - 2015 SP - 40-52 AU - Wirthmueller, L. AU - Roth, C. AU - Fabro, G. AU - Caillaud, M.-C. AU - Rallapalli, G. AU - Asai, S. AU - Sklenar, J. AU - Jones, A. M. E. AU - Wiermer, M. AU - Jones, J. D. G. AU - Banfield, M. J. AU - VL - 81 UR - DO - 10.1111/tpj.12691 AB - Importin‐αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin‐α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin‐α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin‐α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co‐opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin‐α paralogs from Arabidopsis thaliana. A crystal structure of the importin‐α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin‐αs expressed in rosette leaves have an almost identical NLS‐binding site. Comparison of the importin‐α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin‐α, sequence variation at the importin‐α NLS‐binding sites and tissue‐specific expression levels of importin‐αs determine formation of cargo/importin‐α transport complexes in plant cells. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1148 TI - A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors JO - Cell PY - 2015 SP - 1089-1100 AU - Sarris, P. AU - Duxbury, Z. AU - Huh, S. AU - Ma, Y. AU - Segonzac, C. AU - Sklenar, J. AU - Derbyshire, P. AU - Cevik, V. AU - Rallapalli, G. AU - Saucet, S. AU - Wirthmueller, L. AU - Menke, F. H. AU - Sohn, K. AU - Jones, J. G. AU - VL - 161 UR - DO - 10.1016/j.cell.2015.04.024 AB - Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a “decoy” domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1138 TI - A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana JO - Nat. Immunol. PY - 2015 SP - 426-433 AU - Ranf, S. AU - Gisch, N. AU - Schäffer, M. AU - Illig, T. AU - Westphal, L. AU - Knirel, Y. A. AU - Sánchez-Carballo, P. M. AU - Zähringer, U. AU - Hückelhoven, R. AU - Lee, J. AU - Scheel, D. AU - VL - 16 UR - DO - 10.1038/ni.3124 AB - The sensing of microbe-associated molecular patterns (MAMPs) triggers innate immunity in animals and plants. Lipopolysaccharide (LPS) from Gram-negative bacteria is a potent MAMP for mammals, with the lipid A moiety activating proinflammatory responses via Toll-like receptor 4 (TLR4). Here we found that the plant Arabidopsis thaliana specifically sensed LPS of Pseudomonas and Xanthomonas. We isolated LPS-insensitive mutants defective in the bulb-type lectin S-domain-1 receptor–like kinase LORE (SD1-29), which were hypersusceptible to infection with Pseudomonas syringae. Targeted chemical degradation of LPS from Pseudomonas species suggested that LORE detected mainly the lipid A moiety of LPS. LORE conferred sensitivity to LPS onto tobacco after transient expression, which demonstrated a key function in LPS sensing and indicated the possibility of engineering resistance to bacteria in crop species. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1124 TI - The Calcium-Dependent Protein Kinase CPK28 Regulates Development by Inducing Growth Phase-Specific, Spatially Restricted Alterations in Jasmonic Acid Levels Independent of Defense Responses in Arabidopsis JO - Plant Cell PY - 2015 SP - 591-606 AU - Matschi, S. AU - Hake, K. AU - Herde, M. AU - Hause, B. AU - Romeis, T. AU - VL - 27 UR - DO - 10.1105/tpc.15.00024 AB - Phytohormones play an important role in development and stress adaptations in plants, and several interacting hormonal pathways have been suggested to accomplish fine-tuning of stress responses at the expense of growth. This work describes the role played by the CALCIUM-DEPENDENT PROTEIN KINASE CPK28 in balancing phytohormone-mediated development in Arabidopsis thaliana, specifically during generative growth. cpk28 mutants exhibit growth reduction solely as adult plants, coinciding with altered balance of the phytohormones jasmonic acid (JA) and gibberellic acid (GA). JA-dependent gene expression and the levels of several JA metabolites were elevated in a growth phase-dependent manner in cpk28, and accumulation of JA metabolites was confined locally to the central rosette tissue. No elevated resistance toward herbivores or necrotrophic pathogens was detected for cpk28 plants, either on the whole-plant level or specifically within the tissue displaying elevated JA levels. Abolishment of JA biosynthesis or JA signaling led to a full reversion of the cpk28 growth phenotype, while modification of GA signaling did not. Our data identify CPK28 as a growth phase-dependent key negative regulator of distinct processes: While in seedlings, CPK28 regulates reactive oxygen species-mediated defense signaling; in adult plants, CPK28 confers developmental processes by the tissue-specific balance of JA and GA without affecting JA-mediated defense responses. A2 - C1 - Cell and Metabolic Biology; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1114 TI - Cellular reprogramming through mitogen-activated protein kinases JO - Front. Plant Sci. PY - 2015 SP - 940 AU - Lee, J. AU - Eschen-Lippold, L. AU - Lassowskat, I. AU - Böttcher, C. AU - Scheel, D. AU - VL - 6 UR - DO - 10.3389/fpls.2015.00940 AB - Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1098 TI - Mutations in the EDR1 Gene Alter the Response of Arabidopsis thaliana to Phytophthora infestans and the Bacterial PAMPs flg22 and elf18 JO - Mol. Plant Microbe Interact. PY - 2015 SP - 122-133 AU - Geissler, K. AU - Eschen-Lippold, L. AU - Naumann, K. AU - Schneeberger, K. AU - Weigel, D. AU - Scheel, D. AU - Rosahl, S. AU - Westphal, L. AU - VL - 28 UR - DO - 10.1094/MPMI-09-14-0282-R AB - Mechanistically, nonhost resistance of Arabidopsis thaliana against the oomycete Phytophthora infestans is not well understood. Besides PEN2 and PEN3, which contribute to penetration resistance, no further components have been identified so far. In an ethylmethane sulphonate–mutant screen, we mutagenized pen2-1 and screened for mutants with an altered response to infection by P. infestans. One of the mutants obtained, enhanced response to Phytophthora infestans6 (erp6), was analyzed. Whole-genome sequencing of erp6 revealed a single nucleotide polymorphism in the coding region of the kinase domain of At1g08720, which encodes the putative MAPKKK ENHANCED DISEASE RESISTANCE1 (EDR1). We demonstrate that three independent lines with knock-out alleles of edr1 mount an enhanced response to P. infestans inoculation, mediated by increased salicylic acid signaling and callose deposition. Moreover, we show that the single amino acid substitution in erp6 causes the loss of in vitro autophosphorylation activity of EDR1. Furthermore, growth inhibition experiments suggest a so-far-unknown involvement of EDR1 in the response to the pathogen-associated molecular patterns flg22 and elf18. We conclude that EDR1 contributes to the defense response of A. thaliana against P. infestans. Our data position EDR1 as a negative regulator in postinvasive nonhost resistance. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1112 TI - Expression of Caenorhabditis elegans PCS in the AtPCS1-deficient Arabidopsis thaliana cad1-3 mutant separates the metal tolerance and non-host resistance functions of phytochelatin synthases JO - Plant Cell Environ. PY - 2015 SP - 2239-2247 AU - Kühnlenz, T. AU - Westphal, L. AU - Schmidt, H. AU - Scheel, D. AU - Clemens, S. AU - VL - 38 UR - DO - 10.1111/pce.12534 AB - Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal‐binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non‐host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non‐host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non‐host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1273 TI - Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants JO - J. Cell Biol. PY - 2014 SP - 677-692 AU - Zhou, X. AU - Graumann, K. AU - Wirthmueller, L. AU - Jones, J. D. G. AU - Meier, I. AU - VL - 205 UR - DO - 10.1083/jcb.201401138 AB - Although a plethora of nuclear envelope (NE) transmembrane proteins (NETs) have been identified in opisthokonts, plant NETs are largely unknown. The only known NET homologues in plants are Sad1/UNC-84 (SUN) proteins, which bind Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. Therefore, de novo identification of plant NETs is necessary. Based on similarities between opisthokont KASH proteins and the only known plant KASH proteins, WPP domain–interacting proteins, we used a computational method to identify the KASH subset of plant NETs. Ten potential plant KASH protein families were identified, and five candidates from four of these families were verified for their NE localization, depending on SUN domain interaction. Of those, Arabidopsis thaliana SINE1 is involved in actin-dependent nuclear positioning in guard cells, whereas its paralogue SINE2 contributes to innate immunity against an oomycete pathogen. This study dramatically expands our knowledge of plant KASH proteins and suggests that plants and opisthokonts have recruited different KASH proteins to perform NE regulatory functions. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1272 TI - Ménage à trois: The complex relationships between mitogen-activated protein kinases, WRKY transcription factors, and VQ-motif-containing proteins JO - Plant Signal Behav. PY - 2014 SP - e29519 AU - Weyhe, M. AU - Eschen-Lippold, L. AU - Pecher, P. AU - Scheel, D. AU - Lee, J. AU - VL - 9 UR - DO - 10.4161/psb.29519 AB - Out of the 34 members of the VQ-motif-containing protein (VQP) family, 10 are phosphorylated by the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6. Most of these MPK3/6-targeted VQPs (MVQs) interacted with specific sub-groups of WRKY transcription factors in a VQ-motif-dependent manner. In some cases, the MAPK appears to phosphorylate either the MVQ or the WRKY, while in other cases, both proteins have been reported to act as MAPK substrates. We propose a network of dynamic interactions between members from the MAPK, MVQ and WRKY families – either as binary or as tripartite interactions. The compositions of the WRKY-MVQ transcriptional protein complexes may change – for instance, through MPK3/6-mediated modulation of protein stability – and therefore control defense gene transcription. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1266 TI - Fast Retrograde Signaling in Response to High Light Involves Metabolite Export, MITOGEN-ACTIVATED PROTEIN KINASE6, and AP2/ERF Transcription Factors in Arabidopsis JO - Plant Cell PY - 2014 SP - 1151-1165 AU - Vogel, M. O. AU - Moore, M. AU - König, K. AU - Pecher, P. AU - Alsharafa, K. AU - Lee, J. AU - Dietz, K.-J. AU - VL - 26 UR - DO - 10.1105/tpc.113.121061 AB - Regulation of the expression of nuclear genes encoding chloroplast proteins allows for metabolic adjustment in response to changing environmental conditions. This regulation is linked to retrograde signals that transmit information on the metabolic state of the chloroplast to the nucleus. Transcripts of several APETALA2/ETHYLENE RESPONSE FACTOR transcription factors (AP2/ERF-TFs) were found to respond within 10 min after transfer of low-light-acclimated Arabidopsis thaliana plants to high light. Initiation of this transcriptional response was completed within 1 min after transfer to high light. The fast responses of four AP2/ERF genes, ERF6, RRTF1, ERF104, and ERF105, were entirely deregulated in triose phosphate/phosphate translocator (tpt) mutants. Similarly, activation of MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) was upregulated after 1 min in the wild type but not in the tpt mutant. Based on this, together with altered transcript regulation in mpk6 and erf6 mutants, a retrograde signal transmission model is proposed starting with metabolite export through the triose phosphate/phosphate translocator with subsequent MPK6 activation leading to initiation of AP2/ERF-TF gene expression and other downstream gene targets. The results show that operational retrograde signaling in response to high light involves a metabolite-linked pathway in addition to previously described redox and hormonal pathways. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1265 TI - Comparative analysis of mitochondrial genomes from closely related Rhynchosporium species reveals extensive intron invasion JO - Fungal Genet. Biol. PY - 2014 SP - 34-42 AU - Torriani, S. F. F. AU - Penselin, D. AU - Knogge, W. AU - Felder, M. AU - Taudien, S. AU - Platzer, M. AU - McDonald, B. A. AU - Brunner, P. C. AU - VL - 62 UR - DO - 10.1016/j.fgb.2013.11.001 AB - We sequenced and annotated the complete mitochondrial (mt) genomes of four closely related Rhynchosporium species that diverged ∼14,000–35,000 years ago. During this time frame, three of the mt genomes expanded significantly due to an invasion of introns into three genes (cox1, cox2, and nad5). The enlarged mt genomes contained ∼40% introns compared to 8.1% in uninvaded relatives. Many intron gains were accompanied by co-conversion of flanking exonic regions. The comparative analysis revealed a highly variable set of non-intronic, free-standing ORFs of unknown function (uORFs). This is consistent with a rapidly evolving accessory compartment in the mt genome of these closely related species. Only one free-standing uORF was shared among all mt genomes analyzed. This uORF had a mutation rate similar to the core mt protein-encoding genes, suggesting conservation of function among the species. The nucleotide composition of the core protein-encoding genes significantly differed from those of introns and uORFs. The mt mutation rate was 77 times higher than the nuclear mutation rate, indicating that the phylogeny inferred from mt genes may better resolve the phylogenetic relationships among closely related Rhynchosporium species than phylogenies inferred from nuclear genes. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1259 TI - PFP1, a Gene Encoding an Epc-N Domain-Containing Protein, Is Essential for Pathogenicity of the Barley Pathogen Rhynchosporium commune JO - Eukaryot. Cell PY - 2014 SP - 1026-1035 AU - Siersleben, S. AU - Penselin, D. AU - Wenzel, C. AU - Albert, S. AU - Knogge, W. AU - VL - 13 UR - DO - 10.1128/EC.00043-14 AB - Scald caused by Rhynchosporium commune is an important foliar disease of barley. Insertion mutagenesis of R. commune generated a nonpathogenic fungal mutant which carries the inserted plasmid in the upstream region of a gene named PFP1. The characteristic feature of the gene product is an Epc-N domain. This motif is also found in homologous proteins shown to be components of histone acetyltransferase (HAT) complexes of fungi and animals. Therefore, PFP1 is suggested to be the subunit of a HAT complex in R. commune with an essential role in the epigenetic control of fungal pathogenicity. Targeted PFP1 disruption also yielded nonpathogenic mutants which showed wild-type-like growth ex planta, except for the occurrence of hyphal swellings. Complementation of the deletion mutants with the wild-type gene reestablished pathogenicity and suppressed the hyphal swellings. However, despite wild-type-level PFP1 expression, the complementation mutants did not reach wild-type-level virulence. This indicates that the function of the protein complex and, thus, fungal virulence are influenced by a position-affected long-range control of PFP1 expression. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1258 TI - Agroinfiltration by Cytokinin-Producing Agrobacterium sp. Strain GV3101 Primes Defense Responses in Nicotiana tabacum JO - Mol. Plant Microbe Interact. PY - 2014 SP - 1175-1185 AU - Sheikh, A. H. AU - Raghuram, B. AU - Eschen-Lippold, L. AU - Scheel, D. AU - Lee, J. AU - Sinha, A. K. AU - VL - 27 UR - DO - 10.1094/MPMI-04-14-0114-R AB - Transient infiltrations in tobacco are commonly used in plant studies, but the host response to different disarmed Agrobacterium strains is not fully understood. The present study shows that pretreatment with disarmed Agrobacterium tumefaciens GV3101 primes the defense response to subsequent infection by Pseudomonas syringae in Nicotiana tabacum. The presence of a trans-zeatin synthase (tzs) gene in strain GV3101 may be partly responsible for the priming response, as the tzs-deficient Agrobacterium sp. strain LBA4404 only weakly imparts such responses. Besides inducing the expression of defense-related genes like PR-1 and NHL10, GV3101 pretreatment increased the expression of tobacco mitogen-activated protein kinase (MAPK) pathway genes like MEK2, WIPK (wound-induced protein kinase), and SIPK (salicylic acid-induced protein kinase). Furthermore, the GV3101 strain showed a stronger effect than the LBA4404 strain in activating phosphorylation of the tobacco MAPK, WIPK and SIPK, which presumably prime the plant immune machinery. Lower doses of exogenously applied cytokinins increased the activation of MAPK, while higher doses decreased the activation, suggesting a balanced level of cytokinins is required to generate defense response in planta. The current study serves as a cautionary warning for plant researchers over the choice of Agrobacterium strains and their possible consequences on subsequent pathogen-related studies. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1257 TI - Ca2+ signalling in plant immune response: from pattern recognition receptors to Ca2+ decoding mechanisms JO - New Phytol. PY - 2014 SP - 782-790 AU - Seybold, H. AU - Trempel, F. AU - Ranf, S. AU - Scheel, D. AU - Romeis, T. AU - Lee, J. AU - VL - 204 UR - DO - 10.1111/nph.13031 AB - Ca2+ is a ubiquitous second messenger for cellular signalling in various stresses and developmental processes. Here, we summarize current developments in the roles of Ca2+ during plant immunity responses. We discuss the early perception events preceding and necessary for triggering cellular Ca2+ fluxes, the potential Ca2+‐permeable channels, the decoding of Ca2+ signals predominantly via Ca2+‐dependent phosphorylation events and transcriptional reprogramming. To highlight the complexity of the cellular signal network, we briefly touch on the interplay between Ca2+‐dependent signalling and selected major signalling mechanisms – with special emphasis on reactive oxygen species at local and systemic levels. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1256 TI - Abscisic Acid Flux Alterations Result in Differential Abscisic Acid Signaling Responses and Impact Assimilation Efficiency in Barley under Terminal Drought Stress JO - Plant Physiol. PY - 2014 SP - 1677-1696 AU - Seiler, C. AU - Harshavardhan, V. T. AU - Reddy, P. S. AU - Hensel, G. AU - Kumlehn, J. AU - Eschen-Lippold, L. AU - Rajesh, K. AU - Korzun, V. AU - Wobus, U. AU - Lee, J. AU - Selvaraj, G. AU - Sreenivasulu, N. AU - VL - 164 UR - DO - 10.1104/pp.113.229062 AB - Abscisic acid (ABA) is a central player in plant responses to drought stress. How variable levels of ABA under short-term versus long-term drought stress impact assimilation and growth in crops is unclear. We addressed this through comparative analysis, using two elite breeding lines of barley (Hordeum vulgare) that show senescence or stay-green phenotype under terminal drought stress and by making use of transgenic barley lines that express Arabidopsis (Arabidopsis thaliana) 9-cis-epoxycarotenoid dioxygenase (AtNCED6) coding sequence or an RNA interference (RNAi) sequence of ABA 8′-hydroxylase under the control of a drought-inducible barley promoter. The high levels of ABA and its catabolites in the senescing breeding line under long-term stress were detrimental for assimilate productivity, whereas these levels were not perturbed in the stay-green type that performed better. In transgenic barley, drought-inducible AtNCED expression afforded temporal control in ABA levels such that the ABA levels rose sooner than in wild-type plants but also subsided, unlike as in the wild type , to near-basal levels upon prolonged stress treatment due to down-regulation of endogenous HvNCED genes. Suppressing of ABA catabolism with the RNA interference approach of ABA 8′-hydroxylase caused ABA flux during the entire period of stress. These transgenic plants performed better than the wild type under stress to maintain a favorable instantaneous water use efficiency and better assimilation. Gene expression analysis, protein structural modeling, and protein-protein interaction analyses of the members of the PYRABACTIN RESISTANCE1/PYRABACTIN RESISTANCE1-LIKE/REGULATORY COMPONENT OF ABA RECEPTORS, TYPE 2C PROTEIN PHOSPHATASE Sucrose non-fermenting1-related protein kinase2, and ABA-INSENSITIVE5/ABA-responsive element binding factor family identified specific members that could potentially impact ABA metabolism and stress adaptation in barley. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1249 TI - Suppression of UV-B stress responses by flg22 is regulated at the chromatin level via histone modification JO - Plant Cell Environ. PY - 2014 SP - 1716-1721 AU - Schenke, D. AU - Cai, D. AU - Scheel, D. AU - VL - 37 UR - DO - 10.1111/pce.12283 AB - Genes of the flavonol pathway are activated by UV‐B, but suppressed by concomitant flg22 application in Arabidopsis. Analysis at the metabolite level suggested that this regulation allows the plant to focus its secondary metabolism on the plant defence towards pathogen attack. We now demonstrate by chromatin immunoprecipitation followed by quantitative PCR, that this antagonistic gene regulation is mediated at the chromatin level by differential regulation of histone 3 lysine 9 acetylation (H3K9ac), which is a hallmark for gene activation. Since H3K9ac levels were altered at least at four independent gene loci, namely, chalcone synthase, chalcone‐flavone isomerase, flavanone 3‐hydroxylase and the positive regulator MYB12, which correlates with the observed gene activation/suppression reported previously, it appears that this process is mediated by chromatin remodelling. Since suppression of H3K9ac prevents gene expression, we conclude H3K9ac is rather cause than consequence of gene activation. This finding allows us also to extend our working model, involving the two opposing MYB transcription factors of the flavonol pathway, MYB12 (being UV‐B‐activated and flg22‐suppressed) and MYB4 (a negative regulator, which is activated by both flg22 and UV‐B stress). A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1244 TI - Unraveling Regulation of the Small Heat Shock Proteins by the Heat Shock Factor HvHsfB2c in Barley: Its Implications in Drought Stress Response and Seed Development JO - PLOS ONE PY - 2014 SP - e89125 AU - Reddy, P. S. AU - Kavi Kishor, P. B. AU - Seiler, C. AU - Kuhlmann, M. AU - Eschen-Lippold, L. AU - Lee, J. AU - Reddy, M. K. AU - Sreenivasulu, N. AU - VL - 9 UR - DO - 10.1371/journal.pone.0089125 AB - The rapid increase in heat shock proteins upon exposure to damaging stresses and during plant development related to desiccation events reveal their dual importance in plant development and stress tolerance. Genome-wide sequence survey identified 20 non-redundant small heat shock proteins (sHsp) and 22 heat shock factor (Hsf) genes in barley. While all three major classes (A, B, C) of Hsfs are localized in nucleus, the 20 sHsp gene family members are localized in different cell organelles like cytoplasm, mitochondria, plastid and peroxisomes. Hsf and sHsp members are differentially regulated during drought and at different seed developmental stages suggesting the importance of chaperone role under drought as well as seed development. In silico cis-regulatory motif analysis of Hsf promoters showed an enrichment with abscisic acid responsive cis-elements (ABRE), implying regulatory role of ABA in mediating transcriptional response of HvsHsf genes. Gene regulatory network analysis identified HvHsfB2c as potential central regulator of the seed-specific expression of several HvsHsps including 17.5CI sHsp. These results indicate that HvHsfB2c is co-expressed in the central hub of small Hsps and therefore it may be regulating the expression of several HvsHsp subclasses HvHsp16.88-CI, HvHsp17.5-CI and HvHsp17.7-CI. The in vivo relevance of binding specificity of HvHsfB2C transcription factor to HSE-element present in the promoter of HvSHP17.5-CI under heat stress exposure is confirmed by gel shift and LUC-reporter assays. Further, we isolated 477 bp cDNA from barley encoding a 17.5 sHsp polypeptide, which was predominantly upregulated under drought stress treatments and also preferentially expressed in developing seeds. Recombinant HvsHsp17.5-CI protein was expressed in E. coli and purified to homogeneity, which displayed in vitro chaperone activity. The predicted structural model of HvsHsp-17.5-CI protein suggests that the α-crystallin domain is evolutionarily highly conserved. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1242 TI - Microbe-associated molecular pattern-induced calcium signaling requires the receptor-like cytoplasmic kinases, PBL1 and BIK1 JO - BMC Plant Biol. PY - 2014 SP - 374 AU - Ranf, S. AU - Eschen-Lippold, L. AU - Fröhlich, K. AU - Westphal, L. AU - Scheel, D. AU - Lee, J. AU - VL - 14 UR - DO - 10.1186/s12870-014-0374-4 AB - BackgroundPlant perception of conserved microbe-derived or damage-derived molecules (so-called microbe- or damage-associated molecular patterns, MAMPs or DAMPs, respectively) triggers cellular signaling cascades to initiate counteracting defence responses. Using MAMP-induced rise in cellular calcium levels as one of the earliest biochemical readouts, we initiated a genetic screen for components involved in early MAMP signaling in Arabidopsis thaliana.ResultsWe characterized here the “changed calcium elevation 5” (cce5) mutant, where five allelic cce5 mutants were isolated. They all show reduced calcium levels after elicitation with peptides representing bacteria-derived MAMPs (flg22 and elf18) and endogenous DAMP (AtPep1), but a normal response to chitin octamers. Mapping, sequencing of the mutated locus and complementation studies revealed CCE5 to encode the receptor-like cytoplasmic kinase (RLCK), avrPphB sensitive 1-like 1 (PBL1). Kinase activities of PBL1 derived from three of the cce5 alleles are abrogated in vivo. Validation with T-DNA mutants revealed that, besides PBL1, another RLCK, Botrytis-induced kinase 1 (BIK1), is also required for MAMP/DAMP-induced calcium elevations.ConclusionsHence, PBL1 and BIK1 (but not two related RLCKs, PBS1 and PBL2) are required for MAMP/DAMP-induced calcium signaling. It remains to be investigated if the many other RLCKs encoded in the Arabidopsis genome affect early calcium signal transduction – perhaps in dependence on the type of MAMP/DAMP ligands. A future challenge would be to identify the substrates of these various RLCKs, in order to elucidate their signaling role between the receptor complexes at the plasma membrane and downstream cellular signaling components. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1176 TI - Expression Profiling during Arabidopsis/Downy Mildew Interaction Reveals a Highly-Expressed Effector That Attenuates Responses to Salicylic Acid JO - PLOS Pathog. PY - 2014 SP - e1004443 AU - Asai, S. AU - Rallapalli, G. AU - Piquerez, S. J. M. AU - Caillaud, M.-C. AU - Furzer, O. J. AU - Ishaque, N. AU - Wirthmueller, L. AU - Fabro, G. AU - Shirasu, K. AU - Jones, J. D. G. AU - VL - 10 UR - DO - 10.1371/journal.ppat.1004443 AB - Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1238 TI - The Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6 target a subclass of ‘VQ-motif’-containing proteins to regulate immune responses JO - New Phytol. PY - 2014 SP - 592-606 AU - Pecher, P. AU - Eschen-Lippold, L. AU - Herklotz, S. AU - Kuhle, K. AU - Naumann, K. AU - Bethke, G. AU - Uhrig, J. AU - Weyhe, M. AU - Scheel, D. AU - Lee, J. AU - VL - 203 UR - DO - 10.1111/nph.12817 AB - Mitogen‐activated protein kinase (MAPK) cascades play key roles in plant immune signalling, and elucidating their regulatory functions requires the identification of the pathway‐specific substrates.We used yeast two‐hybrid interaction screens, in vitro kinase assays and mass spectrometry‐based phosphosite mapping to study a family of MAPK substrates. Site‐directed mutagenesis and promoter‐reporter fusion studies were performed to evaluate the impact of substrate phosphorylation on downstream signalling.A subset of the Arabidopsis thaliana VQ‐motif‐containing proteins (VQPs) were phosphorylated by the MAPKs MPK3 and MPK6, and renamed MPK3/6‐targeted VQPs (MVQs). When plant protoplasts (expressing these MVQs) were treated with the flagellin‐derived peptide flg22, several MVQs were destabilized in vivo. The MVQs interact with specific WRKY transcription factors. Detailed analysis of a representative member of the MVQ subset, MVQ1, indicated a negative role in WRKY‐mediated defence gene expression – with mutation of the VQ‐motif abrogating WRKY binding and causing mis‐regulation of defence gene expression.We postulate the existence of a variety of WRKY‐VQP‐containing transcriptional regulatory protein complexes that depend on spatio‐temporal VQP and WRKY expression patterns. Defence gene transcription can be modulated by changing the composition of these complexes – in part – through MAPK‐mediated VQP degradation. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1228 TI - The Arabidopsis Tandem Zinc Finger 9 Protein Binds RNA and Mediates Pathogen-Associated Molecular Pattern-Triggered Immune Responses JO - Plant Cell Physiol. PY - 2014 SP - 412-425 AU - Maldonado-Bonilla, L. D. AU - Eschen-Lippold, L. AU - Gago-Zachert, S. AU - Tabassum, N. AU - Bauer, N. AU - Scheel, D. AU - Lee, J. AU - VL - 55 UR - DO - 10.1093/pcp/pct175 AB - Recognition of pathogen-associated molecular patterns (PAMPs) induces multiple defense mechanisms to limit pathogen growth. Here, we show that the Arabidopsis thaliana tandem zinc finger protein 9 (TZF9) is phosphorylated by PAMP-responsive mitogen-activated protein kinases (MAPKs) and is required to trigger a full PAMP-triggered immune response. Analysis of a tzf9 mutant revealed attenuation in specific PAMP-triggered reactions such as reactive oxygen species accumulation, MAPK activation and, partially, the expression of several PAMP-responsive genes. In accordance with these weaker PAMP-triggered responses, tzf9 mutant plants exhibit enhanced susceptibility to virulent Pseudomonas syringae pv. tomato DC3000. Visualization of TZF9 localization by fusion to green fluorescent protein revealed cytoplasmic foci that co-localize with marker proteins of processing bodies (P-bodies). This localization pattern is affected by inhibitor treatments that limit mRNA availability (such as cycloheximide or actinomycin D) or block nuclear export (leptomycin B). Coupled with its ability to bind the ribohomopolymers poly(rU) and poly(rG), these results suggest involvement of TZF9 in post-transcriptional regulation, such as mRNA processing or storage pathways, to regulate plant innate immunity. A2 - C1 - Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1227 TI - Targeted Proteomics Analysis of Protein Degradation in Plant Signaling on an LTQ-Orbitrap Mass Spectrometer JO - J. Proteome Res. PY - 2014 SP - 4246-4258 AU - Majovsky, P. AU - Naumann, C. AU - Lee, C.-W. AU - Lassowskat, I. AU - Trujillo, M. AU - Dissmeyer, N. AU - Hoehenwarter, W. AU - VL - 13 UR - DO - 10.1021/pr500164j AB - Targeted proteomics has become increasingly popular recently because of its ability to precisely quantify selected proteins in complex cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap Velos Pro mass spectrometer in targeted parallel reaction monitoring (PRM) despite its unconventional dual ion trap configuration. We evaluated absolute specificity (>99%) and sensitivity (100 amol on column in 1 μg of total cellular extract) using full and mass range scans as survey scans together with data-dependent (DDA) and targeted MS/MS acquisition. The instrument duty cycle was a critical parameter limiting sensitivity, necessitating peptide retention time scheduling. We assessed synthetic peptide and recombinant peptide standards to predict or experimentally determine target peptide retention times. We applied optimized PRM to protein degradation in signaling regulation, an area that is receiving increased attention in plant physiology. We quantified relative abundance of selected proteins in plants that are mutant for enzymatic components of the N-end rule degradation (NERD) pathway such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated proteins, which might represent degradation targets. We also targeted FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation of plant immunity by degradation of the receptor. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1224 TI - Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana JO - Front. Plant Sci. PY - 2014 SP - 554 AU - Lassowskat, I. AU - Böttcher, C. AU - Eschen-Lippold, L. AU - Scheel, D. AU - Lee, J. AU - VL - 5 UR - DO - 10.3389/fpls.2014.00554 AB - Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the “PEN” pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org). A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1223 TI - The ABC Transporter ABCG1 Is Required for Suberin Formation in Potato Tuber Periderm JO - Plant Cell PY - 2014 SP - 3403-3415 AU - Landgraf, R. AU - Smolka, U. AU - Altmann, S. AU - Eschen-Lippold, L. AU - Senning, M. AU - Sonnewald, S. AU - Weigel, B. AU - Frolova, N. AU - Strehmel, N. AU - Hause, G. AU - Scheel, D. AU - Böttcher, C. AU - Rosahl, S. AU - VL - 26 UR - DO - 10.1105/tpc.114.124776 AB - The lipid biopolymer suberin plays a major role as a barrier both at plant-environment interfaces and in internal tissues, restricting water and nutrient transport. In potato (Solanum tuberosum), tuber integrity is dependent on suberized periderm. Using microarray analyses, we identified ABCG1, encoding an ABC transporter, as a gene responsive to the pathogen-associated molecular pattern Pep-13. Further analyses revealed that ABCG1 is expressed in roots and tuber periderm, as well as in wounded leaves. Transgenic ABCG1-RNAi potato plants with downregulated expression of ABCG1 display major alterations in both root and tuber morphology, whereas the aerial part of the ABCG1-RNAi plants appear normal. The tuber periderm and root exodermis show reduced suberin staining and disorganized cell layers. Metabolite analyses revealed reduction of esterified suberin components and hyperaccumulation of putative suberin precursors in the tuber periderm of RNA interference plants, suggesting that ABCG1 is required for the export of suberin components. A2 - C1 - Biochemistry of Plant Interactions; Bioorganic Chemistry ER - TY - JOUR ID - 1221 TI - N-Terminal Phosphorylation of Parathyroid Hormone (PTH) Abolishes Its Receptor Activity JO - ACS Chem. Biol. PY - 2014 SP - 2465-2470 AU - Kumar, A. AU - Gopalswamy, M. AU - Wishart, C. AU - Henze, M. AU - Eschen-Lippold, L. AU - Donnelly, D. AU - Balbach, J. AU - VL - 9 UR - DO - 10.1021/cb5004515 AB - The parathyroid hormone (PTH) is an 84-residue peptide, which regulates the blood Ca2+ level via GPCR binding and subsequent activation of intracellular signaling cascades. PTH is posttranslationally phosphorylated in the parathyroid glands; however, the functional significance of this processes is not well characterized. In the present study, mass spectrometric analysis revealed three sites of phosphorylation, and NMR spectroscopy assigned Ser1, Ser3, and Ser17 as modified sites. These sites are located at the N-terminus of the hormone, which is important for receptor recognition and activation. NMR shows further that the three phosphate groups remotely disturb the α-helical propensity up to Ala36. An intracellular cAMP accumulation assay elucidated the biological significance of this phosphorylation because it ablated the PTH-mediated signaling. Our studies thus shed light on functional implications of phosphorylation at native PTH as an additional level of regulation. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1216 TI - Growing and Processing Conditions Lead to Changes in the Carotenoid Profile of Spinach JO - J. Agr. Food Chem. PY - 2014 SP - 4960-4967 AU - Heymann, T. AU - Westphal, L. AU - Wessjohann, L. AU - Glomb, M. A. AU - VL - 62 UR - DO - 10.1021/jf501136g AB - This study aimed to evaluate the influence of different light regimens during spinach cultivation on the isomeric composition of β-carotene. Irradiation with a halogen lamp, which has a wavelength spectrum close to that of daylight, was used to mimic field-grown conditions. The additional use of optical filters was established as a model system for greenhouse cultivation. Field-grown model systems led to a preferential increase of 9-cis-β-carotene, whereas 13-cis-β-carotene was just formed at the beginning of irradiation. Additionally 9,13-di-cis-β-carotene decreased significantly in the presence of energy-rich light. Isomerization of β-carotene was strongly suppressed during irradiation in greenhouse-grown model systems and led to significant differences. These results were verified in biological samples. Authentic field-grown spinach (Spinacia oleracea L.) showed among changes of other isomers a significantly higher level of 9-cis-isomers (7.52 ± 0.14%) and a significantly lower level of 9,13-di-cis-isomers (0.25 ± 0.03%) compared to authentic greenhouse-grown spinach (6.49 ± 0.11 and 0.76 ± 0.05%). Almost all analyzed commercial spinach samples (fresh and frozen) were identified as common field-grown cultivation. Further investigations resulted in a clear differentiation of frozen commercial samples from fresh spinach, caused by significantly higher levels of 13-cis- and 15-cis-β-carotene as a result of industrial blanching processes. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1214 TI - Paralog Re-Emergence: A Novel, Historically Contingent Mechanism in the Evolution of Antimicrobial Resistance JO - Mol. Biol. Evol. PY - 2014 SP - 1793-1802 AU - Hawkins, N. J. AU - Cools, H. J. AU - Sierotzki, H. AU - Shaw, M. W. AU - Knogge, W. AU - Kelly, S. L. AU - Kelly, D. E. AU - Fraaije, B. A. AU - VL - 31 UR - DO - 10.1093/molbev/msu134 AB - Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1191 TI - Arabidopsis Protein Phosphatase DBP1 Nucleates a Protein Network with a Role in Regulating Plant Defense JO - PLOS ONE PY - 2014 SP - e90734 AU - Carrasco, J. L. AU - Castelló, M. J. AU - Naumann, K. AU - Lassowskat, I. AU - Navarrete-Gómez, M. AU - Scheel, D. AU - Vera, P. AU - VL - 9 UR - DO - 10.1371/journal.pone.0090734 AB - Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6), a previously reported DBP1 interactor, and MAP kinase (MAPK) MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV), and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1190 TI - The Plasmodesmal Protein PDLP1 Localises to Haustoria-Associated Membranes during Downy Mildew Infection and Regulates Callose Deposition JO - PLOS Pathog. PY - 2014 SP - e1004496 AU - Caillaud, M.-C. AU - Wirthmueller, L. AU - Sklenar, J. AU - Findlay, K. AU - Piquerez, S. J. M. AU - Jones, A. M. E. AU - Robatzek, S. AU - Jones, J. D. G. AU - Faulkner, C. AU - VL - 10 UR - DO - 10.1371/journal.ppat.1004496 AB - The downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) is a filamentous oomycete that invades plant cells via sophisticated but poorly understood structures called haustoria. Haustoria are separated from the host cell cytoplasm and surrounded by an extrahaustorial membrane (EHM) of unknown origin. In some interactions, including Hpa-Arabidopsis, haustoria are progressively encased by host-derived, callose-rich materials but the molecular mechanisms by which callose accumulates around haustoria remain unclear. Here, we report that PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1) is expressed at high levels in Hpa infected cells. Unlike other plasma membrane proteins, which are often excluded from the EHM, PDLP1 is located at the EHM in Hpa-infected cells prior to encasement. The transmembrane domain and cytoplasmic tail of PDLP1 are sufficient to convey this localization. PDLP1 also associates with the developing encasement but this association is lost when encasements are fully mature. We found that the pdlp1,2,3 triple mutant is more susceptible to Hpa while overexpression of PDLP1 enhances plant resistance, suggesting that PDLPs enhance basal immunity against Hpa. Haustorial encasements are depleted in callose in pdlp1,2,3 mutant plants whereas PDLP1 over-expression elevates callose deposition around haustoria and across the cell surface. These data indicate that PDLPs contribute to callose encasement of Hpa haustoria and suggests that the deposition of callose at haustoria may involve similar mechanisms to callose deposition at plasmodesmata. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1341 TI - A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture JO - PLOS Pathog. PY - 2013 SP - e1003121 AU - Singer, A. U. AU - Schulze, S. AU - Skarina, T. AU - Xu, X. AU - Cui, H. AU - Eschen-Lippold, L. AU - Egler, M. AU - Srikumar, T. AU - Raught, B. AU - Lee, J. AU - Scheel, D. AU - Savchenko, A. AU - Bonas, U. AU - VL - 9 UR - DO - 10.1371/journal.ppat.1003121 AB - Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1338 TI - Analyses of wrky18 wrky40 Plants Reveal Critical Roles of SA/EDS1 Signaling and Indole-Glucosinolate Biosynthesis for Golovinomyces orontii Resistance and a Loss-of Resistance Towards Pseudomonas syringae pv. tomato AvrRPS4 JO - Mol. Plant Microbe Interact. PY - 2013 SP - 758-767 AU - Schön, M. AU - Töller, A. AU - Diezel, C. AU - Roth, C. AU - Westphal, L. AU - Wiermer, M. AU - Somssich, I. E. AU - VL - 26 UR - DO - 10.1094/MPMI-11-12-0265-R AB - Simultaneous mutation of two WRKY-type transcription factors, WRKY18 and WRKY40, renders otherwise susceptible wild-type Arabidopsis plants resistant towards the biotrophic powdery mildew fungus Golovinomyces orontii. Resistance in wrky18 wrky40 double mutant plants is accompanied by massive transcriptional reprogramming, imbalance in salicylic acid (SA) and jasmonic acid (JA) signaling, altered ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) expression, and accumulation of the phytoalexin camalexin. Genetic analyses identified SA biosynthesis and EDS1 signaling as well as biosynthesis of the indole-glucosinolate 4MI3G as essential components required for loss-of-WRKY18 WRKY40–mediated resistance towards G. orontii. The analysis of wrky18 wrky40 pad3 mutant plants impaired in camalexin biosynthesis revealed an uncoupling of pre- from postinvasive resistance against G. orontii. Comprehensive infection studies demonstrated the specificity of wrky18 wrky40–mediated G. orontii resistance. Interestingly, WRKY18 and WRKY40 act as positive regulators in effector-triggered immunity, as the wrky18 wrky40 double mutant was found to be strongly susceptible towards the bacterial pathogen Pseudomonas syringae DC3000 expressing the effector AvrRPS4 but not against other tested Pseudomonas strains. We hypothesize that G. orontii depends on the function of WRKY18 and WRKY40 to successfully infect Arabidopsis wild-type plants while, in the interaction with P. syringae AvrRPS4, they are required to mediate effector-triggered immunity. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1334 TI - Mapping FLS2 function to structure: LRRs, kinase and its working bits JO - Protoplasma PY - 2013 SP - 671-681 AU - Robatzek, S. AU - Wirthmueller, L. AU - VL - 250 UR - DO - 10.1007/s00709-012-0459-6 AB - The plasma membrane-localised FLAGELLIN SENSING 2 (FLS2) receptor is an important component of plant immunity against potentially pathogenic bacteria, acting to recognise the conserved flg22 peptide of flagellin. FLS2 shares the common structure of transmembrane receptor kinases with a receptor-like ectodomain composed of leucine-rich repeats (LRR) and an active intracellular kinase domain. Upon ligand binding, FLS2 dimerises with the regulatory LRR-receptor kinase BRI1-associated kinase 1, which in turn triggers downstream signalling cascades. Although lacking crystal structure data, recent advances have been made in our understanding of flg22 recognition based on structural and functional analyses of FLS2. These studies have revealed critical regions/residues of FLS2 and post-translational modifications that regulate the abundance and activity of this receptor. In this review, we present the current knowledge on the structural mechanism of the FLS2–flg22 interaction and subsequent receptor-mediated signalling. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1327 TI - Plant F-Box Protein Evolution Is Determined by Lineage-Specific Timing of Major Gene Family Expansion Waves JO - PLOS ONE PY - 2013 SP - e68672 AU - Navarro-Quezada, A. AU - Schumann, N. AU - Quint, M. AU - VL - 8 UR - DO - 10.1371/journal.pone.0068672 AB - F-box proteins (FBPs) represent one of the largest and fastest evolving gene/protein families in the plant kingdom. The FBP superfamily can be divided in several subfamilies characterized by different C-terminal protein-protein interaction domains that recruit targets for proteasomal degradation. Hence, a clear picture of their phylogeny and molecular evolution is of special interest for the general understanding of evolutionary histories of multi-domain and/or large protein families in plants. In an effort to further understand the molecular evolution of F-box family proteins, we asked whether the largest subfamily in Arabidopsis thaliana, which carries a C-terminal F-box associated domain (FBA proteins) shares evolutionary patterns and signatures of selection with other FBPs. To address this question, we applied phylogenetic and molecular evolution analyses in combination with the evaluation of transcriptional profiles. Based on the 2219 FBA proteins we de novo identified in 34 completely sequenced plant genomes, we compared their evolutionary patterns to a previously analyzed large subfamily carrying C-terminal kelch repeats. We found that these two large FBP subfamilies generally tend to evolve by massive waves of duplication, followed by sequence conservation of the F-box domain and sequence diversification of the target recruiting domain. We conclude that the earlier in evolutionary time a major wave of expansion occurred, the more pronounced these selection signatures are. As a consequence, when performing cross species comparisons among FBP subfamilies, significant differences will be observed in the selective signatures of protein-protein interaction domains. Depending on the species, the investigated subfamilies comprise up to 45% of the complete superfamily, indicating that other subfamilies possibly follow similar modes of evolution. A2 - C1 - Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1325 TI - Reprogramming the Phenylpropanoid Metabolism in Seeds of Oilseed Rape by Suppressing the Orthologs of REDUCED EPIDERMAL FLUORESCENCE1 JO - J. Plant Physiol. PY - 2013 SP - 1656-1669 AU - Mittasch, J. AU - Böttcher, C. AU - Frolov, A. AU - Strack, D. AU - Milkowski, C. AU - VL - 161 UR - DO - 10.1104/pp.113.215491 AB - As a result of the phenylpropanoid pathway, many Brassicaceae produce considerable amounts of soluble hydroxycinnamate conjugates, mainly sinapate esters. From oilseed rape (Brassica napus), we cloned two orthologs of the Arabidopsis (Arabidopsis thaliana) gene REDUCED EPIDERMAL FLUORESCENCE1 (REF1) encoding a coniferaldehyde/sinapaldehyde dehydrogenase. The enzyme is involved in the formation of ferulate and sinapate from the corresponding aldehydes, thereby linking lignin and hydroxycinnamate biosynthesis as a potential branch-point enzyme. We used RNA interference to silence REF1 genes in seeds of oilseed rape. Nontargeted metabolite profiling showed that BnREF1-suppressing seeds produced a novel chemotype characterized by reduced levels of sinapate esters, the appearance of conjugated monolignols, dilignols, and trilignols, altered accumulation patterns of kaempferol glycosides, and changes in minor conjugates of caffeate, ferulate, and 5-hydroxyferulate. BnREF1 suppression affected the level of minor sinapate conjugates more severely than that of the major component sinapine. Mapping of the changed metabolites onto the phenylpropanoid metabolic network revealed partial redirection of metabolic sequences as a major impact of BnREF1 suppression. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1321 TI - PAPE (Prefractionation-Assisted Phosphoprotein Enrichment): A Novel Approach for Phosphoproteomic Analysis of Green Tissues from Plants JO - Proteomes PY - 2013 SP - 254-274 AU - Lassowskat, I. AU - Naumann, K. AU - Lee, J. AU - Scheel, D. AU - VL - 1 UR - DO - 10.3390/proteomes1030254 AB - Phosphorylation is an important post-translational protein modification with regulatory roles in diverse cellular signaling pathways. Despite recent advances in mass spectrometry, the detection of phosphoproteins involved in signaling is still challenging, as protein phosphorylation is typically transient and/or occurs at low levels. In green plant tissues, the presence of highly abundant proteins, such as the subunits of the RuBisCO complex, further complicates phosphoprotein analysis. Here, we describe a simple, but powerful, method, which we named prefractionation-assisted phosphoprotein enrichment (PAPE), to increase the yield of phosphoproteins from Arabidopsis thaliana leaf material. The first step, a prefractionation via ammonium sulfate precipitation, not only depleted RuBisCO almost completely, but, serendipitously, also served as an efficient phosphoprotein enrichment step. When coupled with a subsequent metal oxide affinity chromatography (MOAC) step, the phosphoprotein content was highly enriched. The reproducibility and efficiency of phosphoprotein enrichment was verified by phospho-specific staining and, further, by mass spectrometry, where it could be shown that the final PAPE fraction contained a significant number of known and additionally novel (potential) phosphoproteins. Hence, this facile two-step procedure is a good prerequisite to probe the phosphoproteome and gain deeper insight into plant phosphorylation-based signaling events. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1319 TI - Impaired sterol ester synthesis alters the response of Arabidopsis thaliana to Phytophthora infestans JO - Plant J. PY - 2013 SP - 456-468 AU - Kopischke, M. AU - Westphal, L. AU - Schneeberger, K. AU - Clark, R. AU - Ossowski, S. AU - Wewer, V. AU - Fuchs, R. AU - Landtag, J. AU - Hause, G. AU - Dörmann, P. AU - Lipka, V. AU - Weigel, D. AU - Schulze-Lefert, P. AU - Scheel, D. AU - Rosahl, S. AU - VL - 73 UR - DO - 10.1111/tpj.12046 AB - Non‐host resistance of Arabidopsis thaliana against Phytophthora infestans, the causal agent of late blight disease of potato, depends on efficient extracellular pre‐ and post‐invasive resistance responses. Pre‐invasive resistance against P. infestans requires the myrosinase PEN2. To identify additional genes involved in non‐host resistance to P. infestans, a genetic screen was performed by re‐mutagenesis of pen2 plants. Fourteen independent mutants were isolated that displayed an enhanced response to Phytophthora (erp) phenotype. Upon inoculation with P. infestans, two mutants, pen2‐1 erp1‐3 and pen2‐1 erp1‐4, showed an enhanced rate of mesophyll cell death and produced excessive callose deposits in the mesophyll cell layer. ERP1 encodes a phospholipid:sterol acyltransferase (PSAT1) that catalyzes the formation of sterol esters. Consistent with this, the tested T‐DNA insertion lines of PSAT1 are phenocopies of erp1 plants. Sterol ester levels are highly reduced in all erp1/psat1 mutants, whereas sterol glycoside levels are increased twofold. Excessive callose deposition occurred independently of PMR4/GSL5 activity, a known pathogen‐inducible callose synthase. A similar formation of aberrant callose deposits was triggered by the inoculation of erp1psat1 plants with powdery mildew. These results suggest a role for sterol conjugates in cell non‐autonomous defense responses against invasive filamentous pathogens. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1360 TI - On the front line: structural insights into plant–pathogen interactions JO - Nat. Rev. Microbiol. PY - 2013 SP - 761-776 AU - Wirthmueller, L. AU - Maqbool, A. AU - Banfield, M. J. AU - VL - 11 UR - DO - 10.1038/nrmicro3118 AB - Over the past decade, considerable advances have been made in understanding the molecular mechanisms that underpin the arms race between plant pathogens and their hosts. Alongside genomic, bioinformatic, proteomic, biochemical and cell biological analyses of plant–pathogen interactions, three-dimensional structural studies of virulence proteins deployed by pathogens to promote infection, in some cases complexed with their plant cell targets, have uncovered key insights into the functions of these molecules. Structural information on plant immune receptors, which regulate the response to pathogen attack, is also starting to emerge. Structural studies of bacterial plant pathogen–host systems have been leading the way, but studies of filamentous plant pathogens are gathering pace. In this Review, we summarize the key developments in the structural biology of plant pathogen–host interactions. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1359 TI - Hop-on hop-off: importin-α-guided tours to the nucleus in innate immune signaling JO - Front. Plant Sci. PY - 2013 SP - 149 AU - Wirthmueller, L. AU - Roth, C. AU - Banfield, M. J. AU - Wiermer, M. AU - VL - 4 UR - DO - 10.3389/fpls.2013.00149 AB - Nuclear translocation of immune regulatory proteins and signal transducers is an essential process in animal and plant defense signaling against pathogenic microbes. Import of proteins containing a nuclear localization signal (NLS) into the nucleus is mediated by nuclear transport receptors termed importins, typically dimers of a cargo-binding α-subunit and a β-subunit that mediates translocation through the nuclear pore complex. Here, we review recent reports of importin-α cargo specificity and mutant phenotypes in plant- and animal–microbe interactions. Using homology modeling of the NLS-binding cleft of nine predicted Arabidopsis α-importins and analyses of their gene expression patterns, we discuss functional redundancy and specialization within this transport receptor family. In addition, we consider how pathogen effector proteins that promote infection by manipulating host cell nuclear processes might compete with endogenous cargo proteins for nuclear uptake. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1346 TI - The exocyst subunit Exo70B1 is involved in the immune response of Arabidopsis thaliana to different pathogens and cell death JO - Plant Signal Behav. PY - 2013 SP - e27421 AU - Stegmann, M. AU - Anderson, R. G. AU - Westphal, L. AU - Rosahl, S. AU - McDowell, J. M. AU - Trujillo, M. AU - VL - 8 UR - DO - 10.4161/psb.27421 AB - Components of the vesicle trafficking machinery are central to the immune response in plants. The role of vesicle trafficking during pre-invasive penetration resistance has been well documented. However, emerging evidence also implicates vesicle trafficking in early immune signaling. Here we report that Exo70B1, a subunit of the exocyst complex which mediates early tethering during exocytosis is involved in resistance. We show that exo70B1 mutants display pathogen-specific immuno-compromised phenotypes. We also show that exo70B1 mutants display lesion-mimic cell death, which in combination with the reduced responsiveness to pathogen-associated molecular patterns (PAMPs) results in complex immunity-related phenotypes. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1309 TI - Arabidopsis TNL-WRKY domain receptor RRS1 contributes to temperature-conditioned RPS4 auto-immunity JO - Front. Plant Sci. PY - 2013 SP - 403 AU - Heidrich, K. AU - Tsuda, K. AU - Blanvillain-Baufumé, S. AU - Wirthmueller, L. AU - Bautor, J. AU - Parker, J. E. AU - VL - 4 UR - DO - 10.3389/fpls.2013.00403 AB - In plant effector-triggered immunity (ETI), intracellular nucleotide binding-leucine rich repeat (NLR) receptors are activated by specific pathogen effectors. The ArabidopsisTIR (Toll-Interleukin-1 receptor domain)-NLR (denoted TNL) gene pair, RPS4 and RRS1, confers resistance to Pseudomonas syringae pv tomato (Pst) strain DC3000 expressing the Type III-secreted effector, AvrRps4. Nuclear accumulation of AvrRps4, RPS4, and the TNL resistance regulator EDS1 is necessary for ETI. RRS1 possesses a C-terminal “WRKY” transcription factor DNA binding domain suggesting that important RPS4/RRS1 recognition and/or resistance signaling events occur at the nuclear chromatin. In Arabidopsis accession Ws-0, the RPS4Ws/RRS1Ws allelic pair governs resistance to Pst/AvrRps4 accompanied by host programed cell death (pcd). In accession Col-0, RPS4Col/RRS1Col effectively limits Pst/AvrRps4 growth without pcd. Constitutive expression of HA-StrepII tagged RPS4Col (in a 35S:RPS4-HS line) confers temperature-conditioned EDS1-dependent auto-immunity. Here we show that a high (28°C, non-permissive) to moderate (19°C, permissive) temperature shift of 35S:RPS4-HS plants can be used to follow defense-related transcriptional dynamics without a pathogen effector trigger. By comparing responses of 35S:RPS4-HS with 35S:RPS4-HSrrs1-11 and 35S:RPS4-HSeds1-2 mutants, we establish that RPS4Col auto-immunity depends entirely on EDS1 and partially on RRS1Col. Examination of gene expression microarray data over 24 h after temperature shift reveals a mainly quantitative RRS1Col contribution to up- or down-regulation of a small subset of RPS4Col-reprogramed, EDS1-dependent genes. We find significant over-representation of WRKY transcription factor binding W-box cis-elements within the promoters of these genes. Our data show that RRS1Col contributes to temperature-conditioned RPS4Col auto-immunity and are consistent with activated RPS4Col engaging RRS1Col for resistance signaling. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1377 TI - Fast and efficient MCR-based synthesis of clickable rhodamine tags for protein profiling JO - Org. Biomol. Chem. PY - 2012 SP - 958-965 AU - Brauch, S. AU - Henze, M. AU - Osswald, B. AU - Naumann, K. AU - Wessjohann, L. A. AU - van Berkel, S. S. AU - Westermann, B. AU - VL - 10 UR - DO - 10.1039/C1OB06581E AB - Protein profiling probes are important tools for studying the composition of the proteome and as such have contributed greatly to the understanding of various complex biological processes in higher organisms. For this purpose the application of fluorescently labeled activity or affinity probes is highly desirable. Especially for in vivodetection of low abundant target proteins, otherwise difficult to analyse by standard blotting techniques, fluorescently labeled profiling probes are of high value. Here, a one-pot protocol for the synthesis of activated fluorescent labels (i.e.azide, alkynyl or NHS), based on the Ugi-4-component reaction (Ugi-4CR), is presented. As a result of the peptoidic structure formed, the fluorescent properties of the products are pH insensitive. Moreover, the applicability of these probes, as exemplified by the labeling of model protein BSA, will be discussed. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1373 TI - Activation of the Arabidopsis thaliana Mitogen-Activated Protein Kinase MPK11 by the Flagellin-Derived Elicitor Peptide, flg22 JO - Mol. Plant Microbe Interact. PY - 2012 SP - 471-480 AU - Bethke, G. AU - Pecher, P. AU - Eschen-Lippold, L. AU - Tsuda, K. AU - Katagiri, F. AU - Glazebrook, J. AU - Scheel, D. AU - Lee, J. AU - VL - 25 UR - DO - 10.1094/MPMI-11-11-0281 AB - Mitogen-activated protein kinases (MAPK) mediate cellular signal transduction during stress responses, as well as diverse growth and developmental processes in eukaryotes. Pathogen infection or treatments with conserved pathogen-associated molecular patterns (PAMPs) such as the bacterial flagellin-derived flg22 peptide are known to activate three Arabidopsis thaliana MAPK: MPK3, MPK4, and MPK6. Several stresses, including flg22 treatment, are known to increase MPK11 expression but activation of MPK11 has not been shown. Here, we show that MPK11 activity can, indeed, be increased through flg22 elicitation. A small-scale microarray for profiling defense-related genes revealed that cinnamyl alcohol dehyrogenase 5 requires MPK11 for full flg22-induced expression. An mpk11 mutant showed increased flg22-mediated growth inhibition but no altered susceptibility to Pseudomonas syringae, Botrytis cinerea, or Alternaria brassicicola. In mpk3, mpk6, or mpk4 backgrounds, MPK11 is required for embryo or seed development or general viability. Although this developmental deficiency in double mutants and the lack of or only subtle mpk11 phenotypes suggest functional MAPK redundancies, comparison with the paralogous MPK4 reveals distinct functions. Taken together, future investigations of MAPK roles in stress signaling should include MPK11 as a fourth PAMP-activated MAPK. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1369 TI - Rhynchosporium commune: a persistent threat to barley cultivation JO - Mol. Plant Pathol. PY - 2012 SP - 986-997 AU - Avrova, A. AU - Knogge, W. AU - VL - 13 UR - DO - 10.1111/j.1364-3703.2012.00811.x AB - Rhynchosporium commune is a haploid fungus causing scald or leaf blotch on barley, other Hordeum spp. and Bromus diandrus.TaxonomyRhynchosporium commune is an anamorphic Ascomycete closely related to the teleomorph Helotiales genera Oculimacula and Pyrenopeziza.Disease symptomsRhynchosporium commune causes scald‐like lesions on leaves, leaf sheaths and ears. Early symptoms are generally pale grey oval lesions. With time, the lesions acquire a dark brown margin with the centre of the lesion remaining pale green or pale brown. Lesions often merge to form large areas around which leaf yellowing is common. Infection frequently occurs in the leaf axil, which can lead to chlorosis and eventual death of the leaf.Life cycleRhynchosporium commune is seed borne, but the importance of this phase of the disease is not fully understood. Debris from previous crops and volunteers, infected from the stubble from previous crops, are considered to be the most important sources of the disease. Autumn‐sown crops can become infected very soon after sowing. Secondary spread of disease occurs mainly through splash dispersal of conidia from infected leaves. Rainfall at the stem extension growth stage is the major environmental factor in epidemic development.Detection and quantificationRhynchosporium commune produces unique beak‐shaped, one‐septate spores both on leaves and in culture. The development of a specific polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) has allowed the identification of asymptomatic infection in seeds and during the growing season.Disease controlThe main measure for the control of R. commune is the use of fungicides with different modes of action, in combination with the use of resistant cultivars. However, this is constantly under review because of the ability of the pathogen to adapt to host plant resistance and to develop fungicide resistance. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1389 TI - MPK11—a fourth elicitor-responsive mitogen-activated protein kinase in Arabidopsis thaliana JO - Plant Signal Behav. PY - 2012 SP - 1203-1205 AU - Eschen-Lippold, L. AU - Bethke, G. AU - Palm-Forster, M. A. T. AU - Pecher, P. AU - Bauer, N. AU - Glazebrook, J. AU - Scheel, D. AU - Lee, J. AU - VL - 7 UR - DO - 10.4161/psb.21323 AB - Recognition of pathogen attack or elicitation with pathogen-associated molecular patterns (PAMPs) leads to defense signaling that includes activation of the three mitogen-activated protein kinases (MPKs), MPK3, MPK4 and MPK6 in Arabidopsis. Recently, we demonstrated the activation of a fourth MPK, MPK11, after treatment with flg22, a 22 amino acid PAMP derived from bacterial flagellin. Here, we extended the study by examining elicitation with two other PAMPs, elf18 (derived from bacterial elongation factor EF-Tu) and ch8 (N-acetylchitooctaose derived from fungal chitin). Both PAMPs led to rapid MPK11 transcript accumulation and increased MPK11 kinase activity, suggesting that multiple PAMPs (or stresses) can activate MPK11. However, probably due to functional redundancies, bacteria-induced phytoalexin accumulation does not absolutely require MPK11. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1388 TI - Die Abwehr der Kartoffel gegen den Erreger der Kraut- und Knollenfäule JO - Kartoffelbau PY - 2012 SP - 38-39 AU - Eschen-Lippold, L. AU - Rosahl, S. AU - VL - 63 UR - https://www.dlg-agrofoodmedien.de/fileadmin/download/jahresindex/Kartoffelbau/IND_KB_2012.pdf AB - A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1387 TI - Characterization of potato plants with reduced StSYR1 expression JO - Plant Signal Behav. PY - 2012 SP - 559-562 AU - Eschen-Lippold, L. AU - Lübken, T. AU - Smolka, U. AU - Rosahl, S. AU - VL - 7 UR - DO - 10.4161/psb.19866 AB - Vesicle fusion processes in plants are important for both development and stress responses. Transgenic potato plants with reduced expression of SYNTAXIN-RELATED1 (StSYR1), a gene encoding the potato homolog of Arabidopsis PENETRATION1 (AtPEN1), display spontaneous necrosis and chlorosis at later stages of development. In accordance with this developmental defect, tuber number, weight and overall yield are significantly reduced in StSYR1-RNAi lines. Enhanced resistance of StSYR1-RNAi plants to Phytophthora infestans, the causal agent of late blight disease of potato, correlates with enhanced levels of salicylic acid, whereas levels of 12-oxophytodienoic acid and jasmonic acid are unaltered. Cultured cells of StSYR1-RNAi lines secrete at least two compounds which are not detectable in the supernatant of control cells, suggesting an involvement of StSYR1 in secretion processes to the apoplast. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1386 TI - Aus Pilzen isolierte Substanz gegen den Erreger der Kraut- und Knollenfäule JO - Kartoffelbau PY - 2012 SP - 18-21 AU - Eschen-Lippold, L. AU - Rosahl, S. AU - Westermann, B. AU - Arnold, N. AU - VL - 63 UR - https://www.dlg-agrofoodmedien.de/fileadmin/download/jahresindex/Kartoffelbau/IND_KB_2012.pdf AB - A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1385 TI - Activation of defense against Phytophthora infestans in potato by down-regulation of syntaxin gene expression JO - New Phytol. PY - 2012 SP - 985-996 AU - Eschen-Lippold, L. AU - Landgraf, R. AU - Smolka, U. AU - Schulze, S. AU - Heilmann, M. AU - Heilmann, I. AU - Hause, G. AU - Rosahl, S. AU - VL - 193 UR - DO - 10.1111/j.1469-8137.2011.04024.x AB - • The oomycete Phytophthora infestans is the causal agent of late blight, the most devastating disease of potato. The importance of vesicle fusion processes and callose deposition for defense of potato against Phytophthora infestans was analyzed.• Transgenic plants were generated, which express RNA interference constructs targeted against plasma membrane‐localized SYNTAXIN‐RELATED 1 (StSYR1) and SOLUBLE N‐ETHYLMALEIMIDE‐SENSITIVE FACTOR ADAPTOR PROTEIN 33 (StSNAP33), the potato homologs of Arabidopsis AtSYP121 and AtSNAP33, respectively.• Phenotypically, transgenic plants grew normally, but showed spontaneous necrosis and chlorosis formation at later stages. In response to infection with Phytophthora infestans, increased resistance of StSYR1‐RNAi plants, but not StSNAP33‐RNAi plants, was observed. This increased resistance correlated with the constitutive accumulation of salicylic acid and PR1 transcripts. Aberrant callose deposition in Phytophthora infestans‐infected StSYR1‐RNAi plants coincided with decreased papilla formation at penetration sites. Resistance against the necrotrophic fungus Botrytis cinerea was not significantly altered. Infiltration experiments with bacterial solutions of Agrobacterium tumefaciens and Escherichia coli revealed a hypersensitive phenotype of both types of RNAi lines.• The enhanced defense status and the reduced growth of Phytophthora infestans on StSYR1‐RNAi plants suggest an involvement of syntaxins in secretory defense responses of potato and, in particular, in the formation of callose‐containing papillae. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1378 TI - Mechanisms of Nuclear Suppression of Host Immunity by Effectors from the Arabidopsis Downy Mildew Pathogen Hyaloperonospora arabidopsidis (Hpa) JO - Cold Spring Harb. Symp. Quant. Biol. PY - 2012 SP - 285-293 AU - Caillaud, M.-. C. AU - Wirthmueller, L. AU - Fabro, G. AU - Piquerez, S. J. M. AU - Asai, S. AU - Ishaque, N. AU - Jones, J. D. G. AU - VL - 77 UR - DO - 10.1101/sqb.2012.77.015115 AB - Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria additional to their more characterized role of suppressing plant defense. Recent studies suggest that effectors may manipulate host transcription or other nuclear regulatory components for the benefit of pathogen development. However, the specific mechanisms by which these effectors promote susceptibility remain unclear. Of two recent screenings, we identified 15 nuclear-localized Hpa effectors (HaRxLs) that interact directly or indirectly with host nuclear components. When stably expressed in planta, nuclear HaRxLs cause diverse developmental phenotypes highlighting that nuclear effectors might interfere with fundamental plant regulatory mechanisms. Here, we report recent advances in understanding how a pathogen can manipulate nuclear processes in order to cause disease. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1435 TI - Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity JO - New Phytol. PY - 2012 SP - 894-911 AU - Schulze, S. AU - Kay, S. AU - Büttner, D. AU - Egler, M. AU - Eschen-Lippold, L. AU - Hause, G. AU - Krüger, A. AU - Lee, J. AU - Müller, O. AU - Scheel, D. AU - Szczesny, R. AU - Thieme, F. AU - Bonas, U. AU - VL - 195 UR - DO - 10.1111/j.1469-8137.2012.04210.x AB - The pathogenicity of the Gram‐negative plant‐pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is dependent on type III effectors (T3Es) that are injected into plant cells by a type III secretion system and interfere with cellular processes to the benefit of the pathogen.In this study, we analyzed eight T3Es from Xcv strain 85‐10, six of which were newly identified effectors. Genetic studies and protoplast expression assays revealed that XopB and XopS contribute to disease symptoms and bacterial growth, and suppress pathogen‐associated molecular pattern (PAMP)‐triggered plant defense gene expression.In addition, XopB inhibits cell death reactions induced by different T3Es, thus suppressing defense responses related to both PAMP‐triggered immunity (PTI) and effector‐triggered immunity (ETI).XopB localizes to the Golgi apparatus and cytoplasm of the plant cell and interferes with eukaryotic vesicle trafficking. Interestingly, a XopB point mutant derivative was defective in the suppression of ETI‐related responses, but still interfered with vesicle trafficking and was only slightly affected with regard to the suppression of defense gene induction. This suggests that XopB‐mediated suppression of PTI and ETI is dependent on different mechanisms that can be functionally separated. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1425 TI - Defense-Related Calcium Signaling Mutants Uncovered via a Quantitative High-Throughput Screen in Arabidopsis thaliana JO - Mol. Plant PY - 2012 SP - 115-130 AU - Ranf, S. AU - Grimmer, J. AU - Pöschl, Y. AU - Pecher, P. AU - Chinchilla, D. AU - Scheel, D. AU - Lee, J. AU - VL - 5 UR - DO - 10.1093/mp/ssr064 AB - Calcium acts as a second messenger for signaling to a variety of stimuli including MAMPs (Microbe-Associated Molecular Patterns), such as flg22 and elf18 that are derived from bacterial flagellin and elongation factor Tu, respectively. Here, Arabidopsis thaliana mutants with changed calcium elevation (cce) in response to flg22 treatment were isolated and characterized. Besides novel mutant alleles of the flg22 receptor, FLS2 (Flagellin-Sensitive 2), and the receptor-associated kinase, BAK1 (Brassinosteroid receptor 1-Associated Kinase 1), the new cce mutants can be categorized into two main groups—those with a reduced or an enhanced calcium elevation. Moreover, cce mutants from both groups show differential phenotypes to different sets of MAMPs. Thus, these mutants will facilitate the discovery of novel components in early MAMP signaling and bridge the gaps in current knowledge of calcium signaling during plant–microbe interactions. Last but not least, the screening method is optimized for speed (covering 384 plants in 3 or 10 h) and can be adapted to genetically dissect any other stimuli that induce a change in calcium levels. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1420 TI - A mutagenesis-based screen to rapidly identify phosphorylation sites in mitogen-activated protein kinase substrates JO - Anal. Biochem. PY - 2012 SP - 127-129 AU - Palm-Forster, M. A. T. AU - Eschen-Lippold, L. AU - Lee, J. AU - VL - 427 UR - DO - 10.1016/j.ab.2012.05.015 AB - Identification and characterization of protein phosphorylation sites often requires mass spectrometric analysis, which is not trivial or accessible to many laboratories. Here, a targeted strategy to mutagenize putative phosphorylation sites within mitogen-activated protein kinase (MAPK) substrates is described. This employs a combination of standard type II with type IIs restriction enzymes to rapidly create individual or multiple phosphorylation site mutant versions of kinase substrates with high efficiency, thereby reducing the cost for screening mutated clones. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1409 TI - Necrosis-Inducing Proteins of Rhynchosporium commune, Effectors in Quantitative Disease Resistance JO - Mol. Plant Microbe Interact. PY - 2012 SP - 1314-1325 AU - Kirsten, S. AU - Navarro-Quezada, A. AU - Penselin, D. AU - Wenzel, C. AU - Matern, A. AU - Leitner, A. AU - Baum, T. AU - Seiffert, U. AU - Knogge, W. AU - VL - 25 UR - DO - 10.1094/MPMI-03-12-0065-R AB - The barley pathogen Rhynchosporium commune secretes necrosis-inducing proteins NIP1, NIP2, and NIP3. Expression analysis revealed that NIP1 transcripts appear to be present in fungal spores already, whereas NIP2 and NIP3 are synthesized after inoculation of host plants. To assess the contribution of the three effector proteins to disease development, deletion mutants were generated. The development of these fungal mutants on four barley cultivars was quantified in comparison with that of the parent wild-type strain and with two fungal strains failing to secrete an “active” NIP1 avirulence protein, using quantitative polymerase chain reaction as well as microscopic imaging after fungal green fluorescent protein tagging. The impact of the three deletions varied quantitatively depending on the host genotype, suggesting that the activities of the fungal effectors add up to produce stronger growth patterns and symptom development. Alternatively, recognition events of differing intensities may be converted into defense gene expression in a quantitative manner. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1457 TI - mADP-RTs: versatile virulence factors from bacterial pathogens of plants and mammals JO - Front. Plant Sci. PY - 2012 SP - 142 AU - Wirthmueller, L. AU - Banfield, M. J. AU - VL - 3 UR - DO - 10.3389/fpls.2012.00142 AB - Mono ADP-ribosyltransferases (mADP-RTs) are a family of enzymes that cleave NAD+ and covalently attach the ADP-ribosyl moiety to target proteins. mADP-RTs are well established as important virulence factors of bacteria that infect mammals. Cholera toxin, pertussis toxin, and diphtheria toxin are three of the best-known examples of mADP-RTs. They modify host target proteins in order to promote infection and/or killing of the host cell. Despite low sequence similarity at the primary amino acid level, mADP-RTs share a conserved core catalytic fold and structural biology has made important contributions to elucidating how mADP-RTs modify mammalian host targets. Recently, mADP-RTs were shown to be present in plant pathogenic bacteria, suggesting that mADP-RTs are also important virulence factors of plant pathogens. Crystal structures of plant pathogenic bacterial mADP-RTs are also now available. Here we review the structure/function of mADP-RTs from pathogens of mammals and plants, highlighting both commonalities and differences. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1467 TI - HyphArea—Automated analysis of spatiotemporal fungal patterns JO - J. Plant Physiol. PY - 2011 SP - 72-78 AU - Baum, T. AU - Navarro-Quezada, A. AU - Knogge, W. AU - Douchkov, D. AU - Schweizer, P. AU - Seiffert, U. AU - VL - 168 UR - DO - 10.1016/j.jplph.2010.08.004 AB - In phytopathology quantitative measurements are rarely used to assess crop plant disease symptoms. Instead, a qualitative valuation by eye is often the method of choice. In order to close the gap between subjective human inspection and objective quantitative results, the development of an automated analysis system that is capable of recognizing and characterizing the growth patterns of fungal hyphae in micrograph images was developed. This system should enable the efficient screening of different host–pathogen combinations (e.g., barley—Blumeria graminis, barley—Rhynchosporium secalis) using different microscopy technologies (e.g., bright field, fluorescence). An image segmentation algorithm was developed for gray-scale image data that achieved good results with several microscope imaging protocols. Furthermore, adaptability towards different host–pathogen systems was obtained by using a classification that is based on a genetic algorithm. The developed software system was named HyphArea, since the quantification of the area covered by a hyphal colony is the basic task and prerequisite for all further morphological and statistical analyses in this context. By means of a typical use case the utilization and basic properties of HyphArea could be demonstrated. It was possible to detect statistically significant differences between the growth of an R. secalis wild-type strain and a virulence mutant. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1465 TI - Light exposure of Arabidopsis seedlings causes rapid de-stabilization as well as selective post-translational inactivation of the repressor of photomorphogenesis SPA2 JO - Plant J. PY - 2011 SP - 712-723 AU - Balcerowicz, M. AU - Fittinghoff, K. AU - Wirthmueller, L. AU - Maier, A. AU - Fackendahl, P. AU - Fiene, G. AU - Koncz, C. AU - Hoecker, U. AU - VL - 65 UR - DO - 10.1111/j.1365-313X.2010.04456.x AB - The COP1/SPA complex acts as an E3 ubiquitin ligase to repress photomorphogenesis by targeting activators of the light response for degradation. Genetic analysis has shown that the four members of the SPA gene family (SPA1–SPA4) have overlapping but distinct functions. In particular, SPA1 and SPA2 differ in that SPA1 encodes a potent repressor in light‐ and dark‐grown seedlings, but SPA2 fully loses its function when seedlings are exposed to light, indicating that SPA2 function is hyper‐inactivated by light. Here, we have used chimeric SPA1/SPA2 constructs to show that the distinct functions of SPA1 and SPA2 genes in light‐grown seedlings are due to the SPA protein sequences and independent of the SPA promoter sequences. Biochemical analysis of SPA1 and SPA2 protein levels shows that light exposure leads to rapid proteasomal degradation of SPA2, and, more weakly, of SPA1, but not of COP1. This suggests that light inactivates the COP1/SPA complex partly by reducing SPA protein levels. Although SPA2 was more strongly degraded than SPA1, this was not the sole reason for the lack of SPA2 function in the light. We found that the SPA2 protein is inherently incapable of repressing photomorphogenesis in light‐grown seedlings. The data therefore indicate that light inactivates the function of SPA2 through a post‐translational mechanism that eliminates the activity of the remaining SPA2 protein in the cell. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1463 TI - Dynamics of Responses in Compatible Potato - Potato virus Y Interaction Are Modulated by Salicylic Acid JO - PLOS ONE PY - 2011 SP - e29009 AU - Baebler, ?. AU - Stare, K. AU - Kovač, M. AU - Blejec, A. AU - Prezelj, N. AU - Stare, T. AU - Kogovšek, P. AU - Pompe-Novak, M. AU - Rosahl, S. AU - Ravnikar, M. AU - Gruden, K. AU - VL - 6 UR - DO - 10.1371/journal.pone.0029009 AB - To investigate the dynamics of the potato – Potato virus Y (PVY) compatible interaction in relation to salicylic acid - controlled pathways we performed experiments using non-transgenic potato cv. Désirée, transgenic NahG-Désirée, cv. Igor and PVYNTN, the most aggressive strain of PVY. The importance of salicylic acid in viral multiplication and symptom development was confirmed by pronounced symptom development in NahG-Désirée, depleted in salicylic acid, and reversion of the effect after spraying with 2,6-dichloroisonicotinic acid (a salicylic acid - analogue). We have employed quantitative PCR for monitoring virus multiplication, as well as plant responses through expression of selected marker genes of photosynthetic activity, carbohydrate metabolism and the defence response. Viral multiplication was the slowest in inoculated potato of cv. Désirée, the only asymptomatic genotype in the study. The intensity of defence-related gene expression was much stronger in both sensitive genotypes (NahG-Désirée and cv. Igor) at the site of inoculation than in asymptomatic plants (cv. Désirée). Photosynthesis and carbohydrate metabolism gene expression differed between the symptomatic and asymptomatic phenotypes. The differential gene expression pattern of the two sensitive genotypes indicates that the outcome of the interaction does not rely simply on one regulatory component, but similar phenotypical features can result from distinct responses at the molecular level. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1520 TI - Crystallization and preliminary X-ray analysis of the RXLR-type effector RXLR3 from the oomycete pathogen Hyaloperonospora arabidopsidis JO - Acta Crystallogr. F PY - 2011 SP - 1417-1420 AU - Wirthmueller, L. AU - Jones, J. D. AU - Banfield, M. J. AU - VL - 67 UR - DO - 10.1107/S1744309111035901 AB - Manipulating defence responses in infected host cells is a prerequisite for filamentous plant pathogens to complete their life cycle on infected host plants. During infection of its host Arabidopsis thaliana, the oomycete pathogen Hyaloperonospora arabidopsidis secretes numerous RXLR-type effector proteins, some of which are translocated into host cells. RXLR-type effectors share conserved N-terminal translocation motifs but show high diversity in their C-terminal `effector domains' that manipulate host defence mechanisms. Therefore, obtaining structural information on the effector domains of RXLR-type effectors will contribute to elucidating their molecular-virulence functions in infected host cells. Here, the expression, purification and crystallization of the effector domain of RXLR3 from H. arabidopsidis isolate Waco9 are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 61.49, b = 27.99, c = 37.59 Å. X-ray data were collected to a resolution of 1.8 Å from a single crystal using synchrotron radiation. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1519 TI - Arabidopsis methyltransferase fingerprints by affinity-based protein profiling JO - Anal. Biochem. PY - 2011 SP - 220-225 AU - Wirsing, L. AU - Naumann, K. AU - Vogt, T. AU - VL - 408 UR - DO - 10.1016/j.ab.2010.09.029 AB - Precise annotation of time and spatial distribution of enzymes involved in plant secondary metabolism by gel electrophoresis are usually difficult due to their low abundance. Therefore, effective methods to enrich these enzymes are required to correlate available transcript and metabolite data with the actual presence of active enzymes in wild-type and mutant plants or to monitor variations of these enzymes under various types of biotic and abiotic stress conditions. S-Adenosyl-L-methionine-dependent O-methyltransferases play important roles in the modification of natural products such as phenylpropanoids or alkaloids. In plants they occur as small superfamilies with defined roles for each of its members in different organs and tissues. We explored the use of S-adenosyl-L-homocysteine as a selectivity function in affinity-based protein profiling supported by capture compound mass spectrometry. Due to their high affinity to this ligand it was possible to identify developmental changes of flower-specific patterns of plant natural product O-methyltransferases and corroborate the absence of individual O-methyltransferases in the corresponding Arabidopsis knockout lines. Developmental changes in the OMT pattern were correlated with transcript data obtained by qPCR. A2 - C1 - Cell and Metabolic Biology; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1508 TI - Verticillin A is likely not produced by Verticillium sp. JO - J. Antibiot. PY - 2011 SP - 523-524 AU - Schenke, D. AU - Böttcher, C. AU - Lee, J. AU - Scheel, D. AU - VL - 64 UR - DO - 10.1038/ja.2011.36 AB - A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1507 TI - Crosstalk between abiotic ultraviolet-B stress and biotic (flg22) stress signalling in Arabidopsis prevents flavonol accumulation in favor of pathogen defence compound production JO - Plant Cell Environ. PY - 2011 SP - 1849-1864 AU - Schenke, D. AU - Böttcher, C. AU - Scheel, D. AU - VL - 34 UR - DO - 10.1111/j.1365-3040.2011.02381.x AB - Plants respond to both abiotic and biotic stresses with alterations in the expression of genes required to produce protective metabolites. Sometimes plants can be challenged with different stresses simultaneously and as they cannot evade from this situation, priorities have to be set to deal with the most urgent threat. The abiotic stress ultraviolet‐B (UV‐B) light induces the production of UV‐protective flavonols in Arabidopsis Col‐0 cell suspension cultures and this accumulation is attenuated by concurrent application of the bacterial elicitor flg22 (simulating biotic stress). This inhibition correlates with strong suppression of the flavonol biosynthesis genes. In parallel, flg22 induces the production of defence‐related compounds, such as the phytoalexins, camalexin and scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread. This correlated positively with flg22‐mediated expression of enzymes for lignin, scopoletin and camalexin production. As flavonols, lignin and scopoletin are all derived from phenylalanine, it appears that the plant focuses the metabolism on production of scopoletin and lignin at the expense of flavonol production. Furthermore, it appears that this crosstalk involves antagonistic regulation of two opposing MYB transcription factors, the positive regulator of the flavonol pathway MYB12 (UV‐B‐induced and flg22‐suppressed) and the negative regulator MYB4 (UV‐B‐ and flg22‐induced). A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1502 TI - Interplay between calcium signalling and early signalling elements during defence responses to microbe- or damage-associated molecular patterns JO - Plant J. PY - 2011 SP - 100-113 AU - Ranf, S. AU - Eschen-Lippold, L. AU - Pecher, P. AU - Lee, J. AU - Scheel, D. AU - VL - 68 UR - DO - 10.1111/j.1365-313X.2011.04671.x AB - While diverse microbe‐ or damage‐associated molecular patterns (MAMPs/DAMPs) typically trigger a common set of intracellular signalling events, comparative analysis between the MAMPs flg22 and elf18 revealed MAMP‐specific differences in Ca2+ signalling, defence gene expression and MAMP‐mediated growth arrest in Arabidopsis thaliana. Such MAMP‐specific differences are, in part, controlled by BAK1, a kinase associated with several receptors. Whereas defence gene expression and growth inhibition mediated by flg22 were reduced in bak1 mutants, BAK1 had no or minor effects on the same responses elicited by elf18. As the residual Ca2+ elevations induced by diverse MAMPs/DAMPs (flg22, elf18 and Pep1) were virtually identical in bak1 mutants, a differential BAK1‐mediated signal amplification to attain MAMP/DAMP‐specific Ca2+ amplitudes in wild‐type plants may be hypothesized. Furthermore, abrogation of reactive oxygen species (ROS) accumulation, either in the rbohD mutant or through inhibitor application, led to loss of a second Ca2+ peak, demonstrating a feedback effect of ROS on Ca2+ signalling. Conversely, mpk3 mutants showed a prolonged accumulation of ROS but this did not significantly impinge on the overall Ca2+ response. Thus, fine‐tuning of MAMP/DAMP responses involves interplay between diverse signalling elements functioning both up‐ or downstream of Ca2+ signalling. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1492 TI - A GFP-based assay to quantify the impact of effectors on the ex planta development of the slowly growing barley pathogen Rhynchosporium commune JO - Mycologia PY - 2011 SP - 1019-1027 AU - Kirsten, S. AU - Siersleben, S. AU - Knogge, W. AU - VL - 103 UR - DO - 10.3852/10-306 AB - A growth assay was established for the barley pathogen Rhynchosporium commune with EGFP-tagged fungal mutants. This assay was used to study the effect of four antibiotics (hygromycin B, nourseothricin, kanamycin, phleomycin) and a herbicide (phosphinothricin) on fungal development. Fitting the growth curves to the modified Gompertz model allowed calculating growth parameters, such as lag periods of fungal colony formation and mycelial growth rates as well as EC50 values. Except kanamycin all compounds were efficient inhibitors so that the corresponding resistance-conferring genes can be used as markers for selection of fungal transformation-based mutants. In addition the assay was used to quantify the inhibitory activity of a barley secondary metabolite, the indole alkaloid gramine. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1490 TI - When and how to kill a plant cell: Infection strategies of plant pathogenic fungi JO - J. Plant Physiol. PY - 2011 SP - 51-62 AU - Horbach, R. AU - Navarro-Quesada, A. R. AU - Knogge, W. AU - Deising, H. B. AU - VL - 168 UR - DO - 10.1016/j.jplph.2010.06.014 AB - Fungi cause severe diseases on a broad range of crop and ornamental plants, leading to significant economical losses. Plant pathogenic fungi exhibit a huge variability in their mode of infection, differentiation and function of infection structures and nutritional strategy. In this review, advances in understanding mechanisms of biotrophy, necrotrophy and hemibiotrophic lifestyles are described. Special emphasis is given to the biotrophy-necrotrophy switch of hemibiotrophic pathogens, and to biosynthesis, chemical diversity and mode of action of various fungal toxins produced during the infection process. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1484 TI - Arabidopsis EDS1 Connects Pathogen Effector Recognition to Cell Compartment-Specific Immune Responses JO - Science PY - 2011 SP - 1401-1404 AU - Heidrich, K. AU - Wirthmueller, L. AU - Tasset, C. AU - Pouzet, C. AU - Deslandes, L. AU - Parker, J. E. AU - VL - 334 UR - DO - 10.1126/science.1211641 AB - Pathogen effectors are intercepted by plant intracellular nucleotide binding–leucine-rich repeat (NB-LRR) receptors. However, processes linking receptor activation to downstream defenses remain obscure. Nucleo-cytoplasmic basal resistance regulator EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1) is indispensible for immunity mediated by TIR (Toll–interleukin-1 receptor)–NB-LRR receptors. We show that Arabidopsis EDS1 molecularly connects TIR-NB-LRR disease resistance protein RPS4 recognition of bacterial effector AvrRps4 to defense pathways. RPS4-EDS1 and AvrRps4-EDS1 complexes are detected inside nuclei of living tobacco cells after transient coexpression and in Arabidopsis soluble leaf extracts after resistance activation. Forced AvrRps4 localization to the host cytoplasm or nucleus reveals cell compartment–specific RPS4-EDS1 defense branches. Although nuclear processes restrict bacterial growth, programmed cell death and transcriptional resistance reinforcement require nucleo-cytoplasmic coordination. Thus, EDS1 behaves as an effector target and activated TIR-NB-LRR signal transducer for defenses across cell compartments. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1483 TI - Functional mapping of harpin HrpZ of Pseudomonas syringae reveals the sites responsible for protein oligomerization, lipid interactions and plant defence induction JO - Mol. Plant Pathol. PY - 2011 SP - 151-166 AU - Haapalainen, M. AU - Engelhardt, S. AU - Küfner, I. AU - Li, C.-M. AU - Nürnberger, T. AU - Lee, J. AU - Romantschuk, M. AU - Taira, S. AU - VL - 12 UR - DO - 10.1111/j.1364-3703.2010.00655.x AB - Harpin HrpZ is one of the most abundant proteins secreted through the pathogenesis‐associated type III secretion system of the plant pathogen Pseudomonas syringae. HrpZ shows membrane‐binding and pore‐forming activities in vitro, suggesting that it could be targeted to the host cell plasma membrane. We studied the native molecular forms of HrpZ and found that it forms dimers and higher order oligomers. Lipid binding by HrpZ was tested with 15 different membrane lipids, with HrpZ interacting only with phosphatidic acid. Pore formation by HrpZ in artificial lipid vesicles was found to be dependent on the presence of phosphatidic acid. In addition, HrpZ was able to form pores in vesicles prepared from Arabidopsis thaliana plasma membrane, providing evidence for the suggested target of HrpZ in the host. To map the functions associated with HrpZ, we constructed a comprehensive series of deletions in the hrpZ gene derived from P. syringae pv. phaseolicola, and studied the mutant proteins. We found that oligomerization is mainly mediated by a region near the C‐terminus of the protein, and that the same region is also essential for membrane pore formation. Phosphatidic acid binding seems to be mediated by two regions separate in the primary structure. Tobacco, a nonhost plant, recognizes, as a defence elicitor, a 24‐amino‐acid HrpZ fragment which resides in the region indispensable for the oligomerization and pore formation functions of HrpZ. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1511 TI - Molecular Evolution and Selection Patterns of Plant F-Box Proteins with C-Terminal Kelch Repeats JO - Plant Physiol. PY - 2011 SP - 835-850 AU - Schumann, N. AU - Navarro-Quezada, A. AU - Ullrich, K. AU - Kuhl, C. AU - Quint, M. AU - VL - 155 UR - DO - 10.1104/pp.110.166579 AB - The F-box protein superfamily represents one of the largest families in the plant kingdom. F-box proteins phylogenetically organize into numerous subfamilies characterized by their carboxyl (C)-terminal protein-protein interaction domain. Among the largest F-box protein subfamilies in plant genomes are those with C-terminal kelch repeats. In this study, we analyzed the phylogeny and evolution of F-box kelch proteins/genes (FBKs) in seven completely sequenced land plant genomes including a bryophyte, a lycophyte, monocots, and eudicots. While absent in prokaryotes, F-box kelch proteins are widespread in eukaryotes. Nonplant eukaryotes usually contain only a single FBK gene. In land plant genomes, however, FBKs expanded dramatically. Arabidopsis thaliana, for example, contains at least 103 F-box genes with well-conserved C-terminal kelch repeats. The construction of a phylogenetic tree based on the full-length amino acid sequences of the FBKs that we identified in the seven species enabled us to classify FBK genes into unstable/stable/superstable categories. In contrast to superstable genes, which are conserved across all seven species, kelch domains of unstable genes, which are defined as lineage specific, showed strong signatures of positive selection, indicating adaptational potential. We found evidence for conserved protein features such as binding affinities toward A. thaliana SKP1-like adaptor proteins and subcellular localization among closely related FBKs. Pseudogenization seems to occur only rarely, but differential transcriptional regulation of close relatives may result in subfunctionalization. A2 - C1 - Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1551 TI - Photoaffinity-Labeled Peptoids and Depsipeptides by Multicomponent Reactions JO - Synthesis PY - 2010 SP - 2997-3003 AU - Henze, M. AU - Kreye, O. AU - Brauch, S. AU - Nitsche, C. AU - Naumann, K. AU - Wessjohann, L. A. AU - Westermann, B. AU - VL - 2010 UR - DO - 10.1055/s-0030-1258182 AB - Photoaffinity tags can be incorporated easily into peptoids and congeners by the Ugi and Passerini multicomponent reactions. Products related to photo-methionine and photo-leucine can be accomplished by diazirine-containing building blocks. The same protocols can be used to synthesize derivatives with benzophenone photo cross-linkers. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1544 TI - Oxylipins are not required for R gene-mediated resistance in potato JO - Eur. J. Plant Pathol. PY - 2010 SP - 437-442 AU - Eschen-Lippold, L. AU - Altmann, S. AU - Gebhardt, C. AU - Göbel, C. AU - Feussner, I. AU - Rosahl, S. AU - VL - 127 UR - DO - 10.1007/s10658-010-9621-1 AB - The role of 9- and 13-lipoxygenase-derived oxylipins for race-cultivar-specific resistance in potato was analyzed by expressing RNA interference constructs against oxylipin biosynthetic genes in transgenic potato plants carrying the resistance gene R1 against Phytophthora infestans. Down-regulation of 9-lipoxygenase expression resulted in highly reduced levels of 9-hydroxyoctadecatrienoic acid after treatment with the pathogen-associated molecular pattern Pep-13. However, neither 9-lipoxygenase nor 9-divinyl ether synthase RNAi plants exhibited alterations in their resistance to P. infestans. Similarly, successful down-regulation of transcript accumulation of the 13-lipoxygenase pathway genes encoding allene oxide cyclase, 12-oxophytodienoic acid reductase 3 and the jasmonic acid receptor coronatine-insensitive 1 resulted in highly reduced levels of jasmonic acid after Pep-13 treatment. Race-cultivar-specific resistance, however, was not lost in these plants. Our results suggest that neither 9-lipoxygenase-derived oxylipins nor jasmonic acid are required for R-gene-mediated resistance in potato. Importantly, in tobacco, the silencing of 9-lipoxygenase expression was previously demonstrated to suppress race-cultivar-specific resistance. Thus, we conclude a differential requirement of oxylipins for R-gene-mediated resistance in different solanaceous plants. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1543 TI - DL-β-Aminobutyric Acid–Induced Resistance of Potato Against Phytophthora infestans Requires Salicylic Acid but Not Oxylipins JO - Mol. Plant Microbe Interact. PY - 2010 SP - 585-592 AU - Eschen-Lippold, L. AU - Altmann, S. AU - Rosahl, S. AU - VL - 23 UR - DO - 10.1094/MPMI-23-5-0585 AB - Inducing systemic resistance responses in crop plants is a promising alternative way of disease management. To understand the underlying signaling events leading to induced resistance, functional analyses of plants defective in defined signaling pathway steps are required. We used potato, one of the economically most-important crop plants worldwide, to examine systemic resistance against the devastating late blight pathogen Phytophthora infestans, induced by treatment with dl-β-aminobutyric acid (BABA). Transgenic plants impaired in either the 9-lipoxygenase pathway, which produces defense-related compounds, or the 13-lipoxygenase pathway, which generates jasmonic acid–derived signals, expressed wild-type levels of BABA-induced resistance. Plants incapable of accumulating salicylic acid (SA), on the other hand, failed to mount this type of induced resistance. Consistently, treatment of these plants with the SA analog 2,6-dichloroisonicotinic acid restored BABA-induced resistance. Together, these results demonstrate the indispensability of a functional SA pathway for systemic resistance in potato induced by BABA. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1536 TI - Ethylene signalling and ethylene-targeted transcription factors are required to balance beneficial and nonbeneficial traits in the symbiosis between the endophytic fungus Piriformospora indica and Arabidopsis thaliana JO - New Phytol. PY - 2010 SP - 1062-1073 AU - Camehl, I. AU - Sherameti, I. AU - Venus, Y. AU - Bethke, G. AU - Varma, A. AU - Lee, J. AU - Oelmüller, R. AU - VL - 185 UR - DO - 10.1111/j.1469-8137.2009.03149.x AB - The endophytic fungus Piriformospora indica colonizes the roots of the model plant Arabidopsis thaliana and promotes its growth and seed production. The fungus can be cultivated in axenic culture without a host, and therefore this is an excellent system to investigate plant–fungus symbiosis.The growth of etr1, ein2 and ein3/eil1 mutant plants was not promoted or even inhibited by the fungus; the plants produced less seeds and the roots were more colonized compared with the wild‐type. This correlates with a mild activation of defence responses. The overexpression of ETHYLENE RESPONSE FACTOR1 constitutively activated defence responses, strongly reduced root colonization and abolished the benefits for the plants.Piriformospora indica‐mediated stimulation of growth and seed yield was not affected by jasmonic acid, and jasmonic acid‐responsive promoter β‐glucuronidase gene constructs did not respond to the fungus in Arabidopsis roots.We propose that ethylene signalling components and ethylene‐targeted transcription factors are required to balance beneficial and nonbeneficial traits in the symbiosis. The results show that the restriction of fungal growth by ethylene signalling components is required for the beneficial interaction between the two symbionts. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1533 TI - The Arabidopsis Mitogen-Activated Protein Kinase Phosphatase PP2C5 Affects Seed Germination, Stomatal Aperture, and Abscisic Acid-Inducible Gene Expression JO - Plant Physiol. PY - 2010 SP - 1098-1111 AU - Brock, A. K. AU - Willmann, R. AU - Kolb, D. AU - Grefen, L. AU - Lajunen, H. M. AU - Bethke, G. AU - Lee, J. AU - Nürnberger, T. AU - Gust, A. A. AU - VL - 153 UR - DO - 10.1104/pp.110.156109 AB - Abscisic acid (ABA) is an important phytohormone regulating various cellular processes in plants, including stomatal opening and seed germination. Although protein phosphorylation via mitogen-activated protein kinases (MAPKs) has been suggested to be important in ABA signaling, the corresponding phosphatases are largely unknown. Here, we show that a member of the Protein Phosphatase 2C (PP2C) family in Arabidopsis (Arabidopsis thaliana), PP2C5, is acting as a MAPK phosphatase. The PP2C5 protein colocalizes and directly interacts with stress-induced MPK3, MPK4, and MPK6, predominantly in the nucleus. Importantly, altered PP2C5 levels affect MAPK activation. Whereas Arabidopsis plants depleted of PP2C5 show an enhanced ABA-induced activation of MPK3 and MPK6, ectopic expression of PP2C5 in tobacco (Nicotiana benthamiana) resulted in the opposite effect, with the two MAPKs salicylic acid-induced protein kinase and wound-induced protein kinase not being activated any longer after ABA treatment. Moreover, depletion of PP2C5, whose gene expression itself is affected by ABA treatment, resulted in altered ABA responses. Loss-of-function mutation in PP2C5 or AP2C1, a close PP2C5 homolog, resulted in an increased stomatal aperture under normal growth conditions and a partial ABA-insensitive phenotype in seed germination that was most prominent in the pp2c5 ap2c1 double mutant line. In addition, the response of ABA-inducible genes such as ABI1, ABI2, RD29A, and Erd10 was reduced in the mutant plants. Thus, we suggest that PP2C5 acts as a MAPK phosphatase that positively regulates seed germination, stomatal closure, and ABA-inducible gene expression. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1592 TI - A protein phosphatase 2C, responsive to the bacterial effector AvrRpm1 but not to the AvrB effector, regulates defense responses in Arabidopsis JO - Plant J. PY - 2010 SP - 249-258 AU - Widjaja, I. AU - Lassowskat, I. AU - Bethke, G. AU - Eschen-Lippold, L. AU - Long, H.-H. AU - Naumann, K. AU - Dangl, J. L. AU - Scheel, D. AU - Lee, J. AU - VL - 61 UR - DO - 10.1111/j.1365-313X.2009.04047.x AB - Using a proteomics approach, a PP2C‐type phosphatase (renamed PIA1, for PP2C induced by AvrRpm1) was identified that accumulates following infection by Pseudomonas syringae expressing the type III effector AvrRpm1, and subsequent activation of the corresponding plant NB‐LRR disease resistance protein RPM1. No accumulation of PIA1 protein was seen following infection with P. syringae expressing AvrB, another type III effector that also activates RPM1, although PIA transcripts were observed. Accordingly, mutation of PIA1 resulted in enhanced RPM1 function in response to P. syringae pathover tomato (Pto) DC3000 (avrRpm1) but not to Pto DC3000 (avrB). Thus, PIA1 is a protein marker that distinguishes AvrRpm1‐ and AvrB‐dependent activation of RPM1. AvrRpm1‐induced expression of the pathogenesis‐related genes PR1, PR2 and PR3, and salicylic acid accumulation were reduced in two pia1 mutants. By contrast, expression of other defense‐related genes, including PR5 and PDF1.2 (plant defensin), was elevated in unchallenged pia1 mutants. Hence, PIA1 is required for AvrRpm1‐induced responses, and confers dual (both positive and negative) regulation of defense gene expression. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1664 TI - Combining subproteome enrichment and Rubisco depletion enables identification of low abundance proteins differentially regulated during plant defense JO - Proteomics PY - 2009 SP - 138-147 AU - Widjaja, I. AU - Naumann, K. AU - Roth, U. AU - Wolf, N. AU - Mackey, D. AU - Dangl, J. L. AU - Scheel, D. AU - Lee, J. AU - VL - 9 UR - DO - 10.1002/pmic.200800293 AB - Transgenic Arabidopsis conditionally expressing the bacterial avrRpm1 type III effector under the control of a dexamethasone‐responsive promoter were used for proteomics studies. This model system permits study of an individual effector without interference from additional bacterial components. Coupling of different prefractionation approaches to high resolution 2‐DE facilitated the discovery of low abundance proteins – enabling the identification of proteins that have escaped detection in similar experiments. A total of 34 differentially regulated protein spots were identified. Four of these (a remorin, a protein phosphatase 2C (PP2C), an RNA‐binding protein, and a C2‐domain‐containing protein) are potentially early signaling components in the interaction between AvrRpm1 and the cognate disease resistance gene product, resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). For the remorin and RNA‐binding protein, involvement of PTM and post‐transcriptional regulation are implicated, respectively. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1657 TI - A cell wall extract from the endophytic fungus Piriformospora indica promotes growth of Arabidopsis seedlings and induces intracellular calcium elevation in roots JO - Plant J. PY - 2009 SP - 193-206 AU - Vadassery, J. AU - Ranf, S. AU - Drzewiecki, C. AU - Mithöfer, A. AU - Mazars, C. AU - Scheel, D. AU - Lee, J. AU - Oelmüller, R. AU - VL - 59 UR - DO - 10.1111/j.1365-313X.2009.03867.x AB - Calcium (Ca2+), as a second messenger, is crucial for signal transduction processes during many biotic interactions. We demonstrate that cellular [Ca2+] elevations are early events in the interaction between the plant growth‐promoting fungus Piriformospora indica and Arabidopsis thaliana . A cell wall extract (CWE) from the fungus promotes the growth of wild‐type seedlings but not of seedlings from P. indica ‐insensitive mutants. The extract and the fungus also induce a similar set of genes in Arabidopsis roots, among them genes with Ca2+ signalling‐related functions. The CWE induces a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in the roots of Arabidopsis and tobacco (Nicotiana tabacum ) plants, as well as in BY‐2 suspension cultures expressing the Ca2+ bioluminescent indicator aequorin. Nuclear Ca2+ transients were also observed in tobacco BY‐2 cells. The Ca2+ response was more pronounced in roots than in shoots and involved Ca2+ uptake from the extracellular space as revealed by inhibitor studies. Inhibition of the Ca2+ response by staurosporine and the refractory nature of the Ca2+ elevation suggest that a receptor may be involved. The CWE does not stimulate H2O2 production and the activation of defence gene expression, although it led to phosphorylation of mitogen‐activated protein kinases (MAPKs) in a Ca2+‐dependent manner. The involvement of MAPK6 in the mutualistic interaction was shown for an mpk6 line, which did not respond to P. indica . Thus, Ca2+ is likely to be an early signalling component in the mutualistic interaction between P. indica and Arabidopsis or tobacco. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1654 TI - Phytochelatin Synthesis Is Essential for the Detoxification of Excess Zinc and Contributes Significantly to the Accumulation of Zinc JO - Plant Physiol. PY - 2009 SP - 938-948 AU - Tennstedt, P. AU - Peisker, D. AU - Böttcher, C. AU - Trampczynska, A. AU - Clemens, S. AU - VL - 149 UR - DO - 10.1104/pp.108.127472 AB - The synthesis of phytochelatins (PCs) is essential for the detoxification of nonessential metals and metalloids such as cadmium and arsenic in plants and a variety of other organisms. To our knowledge, no direct evidence for a role of PCs in essential metal homeostasis has been reported to date. Prompted by observations in Schizosaccharomyces pombe and Saccharomyces cerevisiae indicating a contribution of PC synthase expression to Zn2+ sequestration, we investigated a known PC-deficient Arabidopsis (Arabidopsis thaliana) mutant, cad1-3, and a newly isolated second strong allele, cad1-6, with respect to zinc (Zn) homeostasis. We found that in a medium with low cation content PC-deficient mutants show pronounced Zn2+ hypersensitivity. This phenotype is of comparable strength to the well-documented Cd2+ hypersensitivity of cad1 mutants. PC deficiency also results in significant reduction in root Zn accumulation. To be able to sensitively measure PC accumulation, we established an assay using capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry of derivatized extracts. Plants grown under control conditions consistently showed PC2 accumulation. Analysis of plants treated with same-effect concentrations revealed that Zn2+-elicited PC2 accumulation in roots reached about 30% of the level of Cd2+-elicited PC2 accumulation. We conclude from these data that PC formation is essential for Zn2+ tolerance and provides driving force for the accumulation of Zn. This function might also help explain the mysterious occurrence of PC synthase genes throughout the plant kingdom and in a wide range of other organisms. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1635 TI - Arabidopsis IRT3 is a zinc-regulated and plasma membrane localized zinc/iron transporter JO - New Phytol. PY - 2009 SP - 392-404 AU - Lin, Y.-F. AU - Liang, H.-M. AU - Yang, S.-Y. AU - Boch, A. AU - Clemens, S. AU - Chen, C.-C. AU - Wu, J.-F. AU - Huang, J.-L. AU - Yeh, K.-C. AU - VL - 182 UR - DO - 10.1111/j.1469-8137.2009.02766.x AB - ZIP transporters (ZRT, IRT‐like proteins) are involved in the transport of iron (Fe), zinc (Zn) and other divalent metal cations. The expression of IRT3, a ZIP transporter, is higher in the Zn/cadmium (Cd) hyperaccumulator Arabidopsis halleri than is that of its ortholog in Arabidopsis thaliana , which implies a positive association of its expression with Zn accumulation in A. halleri. IRT3 genes from both A. halleri and A. thaliana functionally complemented the Zn uptake mutant Spzrt1 in Schizosaccharomyces pombe ; and Zn uptake double mutant zrt1zrt2 , Fe‐uptake mutant fet3fet4 and conferred Zn and Fe uptake activity in Saccharomyces cerevisiae . By contrast, the manganese (Mn) uptake mutant smf1 phenotypes were not rescued. Insufficient Cd uptake for toxicity was found.Expression of IRT3‐green fluorescent protein (GFP) fusion proteins in Arabidopsis root protoplasts indicated localization of both IRT3 proteins in the plasma membrane.Overexpressing AtIRT3 in A. thaliana led to increased accumulation of Zn in the shoot and Fe in the root of transgenic lines. Therefore, IRT3 functions as a Zn and Fe‐uptake transporter in Arabidopsis. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1626 TI - PAMP-induced defense responses in potato require both salicylic acid and jasmonic acid JO - Plant J. PY - 2009 SP - 230-242 AU - Halim, V. A. AU - Altmann, S. AU - Ellinger, D. AU - Eschen-Lippold, L. AU - Miersch, O. AU - Scheel, D. AU - Rosahl, S. AU - VL - 57 UR - DO - 10.1111/j.1365-313X.2008.03688.x AB - To elucidate the molecular mechanisms underlying pathogen‐associated molecular pattern (PAMP)‐induced defense responses in potato (Solanum tuberosum ), the role of the signaling compounds salicylic acid (SA) and jasmonic acid (JA) was analyzed. Pep‐13, a PAMP from Phytophthora , induces the accumulation of SA, JA and hydrogen peroxide, as well as the activation of defense genes and hypersensitive‐like cell death. We have previously shown that SA is required for Pep‐13‐induced defense responses. To assess the importance of JA, RNA interference constructs targeted at the JA biosynthetic genes, allene oxide cyclase and 12‐oxophytodienoic acid reductase, were expressed in transgenic potato plants. In addition, expression of the F‐box protein COI1 was reduced by RNA interference. Plants expressing the RNA interference constructs failed to accumulate the respective transcripts in response to wounding or Pep‐13 treatment, neither did they contain significant amounts of JA after elicitation. In response to infiltration of Pep‐13, the transgenic plants exhibited a highly reduced accumulation of reactive oxygen species as well as reduced hypersensitive cell death. The ability of the JA‐deficient plants to accumulate SA suggests that SA accumulation is independent or upstream of JA accumulation. These data show that PAMP responses in potato require both SA and JA and that, in contrast to Arabidopsis, these compounds act in the same signal transduction pathway. Despite their inability to fully respond to PAMP treatment, the transgenic RNA interference plants are not altered in their basal defense against Phytophthora infestans . A2 - C1 - Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1617 TI - Antioomycete Activity of γ-Oxocrotonate Fatty Acids against P. infestans JO - J. Agr. Food Chem. PY - 2009 SP - 9607-9612 AU - Eschen-Lippold, L. AU - Draeger, T. AU - Teichert, A. AU - Wessjohann, L. AU - Westermann, B. AU - Rosahl, S. AU - Arnold, N. AU - VL - 57 UR - DO - 10.1021/jf902067k AB - Infections with Phytophthora infestans, the causal agent of potato and tomato late blight disease, are difficult to control and can lead to considerable agricultural losses. Thus, the development of new effective agents against the pathogen is of great interest. In previous work, (E)-4-oxohexadec-2-enoic acid (3) was isolated from Hygrophorus eburneus, which exhibited fungicidal activity against Cladosporium cucumerinum. Here, the inhibitory effect of 3 on P. infestans spore germination and mycelium growth in vitro is demonstrated. The in vivo effect on infections of whole potato plants was investigated by spraying plants with the sodium salt of 3, sodium (2E)-4-oxohexadec-2-enoic acid (4), prior to P. infestans inoculation. Additionally, the influence of 3 on mycelium growth of Colletotrichum coccodes, the causal agent of potato black dot disease, was analyzed. In all approaches, a significant inhibition of pathogen development was achieved. Importantly, the unsaturated fatty acid exerted no toxic effect when sprayed on plants, a prerequisite for its commercial use. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1615 TI - Separable roles of the Pseudomonas syringae pv. phaseolicola accessory protein HrpZ1 in ion-conducting pore formation and activation of plant immunity JO - Plant J. PY - 2009 SP - 706-717 AU - Engelhardt, S. AU - Lee, J. AU - Gäbler, Y. AU - Kemmerling, B. AU - Haapalainen, M.-L. AU - Li, C.-M. AU - Wei, Z. AU - Keller, H. AU - Joosten, M. AU - Taira, S. AU - Nürnberger, T. AU - VL - 57 UR - DO - 10.1111/j.1365-313X.2008.03723.x AB - The HrpZ1 gene product from phytopathogenic Pseudomonas syringae is secreted in a type‐III secretion system‐dependent manner during plant infection. The ability of HrpZ1 to form ion‐conducting pores is proposed to contribute to bacterial effector delivery into host cells, or may facilitate the nutrition of bacteria in the apoplast. Furthermore, HrpZ1 is reminiscent of a pathogen‐associated molecular pattern (PAMP) that triggers immunity‐associated responses in a variety of plants. Here, we provide evidence that the ion pore formation and immune activation activities of HrpZ1 have different structure requirements. All HrpZ1 orthologous proteins tested possess pore formation activities, but some of these proteins fail to trigger plant defense‐associated responses. In addition, a C‐terminal fragment of HrpZ1 retains the ability to activate plant immunity, whereas ion pore formation requires intact HrpZ1. Random insertion mutagenesis of HrpZ1 further revealed the C terminus to be important for the PAMP activity of the protein. HrpZ1 binds to plant membranes with high affinity and specificity, suggesting that the activation of plant immunity‐associated responses by HrpZ1 is receptor‐mediated. Our data are consistent with dual roles of HrpZ1 as a virulence factor affecting host membrane integrity, and as a microbial pattern governing the activation of plant immunity during infection. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1603 TI - The Multifunctional Enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) Converts Cysteine-Indole-3-Acetonitrile to Camalexin in the Indole-3-Acetonitrile Metabolic Network of Arabidopsis thaliana JO - Plant Cell PY - 2009 SP - 1830-1845 AU - Böttcher, C. AU - Westphal, L. AU - Schmotz, C. AU - Prade, E. AU - Scheel, D. AU - Glawischnig, E. AU - VL - 21 UR - DO - 10.1105/tpc.109.066670 AB - Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, γ-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate–treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys(IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1599 TI - Sometimes new results raise new questions: the question marks between mitogen-activated protein kinase and ethylene signaling JO - Plant Signal Behav. PY - 2009 SP - 672-674 AU - Bethke, G. AU - Scheel, D. AU - Lee, J. AU - VL - 4 UR - DO - 10.4161/psb.4.7.9039 AB - In Arabidopsis thaliana, mitogen activated protein kinase (MAPK) signaling cascades that contain MPK3, MPK4 and MPK6 have been implicated in various aspects of developmental processes and stress responses. We identified an ethylene response factor (ERF104), which controls innate immunity, to be a specific substrate of MPK6 and showed that ethylene signaling regulates the release of the ERF104 substrate from its kinase. Implications and questions that arise from our findings are addressed. To promote discussions, previously unpublished data, that are rather confounding, are presented and possible explanation provided on how these may fit into our current model. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1598 TI - Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling JO - Proc. Natl. Acad. Sci. U.S.A. PY - 2009 SP - 8067-8072 AU - Bethke, G. AU - Unthan, T. AU - Uhrig, J. F. AU - Pöschl, Y. AU - Gust, A. A. AU - Scheel, D. AU - Lee, J. AU - VL - 106 UR - DO - 10.1073/pnas.0810206106 AB - Mitogen-activated protein kinase (MAPK)–mediated responses are in part regulated by the repertoire of MAPK substrates, which is still poorly elucidated in plants. Here, the in vivo enzyme–substrate interaction of the Arabidopsis thaliana MAP kinase, MPK6, with an ethylene response factor (ERF104) is shown by fluorescence resonance energy transfer. The interaction was rapidly lost in response to flagellin-derived flg22 peptide. This complex disruption requires not only MPK6 activity, which also affects ERF104 stability via phosphorylation, but also ethylene signaling. The latter points to a novel role of ethylene in substrate release, presumably allowing the liberated ERF104 to access target genes. Microarray data show enrichment of GCC motifs in the promoters of ERF104–up-regulated genes, many of which are stress related. ERF104 is a vital regulator of basal immunity, as altered expression in both erf104 and overexpressors led to more growth inhibition by flg22 and enhanced susceptibility to a non-adapted bacterial pathogen. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1597 TI - Crystal Structure and Functional Analysis of the Protein Disulfide Isomerase-Related Protein ERp29 JO - J. Mol. Biol. PY - 2009 SP - 1630-1642 AU - Barak, N. N. AU - Neumann, P. AU - Sevvana, M. AU - Schutkowski, M. AU - Naumann, K. AU - Malešević, M. AU - Reichardt, H. AU - Fischer, G. AU - Stubbs, M. T. AU - Ferrari, D. M. AU - VL - 385 UR - DO - 10.1016/j.jmb.2008.11.052 AB - The protein disulfide isomerase-related protein ERp29 is a putative chaperone involved in processing and secretion of secretory proteins. Until now, however, both the structure and the exact nature of interacting substrates remained unclear. We provide for the first time a crystal structure of human ERp29, refined to 2.9 Å, and show that the protein has considerable structural homology to its Drosophila homolog Wind. We show that ERp29 binds directly not only to thyroglobulin and thyroglobulin-derived peptides in vitro but also to the Wind client protein Pipe and Pipe-derived peptides, although it fails to process Pipe in vivo. A monomeric mutant of ERp29 and a D domain mutant in which the second peptide binding site is inactivated also bind protein substrates, indicating that the monomeric thioredoxin domain is sufficient for client protein binding. Indeed, the b domains of ERp29 or Wind, expressed alone, are sufficient for binding proteins and peptides. Interacting peptides have in common two or more aromatic residues, with stronger binding for sequences with overall basic character. Thus, the data allow a view of the two putative peptide binding sites of ERp29 and indicate that the apparent, different processing activity of the human and Drosophila proteins in vivo does not stem from differences in peptide binding properties. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1596 TI - On the Substrate Binding of Linoleate 9-Lipoxygenases JO - Lipids PY - 2009 SP - 207-215 AU - Andreou, A.-Z. AU - Hornung, E. AU - Kunze, S. AU - Rosahl, S. AU - Feussner, I. AU - VL - 44 UR - DO - 10.1007/s11745-008-3264-4 AB - Lipoxygenases (LOX; linoleate:oxygen oxidoreductase EC 1.13.11.12) consist of a class of enzymes that catalyze the regio‐ and stereo specific dioxygenation of polyunsaturated fatty acids. Here we characterize two proteins that belong to the less studied class of 9‐LOXs, Solanum tuberosum StLOX1 and Arabidopsis thaliana AtLOX1. The proteins were recombinantly expressed in E. coli and the product specificity of the enzymes was tested against different fatty acid substrates. Both enzymes showed high specificity against all tested C18 fatty acids and produced (9S )‐hydroperoxides. However, incubation of the C20 fatty acid arachidonic acid with AtLOX1 gave a mixture of racemic hydroperoxides. On the other hand, with StLOX1 we observed the formation of a mixture of products among which the (5S )‐hydroperoxy eicosatetraenoic acid (5S‐ H(P)ETE) was the most abundant. Esterified fatty acids were no substrates. We used site directed mutagenesis to modify a conserved valine residue in the active site of StLOX1 and examine the importance of space within the active site, which has been shown to play a role in determining the positional specificity. The Val576Phe mutant still catalyzed the formation of (9S )‐hydroperoxides with C18 fatty acids, while it exhibited altered specificity against arachidonic acid and produced mainly (11S )‐H(P)ETE. These data confirm the model that in case of linoleate 9‐LOX binding of the substrate takes place with the carboxyl‐group first. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1608 TI - Nuclear Pore Complex Component MOS7/Nup88 Is Required for Innate Immunity and Nuclear Accumulation of Defense Regulators in Arabidopsis JO - Plant Cell PY - 2009 SP - 2503-2516 AU - Cheng, Y. T. AU - Germain, H. AU - Wiermer, M. AU - Bi, D. AU - Xu, F. AU - García, A. V. AU - Wirthmueller, L. AU - Després, C. AU - Parker, J. E. AU - Zhang, Y. AU - Li, X. AU - VL - 21 UR - DO - 10.1105/tpc.108.064519 AB - Plant immune responses depend on dynamic signaling events across the nuclear envelope through nuclear pores. Nuclear accumulation of certain resistance (R) proteins and downstream signal transducers are critical for their functions, but it is not understood how these processes are controlled. Here, we report the identification, cloning, and analysis of Arabidopsis thalianamodifier of snc1,7 (mos7-1), a partial loss-of-function mutation that suppresses immune responses conditioned by the autoactivated R protein snc1 (for suppressor of npr1-1, constitutive 1). mos7-1 single mutant plants exhibit defects in basal and R protein–mediated immunity and in systemic acquired resistance but do not display obvious pleiotropic defects in development, salt tolerance, or plant hormone responses. MOS7 is homologous to human and Drosophila melanogaster nucleoporin Nup88 and resides at the nuclear envelope. In animals, Nup88 attenuates nuclear export of activated NF-κB transcription factors, resulting in nuclear accumulation of NF-κB. Our analysis shows that nuclear accumulation of snc1 and the defense signaling components Enhanced Disease Susceptibility 1 and Nonexpresser of PR genes 1 is significantly reduced in mos7-1 plants, while nuclear retention of other tested proteins is unaffected. The data suggest that specifically modulating the nuclear concentrations of certain defense proteins regulates defense outputs. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1745 TI - Haplo-Insufficiency of MPK3 in MPK6 Mutant Background Uncovers a Novel Function of These Two MAPKs in Arabidopsis Ovule Development JO - Plant Cell PY - 2008 SP - 602-613 AU - Wang, H. AU - Liu, Y. AU - Bruffett, K. AU - Lee, J. AU - Hause, G. AU - Walker, J. C. AU - Zhang, S. AU - VL - 20 UR - DO - 10.1105/tpc.108.058032 AB - The plant life cycle includes diploid sporophytic and haploid gametophytic generations. Female gametophytes (embryo sacs) in higher plants are embedded in specialized sporophytic structures (ovules). Here, we report that two closely related mitogen-activated protein kinases in Arabidopsis thaliana, MPK3 and MPK6, share a novel function in ovule development: in the MPK6 mutant background, MPK3 is haplo-insufficient, giving female sterility when heterozygous. By contrast, in the MPK3 mutant background, MPK6 does not show haplo-insufficiency. Using wounding treatment, we discovered gene dosage–dependent activation of MPK3 and MPK6. In addition, MPK6 activation is enhanced when MPK3 is null, which may help explain why mpk3−/− mpk6+/− plants are fertile. Genetic analysis revealed that the female sterility of mpk3+/− mpk6−/− plants is a sporophytic effect. In mpk3+/− mpk6−/− mutant plants, megasporogenesis and megagametogenesis are normal and the female gametophyte identity is correctly established. Further analysis demonstrates that the mpk3+/− mpk6−/− ovules have abnormal integument development with arrested cell divisions at later stages. The mutant integuments fail to accommodate the developing embryo sac, resulting in the embryo sacs being physically restricted and female reproductive failure. Our results highlight an essential function of MPK3 and MPK6 in promoting cell division in the integument specifically during ovule development. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1720 TI - Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic and biotic stresses JO - Plant J. PY - 2008 SP - 287-299 AU - Ranf, S. AU - Wünnenberg, P. AU - Lee, J. AU - Becker, D. AU - Dunkel, M. AU - Hedrich, R. AU - Scheel, D. AU - Dietrich, P. AU - VL - 53 UR - DO - 10.1111/j.1365-313X.2007.03342.x AB - The putative two‐pore Ca2+ channel TPC1 has been suggested to be involved in responses to abiotic and biotic stresses. We show that AtTPC1 co‐localizes with the K+‐selective channel AtTPK1 in the vacuolar membrane. Loss of AtTPC1 abolished Ca2+‐activated slow vacuolar (SV) currents, which were increased in AtTPC1 ‐over‐expressing Arabidopsis compared to the wild‐type. A Ca2+‐insensitive vacuolar cation channel, as yet uncharacterized, could be resolved in tpc1‐2 knockout plants. The kinetics of ABA‐ and CO2‐induced stomatal closure were similar in wild‐type and tpc1‐2 knockout plants, excluding a role of SV channels in guard‐cell signalling in response to these physiological stimuli. ABA‐, K+‐, and Ca2+‐dependent root growth phenotypes were not changed in tpc1‐2 compared to wild‐type plants. Given the permeability of SV channels to mono‐ and divalent cations, the question arises as to whether TPC1 in vivo represents a pathway for Ca2+ entry into the cytosol. Ca2+ responses as measured in aequorin‐expressing wild‐type, tpc1‐2 knockout and TPC1 ‐over‐expressing plants disprove a contribution of TPC1 to any of the stimulus‐induced Ca2+ signals tested, including abiotic stresses (cold, hyperosmotic, salt and oxidative), elevation in extracellular Ca2+ concentration and biotic factors (elf18, flg22). In good agreement, stimulus‐ and Ca2+‐dependent gene activation was not affected by alterations in TPC1 expression. Together with our finding that the loss of TPC1 did not change the activity of hyperpolarization‐activated Ca2+‐permeable channels in the plasma membrane, we conclude that TPC1, under physiological conditions, functions as a vacuolar cation channel without a major impact on cytosolic Ca2+ homeostasis. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1717 TI - Natural variation of potato allene oxide synthase 2 causes differential levels of jasmonates and pathogen resistance in Arabidopsis JO - Planta PY - 2008 SP - 293-306 AU - Pajerowska-Mukhtar, K. M. AU - Mukhtar, M. S. AU - Guex, N. AU - Halim, V. A. AU - Rosahl, S. AU - Somssich, I. E. AU - Gebhardt, C. AU - VL - 228 UR - DO - 10.1007/s00425-008-0737-x AB - Natural variation of plant pathogen resistance is often quantitative. This type of resistance can be genetically dissected in quantitative resistance loci (QRL). To unravel the molecular basis of QRL in potato (Solanum tuberosum), we employed the model plant Arabidopsis thaliana for functional analysis of natural variants of potato allene oxide synthase 2 (StAOS2). StAOS2 is a candidate gene for QRL on potato chromosome XI against the oömycete Phytophthora infestans causing late blight, and the bacterium Erwinia carotovora ssp. atroseptica causing stem black leg and tuber soft rot, both devastating diseases in potato cultivation. StAOS2 encodes a cytochrome P450 enzyme that is essential for biosynthesis of the defense signaling molecule jasmonic acid. Allele non-specific dsRNAi-mediated silencing of StAOS2 in potato drastically reduced jasmonic acid production and compromised quantitative late blight resistance. Five natural StAOS2 alleles were expressed in the null Arabidopsis aos mutant under control of the Arabidopsis AOS promoter and tested for differential complementation phenotypes. The aos mutant phenotypes evaluated were lack of jasmonates, male sterility and susceptibility to Erwinia carotovora ssp. carotovora. StAOS2 alleles that were associated with increased disease resistance in potato complemented all aos mutant phenotypes better than StAOS2 alleles associated with increased susceptibility. First structure models of ‘quantitative resistant’ versus ‘quantitative susceptible’ StAOS2 alleles suggested potential mechanisms for their differential activity. Our results demonstrate how a candidate gene approach in combination with using the homologous Arabidopsis mutant as functional reporter can help to dissect the molecular basis of complex traits in non model crop plants. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1751 TI - The coi1-16 Mutant Harbors a Second Site Mutation Rendering PEN2 Nonfunctional JO - Plant Cell PY - 2008 SP - 824-826 AU - Westphal, L. AU - Scheel, D. AU - Rosahl, S. AU - VL - 20 UR - DO - 10.1105/tpc.107.056895 AB - A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1714 TI - A cysteine-rich receptor-like kinase NCRK and a pathogen-induced protein kinase RBK1 are Rop GTPase interactors JO - Plant J. PY - 2008 SP - 909-923 AU - Molendijk, A. J. AU - Ruperti, B. AU - Singh, M. K. AU - Dovzhenko, A. AU - Ditengou, F. A. AU - Milia, M. AU - Westphal, L. AU - Rosahl, S. AU - Soellick, T.-R. AU - Uhrig, J. AU - Weingarten, L. AU - Huber, M. AU - Palme, K. AU - VL - 53 UR - DO - 10.1111/j.1365-313X.2007.03384.x AB - In plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two‐hybrid screening we have found three previously uncharacterized receptor‐like protein kinases to be Rop GTPase‐interacting molecules: a cysteine‐rich receptor kinase, named NCRK, and two receptor‐like cytosolic kinases from the Arabidopsis RLCK‐VIb family, named RBK1 and RBK2. Uniquely for Rho‐family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP‐bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro , suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co‐localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single‐copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK‐VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2 . We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross‐talk between Rop GTPases and specific receptor‐like kinases through direct molecular interaction. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1673 TI - Loss of Zhf and the tightly regulated zinc-uptake system SpZrt1 in Schizosaccharomyces pombe reveals the delicacy of cellular zinc balance JO - FEMS Yeast Res. PY - 2008 SP - 883-896 AU - Boch, A. AU - Trampczynska, A. AU - Simm, C. AU - Taudte, N. AU - Krämer, U. AU - Clemens, S. AU - VL - 8 UR - DO - 10.1111/j.1567-1364.2008.00414.x AB - Zinc is an essential micronutrient, and yet it can be toxic when present in excess. Zinc acquisition and distribution are dependent on tightly controlled transport of Zn2+ ions. Schizosaccharomyces pombe represents a second eukaryotic model to study cellular metal homeostasis. In several ways its micronutrient metabolism is fundamentally different from Saccharomyces cerevisiae. We identified the first Zn2+-uptake system in S. pombe and named it SpZrt1. Knock-out strains for all three ZIP (Zrt, Irt-like protein) transporters in fission yeast were constructed. Only zrt1Δ cells were unable to grow at low Zn2+ and showed reduced65Zn2+ uptake. Elemental profiles revealed a strong decrease in zinc accumulation. Cd2+ ions inhibited uptake but Fe2+ or Mn2+ did not. Both mRNA abundance and protein amount are tightly regulated. Zrt1 activity is rapidly shut down upon transfer of zinc-deficient cells to zinc-replete conditions. In cells lacking Zhf, a transporter mediating endoplasmic reticulum storage of zinc, this response is about 100-fold more sensitive. Thus, removal of excess of zinc from the cytosol is largely Zhf dependent. Moreover, cells deficient for both transporters are no longer able to adjust to changing external Zn2+ concentrations. Optimal growth is restricted to a narrow range of Zn2+ concentrations, illustrating the fine balance between micronutrient deficiency and toxicity. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1778 TI - Asexual Genetic Exchange in the Barley Pathogen Rhynchosporium secalis JO - Phytopathology PY - 2007 SP - 650-654 AU - Forgan, A. H. AU - Knogge, W. AU - Anderson, P. A. AU - VL - 97 UR - DO - 10.1094/PHYTO-97-5-0650 AB - The causal agent of barley scald, Rhynchosporium secalis, is a haploid anamorphic ascomycete with no known sexual stage. Nevertheless, a high degree of genetic variation has been observed in fungal populations on commercial barley cultivars and parasexuality has been suggested to contribute to this variation. In order to test whether asexual genetic exchange can occur, isolates of R. secalis were transformed to hygromycin B resistance or phleomycin resistance. Mixtures of transformants were co-inoculated either on agar or in planta and screened for the occurrence of dual-antibiotic-resistant colonies. No dual-antibiotic-resistant colonies resulted from mixing transformants of different fungal isolates. In contrast, with transformants originating from the same fungal isolate, asexual exchange of markers was demonstrated on agar plates and in planta. This is the first definitive evidence of asexual genetic exchange in R. secalis. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1774 TI - Reduction of divinyl ether-containing polyunsaturated fatty acids in transgenic potato plants JO - Phytochemistry PY - 2007 SP - 797-801 AU - Eschen-Lippold, L. AU - Rothe, G. AU - Stumpe, M. AU - Göbel, C. AU - Feussner, I. AU - Rosahl, S. AU - VL - 68 UR - DO - 10.1016/j.phytochem.2006.12.010 AB - Oxygenated polyunsaturated fatty acids synthesized via the lipoxygenase pathway play a role in plant responses to pathogen attack. In solanaceous plants, the preferential stimulation of the 9-lipoxygenase pathway in response to pathogen infection leads to the formation of the divinyl ether-containing polyunsaturated fatty acids colneleic and colnelenic acid, as well as hydroxy and trihydroxy polyunsaturated fatty acids. To functionally assess the role of divinyl ethers, transgenic potato plants were generated which express an RNA interference construct directed against the pathogen-inducible 9-divinyl ether synthase. Efficient reduction of 9-divinyl ether synthase transcript accumulation correlated with reduced levels of colneleic and colnelenic acid. However, in response to infection with virulent Phytophthora infestans, the causal agent of late blight disease, no significant differences in pathogen biomass could be detected suggesting that the levels of antimicrobial divinyl ethers are not critical for defense against Phytophthora infestans in a compatible interaction. A2 - C1 - Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1761 TI - Arabidopsis thaliana is a susceptible host plant for the holoparasite Cuscuta spec JO - Planta PY - 2007 SP - 1231-1241 AU - Birschwilks, M. AU - Sauer, N. AU - Scheel, D. AU - Neumann, S. AU - VL - 226 UR - DO - 10.1007/s00425-007-0571-6 AB - Arabidopsis thaliana and Cuscuta spec. represent a compatible host–parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host–parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP–ubiquitin fusion (36 kDa), respectively, under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP–ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP–ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A.thaliana covering the entire range of its genetic diversity, as well as Arabidopsishalleri, were found to be susceptible towards Cuscutareflexa. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1801 TI - Interaction between SGT1 and Cytosolic/Nuclear HSC70 Chaperones Regulates Arabidopsis Immune Responses JO - Plant Cell PY - 2007 SP - 4061-4076 AU - Noël, L. D. AU - Cagna, G. AU - Stuttmann, J. AU - Wirthmüller, L. AU - Betsuyaku, S. AU - Witte, C.-P. AU - Bhat, R. AU - Pochon, N. AU - Colby, T. AU - Parker, J. E. AU - VL - 19 UR - DO - 10.1105/tpc.107.051896 AB - The conserved eukaryotic protein SGT1 (for Suppressor of G2 allele of skp1) has characteristics of an HSP90 (for heat shock protein 90 kD) cochaperone and in plants regulates hormone responses and Resistance gene–triggered immunity. We affinity-purified SGT1-interacting proteins from Arabidopsis thaliana leaf extracts and identified by mass spectrometry cytosolic heat shock cognate 70 (HSC70) chaperones as the major stable SGT1 interactors. Arabidopsis SGT1a and SGT1b proteins associate with HSC70 in vivo and distribute with HSC70 in the cytosol and nucleus. An intact C-terminal SGT1-specific (SGS) domain that is required for all known SGT1b functions in immunity and development is needed for HSC70 interaction and for the nuclear accumulation of SGT1b. Interaction assays of transiently expressed proteins or their domains in Nicotiana benthamiana point to a role of SGT1 as a HSC70 cofactor. Expression of two HSC70 isoforms is upregulated by pathogen challenge, and while loss of function of individual cytosolic HSC70 genes has no defense phenotype, HSC70-1 overexpression disables resistance to virulent and avirulent pathogens. Moreover, mutations in SGT1b lead to a similar degree of heat shock tolerance as deregulation of HSC70-1. We conclude that an HSC70-SGT1 chaperone complex is important for multiple plant environmental responses and that the evolutionarily conserved SGS domain of SGT1 is a key determinant of the HSC70–SGT1 association. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1787 TI - Salicylic Acid Is Important for Basal Defense of Solanum tuberosum Against Phytophthora infestans JO - Mol. Plant Microbe Interact. PY - 2007 SP - 1346-1352 AU - Halim, V. A. AU - Eschen-Lippold, L. AU - Altmann, S. AU - Birschwilks, M. AU - Scheel, D. AU - Rosahl, S. AU - VL - 20 UR - DO - 10.1094/MPMI-20-11-1346 AB - The importance of the signaling compound salicylic acid for basal defense of potato (Solanum tuberosum L. cv. Désirée) against Phytophthora infestans, the causal agent of late blight disease, was assessed using transgenic NahG potato plants which are unable to accumulate salicylic acid. Although the size of lesions caused by P. infestans was not significantly different in wild-type and transgenic NahG plants, real-time polymerase chain reaction analyses revealed a drastic enhancement of pathogen growth in potato plants depleted of salicylic acid. Increased susceptibility of NahG plants correlated with compromised callose formation and reduced early defense gene expression. NahG plants pretreated with the salicylic acid analog 2,6-dichloro-isonicotinic acid allowed pathogen growth to a similar extent as did wild-type plants, indicating that salicylic acid is an important compound required for basal defense of potato against P. infestans. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1786 TI - Bacteria-derived Peptidoglycans Constitute Pathogen-associated Molecular Patterns Triggering Innate Immunity in Arabidopsis JO - J. Biol. Chem. PY - 2007 SP - 32338-32348 AU - Gust, A. A. AU - Biswas, R. AU - Lenz, H. D. AU - Rauhut, T. AU - Ranf, S. AU - Kemmerling, B. AU - Götz, F. AU - Glawischnig, E. AU - Lee, J. AU - Felix, G. AU - Nürnberger, T. AU - VL - 282 UR - DO - 10.1074/jbc.M704886200 AB - Pathogen-associated molecular pattern (PAMP)-triggered immunity constitutes the primary plant immune response that has evolved to recognize invariant structures of microbial surfaces. Here we show that Gram-positive bacteria-derived peptidoglycan (PGN) constitutes a novel PAMP of immune responses in Arabidopsis thaliana. Treatment with PGN from Staphylococcus aureus results in the activation of plant responses, such as medium alkalinization, elevation of cytoplasmic calcium concentrations, nitric oxide, and camalexin production and the post-translational induction of MAPK activities. Microarray analysis performed with RNA prepared from PGN-treated Arabidopsis leaves revealed enhanced transcript levels for 236 genes, many of which are also altered upon administration of flagellin. Comparison of cellular responses after treatment with bacteria-derived PGN and structurally related fungal chitin indicated that both PAMPs are perceived via different perception systems. PGN-mediated immune stimulation in Arabidopsis is based upon recognition of the PGN sugar backbone, while muramyl dipeptide, which is inactive in this plant, triggers immunity-associated responses in animals. PGN adds to the list of PAMPs that induce innate immune programs in both plants and animals. However, we propose that PGN perception systems arose independently in both lineages and are the result of convergent evolution. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1839 TI - Nuclear Accumulation of the Arabidopsis Immune Receptor RPS4 Is Necessary for Triggering EDS1-Dependent Defense JO - Curr. Biol. PY - 2007 SP - 2023-2029 AU - Wirthmueller, L. AU - Zhang, Y. AU - Jones, J. D. G. AU - Parker, J. E. AU - VL - 17 UR - DO - 10.1016/j.cub.2007.10.042 AB - Recognition of specific pathogen molecules inside the cell by nucleotide-binding domain and leucine-rich repeat (NB-LRR) receptors constitutes an important layer of innate immunity in plants [1]. Receptor activation triggers host cellular reprogramming involving transcriptional potentiation of basal defenses and localized programmed cell death 1, 2, 3. The sites and modes of action of NB-LRR receptors are, however, poorly understood. Arabidopsis Toll/Interleukin-1 (TIR) type NB-LRR receptor RPS4 recognizes the bacterial type III effector AvrRps4 [4]. We show that epitope-tagged RPS4 expressed under its native regulatory sequences distributes between endomembranes and nuclei in healthy and AvrRps4-triggered tissues. RPS4 accumulation in the nucleus, mediated by a bipartite nuclear localization sequence (NLS) at its C terminus, is necessary for triggering immunity through authentic activation by AvrRps4 in Arabidopsis or as an effector-independent “deregulated” receptor in tobacco. A strikingly conserved feature of TIR-NB-LRR receptors is their recruitment of the nucleocytoplasmic basal-defense regulator EDS1 in resistance to diverse pathogens 5, 6. We find that EDS1 is an indispensable component of RPS4 signaling and that it functions downstream of RPS4 activation but upstream of RPS4-mediated transcriptional reprogramming in the nucleus. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1829 TI - A Single Binding Site Mediates Resistance- and Disease-Associated Activities of the Effector Protein NIP1 from the Barley Pathogen Rhynchosporium secalis JO - Plant Physiol. PY - 2007 SP - 1654-1666 AU - van't Slot, K. A. AU - Gierlich, A. AU - Knogge, W. AU - VL - 144 UR - DO - 10.1104/pp.106.094912 AB - The effector protein NIP1 from the barley (Hordeum vulgare) pathogen Rhynchosporium secalis specifically induces the synthesis of defense-related proteins in cultivars of barley expressing the complementary resistance gene, Rrs1. In addition, it stimulates the activity of the barley plasma membrane H+-ATPase in a genotype-unspecific manner and it induces necrotic lesions in leaf tissues of barley and other cereal plant species. NIP1 variants type I and II, which display quantitative differences in their activities as elicitor and H+-ATPase stimulator, and the inactive mutant variants type III* and type IV*, were produced in Escherichia coli. Binding studies using 125I-NIP1 type I revealed a single class of binding sites with identical binding characteristics in microsomes from near-isogenic resistant (Rrs1) and susceptible (rrs1) barley. Binding was specific, reversible, and saturable, and saturation ligand-binding experiments yielded a Kd of 5.6 nm. A binding site was also found in rye (Secale cereale) and the nonhost species wheat (Triticum aestivum), oat (Avena sativa), and maize (Zea mays), but not in Arabidopsis (Arabidopsis thaliana). For NIP1 types I and II, equilibrium competition-binding experiments revealed a correlation between the difference in their affinities to the binding site and the differences in their elicitor activity and H+-ATPase stimulation, indicating a single target molecule to mediate both activities. In contrast, the inactive proteins type III* and type IV* are both characterized by high affinities similar to type I, suggesting that binding of NIP1 to this target is not sufficient for its activities. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1823 TI - Immunomodulation of jasmonate to manipulate the wound response JO - J. Exp. Bot. PY - 2007 SP - 2525-2535 AU - ten Hoopen, P. AU - Hunger, A. AU - Muller, A. AU - Hause, B. AU - Kramell, R. AU - Wasternack, C. AU - Rosahl, S. AU - Conrad, U. AU - VL - 58 UR - DO - 10.1093/jxb/erm122 AB - Jasmonates are signals in plant stress responses and development. The exact mode of their action is still controversial. To modulate jasmonate levels intracellularly as well as compartment-specifically, transgenic Nicotiana tabacum plants expressing single-chain antibodies selected against the naturally occurring (3R,7R)-enantiomer of jasmonic acid (JA) were created in the cytosol and the endoplasmic reticulum. Consequently, the expression of anti-JA antibodies in planta caused JA-deficient phenotypes such as insensitivity of germinating transgenic seedlings towards methyl jasmonate and the loss of wound-induced gene expression. Results presented here suggest an essential role for cytosolic JA in the wound response of tobacco plants. The findings support the view that substrate availability takes part in regulating JA biosynthesis upon wounding. Moreover, high JA levels observed in immunomodulated plants in response to wounding suggest that tobacco plants are able to perceive a reduced level of physiologically active JA and attempt to compensate for this by increased JA accumulation. A2 - C1 - Cell and Metabolic Biology; Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1913 TI - Comparative transcriptome analysis of toxic metal responses in Arabidopsis thaliana and the Cd2+-hypertolerant facultative metallophyte Arabidopsis halleri JO - Plant Cell Environ. PY - 2006 SP - 950-963 AU - Weber, M. AU - Trampczynska, A. AU - Clemens, S. AU - VL - 29 UR - DO - 10.1111/j.1365-3040.2005.01479.x AB - Toxic effects of both essential and non‐essential heavy metals are well documented in plants. Very little is known, however, about their modes of toxicity, about tolerance mechanisms and the signalling cascades involved in mediating transcriptional responses to toxic metal excess. We analysed transcriptome changes upon Cd2+ and Cu2+ exposure in roots of Arabidopsis thaliana and the Cd2+‐hypertolerant metallophyte Arabidopsis halleri . Particularly, three categories of genes were identified with the help of this comparative approach: (1) common responses, which might indicate stable and functionally relevant changes conserved across plant species; (2) metallophyte‐specific responses as well as transcripts differentially regulated between the two species, representing candidate genes for Cd2+ hypertolerance; and (3) those specifically responsive to Cd2+ and therefore indicative of toxicity mechanisms or potentially involved in signalling cascades. Our data define, for instance, Arabidopsis core responses to Cd2+ and Cu2+. In addition, they suggest that Cd2+ exposure very rapidly results in apparent Zn deficiency, and they show the existence of highly specific Cd2+ responses and distinct signalling cascades. Array results were independently confirmed by real‐time quantitative PCR, thereby further validating cross‐species transcriptome analysis with oligonucleotide microarrays. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1908 TI - The transition metal chelator nicotianamine is synthesized by filamentous fungi JO - FEBS Lett. PY - 2006 SP - 3173-3178 AU - Trampczynska, A. AU - Böttcher, C. AU - Clemens, S. AU - VL - 580 UR - DO - 10.1016/j.febslet.2006.04.073 AB - Nicotianamine is an important metal ligand in plants. Surprisingly, recent genome sequencing revealed that ascomycetes encode proteins with similarity to plant nicotianamine synthases (NAS). By expression in a Zn2+‐hypersensitive fission yeast mutant we show for a protein from Neurospora crassa that it indeed possesses NAS activity. Using electrospray‐ionization‐quadrupole‐time‐of‐flight mass spectrometry we prove the formation of nicotianamine in N. crassa . Transcript level is strongly upregulated under Zn deficiency as shown by real‐time PCR. These findings demonstrate that nicotianamine is more widespread in nature than anticipated and provide further evidence for a function of nicotianamine as a cytosolic chelator of Zn2+ ions. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1890 TI - Trichomes of Tobacco Excrete Zinc as Zinc-Substituted Calcium Carbonate and Other Zinc-Containing Compounds JO - Plant Physiol. PY - 2006 SP - 1021-1034 AU - Sarret, G. AU - Harada, E. AU - Choi, Y.-E. AU - Isaure, M.-P. AU - Geoffroy, N. AU - Fakra, S. AU - Marcus, M. A. AU - Birschwilks, M. AU - Clemens, S. AU - Manceau, A. AU - VL - 141 UR - DO - 10.1104/pp.106.082743 AB - Tobacco (Nicotiana tabacum L. cv Xanthi) plants were exposed to toxic levels of zinc (Zn). Zn exposure resulted in toxicity signs in plants, and these damages were partly reduced by a calcium (Ca) supplement. Confocal imaging of intracellular Zn using Zinquin showed that Zn was preferentially accumulated in trichomes. Exposure to Zn and Zn + Ca increased the trichome density and induced the production of Ca/Zn mineral grains on the head cells of trichomes. These grains were aggregates of submicrometer-sized crystals and poorly crystalline material and contained Ca as major element, along with subordinate amounts of Zn, manganese, potassium, chlorine, phosphorus, silicon, and magnesium. Micro x-ray diffraction revealed that the large majority of the grains were composed essentially of metal-substituted calcite (CaCO3). CaCO3 polymorphs (aragonite and vaterite) and CaC2O4 (Ca oxalate) mono- and dihydrate also were identified, either as an admixture to calcite or in separate grains. Some grains did not diffract, although they contained Ca, suggesting the presence of amorphous form of Ca. The presence of Zn-substituted calcite was confirmed by Zn K-edge micro-extended x-ray absorption fine structure spectroscopy. Zn bound to organic compounds and Zn-containing silica and phosphate were also identified by this technique. The proportion of Zn-substituted calcite relative to the other species increased with Ca exposure. The production of Zn-containing biogenic calcite and other Zn compounds through the trichomes is a novel mechanism involved in Zn detoxification. This study illustrates the potential of laterally resolved x-ray synchrotron radiation techniques to study biomineralization and metal homeostasis processes in plants. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1889 TI - Proteome changes in Arabidopsis thaliana roots upon exposure to Cd2+ JO - J. Exp. Bot. PY - 2006 SP - 4003-4013 AU - Roth, U. AU - von Roepenack-Lahaye, E. AU - Clemens, S. AU - VL - 57 UR - DO - 10.1093/jxb/erl170 AB - Cadmium is a major environmental pollutant that enters human food via accumulation in crop plants. Responses of plants to cadmium exposure—which directly influence accumulation rates—are not well understood. In general, little is known about stress-elicited changes in plants at the proteome level. Alterations in the root proteome of hydroponically grown Arabidopsis thaliana plants treated with 10 μM Cd2+ for 24 h are reported here. These conditions trigger the synthesis of phytochelatins (PCs), glutathione-derived metal-binding peptides, shown here as PC2 accumulation. Two-dimensional gel electrophoresis using different pH gradients in the first dimension detected on average ∼1100 spots per gel type. Forty-one spots indicated significant changes in protein abundance upon Cd2+ treatment. Seventeen proteins found in 25 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Selected results were independently confirmed by western analysis and selective enrichment of a protein family (glutathione S-transferases) through affinity chromatography. Most of the identified proteins belong to four different classes: metabolic enzymes such as ATP sulphurylase, glycine hydroxymethyltransferase, and trehalose-6-phosphate phosphatase; glutathione S-transferases; latex allergen-like proteins; and unknown proteins. These results represent a basis for reverse genetics studies to better understand plant responses to toxic metal exposure and to the generation of internal sinks for reduced sulphur. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1886 TI - Phytotoxicity and Innate Immune Responses Induced by Nep1-Like Proteins JO - Plant Cell PY - 2006 SP - 3721-3744 AU - Qutob, D. AU - Kemmerling, B. AU - Brunner, F. AU - Küfner, I. AU - Engelhardt, S. AU - Gust, A. A. AU - Luberacki, B. AU - Seitz, H. U. AU - Stahl, D. AU - Rauhut, T. AU - Glawischnig, E. AU - Schween, G. AU - Lacombe, B. AU - Watanabe, N. AU - Lam, E. AU - Schlichting, R. AU - Scheel, D. AU - Nau, K. AU - Dodt, G. AU - Hubert, D. AU - Gijzen, M. AU - Nürnberger, T. AU - VL - 18 UR - DO - 10.1105/tpc.106.044180 AB - We show that oomycete-derived Nep1 (for necrosis and ethylene-inducing peptide1)–like proteins (NLPs) trigger a comprehensive immune response in Arabidopsis thaliana, comprising posttranslational activation of mitogen-activated protein kinase activity, deposition of callose, production of nitric oxide, reactive oxygen intermediates, ethylene, and the phytoalexin camalexin, as well as cell death. Transcript profiling experiments revealed that NLPs trigger extensive reprogramming of the Arabidopsis transcriptome closely resembling that evoked by bacteria-derived flagellin. NLP-induced cell death is an active, light-dependent process requiring HSP90 but not caspase activity, salicylic acid, jasmonic acid, ethylene, or functional SGT1a/SGT1b. Studies on animal, yeast, moss, and plant cells revealed that sensitivity to NLPs is not a general characteristic of phospholipid bilayer systems but appears to be restricted to dicot plants. NLP-induced cell death does not require an intact plant cell wall, and ectopic expression of NLP in dicot plants resulted in cell death only when the protein was delivered to the apoplast. Our findings strongly suggest that NLP-induced necrosis requires interaction with a target site that is unique to the extracytoplasmic side of dicot plant plasma membranes. We propose that NLPs play dual roles in plant pathogen interactions as toxin-like virulence factors and as triggers of plant innate immune responses. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1877 TI - LysM receptors recognize friend and foe JO - Proc. Natl. Acad. Sci. U.S.A. PY - 2006 SP - 10829-10830 AU - Knogge, W. AU - Scheel, D. AU - VL - 103 UR - DO - 10.1073/pnas.0604601103 AB - A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1866 TI - Ancient signals: comparative genomics of plant MAPK and MAPKK gene families JO - Trends Plant Sci. PY - 2006 SP - 192-198 AU - Hamel, L.-P. AU - Nicole, M.-C. AU - Sritubtim, S. AU - Morency, M.-J. AU - Ellis, M. AU - Ehlting, J. AU - Beaudoin, N. AU - Barbazuk, B. AU - Klessig, D. AU - Lee, J. AU - Martin, G. AU - Mundy, J. AU - Ohashi, Y. AU - Scheel, D. AU - Sheen, J. AU - Xing, T. AU - Zhang, S. AU - Seguin, A. AU - Ellis, B. E. AU - VL - 11 UR - DO - 10.1016/j.tplants.2006.02.007 AB - MAPK signal transduction modules play crucial roles in regulating many biological processes in plants, and their components are encoded by highly conserved genes. The recent availability of genome sequences for rice and poplar now makes it possible to examine how well the previously described Arabidopsis MAPK and MAPKK gene family structures represent the broader evolutionary situation in plants, and analysis of gene expression data for MPK and MKK genes in all three species allows further refinement of those families, based on functionality. The Arabidopsis MAPK nomenclature appears sufficiently robust to allow it to be usefully extended to other well-characterized plant systems. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1865 TI - The Role of Salicylic Acid and Jasmonic Acid in Pathogen Defence JO - Plant Biol. PY - 2006 SP - 307-313 AU - Halim, V. A. AU - Vess, A. AU - Scheel, D. AU - Rosahl, S. AU - VL - 8 UR - DO - 10.1055/s-2006-924025 AB - Phytohormones are not only instrumental in regulating developmental processes in plants but also play important roles for the plant's responses to biotic and abiotic stresses. In particular, abscisic acid, ethylene, jasmonic acid, and salicylic acid have been shown to possess crucial functions in mediating or orchestrating stress responses in plants. Here, we review the role of salicylic acid and jasmonic acid in pathogen defence responses with special emphasis on their function in the solanaceous plant potato. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1864 TI - Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase JO - FEBS Lett. PY - 2006 SP - 6384-6390 AU - Grzam, A. AU - Tennstedt, P. AU - Clemens, S. AU - Hell, R. AU - Meyer, A. J. AU - VL - 580 UR - DO - 10.1016/j.febslet.2006.10.050 AB - Monochlorobimane was used as a model xenobiotic for Arabidopsis to directly monitor the compartmentation of glutathione‐bimane conjugates in situ and to quantify degradation intermediates in vitro. Vacuolar sequestration of the conjugate was very fast and outcompeted carboxypeptidation to the γ‐glutamylcysteine‐bimane intermediate (γ‐EC‐B) by phytochelatin synthase (PCS) in the cytosol. Following vacuolar sequestration, degradation proceeded to cysteine‐bimane without intermediate. Only co‐infiltration of monochlorobimane with Cd2+ and Cu2+ increased γ‐EC‐B formation to 4% and 25%, respectively, within 60 min. The role of PCS under simultaneous heavy metal stress was confirmed by investigation of different pcs1 null‐mutants. In the absence of elevated heavy metal concentrations glutathione‐conjugates are therefore first sequestered to the vacuole and subsequently degraded with the initial breakdown step being rate‐limiting. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1850 TI - Conserved requirement for a plant host cell protein in powdery mildew pathogenesis JO - Nat. Genet. PY - 2006 SP - 716-720 AU - Consonni, C. AU - Humphry, M. E. AU - Hartmann, H. A. AU - Livaja, M. AU - Durner, J. AU - Westphal, L. AU - Vogel, J. AU - Lipka, V. AU - Kemmerling, B. AU - Schulze-Lefert, P. AU - Somerville, S. C. AU - Panstruga, R. AU - VL - 38 UR - DO - 10.1038/ng1806 AB - In the fungal phylum Ascomycota, the ability to cause disease in plants and animals has been gained and lost repeatedly during phylogenesis1. In monocotyledonous barley, loss-of-function mlo alleles result in effective immunity against the Ascomycete Blumeria graminis f. sp. hordei, the causal agent of powdery mildew disease2,3. However, mlo-based disease resistance has been considered a barley-specific phenomenon to date. Here, we demonstrate a conserved requirement for MLO proteins in powdery mildew pathogenesis in the dicotyledonous plant species Arabidopsis thaliana. Epistasis analysis showed that mlo resistance in A. thaliana does not involve the signaling molecules ethylene, jasmonic acid or salicylic acid, but requires a syntaxin, glycosyl hydrolase and ABC transporter4,5,6. These findings imply that a common host cell entry mechanism of powdery mildew fungi evolved once and at least 200 million years ago, suggesting that within the Erysiphales (powdery mildews) the ability to cause disease has been a stable trait throughout phylogenesis. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1849 TI - Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants JO - Biochimie PY - 2006 SP - 1707-1719 AU - Clemens, S. AU - VL - 88 UR - DO - 10.1016/j.biochi.2006.07.003 AB - Over the past 200 years emissions of toxic heavy metals have risen tremendously and significantly exceed those from natural sources for practically all metals. Uptake and accumulation by crop plants represents the main entry pathway for potentially health-threatening toxic metals into human and animal food. Of major concern are the metalloids arsenic (As) and selenium (Se), and the metals cadmium (Cd), mercury (Hg), and lead (Pb). This review discusses the molecular mechanisms of toxic metal accumulation in plants and algae, the responses to metal exposure, as well as our understanding of metal tolerance and its evolution. The main emphasis will be on cadmium, which is by far the most widely studied of the non-essential toxic metals/metalloids. Entry via Zn2+, Fe2+, and Ca2+ transporters is the molecular basis of Cd2+ uptake into plant cells. Much less is known about the partitioning of non-essential metals and about the genes underlying the enormous diversity among plants with respect to Cd accumulation in different tissues. Numerous studies have described symptoms and responses of plants upon toxic metal exposure. Mysterious are primary targets of toxicity, the degree of specificity of responses, the sensing and the signaling events that lead to transcriptional activation. All plants apparently possess a basal tolerance of toxic non-essential metals. For Cd and As, this is largely dependent on the phytochelatin pathway. Not understood is the molecular biology of Cd hypertolerance in certain plant species such as the metallophytes Arabidopsis halleri or Thlaspi caerulescens. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1848 TI - Evolution and function of phytochelatin synthases JO - J. Plant Physiol. PY - 2006 SP - 319-332 AU - Clemens, S. AU - VL - 163 UR - DO - 10.1016/j.jplph.2005.11.010 AB - Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of phytochelatin synthases and our current knowledge about the enzymology of phytochelatin synthesis are reviewed. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1844 TI - Transfer of phloem-mobile substances from the host plants to the holoparasite Cuscuta sp. JO - J. Exp. Bot. PY - 2006 SP - 911-921 AU - Birschwilks, M. AU - Haupt, S. AU - Hofius, D. AU - Neumann, S. AU - VL - 57 UR - DO - 10.1093/jxb/erj076 AB - During the development of the haustorium, searching hyphae of the parasite and the host parenchyma cells are connected by plasmodesmata. Using transgenic tobacco plants expressing a GFP-labelled movement protein of the tobacco mosaic virus, it was demonstrated that the interspecific plasmodesmata are open. The transfer of substances in the phloem from host to the parasite is not selective. After simultaneous application of 3H-sucrose and 14C-labelled phloem-mobile amino acids, phytohormones, and xenobiotica to the host, corresponding percentages of the translocated compounds are found in the parasite. An open continuity between the host phloem and the Cuscuta phloem via the haustorium was demonstrated in CLSM pictures after application of the phloem-mobile fluorescent probes, carboxyfluorescein (CF) and hydroxypyrene trisulphonic acid (HPTS), to the host. Using a Cuscuta bridge 14C-sucrose and the virus PVYN were transferred from one host plant to the another. The results of translocation experiments with labelled compounds, phloem-mobile dyes and the virus should be considered as unequivocal evidence for a symplastic transfer of phloem solutes between Cuscuta species and their compatible hosts. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1939 TI - High Throughput Identification of Potential Arabidopsis Mitogen-activated Protein Kinases Substrates JO - Mol. Cell. Proteomics PY - 2005 SP - 1558-1568 AU - Feilner, T. AU - Hultschig, C. AU - Lee, J. AU - Meyer, S. AU - Immink, R. G. H. AU - Koenig, A. AU - Possling, A. AU - Seitz, H. AU - Beveridge, A. AU - Scheel, D. AU - Cahill, D. J. AU - Lehrach, H. AU - Kreutzberger, J. AU - Kersten, B. AU - VL - 4 UR - DO - 10.1074/mcp.M500007-MCP200 AB - Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1977 TI - Ca2+-dependent lipid binding and membrane integration of PopA, a harpin-like elicitor of the hypersensitive response in tobacco JO - Mol. Microbiol. PY - 2005 SP - 1406-1420 AU - Racapé, J. AU - Belbahri, L. AU - Engelhardt, S. AU - Lacombe, B. AU - Lee, J. AU - Lochman, J. AU - Marais, A. AU - Nicole, M. AU - Nürnberger, T. AU - Parlange, F. AU - Puverel, S. AU - Keller, H. AU - VL - 58 UR - DO - 10.1111/j.1365-2958.2004.04910.x AB - PopA is released by type III secretion from the bacterial plant pathogen Ralstonia solanacearum and triggers the hypersensitive response (HR) in tobacco. The function of PopA remains obscure, mainly because mutants lacking this protein are not altered in their ability to interact with plants. In an attempt to identify the site of PopA activity in plant cells, we generated transgenic tobacco plants expressing the popA gene under the control of an inducible promoter. Immunocytologic analysis revealed that the HR phenotype of these plants correlated with the presence of PopA at the plant plasma membrane. Membrane localization was observed irrespective of whether the protein was designed to accumulate in the cytoplasm or to be secreted by the plant cell, suggesting a general lipid‐binding ability. We found that the protein had a high affinity for sterols and sphingolipids in vitro and that it required Ca2+ for both lipid binding and oligomerization. In addition, the protein was integrated into liposomes and membranes from Xenopus laevis oocytes where it formed ion‐conducting pores. These characteristics suggest that PopA is part of a system that aims to attach the host cell plasma membrane and to allow molecules cross this barrier. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1975 TI - Evaluation of the Antimicrobial Activities of Plant Oxylipins Supports Their Involvement in Defense against Pathogens JO - Plant Physiol. PY - 2005 SP - 1902-1913 AU - Prost, I. AU - Dhondt, S. AU - Rothe, G. AU - Vicente, J. AU - Rodriguez, M. J. AU - Kift, N. AU - Carbonne, F. AU - Griffiths, G. AU - Esquerré-Tugayé, M.-T. AU - Rosahl, S. AU - Castresana, C. AU - Hamberg, M. AU - Fournier, J. AU - VL - 139 UR - DO - 10.1104/pp.105.066274 AB - Plant oxylipins are a large family of metabolites derived from polyunsaturated fatty acids. The characterization of mutants or transgenic plants affected in the biosynthesis or perception of oxylipins has recently emphasized the role of the so-called oxylipin pathway in plant defense against pests and pathogens. In this context, presumed functions of oxylipins include direct antimicrobial effect, stimulation of plant defense gene expression, and regulation of plant cell death. However, the precise contribution of individual oxylipins to plant defense remains essentially unknown. To get a better insight into the biological activities of oxylipins, in vitro growth inhibition assays were used to investigate the direct antimicrobial activities of 43 natural oxylipins against a set of 13 plant pathogenic microorganisms including bacteria, oomycetes, and fungi. This study showed unequivocally that most oxylipins are able to impair growth of some plant microbial pathogens, with only two out of 43 oxylipins being completely inactive against all the tested organisms, and 26 oxylipins showing inhibitory activity toward at least three different microbes. Six oxylipins strongly inhibited mycelial growth and spore germination of eukaryotic microbes, including compounds that had not previously been ascribed an antimicrobial activity, such as 13-keto-9(Z),11(E),15(Z)-octadecatrienoic acid and 12-oxo-10,15(Z)-phytodienoic acid. Interestingly, this first large-scale comparative assessment of the antimicrobial effects of oxylipins reveals that regulators of plant defense responses are also the most active oxylipins against eukaryotic microorganisms, suggesting that such oxylipins might contribute to plant defense through their effects both on the plant and on pathogens, possibly through related mechanisms. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1974 TI - Ozone-Induced Programmed Cell Death in the Arabidopsis radical-induced cell death1 Mutant JO - Plant Physiol. PY - 2005 SP - 1092-1104 AU - Overmyer, K. AU - Brosché, M. AU - Pellinen, R. AU - Kuittinen, T. AU - Tuominen, H. AU - Ahlfors, R. AU - Keinänen, M. AU - Saarma, M. AU - Scheel, D. AU - Kangasjärvi, J. AU - VL - 137 UR - DO - 10.1104/pp.104.055681 AB - Short, high-concentration peaks of the atmospheric pollutant ozone (O3) cause the formation of cell death lesions on the leaves of sensitive plants. Numerous similarities between the plant responses to O3 and pathogens suggest that O3 triggers hypersensitive response-like programmed cell death (PCD). We examined O3 and superoxide-induced cell death in the O3-sensitive radical-induced cell death1 (rcd1) mutant. Dying cells in O3-exposed rcd1 exhibited several of the typical morphological characteristics of the hypersensitive response and PCD. Double-mutant analyses indicated a requirement for salicylic acid and the function of the cyclic nucleotide-gated ion channel AtCNGC2 in cell death. Furthermore, a requirement for ATPases, kinases, transcription, Ca2+ flux, caspase-like proteolytic activity, and also one or more phenylmethylsulfonyl fluoride-sensitive protease activities was shown for the development of cell death lesions in rcd1. Furthermore, mitogen-activated protein kinases showed differential activation patterns in rcd1 and Columbia. Taken together, these results directly demonstrate the induction of PCD by O3. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1964 TI - Ethylene-mediated cross-talk between calcium-dependent protein kinase and MAPK signaling controls stress responses in plants JO - Proc. Natl. Acad. Sci. U.S.A. PY - 2005 SP - 10736-10741 AU - Ludwig, A. A. AU - Saitoh, H. AU - Felix, G. AU - Freymark, G. AU - Miersch, O. AU - Wasternack, C. AU - Boller, T. AU - Jones, J. D. G. AU - Romeis, T. AU - VL - 102 UR - DO - 10.1073/pnas.0502954102 AB - Plants are constantly exposed to environmental changes and need to integrate multiple external stress cues. Calcium-dependent protein kinases (CDPKs) are implicated as major primary Ca2+ sensors in plants. CDPK activation, like activation of mitogen-activated protein kinases (MAPKs), is triggered by biotic and abiotic stresses, although distinct stimulus-specific stress responses are induced. To investigate whether CDPKs are part of an underlying mechanism to guarantee response specificity, we identified CDPK-controlled signaling pathways. A truncated form of Nicotiana tabacum CDPK2 lacking its regulatory autoinhibitor and calcium-binding domains was ectopically expressed in Nicotiana benthamiana. Infiltrated leaves responded to an abiotic stress stimulus with the activation of biotic stress reactions. These responses included synthesis of reactive oxygen species, defense gene induction, and SGT1-dependent cell death. Furthermore, N-terminal CDPK2 signaling triggered enhanced levels of the phytohormones jasmonic acid, 12-oxo-phytodienoic acid, and ethylene but not salicylic acid. These responses, commonly only observed after challenge with a strong biotic stimulus, were prevented when the CDPK's intrinsic autoinhibitory peptide was coexpressed. Remarkably, elevated CDPK signaling compromised stress-induced MAPK activation, and this inhibition required ethylene synthesis and perception. These data indicate that CDPK and MAPK pathways do not function independently and that a concerted activation of both pathways controls response specificity to biotic and abiotic stress. A2 - C1 - Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1961 TI - Pre- and Postinvasion Defenses Both Contribute to Nonhost Resistance in Arabidopsis JO - Science PY - 2005 SP - 1180-1183 AU - Lipka, V. AU - Dittgen, J. AU - Bednarek, P. AU - Bhat, R. AU - Wiermer, M. AU - Stein, M. AU - Landtag, J. AU - Brandt, W. AU - Rosahl, S. AU - Scheel, D. AU - Llorente, F. AU - Molina, A. AU - Parker, J. AU - Somerville, S. AU - Schulze-Lefert, P. AU - VL - 310 UR - DO - 10.1126/science.1119409 AB - Nonhost resistance describes the immunity of an entire plant species against nonadapted pathogen species. We report that Arabidopsis PEN2 restricts pathogen entry of two ascomycete powdery mildew fungi that in nature colonize grass and pea species. The PEN2 glycosyl hydrolase localizes to peroxisomes and acts as a component of an inducible preinvasion resistance mechanism. Postinvasion fungal growth is blocked by a separate resistance layer requiring the EDS1-PAD4-SAG101 signaling complex, which is known to function in basal and resistance (R) gene–triggered immunity. Concurrent impairment of pre- and postinvasion resistance renders Arabidopsis a host for both nonadapted fungi. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER - TY - JOUR ID - 1960 TI - Harpin of Pseudomonas syringae pv. phaseolicola Harbors a Protein Binding Site JO - Mol. Plant Microbe Interact. PY - 2005 SP - 60-66 AU - Li, C.-M. AU - Haapalainen, M. AU - Lee, J. AU - Nürnberger, T. AU - Romantschuk, M. AU - Taira, S. AU - VL - 18 UR - DO - 10.1094/MPMI-18-0060 AB - Harpin HrpZ of plant-pathogenic bacterium Pseudomonas syringae elicits a hypersensitive response (HR) in some nonhost plants, but its function in the pathogenesis process is still obscure. HrpZ-interacting proteins were identified by screening a phage-display library of random peptides. HrpZ of the bean pathogen P. syringae pv. Phaseolicola (HrpZPph) shows affinity to peptides with a consensus amino acid motif W(L)ARWLL(G/L). To localize the peptide-binding site, the hrpZPph gene was mutagenized with randomly placed 15-bp insertions, and the mutant proteins were screened for the peptide-binding ability. Mutations that inhibited peptide-binding localized to the central region of hrpZPph, which is separate from the previously determined HR-inducing region. Antiserum raised against one of the hrpZPph-binding peptides recognized small proteins in bean, tomato, parsley, and Arabidopsis thaliana but none in tobacco. On native protein blots, hrpZPph bound to a bean protein with similar pI as the protein recognized by the peptide antiserum. The result suggests a protein-protein interaction between the harpin and a host plant protein, possibly involved in the bacterial pathogenesis. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2049 TI - BRU1, a novel link between responses to DNA damage and epigenetic gene silencing in Arabidopsis JO - Genes Dev. PY - 2004 SP - 782-793 AU - Takeda, S. AU - Tadele, Z. AU - Hofmann, I. AU - Probst, A. V. AU - Angelis, K. J. AU - Kaya, H. AU - Araki, T. AU - Mengiste, T. AU - Scheid, O. M. AU - Shibahara, K.-i. AU - Scheel, D. AU - Paszkowski, J. AU - VL - 18 UR - DO - 10.1101/gad.295404 AB - DNA repair associated with DNA replication is important for the conservation of genomic sequence information, whereas reconstitution of chromatin after replication sustains epigenetic information. We have isolated and characterized mutations in the BRU1 gene of Arabidopsis that suggest a novel link between these underlying maintenance mechanisms. Bru1 plants are highly sensitive to genotoxic stress and show stochastic release of transcriptional gene silencing. They also show increased intrachromosomal homologous recombination and constitutively activated expression of poly (ADP-ribose) polymerase-2 (AtPARP-2), the induction of which is associated with elevated DNA damage. Bru1 mutations affect the stability of heterochromatin organization but do not interfere with genome-wide DNA methylation. BRU1 encodes a novel nuclear protein with two predicted protein–protein interaction domains. The developmental abnormalities characteristic of bru1 mutant plants resemble those triggered by mutations in genes encoding subunits of chromatin assembly factor (CAF-1), the condensin complex, or MRE11. Comparison of bru1 with these mutants indicates cooperative roles in the replication and stabilization of chromatin structure, providing a novel link between chromatin replication, epigenetic inheritance, S-phase DNA damage checkpoints, and the regulation of meristem development. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2046 TI - Molecular Population Genetic Analysis Differentiates Two Virulence Mechanisms of the Fungal Avirulence Gene NIP1 JO - Mol. Plant Microbe Interact. PY - 2004 SP - 1114-1125 AU - Schürch, S. AU - Linde, C. C. AU - Knogge, W. AU - Jackson, L. F. AU - McDonald, B. A. AU - VL - 17 UR - DO - 10.1094/MPMI.2004.17.10.1114 AB - Deletion or alteration of an avirulence gene are two mechanisms that allow pathogens to escape recognition mediated by the corresponding resistance gene in the host. We studied these two mechanisms for the NIP1 avirulence gene in field populations of the fungal barley pathogen Rhynchosporium secalis. The product of the avirulence gene, NIP1, causes leaf necrosis and elicits a defense response on plants with the Rrs1 resistance gene. A high NIP1 deletion frequency (45%) was found among 614 isolates from different geographic populations on four continents. NIP1 was also sequenced for 196 isolates, to identify DNA polymorphisms and corresponding NIP1 types. Positive diversifying selection was found to act on NIP1. A total of 14 NIP1 types were found, 11 of which had not been described previously. The virulence of the NIP1 types was tested on Rrs1 and rrs1 barley lines. Isolates carrying three of these types were virulent on the Rrs1 cultivar. One type each was found in California, Western Europe, and Jordan. Additionally, a field experiment with one pair of near-isogenic lines was conducted to study the selection pressure imposed by Rrs1 on field populations of R. secalis. Deletion of NIP1 was the only mechanism used to infect the Rrs1 cultivar in the field experiment. In this first comprehensive study on the population genetics of a fungal avirulence gene, virulence to Rrs1 in R. secalis was commonly achieved through deletion of the NIP1 avirulence gene but rarely also through point mutations in NIP1. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2036 TI - The jasmonate-insensitive mutant jin1 shows increased resistance to biotrophic as well as necrotrophic pathogens JO - Mol. Plant Pathol. PY - 2004 SP - 425-434 AU - NICKSTADT, A. AU - THOMMA, B. P. H. J. AU - Feussner, I. AU - Kangasjärvi, J. AU - ZEIER, J. AU - LOEFFLER, C. AU - Scheel, D. AU - BERGER, S. AU - VL - 5 UR - DO - 10.1111/j.1364-3703.2004.00242.x AB - Jasmonic acid and related oxylipin compounds are plant signalling molecules that are involved in the response to pathogens, insects, wounding and ozone. To explore further the role of jasmonates in stress signal transduction, the response of two jasmonate‐signalling mutants, jin1 and jin4 , to pathogens and ozone was analysed in this study. Upon treatment with the biotrophic bacterial pathogen Pseudomonas syringae , endogenous jasmonate levels increased in jin1 and jin4 similar to wild‐type, demonstrating that these mutants are not defective in jasmonate biosynthesis. Jin1 but not jin4 is more resistant to P. syringae and this higher resistance is accompanied by higher levels of salicylic acid. Jin1 is also more resistant to the necrotrophic fungal pathogen Botrytis cinerea and shows wild‐type sensitivity to ozone whereas jin4 is more susceptible to B. cinerea and ozone. These results indicate that the mutations in jin1 and jin4 affect different branches of the jasmonate signalling pathway. Additionally, in this combination of phenotypes, jin1 is unique among all other jasmonate‐related mutants described thus far. These data also provide support for a crosstalk between the jasmonate and salicylate pathways. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2024 TI - Dynamic Changes in the Localization of MAPK Cascade Components Controlling Pathogenesis-related (PR) Gene Expression during Innate Immunity in Parsley JO - J. Biol. Chem. PY - 2004 SP - 22440-22448 AU - Lee, J. AU - Rudd, J. J. AU - Macioszek, V. K. AU - Scheel, D. AU - VL - 279 UR - DO - 10.1074/jbc.M401099200 AB - The activation of mitogen-activated protein kinase (MAPK) cascades is an important mechanism for stress adaptation through the control of gene expression in mammals, yeast, and plants. MAPK activation has emerged as a common mechanism by which plants trigger pathogen defense responses following innate immune recognition of potential microbial pathogens. We are studying the non-host plant defense response of parsley to attempted infection by Phytophthora species using an experimental system of cultured parsley cells and the Phytophthora-derived Pep-13 peptide elicitor. Following receptor-mediated recognition of this peptide, parsley cells trigger a multifaceted innate immune response, involving the activation of three MAPKs that have been shown to function in the oxidative burst-independent activation of defense gene expression. Using this same experimental model we now report the identification of a MAPK kinase (MAPKK) that functions upstream in this pathway. This kinase, referred to as PcMKK5 based on sequence similarity to Arabidopsis thaliana AtMKK5, is activated in parsley cells following Pep-13 treatment and functions as an in vivo activator of all three MAPKs previously shown to be involved in this response. Gain- and loss-of-function mutant versions of PcMKK5, when used in protoplast co-transfection assays, demonstrated that kinase activity of PcMKK5 is required for PR gene promoter activation following Pep-13 treatment. Furthermore, using specific antibodies and immunofluorescent labeling, we demonstrate that activation of MAPKs in parsley cells correlates with an increase in their nuclear localization, which is not detectable for activated PcMKK5. These results suggest that activation of gene expression through MAPK cascades during innate immune responses in plants involves dynamic changes in the localization of the proteins involved, which may reflect the distribution of key protein substrates for the activated MAPKs. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2018 TI - A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to γ-glutamylcysteine and lacks phytochelatin synthase activity JO - Phytochemistry PY - 2004 SP - 3179-3185 AU - Harada, E. AU - von Roepenack-Lahaye, E. AU - Clemens, S. AU - VL - 65 UR - DO - 10.1016/j.phytochem.2004.09.017 AB - Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to γ-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC–MSMS analysis was unequivocally identified as γ-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to γ-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2058 TI - Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors JO - Plant J. PY - 2004 SP - 269-281 AU - Weber, M. AU - Harada, E. AU - Vess, C. AU - Roepenack-Lahaye, E. v. AU - Clemens, S. AU - VL - 37 UR - DO - 10.1046/j.1365-313x.2003.01960.x AB - The hyperaccumulation of zinc (Zn) and cadmium (Cd) is a constitutive property of the metallophyte Arabidopsis halleri . We therefore used Arabidopsis GeneChips to identify genes more active in roots of A. halleri as compared to A. thaliana under control conditions. The two genes showing highest expression in A. halleri roots relative to A. thaliana roots out of more than 8000 genes present on the chip encode a nicotianamine (NA) synthase and a putative Zn2+ uptake system. The significantly higher activity of these and other genes involved in metal homeostasis under various growth conditions was confirmed by Northern and RT‐PCR analyses. A. halleri roots also show higher NA synthase protein levels. Furthermore, we developed a capillary liquid chromatography electrospray ionization quadrupole time‐of‐flight mass spectrometry (CapLC‐ESI‐QTOF‐MS)‐based NA analysis procedure and consistently found higher NA levels in roots of A. halleri . Expression of a NA synthase in Zn2+‐hypersensitive Schizosaccharomyces pombe cells demonstrated that formation of NA can confer Zn2+ tolerance. Taken together, these observations implicate NA in plant Zn homeostasis and NA synthase in the hyperaccumulation of Zn by A. halleri . Furthermore, the results show that comparative microarray analysis of closely related species can be a valuable tool for the elucidation of phenotypic differences between such species. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2056 TI - Profiling of Arabidopsis Secondary Metabolites by Capillary Liquid Chromatography Coupled to Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry JO - Plant Physiol. PY - 2004 SP - 548-559 AU - von Roepenack-Lahaye, E. AU - Degenkolb, T. AU - Zerjeski, M. AU - Franz, M. AU - Roth, U. AU - Wessjohann, L. AU - Schmidt, J. AU - Scheel, D. AU - Clemens, S. AU - VL - 134 UR - DO - 10.1104/pp.103.032714 AB - Large-scale metabolic profiling is expected to develop into an integral part of functional genomics and systems biology. The metabolome of a cell or an organism is chemically highly complex. Therefore, comprehensive biochemical phenotyping requires a multitude of analytical techniques. Here, we describe a profiling approach that combines separation by capillary liquid chromatography with the high resolution, high sensitivity, and high mass accuracy of quadrupole time-of-flight mass spectrometry. About 2,000 different mass signals can be detected in extracts of Arabidopsis roots and leaves. Many of these originate from Arabidopsis secondary metabolites. Detection based on retention times and exact masses is robust and reproducible. The dynamic range is sufficient for the quantification of metabolites. Assessment of the reproducibility of the analysis showed that biological variability exceeds technical variability. Tools were optimized or established for the automatic data deconvolution and data processing. Subtle differences between samples can be detected as tested with the chalcone synthase deficient tt4 mutant. The accuracy of time-of-flight mass analysis allows to calculate elemental compositions and to tentatively identify metabolites. In-source fragmentation and tandem mass spectrometry can be used to gain structural information. This approach has the potential to significantly contribute to establishing the metabolome of Arabidopsis and other model systems. The principles of separation and mass analysis of this technique, together with its sensitivity and resolving power, greatly expand the range of metabolic profiling. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER - TY - JOUR ID - 2011 TI - Expression Patterns of Defense-Related Genes in Different Types of Arbuscular Mycorrhizal Development in Wild-Type and Mycorrhiza-Defective Mutant Tomato JO - Mol. Plant Microbe Interact. PY - 2004 SP - 1103-1113 AU - Gao, L.-L. AU - Knogge, W. AU - Delp, G. AU - Smith, F. A. AU - Smith, S. E. AU - VL - 17 UR - DO - 10.1094/MPMI.2004.17.10.1103 AB - The expression of defense-related genes was analyzed in the interactions of six arbuscular mycorrhizal (AM) fungi with the roots of wild-type tomato (Lycopersicon esculentum Mill.) cv. 76R and of the near-isogenic mycorrhiza-defective mutant rmc. Depending on the fungal species, wild-type tomato forms both major morphological AM types, Arum and Paris. The mutant rmc blocks the penetration of the root surface or invasion of the root cortex by most species of AM fungi, but one fungus has been shown to develop normal mycorrhizas. In the wild-type tomato, accumulation of mRNA representing a number of defense-related genes was low in Arum-type interactions, consistent with findings for this AM morphotype in other plant species. In contrast, Paris-type colonization, particularly by members of the family Gigasporaceae, was accompanied by a substantial transient increase in expression of some defense-related genes. However, the extent of root colonization did not differ significantly in the two wild-type AM morpho-types, suggesting that accumulation of defense gene products per se does not limit mycorrhiza development. In the mutant, interactions in which the fungus failed to penetrate the root lacked significant accumulation of defense gene mRNAs. However, phenotypes in which the fungus penetrated epidermal or hypodermal cells were associated with an enhanced and more prolonged gene expression. These results are discussed in relation to the mechanisms that may underlie the specificity of the interactions between AM fungi and the rmc mutant. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2000 TI - Stress hormone-independent activation and nuclear translocation of mitogen-activated protein kinases in Arabidopsis thaliana during ozone exposure JO - Plant J. PY - 2004 SP - 512-522 AU - Ahlfors, R. AU - Macioszek, V. AU - Rudd, J. AU - Brosché, M. AU - Schlichting, R. AU - Scheel, D. AU - Kangasjärvi, J. AU - VL - 40 UR - DO - 10.1111/j.1365-313X.2004.02229.x AB - Changing environmental conditions, atmospheric pollutants and resistance reactions to pathogens cause production of reactive oxygen species (ROS) in plants. ROS in turn trigger the activation of signaling cascades such as the mitogen‐activated protein kinase (MAPK) cascade and accumulation of plant hormones, jasmonic acid, salicylic acid (SA), and ethylene (ET). We have used ozone (O3) to generate ROS in the apoplast of wild‐type Col‐0 and hormonal signaling mutants of Arabidopsis thaliana and show that this treatment caused a transient activation of 43 and 45 kDa MAPKs. These were identified as AtMPK3 and AtMPK6. We also demonstrate that initial AtMPK3 and AtMPK6 activation in response to O3 was not dependent on ET signaling, but that ET is likely to have secondary effects on AtMPK3 and AtMPK6 function, whereas functional SA signaling was needed for full‐level AtMPK3 activation by O3. In addition, we show that AtMPK3 , but not AtMPK6 , responded to O3 transcriptionally and translationally during O3 exposure. Finally, we show in planta that activated AtMPK3 and AtMPK6 are translocated to the nucleus during the early stages of O3 treatment. The use of O3 to induce apoplastic ROS formation offers a non‐invasive in planta system amenable to reverse genetics that can be used for the study of stress‐responsive MAPK signaling in plants. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2014 TI - The oligopeptide elicitor Pep-13 induces salicylic acid-dependent and -independent defense reactions in potato JO - Physiol. Mol. Plant Pathol. PY - 2004 SP - 311-318 AU - Halim, V. A. AU - Hunger, A. AU - Macioszek, V. AU - Landgraf, P. AU - Nürnberger, T. AU - Scheel, D. AU - Rosahl, S. AU - VL - 64 UR - DO - 10.1016/j.pmpp.2004.10.003 AB - The Phytophthora-derived oligopeptide elicitor, Pep-13, originally identified as an inducer of plant defense in the nonhost–pathogen interaction of parsley and Phytophthora sojae, triggers defense responses in potato. In cultured potato cells, Pep-13 treatment results in an oxidative burst and activation of defense genes. Infiltration of Pep-13 into leaves of potato plants induces the accumulation of hydrogen peroxide, defense gene expression and the accumulation of jasmonic and salicylic acids. Derivatives of Pep-13 show similar elicitor activity in parsley and potato, suggesting a receptor-mediated induction of defense response in potato similar to that observed in parsley. However, unlike in parsley, infiltration of Pep-13 into leaves leads to the development of hypersensitive response-like cell death in potato. Interestingly, Pep-13-induced necrosis formation, hydrogen peroxide formation and accumulation of jasmonic acid, but not activation of a subset of defense genes, is dependent on salicylic acid, as shown by infiltration of Pep-13 into leaves of potato plants unable to accumulate salicylic acid. Thus, in a host plant of Phytophthora infestans, Pep-13 is able to elicit salicylic acid-dependent and -independent defense responses. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2126 TI - Metalloid tolerance based on phytochelatins is not functionally equivalent to the arsenite transporter Acr3p JO - Biochem. Biophys. Res. Commun. PY - 2003 SP - 293-300 AU - Wysocki, R. AU - Clemens, S. AU - Augustyniak, D. AU - Golik, P. AU - Maciaszczyk, E. AU - Tamás, M. J. AU - Dziadkowiec, D. AU - VL - 304 UR - DO - 10.1016/S0006-291X(03)00584-9 AB - Active transport of metalloids by Acr3p and Ycf1p in Saccharomyces cerevisiae and chelation by phytochelatins in Schizosaccharomyces pombe, nematodes, and plants represent distinct strategies of metalloid detoxification. In this report, we present results of functional comparison of both resistance mechanisms. The S. pombe and wheat phytochelatin synthase (PCS) genes, when expressed in S. cerevisiae, mediate only modest resistance to arsenite and thus cannot functionally compensate for Acr3p. On the other hand, we show for the first time that phytochelatins also contribute to antimony tolerance as PCS fully complement antimonite sensitivity of ycf1Δ mutant. Remarkably, heterologous expression of PCS sensitizes S. cerevisiae to arsenate, while ACR3 confers much higher arsenic resistance in pcsΔ than in wild-type S. pombe. The analysis of PCS and ACR3 homologues distribution in various organisms and our experimental data suggest that separation of ACR3 and PCS genes may lead to the optimal tolerance status of the cell. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2123 TI - The Arabidopsis NHL3 Gene Encodes a Plasma Membrane Protein and Its Overexpression Correlates with Increased Resistance to Pseudomonas syringae pv. tomato DC3000 JO - Plant Physiol. PY - 2003 SP - 2023-2033 AU - Varet, A. AU - Hause, B. AU - Hause, G. AU - Scheel, D. AU - Lee, J. AU - VL - 132 UR - DO - 10.1104/pp.103.020438 AB - The Arabidopsis genome contains a family of NDR1/HIN1-like (NHL) genes that show homology to the nonrace-specific disease resistance (NDR1) and the tobacco (Nicotiana tabacum) harpin-induced (HIN1) genes. NHL3 is a pathogen-responsive member of this NHL gene family that is potentially involved in defense. In independent transgenic NHL3-overexpressing plant lines, a clear correlation between increased resistance to virulent Pseudomonas syringae pv. tomato DC3000 and enhanced NHL3 transcript levels was seen. These transgenic plants did not show enhanced pathogenesis-related gene expression or reactive oxygen species accumulation. Biochemical and localization experiments were performed to assist elucidation of how NHL3 may confer enhanced disease resistance. Gene constructs expressing amino-terminal c-myc-tagged or carboxyl-terminal hemagglutinin epitope (HA)-tagged NHL3 demonstrated membrane localization in transiently transformed tobacco leaves. Stable Arabidopsis transformants containing the NHL3-HA construct corroborated the findings observed in tobacco. The detected immunoreactive proteins were 10 kD larger than the calculated size and could be partially accounted for by the glycosylation state. However, the expected size was not attained with deglycosylation, suggesting possibly additional posttranslational modification. Detergent treatment, but not chemicals used to strip membrane-associated proteins, could displace the immunoreactive signal from microsomal fractions, showing that NHL3 is tightly membrane associated. Furthermore, immunofluorescence and immunogold labeling, coupled with two-phase partitioning techniques, revealed plasma membrane localization of NHL3-HA. This subcellular localization of NHL3 positions it at an initial contact site to pathogens and may be important in facilitating interception of pathogen-derived signals. A2 - C1 - Cell and Metabolic Biology; Biochemistry of Plant Interactions ER - TY - JOUR ID - 2122 TI - Solution Structure of the Plant Disease Resistance-triggering Protein NIP1 from the Fungus Rhynchosporium secalis Shows a Novel β-Sheet Fold JO - J. Biol. Chem. PY - 2003 SP - 45730-45736 AU - van't Slot, K. A. E. AU - van den Burg, H. A. AU - Kloks, C. P. A. M. AU - Hilbers, C. W. AU - Knogge, W. AU - Papavoine, C. H. M. AU - VL - 278 UR - DO - 10.1074/jbc.M308304200 AB - Activation of the disease resistance response in a host plant frequently requires the interaction of a plant resistance gene product with a corresponding, pathogenderived signal encoded by an avirulence gene. The products of resistance genes from diverse plant species show remarkable structural similarity. However, due to the general paucity of information on pathogen avirulence genes the recognition process remains in most cases poorly understood. NIP1, a small protein secreted by the fungal barley pathogen Rhynchosporium secalis, is one of only a few fungal avirulence proteins identified and characterized to date. The defense-activating activity of NIP1 is mediated by barley resistance gene Rrs1. In addition, a role of the protein in fungal virulence is suggested by its nonspecific toxicity in leaf tissues of host and non-host cereals as well as its resistance gene-independent stimulatory effect on the plant plasma membrane H+-ATPase. Four naturally occurring NIP1 isoforms are characterized by single amino acid alterations that affect the different activities in a similar way. As a step toward unraveling the signal perception/transduction mechanism, the solution structure of NIP1 was determined. The protein structure is characterized by a novel fold. It consists of two parts containing β-sheets of two and three anti-parallel strands, respectively. Five intramolecular disulfide bonds, comprising a novel disulfide bond pattern, stabilize these parts and their position with respect to each other. A comparative analysis of the protein structure with the properties of the NIP1 isoforms suggests two loop regions to be crucial for the resistance-triggering activity of NIP1. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2081 TI - Lipid Peroxidation during the Hypersensitive Response in Potato in the Absence of 9-Lipoxygenases JO - J. Biol. Chem. PY - 2003 SP - 52834-52840 AU - Göbel, C. AU - Feussner, I. AU - Rosahl, S. AU - VL - 278 UR - DO - 10.1074/jbc.M310833200 AB - Hypersensitive cell death is an important defense reaction of plants to pathogen infection and is accompanied by lipid peroxidation processes. These may occur non-enzymatically by the action of reactive oxygen species or may be catalyzed by enzymes such as α-dioxygenases, lipoxygenases, or peroxidases. Correlative data showing increases in 9-lipoxygenase products in hyper-sensitively reacting cells have so far suggested that a large part of lipid peroxidation is mediated by a specific set of 9-lipoxygenases. To address the significance of 9-lipoxygenases for this type of pathogen response in potato, RNA interference constructs of a specific pathogen-induced potato 9-lipoxygenase were transferred to potato plants. Significantly reduced 9-lipoxygenase transcript levels were observed in transgenic plants after pathogen treatment. In addition, 9-lipoxygenase activity was hardly detectable, and levels of 9-lipoxygenase-derived oxylipins were reduced up to 12-fold after pathogen infection. In contrast to wild type plants, high levels of non-enzymatically as well as 13-lipoxygenase-derived oxylipins were present in 9-lipoxygenase-deficient plants. From this we conclude that during the normal hypersensitive response in potato, lipid peroxidation may occur as a controlled and directed process that is facilitated by the action of a specific 9-lipoxygenase. If 9-lipoxygenase-mediated formation of hydroperoxides is repressed, autoxidative lipid peroxidation processes and 13-lipoxygenase-mediated oxylipins synthesis become prominent. The unaltered timing and extent of necrosis formation suggests that the origin of lipid hydroperoxides does not influence pathogen-induced cell death in potato. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2079 TI - Development of SCAR markers linked to a scald resistance gene derived from wild barley JO - Euphytica PY - 2003 SP - 149-159 AU - Genger, R. K. AU - Brown, A. H. D. AU - Knogge, W. AU - Nesbitt, K. AU - Burdon, J. J. AU - VL - 134 UR - DO - 10.1023/B:EUPH.0000003833.63547.78 AB - The F2 progeny of a third backcross(BC3) line, BC line 240, derived from a Turkish accession of wild barley (Hordeum vulgare ssp. spontaneum),segregated for resistance to scald (Rhynchosporium secalis) in a manner indicating the presence of a single dominant resistance gene. Two SCAR marker slinked to this resistance were developed from AFLP markers. Screens of disomic and ditelosomic wheat-barley addition lines with the SCAR markers demonstrated that the scald resistance gene is located in the centromeric region of barley chromosome 3H,a region previously reported to contain a major scald resistance locus, Rrs1. Markers that flank the Rrs1 locus were used to screen the wild barley-derivedBC3F2 population. These markers also flank the wild barley-derived scald resistance, indicating that it maps to the same locus as Rrs1; it may beallelic, or a separate gene within a complex locus. However, BC line 240 does not respond to treatment with the Rhynchosporium secalis avirulence factorNIP1 in the same way as the Rrs1-carrying cultivar Atlas46. This suggests that the scald resistance gene derived from wild barley confers a different specificity of response to theRrs1 allele in Atlas46.In order to increase the durability of scald resistance in the field, we suggest that at least two scald resistances should be combined into barley cultivars before release. The scald resistance gene described here will be of value in the Australian environment, and the several markers linked to it will facilitate pyramiding. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2072 TI - Schizosaccharomyces pombe as a model for metal homeostasis in plant cells: the phytochelatin-dependent pathway is the main cadmium detoxification mechanism JO - New Phytol. PY - 2003 SP - 323-330 AU - Clemens, S. AU - Simm, C. AU - VL - 159 UR - DO - 10.1046/j.1469-8137.2003.00811.x AB - Sequestration of metal ions by phytochelatins is an important metal tolerance mechanism in a wide range of organisms including plants and certain fungi. Substantial progress in understanding phytochelatin formation at the molecular level has been made in Schizosaccharomyces pombe . The genome of S. pombe has been completely sequenced and all the necessary tools of functional genomics are available. Since most other proteins implicated in plant metal tolerance and homeostasis are also present in this yeast, it represents a very powerful system to elucidate basic mechanisms of metal buffering, sequestration, and toxicity in cells that form phytochelatins. Here, we summarize the work on phytochelatin formation and metal homeostasis in S. pombe . We describe examples of molecular insights obtained from experiments with S. pombe that will be useful in guiding studies with plants. We also provide evidence for the dominance of the phytochelatin pathway in Cd detoxification in S. pombe. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2115 TI - Differential Defense Reactions in Leaf Tissues of Barley in Response to Infection by Rhynchosporium secalis and to Treatment with a Fungal Avirulence Gene Product JO - Mol. Plant Microbe Interact. PY - 2003 SP - 893-902 AU - Steiner-Lange, S. AU - Fischer, A. AU - Boettcher, A. AU - Rouhara, I. AU - Liedgens, H. AU - Schmelzer, E. AU - Knogge, W. AU - VL - 16 UR - DO - 10.1094/MPMI.2003.16.10.893 AB - Expression of defense-associated genes was analyzed in leaf tissues of near-isogenic resistant and susceptible barley cultivars upon infection by Rhynchosporium secalis. The genes encoding pathogenesis-related (PR) proteins PR-1, PR-5, and PR-9 are specifically expressed in the mesophyll of resistant plants, whereas a germin-like protein (OxOLP) is synthesized in the epidermis irrespective of the resistance genotype. Restriction-mediated differential display was employed to identify additional epidermis-specific genes. This resulted in the detection of another PR gene, PR-10, along with a lipoxygenase gene, LoxA, and a gene of unknown function, pI2-4, which are specifically induced in the epidermis of resistant plants. The gene encoding a putative protease inhibitor, SD10, is preferentially but not exclusively expressed in the epidermis. The fungal avirulence gene product NIP1 triggers the induction of the four PR genes only. At least two additional elicitors, therefore, must be postulated, one for the unspecific induction of OxOLP and one for the resistance-specific induction of LoxA, pI2-4, and SD10. PR-10 expression can be assumed to be the consequence of NIP1 perception by epidermis cells. In contrast, gene expression in the mesophyll is likely to be triggered by an as yet unknown signal that appears to originate in the epidermis and that is strongly amplified in the mesophyll. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2109 TI - Oxidative burst in plant defense JO - Free Radic. Res. PY - 2003 SP - 5 AU - Scheel, D. AU - Handmann, F. AU - Lee, J. AU - Rudd, J. J. AU - Zinecker, H. AU - VL - 37 UR - AB - A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2095 TI - Crystal Structure of a Hypoallergenic Isoform of the Major Birch Pollen Allergen Bet v 1 and its Likely Biological Function as a Plant Steroid Carrier JO - J. Mol. Biol. PY - 2003 SP - 123-133 AU - Marković-Housley, Z. AU - Degano, M. AU - Lamba, D. AU - von Roepenack-Lahaye, E. AU - Clemens, S. AU - Susani, M. AU - Ferreira, F. AU - Scheiner, O. AU - Breiteneder, H. AU - VL - 325 UR - DO - 10.1016/S0022-2836(02)01197-X AB - Bet v 1l is a naturally occurring hypoallergenic isoform of the major birch pollen allergen Bet v 1. The Bet v 1 protein belongs to the ubiquitous family of pathogenesis-related plant proteins (PR-10), which are produced in defense-response to various pathogens. Although the allergenic properties of PR-10 proteins have been extensively studied, their biological function in plants is not known. The crystal structure of Bet v 1l in complex with deoxycholate has been determined to a resolution of 1.9 Å using the method of molecular replacement. The structure reveals a large hydrophobic Y-shaped cavity that spans the protein and is partly occupied by two deoxycholate molecules which are bound in tandem and only partially exposed to solvent. This finding indicates that the hydrophobic cavity may have a role in facilitating the transfer of apolar ligands. The structural similarity of deoxycholate and brassinosteroids (BRs) ubiquitous plant steroid hormones, prompted the mass spectrometry (MS) study in order to examine whether BRs can bind to Bet v 1l. The MS analysis of a mixture of Bet v 1l and BRs revealed a specific non-covalent interaction of Bet v 1l with brassinolide and 24-epicastasterone. Together, our findings are consistent with a general plant-steroid carrier function for Bet v 1 and related PR-10 proteins. The role of BRs transport in PR-10 proteins may be of crucial importance in the plant defense response to pathological situations as well as in growth and development. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2094 TI - Localization and functional characterization of metal-binding sites in phytochelatin synthases JO - Planta PY - 2003 SP - 300-308 AU - Maier, T. AU - Yu, C. AU - Küllertz, G. AU - Clemens, S. AU - VL - 218 UR - DO - 10.1007/s00425-003-1091-7 AB - Metal-binding domains consisting of short, contiguous stretches of amino acids are found in many proteins mediating the transport, buffering, trafficking or detoxification of metal ions. Phytochelatin synthases are metal-activated enzymes that function in the detoxification of Cd2+ and other toxic metal and metalloid ions. In order to localize Cd2+-binding sites, peptide libraries of two diverse phytochelatin synthases were synthesized and incubated with 109Cd2+. Distinct binding sites and binding motifs could be localized based on the patterns of Cd2+-binding. The number of binding sites was consistent with previous findings for recombinant protein. Positions of binding sites appeared to be conserved even among diverse phytochelatin synthases. Mutant peptide analysis was used to assess the contribution of exemplary amino acids to binding. Several binding motifs contain cysteines or glutamates. For cysteines a strong correlation was found between binding activity and degree of conservation among known phytochelatin synthases. These findings indicate the suitability of peptide scanning for the identification of metal-binding sites. The functional role of several cysteines was investigated by expression of hemagglutinin-tagged phytochelatin synthases in phytochelatin synthase-deficient, Cd2+-hypersensitive Schizosaccharomyces pombe cells. The data are consistent with a model suggesting functionally essential metal-binding activation sites in the N-terminal catalytic part of phytochelatin synthases and additional binding sites at the C-terminus not essential for activity. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2089 TI - Mitogen-activated Protein Kinases Play an Essential Role in Oxidative Burst-independent Expression of Pathogenesis-related Genes in Parsley JO - J. Biol. Chem. PY - 2003 SP - 2256-2264 AU - Kroj, T. AU - Rudd, J. J. AU - Nürnberger, T. AU - Gäbler, Y. AU - Lee, J. AU - Scheel, D. AU - VL - 278 UR - DO - 10.1074/jbc.M208200200 AB - Plants are continuously exposed to attack by potential phytopathogens. Disease prevention requires pathogen recognition and the induction of a multifaceted defense response. We are studying the non-host disease resistance response of parsley to the oomycete, Phytophthora sojae using a cell culture-based system. Receptor-mediated recognition of P. sojae may be achieved through a thirteen amino acid peptide sequence (Pep-13) present within an abundant cell wall transglutaminase. Following recognition of this elicitor molecule, parsley cells mount a defense response, which includes the generation of reactive oxygen species (ROS) and transcriptional activation of genes encoding pathogenesis-related (PR) proteins or enzymes involved in the synthesis of antimicrobial phytoalexins. Treatment of parsley cells with the NADPH oxidase inhibitor, diphenylene iodonium (DPI), blocked both Pep-13-induced phytoalexin production and the accumulation of transcripts encoding enzymes involved in their synthesis. In contrast, DPI treatment had no effect upon Pep-13-induced PRgene expression, suggesting the existence of an oxidative burst-independent mechanism for the transcriptional activation ofPR genes. The use of specific antibodies enabled the identification of three parsley mitogen-activated protein kinases (MAPKs) that are activated within the signal transduction pathway(s) triggered following recognition of Pep-13. Other environmental challenges failed to activate these kinases in parsley cells, suggesting that their activation plays a key role in defense signal transduction. Moreover, by making use of a protoplast co-transfection system overexpressing wild-type and loss-of-function MAPK mutants, we show an essential role for post-translational phosphorylation and activation of MAPKs for oxidative burst-independentPR promoter activation. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2184 TI - NHL25 and NHL3, Two NDR1/HIN1-Like Genes in Arabidopsis thaliana with Potential Role(s) in Plant Defense JO - Mol. Plant Microbe Interact. PY - 2002 SP - 608-616 AU - Varet, A. AU - Parker, J. AU - Tornero, P. AU - Nass, N. AU - Nürnberger, T. AU - Dangl, J. L. AU - Scheel, D. AU - Lee, J. AU - VL - 15 UR - DO - 10.1094/MPMI.2002.15.6.608 AB - The Arabidopsis genome contains 28 genes with sequence homology to the Arabidopsis NDR1 gene and the tobacco HIN1 gene. Expression analysis of eight of these genes identified two (NHL25 and NHL3 for NDR1/HIN1-like) that show pathogen-dependent mRNA accumulation. Transcripts did not accumulate during infection with virulent Pseudomonas syringae pv. tomato DC3000 but did accumulate specifically when the bacteria carried any of the four avirulence genes avrRpm1, avrRpt2, avrB, or avrRps4. Furthermore, expression of avrRpt2 in plants containing the corresponding resistance gene, RPS2, was sufficient to induce transcript accumulation. However, during infection with an avirulent oomycete, Peronospora parasitica isolate Cala-2, only NHL25 expression was reproducibly induced. Salicylic acid (SA) treatment can induce expression of NHL25 and NHL3. Studies performed on nahG plants showed that, during interaction with avirulent bacteria, only the expression of NHL25 but not that of NHL3 was affected. This suggests involvement of separate SA-dependent and SA-independent pathways, respectively, in the transcriptional activation of these genes. Bacteria-induced gene expression was not abolished in ethylene- (etr1-3 and ein2-1) and jasmonate- (coi1-1) insensitive mutants or in mutants impaired in disease resistance (ndr1-1 and pad4-1). Interestingly, NHL3 transcripts accumulated after infiltration with the avirulent hrcC mutant of Pseudomonas syringae pv. tomato DC3000 and nonhost bacteria but not with the virulent Pseudomonas syringae pv. tomato DC3000, suggesting that virulent bacteria may suppress NHL3 expression during pathogenesis. Hence, the expression patterns and sequence homology to NDR1 and HIN1 suggest one or more potential roles for these genes in plant resistance. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2183 TI - A Dual Role for Microbial Pathogen-Derived Effector Proteins in Plant Disease and Resistance JO - Crit. Rev. Plant Sci. PY - 2002 SP - 229-271 AU - van't Slot, K. A. E. AU - Knogge, W. AU - VL - 21 UR - DO - 10.1080/0735-260291044223 AB - Many proteins from plant pathogens affecting the interaction with the host plant have dual functions: they promote virulence on the host species and they function as avirulence determinants by eliciting defense reactions in host cultivars expressing the appropriate resistance genes. In viruses all proteins encoded by the small genomes can be expected to be essential for viral development in the host. However, in different plants surveillance systems have evolved that are able to recognize most of these proteins. Bacteria and fungi have specialized pathogenicity and virulence genes. Many of the latter were originally identified through the resistance gene-dependent elicitor activity of their products. Their role in virulence only became apparent when they were inactivated or transferred to different microbes or after their ectopic expression in host plants. Many microbes appear to maintain these genes despite their disadvantageous effect, introducing only few mutations to abolish the interaction of their products with the plant recognition system. This has been interpreted as been indicative of a virulence function of the gene products that is not impaired by the mutations. Alternatively, in particular in bacteria there is now evidence that pathogenicity was acquired through horizontal gene transfer. Genes supporting virulence in the donor organism's original host appear to have traveled along. Being gratuitous in the new situation, they may have been inactivated without loss of any beneficial function for the pathogen. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2174 TI - A Pathogen-Responsive cDNA from Potato Encodes a Protein with Homology to a Phosphate Starvation-Induced Phosphatase JO - Plant Cell Physiol. PY - 2002 SP - 1049-1053 AU - Petters, J. AU - Göbel, C. AU - Scheel, D. AU - Rosahl, S. AU - VL - 43 UR - DO - 10.1093/pcp/pcf117 AB - Infiltration of potato leaves with the phytopathogenic bacteria Pseudomonas syringae pv. maculicola induces local and systemic defense gene expression as well as increased resistance against subsequent pathogen attacks. By cDNA-AFLP a gene was identified that is activated locally in potato leaves in response to bacterial infiltration and after infection with Phytophthora infestans, the causal agent of late blight disease. The encoded protein has high homology to a phosphate starvation-induced acid phosphatase from tomato. Possibly, decreased phosphate availability after pathogen infection acts as a signal for the activation of the potato phosphatase gene. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2170 TI - Calcium-dependent protein kinases: versatile plant signalling components necessary for pathogen defence JO - Trends Plant Sci. PY - 2002 SP - 97-98 AU - Lee, J. AU - Rudd, J. J. AU - VL - 7 UR - DO - 10.1016/S1360-1385(02)02229-X AB - Plant stress adaptation often uses changes in cytosolic Ca2+ to bring about responses via changing the activity of Ca2+-sensor proteins including Ca2+-dependent protein kinases (CDPK). The activity of a tobacco CDPK(s) is essential for elicitation of the hypersensitive reaction, a typical plant defence response. Moreover, it is becoming apparent that CDPKs might also facilitate cross-talk between different Ca2+-mediated stress signalling pathways. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2167 TI - Accumulation of tyrosol glucoside in transgenic potato plants expressing a parsley tyrosine decarboxylase JO - Phytochemistry PY - 2002 SP - 683-689 AU - Landtag, J. AU - Baumert, A. AU - Degenkolb, T. AU - Schmidt, J. AU - Wray, V. AU - Scheel, D. AU - Strack, D. AU - Rosahl, S. AU - VL - 60 UR - DO - 10.1016/S0031-9422(02)00161-9 AB - As part of the response to pathogen infection, potato plants accumulate soluble and cell wall-bound phenolics such as hydroxycinnamic acid tyramine amides. Since incorporation of these compounds into the cell wall leads to a fortified barrier against pathogens, raising the amounts of hydroxycinnamic acid tyramine amides might positively affect the resistance response. To this end, we set out to increase the amount of tyramine, one of the substrates of the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction, by placing a cDNA encoding a pathogen-induced tyrosine decarboxylase from parsley under the control of the 35S promoter and introducing the construct into potato plants via Agrobacterium tumefaciens-mediated transformation. While no alterations were observed in the pattern and quantity of cell wall-bound phenolic compounds in transgenic plants, the soluble fraction contained several new compounds. The major one was isolated and identified as tyrosol glucoside by liquid chromatography–electrospray ionization–high resolution mass spectrometry and NMR analyses. Our results indicate that expression of a tyrosine decarboxylase in potato does not channel tyramine into the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction but rather unexpectedly, into a different pathway leading to the formation of a potential storage compound.Expression of a parsley tyrosine decarboxylase in potato unexpectedly channels tyramine into a pathway leading to the formation of tyrosol glucoside. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER - TY - JOUR ID - 2166 TI - Systemic Accumulation of 12-oxo-phytodienoic Acid in SAR-induced Potato Plants JO - Eur. J. Plant Pathol. PY - 2002 SP - 279-283 AU - Landgraf, P. AU - Feussner, I. AU - Hunger, A. AU - Scheel, D. AU - Rosahl, S. AU - VL - 108 UR - DO - 10.1023/A:1015132615650 AB - In potato plants induced for systemic resistance by infiltration with Pseudomonas syringae pv. maculicola, 12-oxo-phytodienoic acid (OPDA) accumulated in infiltrated leaves as well as in non-treated leaves of infected plants. In contrast, jasmonic acid (JA) levels increased only in infiltrated leaves, suggesting that the biosynthetic precursor of JA, OPDA, might play a role in systemic acquired resistance. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2163 TI - Mitogen-activated protein kinase cascades in plants: a new nomenclature JO - Trends Plant Sci. PY - 2002 SP - 301-308 AU - Ichimura, K. AU - Shinozaki, K. AU - Tena, G. AU - Sheen, J. AU - Henry, Y. AU - Champion, A. AU - Kreis, M. AU - Zhang, S. AU - Hirt, H. AU - Wilson, C. AU - Heberle-Bors, E. AU - Ellis, B. E. AU - Morris, P. C. AU - Innes, R. W. AU - Ecker, J. R. AU - Scheel, D. AU - Klessig, D. F. AU - Machida, Y. AU - Mundy, J. AU - Ohashi, Y. AU - Walker, J. C. AU - VL - 7 UR - DO - 10.1016/S1360-1385(02)02302-6 AB - Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes, including yeasts, animals and plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. In plants, MAPK cascades are involved in responses to various biotic and abiotic stresses, hormones, cell division and developmental processes. Completion of the Arabidopsis genome-sequencing project has revealed the existence of 20 MAPKs, 10 MAPK kinases and 60 MAPK kinase kinases. Here, we propose a simplified nomenclature for Arabidopsis MAPKs and MAPK kinases that might also serve as a basis for standard annotation of these gene families in all plants. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2155 TI - Oxylipin profiling in pathogen-infected potato leaves JO - BBA-Mol. Cell Biol. Lipids PY - 2002 SP - 55-64 AU - Göbel, C. AU - Feussner, I. AU - Hamberg, M. AU - Rosahl, S. AU - VL - 1584 UR - DO - 10.1016/S1388-1981(02)00268-8 AB - Plants respond to pathogen attack with a multicomponent defense response. Synthesis of oxylipins via the lipoxygenase (LOX) pathway appears to be an important factor for establishment of resistance in a number of pathosystems. In potato cells, pathogen-derived elicitors preferentially stimulate the 9-LOX-dependent metabolism of polyunsaturated fatty acids (PUFAs). Here we show by oxylipin profiling that potato plants react to pathogen infection with increases in the amounts of the 9-LOX-derived 9,10,11- and 9,12,13-trihydroxy derivatives of linolenic acid (LnA), the divinyl ethers colnelenic acid (CnA) and colneleic acid (CA) as well as 9-hydroxy linolenic acid. Accumulation of these compounds is faster and more pronounced during the interaction of potato with the phytopathogenic bacterium Pseudomonas syringae pv. maculicola, which does not lead to disease, compared to the infection of potato with Phytophthora infestans, the causal agent of late blight disease. Jasmonic acid (JA), a 13-LOX-derived oxylipin, accumulates in potato leaves after infiltration with P. syringae pv. maculicola, but not after infection with P. infestans. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2151 TI - Amino acid alterations in isoforms of the effector protein NIP1 from Rhynchosporium secalis have similar effects on its avirulence- and virulence-associated activities on barley JO - Physiol. Mol. Plant Pathol. PY - 2002 SP - 299-302 AU - Fiegen, M. AU - Knogge, W. AU - VL - 61 UR - DO - 10.1006/pmpp.2002.0442 AB - The secreted effector protein NIP1 from the barley pathogen Rhynchosporium secalis is a specific elicitor of defense reactions in host plants carrying the resistance gene Rrs1. In addition, it has activities associated with fungal virulence; independent of the plant genotype it stimulates the plant plasma membrane H+-ATPase and induces leaf necrosis. Four NIP1 isoforms differing in single amino acid residues were isolated from various naturally occurring fungal strains. All three activities of the protein (race specificity, H+-ATPase stimulation, necrosis induction) were affected by the amino acid alterations in a similar way suggesting that they are mediated through a single plant receptor. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2143 TI - A Transporter in the Endoplasmic Reticulum of Schizosaccharomyces pombe Cells Mediates Zinc Storage and Differentially Affects Transition Metal Tolerance JO - J. Biol. Chem. PY - 2002 SP - 18215-18221 AU - Clemens, S. AU - Bloss, T. AU - Vess, C. AU - Neumann, D. AU - Nies, D. H. AU - zur Nieden, U. AU - VL - 277 UR - DO - 10.1074/jbc.M201031200 AB - The cation diffusion facilitator (CDF) family represents a class of ubiquitous metal transporters. Inactivation of a CDF in Schizosaccharomyces pombe, Zhf, causes drastically different effects on the tolerance toward various metals. A deletion mutant is Zn2+/Co2+-hypersensitive yet displays significantly enhanced Cd2+ and Ni2+ tolerance. Accumulation of zinc, cobalt, and cadmium is reduced in mutant cells. Non-vacuolar zinc content, as measured by analytical electron microscopy, is lower in zhf− cells compared with wild-type cells in the presence of elevated Zn2+concentrations. The protective effect against cadmium toxicity is independent of the phytochelatin detoxification pathway. Phytochelatin synthase-deficient cells show extremely enhanced (about 200-fold) cadmium tolerance when zhf is disrupted. Immunogold labeling indicates endoplasmic reticulum (ER) localization of Zhf. Electron spectroscopic imaging shows that accumulation of zinc coincides with Zhf localization, demonstrating a major role of the ER for metal storage and the involvement of Zhf in cellular zinc homeostasis. Also, these observations indicate that Cd2+ions exert their toxic effects on cellular metabolism in the ER rather than in the cytosol. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2142 TI - A long way ahead: understanding and engineering plant metal accumulation JO - Trends Plant Sci. PY - 2002 SP - 309-315 AU - Clemens, S. AU - Palmgren, M. G. AU - Krämer, U. AU - VL - 7 UR - DO - 10.1016/S1360-1385(02)02295-1 AB - Some plants can hyperaccumulate metal ions that are toxic to virtually all other organisms at low dosages. This trait could be used to clean up metal-contaminated soils. Moreover, the accumulation of heavy metals by plants determines both the micronutrient content and the toxic metal content of our food. Complex interactions of transport and chelating activities control the rates of metal uptake and storage. In recent years, several key steps have been identified at the molecular level, enabling us to initiate transgenic approaches to engineer the transition metal content of plants. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2139 TI - Pep-13, a plant defense-inducing pathogen-associated pattern from Phytophthora transglutaminases JO - EMBO J. PY - 2002 SP - 6681-6688 AU - Brunner, F. AU - Rosahl, S. AU - Lee, J. AU - Rudd, J. J. AU - Geiler, C. AU - Kauppinen, S. AU - Rasmussen, G. AU - Scheel, D. AU - Nürnberger, T. AU - VL - 21 UR - DO - 10.1093/emboj/cdf667 AB - Innate immunity, an ancient form of defense against microbial infection, is well described for animals and is also suggested to be important for plants. Discrimination from self is achieved through receptors that recognize pathogen‐associated molecular patterns (PAMPs) not found in the host. PAMPs are evolutionarily conserved structures which are functionally important and, thus, not subject to frequent mutation. Here we report that the previously described peptide elicitor of defense responses in parsley, Pep‐13, constitutes a surface‐exposed fragment within a novel calcium‐dependent cell wall transglutaminase (TGase) from Phytophthora sojae . TGase transcripts and TGase activity are detectable in all Phytophthora species analyzed, among which are some of the most destructive plant pathogens. Mutational analysis within Pep‐13 identified the same amino acids indispensable for both TGase and defense‐eliciting activity. Pep‐13, conserved among Phytophthora TGases, activates defense in parsley and potato, suggesting its function as a genus‐specific recognition determinant for the activation of plant defense in host and non‐host plants. In summary, plants may recognize PAMPs with characteristics resembling those known to trigger innate immune responses in animals. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2138 TI - A β-glucosidase/xylosidase from the phytopathogenic oomycete, Phytophthora infestans JO - Phytochemistry PY - 2002 SP - 689-696 AU - Brunner, F. AU - Wirtz, W. AU - Rose, J. K. C. AU - Darvill, A. G. AU - Govers, F. AU - Scheel, D. AU - Nürnberger, T. AU - VL - 59 UR - DO - 10.1016/S0031-9422(02)00045-6 AB - An 85-kDa β-glucosidase/xylosidase (BGX1) was purified from the axenically grown phytopathogenic oomycete, Phytophthora infestans. The bgx1 gene encodes a predicted 61-kDa protein product which, upon removal of a 21 amino acid leader peptide, accumulates in the apoplastic space. Extensive N-mannosylation accounts for part of the observed molecular mass difference. BGX1 belongs to family 30 of the glycoside hydrolases and is the first such oomycete enzyme deposited in public databases. The bgx1 gene was found in various Phytophthora species, but is apparently absent in species of the related genus, Pythium. Despite significant sequence similarity to human and murine lysosomal glucosylceramidases, BGX1 demonstrated neither glucocerebroside nor galactocerebroside-hydrolyzing activity. The native enzyme exhibited glucohydrolytic activity towards 4-methylumbelliferyl (4-MU) β-d-glucopyranoside and, to lesser extent, towards 4-MU-d-xylopyranoside, but not towards 4-MU-β-d-glucopyranoside. BGX1 did not hydrolyze carboxymethyl cellulose, cellotetraose, chitosan or xylan, suggesting high substrate specificity and/or specific cofactor requirements for enzymatic activity.A β-glucosidase/xylosidase was purified from the phytopathogenic oomycete, Phytophthora infestans. The encoding gene is the first such sequence reported from a species of the kingdom chromista. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2133 TI - Characterization of the ZAT1p zinc transporter from Arabidopsis thaliana in microbial model organisms and reconstituted proteoliposomes JO - Planta PY - 2002 SP - 783-791 AU - Bloß, T. AU - Clemens, S. AU - Nies, D. H. AU - VL - 214 UR - DO - 10.1007/s00425-001-0677-1 AB - The ZAT1p zinc transporter from Arabidopsis thaliana (L.) Heynh. is a member of the cation diffusion facilitator (CDF) protein family. When heterologously expressed in Escherichia coli, ZAT1p bound zinc in a metal blot. Binding of zinc occurred mainly to the hydrophilic amino acid region from H182 to H232. A ZAT1p/ZAT1p*Δ(M1–I25) protein mixture was purified and reconstituted into proteoliposomes. Uptake of zinc into the proteoliposomes did not require a proton gradient across the liposomal membrane. ZAT1p did not transport cobalt, and transported cadmium at only 1% of the zinc transport rate. ZAT1p functioned as an uptake system for 65Zn2+ in two strains of the Gram-negative bacterium Ralstonia metallidurans, which were different in their content of zinc-efflux systems. The ZAT1 gene did not rescue increased zinc sensitivity of a ΔZRC1 single-mutant strain or of a ΔZRC1 ΔCOT1 double-mutant strain of Saccharomyces cerevisiae, but ZAT1 complemented this phenotype in a ΔSpZRC1 mutant strain of Schizosaccharomyces pombe. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2232 TI - A pathogen-inducible divinyl ether synthase (CYP74D) from elicitor-treated potato suspension cells JO - FEBS Lett. PY - 2001 SP - 371-376 AU - Stumpe, M. AU - Kandzia, R. AU - Göbel, C. AU - Rosahl, S. AU - Feussner, I. AU - VL - 507 UR - DO - 10.1016/S0014-5793(01)03019-8 AB - In elicitor-treated potato cells, 9-lipoxygenase-derived oxylipins accumulate with the divinyl ether colneleic acid as the major metabolite. Here, the identification of a potato cDNA is described, whose predicted amino acid sequence corresponds to divinyl ether synthases, belonging to the recently identified new P450 subfamily CYP74D. The recombinant protein was expressed in Escherichia coli and shown to metabolize 9-hydroperoxy linoleic acid to colneleic acid at pH 6.5. This fatty acid derivative has been implicated in functioning as a plant antimicrobial compound. RNA blot analyses revealed accumulation of divinyl ether synthase transcripts both upon infiltration of potato leaves with Pseudomonas syringae and after infection with Phytophthora infestans. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2225 TI - Sterol glycosides and cerebrosides accumulate in Pichia pastoris, Rhynchosporium secalis and other fungi under normal conditions or under heat shock and ethanol stress JO - Yeast PY - 2001 SP - 679-695 AU - Sakaki, T. AU - Zähringer, U. AU - Warnecke, D. C. AU - Fahl, A. AU - Knogge, W. AU - Heinz, E. AU - VL - 18 UR - DO - 10.1002/yea.720 AB - The occurrence of glycolipids such as sterol glycosides, acylated sterol glycosides, cerebrosides and glycosyldiacylglycerols was examined in the three yeast species Candida albicans , Pichia pastoris and Pichia anomala , as well as in the six fungal species Sordaria macrospora , Pyrenophora teres, Ustilago maydis , Acremonium chrysogenum , Penicillium olsonii and Rhynchosporium secalis . Cerebroside was found in all organisms tested, whereas acylated sterol glycosides and glycosyldiacylglycerols were not found in any organism. Sterol glycosides were detected in P. pastoris strain GS115, U. maydis , S. macrospora and R. secalis. This glycolipid occurred in both yeast and filamentous forms of U. maydis but in neither form of C. albicans. This suggests that sterol glycoside is not correlated with the separately grown dimorphic forms of these organisms. Cerebrosides and sterol glycosides from P. pastoris and R. secalis were purified and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The cerebrosides are β‐glucosyl ceramides consisting of a saturated α‐hydroxy or non‐hydroxy fatty acid and a Δ4,8‐diunsaturated, C9‐methyl‐branched sphingobase. Sterol glycoside from P. pastoris was identified as ergosterol‐β‐D ‐glucopyranoside, whereas the sterol glucosides from R. secalis contain two derivatives of ergosterol. The biosynthesis of sterol glucoside in P. pastoris CBS7435 and GS115 depended on the culture conditions. The amount of sterol glucoside in cells grown in complete medium was much lower than in cells from minimal medium and a strong increase in the content of sterol glucoside was observed when cells were subjected to stress conditions such as heat shock or increased ethanol concentrations. From these data we suggest that, in addition to Saccharomyces cerevisiae , new yeast and fungal model organisms should be used to study the physiological functions of glycolipids in eukaryotic cells. This suggestion is based on the ubiquitous and frequent occurrence of cerebrosides and sterol glycosides, both of which are rarely detected in S. cerevisiae . We suggest P. pastoris and two plant pathogenic fungi to be selected for this approach. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2224 TI - Signal transmission in the plant immune response JO - Trends Plant Sci. PY - 2001 SP - 372-379 AU - Nürnberger, T. AU - Scheel, D. AU - VL - 6 UR - DO - 10.1016/S1360-1385(01)02019-2 AB - Genetic and biochemical dissection of signaling pathways regulating plant pathogen defense has revealed remarkable similarities with the innate immune system of mammals and Drosophila. Numerous plant proteins resembling eukaryotic receptors have been implicated in the perception of pathogen-derived signal molecules. Receptor-mediated changes in levels of free calcium in the cytoplasm and production of reactive oxygen species and nitric oxide constitute early events generally observed in plant–pathogen interactions. Positive and negative regulation of plant pathogen defense responses has been attributed to mitogen-activated protein kinase cascades. In addition, salicylic acid, jasmonic acid and ethylene are components of signaling networks that provide the molecular basis for specificity of plant defense responses. This article reviews recent advances in our understanding of early signaling events involved in the establishment of plant disease resistance. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2223 TI - Enhanced luciferin entry causes rapid wound-induced light emission in plants expressing high levels of luciferase JO - Planta PY - 2001 SP - 149-154 AU - Nass, N. AU - Scheel, D. AU - VL - 212 UR - DO - 10.1007/s004250000389 AB - In-vivo imaging of transgenic tobacco plants (Nicotiana tobacum L.) expressing firefly luciferase under the control of the Arabidopsis phenylalanine ammonia-lyase 1 (PAL1)-promoter showed that luciferase-catalyzed light emission began immediately after the substrate luciferin was sprayed onto the leaves and reached a plateau phase after approximately 60 min. This luminescence could easily be detected for up to 24 h after luciferin application although the light intensity declined continuously during this period. A strong and rapid increase in light emission was observed within the first minutes after wounding of luciferin-sprayed leaves. However, these data did not correlate with luciferase activity analysed by an in-vitro enzyme assay. In addition, Arabidopsis plants expressing luciferase under the control of the constitutive 35S-promoter showed similar wound-induced light emission. In experiments in which only parts of the leaves were sprayed with luciferin solutions, it was shown that increased uptake of luciferin at the wound site and its transport through vascular tissue were the main reasons for the rapid burst of light produced by preformed luciferase activity. These data demonstrate that there are barriers that restrict luciferin entry into adult plants, and that luciferin availability can be a limiting factor in non-invasive luciferase assays. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2211 TI - Oxylipin Profiling Reveals the Preferential Stimulation of the 9-Lipoxygenase Pathway in Elicitor-treated Potato Cells JO - J. Biol. Chem. PY - 2001 SP - 6267-6273 AU - Göbel, C. AU - Feussner, I. AU - Schmidt, A. AU - Scheel, D. AU - Sanchez-Serrano, J. AU - Hamberg, M. AU - Rosahl, S. AU - VL - 276 UR - DO - 10.1074/jbc.M008606200 AB - Lipoxygenases are key enzymes in the synthesis of oxylipins and play an important role in the response of plants to wounding and pathogen attack. In cultured potato cells treated with elicitor from Phytophthora infestans, the causal agent of late blight disease, transcripts encoding a linoleate 9-lipoxygenase and a linoleate 13-lipoxygenase accumulate. However, lipoxygenase activity assays and oxylipin profiling revealed only increased 9-lipoxygenase activity and formation of products derived therefrom, such as 9-hydroxy octadecadienoic acid and colneleic acid. Furthermore, the 9-lipoxygenase products 9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoic and 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid were identified as novel, elicitor-inducible oxylipins in potato, suggesting a role of these compounds in the defense response against pathogen attack. Neither 13-lipoxygenase activity nor 13-lipoxygenase products were detected in higher amounts in potato cells after elicitation. Thus, formation of products by the 9-lipoxygenase pathway, including the enzymes hydroperoxide reductase, divinyl ether synthase, and epoxy alcohol synthase, is preferentially stimulated in cultured potato cells in response to treatment with P. infestanselicitor. Moreover, elicitor-induced accumulation of desaturase transcripts and increased phospholipase A2 activity after elicitor treatment suggest that substrates for the lipoxygenase pathway might be provided by de novo synthesis and subsequent release from lipids of the endomembrane system. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2204 TI - Molecular mechanisms of plant metal tolerance and homeostasis JO - Planta PY - 2001 SP - 475-486 AU - Clemens, S. AU - VL - 212 UR - DO - 10.1007/s004250000458 AB - Transition metals such as copper are essential for many physiological processes yet can be toxic at elevated levels. Other metals (e.g. lead) are nonessential and potentially highly toxic. Plants – like all other organisms – possess homeostatic mechanisms to maintain the correct concentrations of essential metal ions in different cellular compartments and to minimize the damage from exposure to nonessential metal ions. A regulated network of metal transport, chelation, trafficking and sequestration activities functions to provide the uptake, distribution and detoxification of metal ions. Some of the components of this network have now been identified: a number of uptake transporters have been cloned as well as candidate transporters for the vacuolar sequestration of metals. Chelators and chaperones are known, and evidence for intracellular metal trafficking is emerging. This recent progress in the molecular understanding of plant metal homeostasis and tolerance is reviewed. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2203 TI - Developing tools for phytoremediation:towards a molecular understanding of plant metal tolerance and accumulation JO - Int. J. Occup. Med. Environ. Health PY - 2001 SP - 235-239 AU - Clemens, S. AU - VL - 14 UR - http://www.imp.lodz.pl/home_en/publishing_office/journals_/_ijomeh/&articleId=18086&l=EN AB - Certain plant species and genotypes are able to accumulate large quantities of heavy metals in their shoots. Based on this trait the concept of phytoremediation was developed, i.e. the use of metal hyperaccumulating plants for the cleansing of contaminated soils and water. In order to more efficiently use this capacity, an engineering of plants might be needed. However, very little is known about the underlying molecular mechanisms. Our work is focussing on the identification and characterization of plant genes involved in plant metal uptake, tolerance and accumulation. Phytochelatins are small glutathione-derived metal-binding peptides which are part of the plant metal detoxification system. Genes encoding phytochelatin synthases have been cloned and are now being studied with regard to their regulation, biochemistry and biotechnological potential. Another project aimes at the dissection of metal responses in the metallophyte Arabidopsis halleri. This plant, a close relative to the model plant Arabidopsis thaliana, is Cd hypertolerant and Zn hyperaccumulating. It grows,for instance, on medieval mining sites in the Harz mountains in Germany and in many other metal-contaminated sites in Central Europe. We have isolated metal-regulated genes from A.halleri and molecularly analyzed interesting candidate genes with regard to their function and involvement in metal accumulation and tolerance. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2202 TI - Caenorhabditis elegans expresses a functional phytochelatin synthase JO - Eur. J. Biochem. PY - 2001 SP - 3640-3643 AU - Clemens, S. AU - Schroeder, J. I. AU - Degenkolb, T. AU - VL - 268 UR - DO - 10.1046/j.1432-1327.2001.02293.x AB - The formation of phytochelatins, small metal‐binding glutathione‐derived peptides, is one of the well‐studied responses of plants to toxic metal exposure. Phytochelatins have also been detected in some fungi and some marine diatoms. Genes encoding phytochelatin synthases (PCS) have recently been cloned from Arabidopsis , wheat and Schizosaccharomyces pombe . Surprisingly, database searches revealed the presence of a homologous gene in the Caenorhabditis elegans genome, DDBJ/EMBL/GenBank accession no. 266513. Here we show that C. elegans indeed expresses a gene coding for a functional phytochelatin synthase. CePCS complements the Cd2+ sensitivity of a Schizosaccharomyces pombe PCS knock‐out strain and confers phytochelatin synthase activity to these cells. Thus, phytochelatins may play a role for metal homeostasis also in certain animals. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2201 TI - Arabidopsis thaliana expresses a second functional phytochelatin synthase JO - FEBS Lett. PY - 2001 SP - 215-219 AU - Cazalé, A.-C. AU - Clemens, S. AU - VL - 507 UR - DO - 10.1016/S0014-5793(01)02976-3 AB - Phytochelatins represent a major detoxifying pathway for heavy metals in plants and many other organisms. The Arabidopsis thaliana CAD1 (=AtPCS1 ) gene encodes a phytochelatin synthase and cad1 mutants are phytochelatin deficient and cadmium hypersensitive. The Arabidopsis genome contains a highly homologous gene, AtPCS2 , of which expression and function were studied in order to understand the apparent non‐redundancy of the two genes. Low constitutive AtPCS2 expression is detected in all plant organs analyzed. The AtPCS2 gene encodes a functional phytochelatin synthase as shown by expression in Saccharomyces cerevisiae and the complementation of a Schizosaccharomyces pombe phytochelatin synthase knockout strain. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2219 TI - HrpZPsph from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro JO - Proc. Natl. Acad. Sci. U.S.A. PY - 2001 SP - 289-294 AU - Lee, J. AU - Klüsener, B. AU - Tsiamis, G. AU - Stevens, C. AU - Neyt, C. AU - Tampakaki, A. P. AU - Panopoulos, N. J. AU - Nöller, J. AU - Weiler, E. W. AU - Cornelis, G. R. AU - Mansfield, J. W. AU - Nürnberger, T. AU - VL - 98 UR - DO - 10.1073/pnas.98.1.289 AB - The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. Some hrp gene products constitute elements of the type III secretion system, by which effector proteins are exported and delivered into plant cells. Here, we show that the hrpZ gene product from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (HrpZPsph), is secreted in an hrp-dependent manner in P. syringae pv. phaseolicola and exported by the type III secretion system in the mammalian pathogen Yersinia enterocolitica. HrpZPsph was found to associate stably with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2 nM of purified recombinant HrpZPsph to the cis compartment of planar lipid bilayers provoked an ion current with a large unitary conductivity of 207 pS. HrpZPsph-related proteins from P. syringae pv. tomato or syringae triggered ion currents similar to those stimulated by HrpZPsph. The HrpZPsph-mediated ion-conducting pore was permeable for cations but did not mediate fluxes of Cl−. Such pore-forming activity may allow nutrient release and/or delivery of virulence factors during bacterial colonization of host plants. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2218 TI - A Harpin Binding Site in Tobacco Plasma Membranes Mediates Activation of the Pathogenesis-Related Gene HIN1 Independent of Extracellular Calcium but Dependent on Mitogen-Activated Protein Kinase Activity JO - Plant Cell PY - 2001 SP - 1079-1093 AU - Lee, J. AU - Klessig, D. F. AU - Nürnberger, T. AU - VL - 13 UR - DO - 10.1105/tpc.13.5.1079 AB - Harpin from the bean halo-blight pathogen Pseudomonas syringae pv phaseolicola (harpinPsph) elicits the hypersensitive response and the accumulation of pathogenesis-related gene transcripts in the nonhost plant tobacco. Here, we report the characterization of a nonproteinaceous binding site for harpinPsph in tobacco plasma membranes, which is assumed to mediate the activation of plant defense responses in a receptor-like manner. Binding of 125I-harpinPsph to tobacco microsomal membranes (dissociation constant = 425 nM) and protoplasts (dissociation constant = 380 nM) was specific, reversible, and saturable. A close correlation was found between the abilities of harpinPsph fragments to elicit the transcript accumulation of the pathogenesis-related tobacco gene HIN1 and to compete for binding of 125I-harpinPsph to its binding site. Another elicitor of the hypersensitive response and HIN1 induction in tobacco, the Phytophthora megasperma–derived β-elicitin β-megaspermin, failed to bind to the putative harpinPsph receptor. In contrast to activation by β-megaspermin, harpinPsph-induced activation of the 48-kD salicylic acid–responsive mitogen-activated protein kinase (MAPK) and HIN1 transcript accumulation were independent of extracellular calcium. Moreover, use of the MAPK kinase inhibitor U0126 revealed that MAPK activity was essential for pathogenesis-related gene expression in harpinPsph-treated tobacco cells. Thus, a receptor-mediated MAPK-dependent signaling pathway may mediate the activation of plant defense responses induced by harpinPsph. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2274 TI - Parasitismus im Pflanzenreich JO - Nova Acta Leopoldina PY - 2000 SP - 25-31 AU - Scheel, D. AU - VL - 316 UR - AB - A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2269 TI - Lipoxygenase isoforms in elicitor-treated parsley cell suspension cultures JO - Biochem. Soc. Trans. PY - 2000 SP - 827-829 AU - Noehringer, C. AU - Scheel, D. AU - Blée, E. AU - VL - 28 UR - DO - 10.1042/bst0280827 AB - Treatment of parsley cell cultures with a fungal elicitor triggered the induction of a lipoxygenase isoform which may be involved in the de novo synthesis of defence-response inducers, such as jasmonic acid or 12-oxo-phytodienoic acid. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2253 TI - Creating lipoxygenases with new positional specificities by site-directed mutagenesis JO - Biochem. Soc. Trans. PY - 2000 SP - 825-826 AU - Hornung, E. AU - Rosahl, S. AU - Kühn, H. AU - Feussner, I. AU - VL - 28 UR - DO - 10.1042/bst0280825 AB - In order to analyse the amino acid determinants which alter the positional specificity of plant lipoxygenases (LOXs), multiple LOX sequence alignments and structural modelling of the enzyme-substrate interactions were carried out. These alignments suggested three amino acid residues as the primary determinants of positional specificity. Here we show the generation of two plant LOXs with new positional specificities, a Δ-linoleneate 6-LOX and an arachidonate 11-LOX, by altering only one of these determinants within the active site of two plant LOXs. In the past, site-directed-mutagenesis studies have mainly been carried out with mammalian lipoxygenases (LOXs) [1]. In these experiments two regions have been identified in the primary structure containing sequence determinants for positional specificity. Amino acids aligning with the Sloane determinants [2] are highly conserved among plant LOXs. In contrast, there is amino acid hetero-geneity among plant LOXs at the position that aligns with P353 of the rabbit reticulocyte 15-LOX (Borngräber determinants) [3]. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2243 TI - Receptor-Mediated Increase in Cytoplasmic Free Calcium Required for Activation of Pathogen Defense in Parsley JO - Plant Cell PY - 2000 SP - 1425-1440 AU - Blume, B. AU - Nürnberger, T. AU - Nass, N. AU - Scheel, D. AU - VL - 12 UR - DO - 10.1105/tpc.12.8.1425 AB - Transient influx of Ca2+ constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae–derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca2+]cyt), which peaked at ∼1 μM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca2+]cyt signature was achieved by elicitor concentrations sufficient to stimulate Ca2+ influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca2+]cyt but not the rapidly induced [Ca2+]cyt transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca2+ influx across the plasma membrane or of Ca2+ release from internal stores suggests that the elicitor-induced sustained increase of [Ca2+]cyt predominantly results from the influx of extracellular Ca2+. Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca2+]cyt, and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca2+]cyt is causally involved in signaling the activation of pathogen defense in parsley. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2238 TI - Induction of an Extracellular Cyclic Nucleotide Phosphodiesterase as an Accessory Ribonucleolytic Activity during Phosphate Starvation of Cultured Tomato Cells JO - Plant Physiol. PY - 2000 SP - 543-552 AU - Abel, S. AU - Nürnberger, T. AU - Ahnert, V. AU - Krauss, G.-J. AU - Glund, K. AU - VL - 122 UR - DO - 10.1104/pp.122.2.543 AB - During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2′:3′-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970–976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2′:3′-cyclic NMP to 3′-NMP and the 3′:5′-cyclic isomers to a mixture of 3′-NMP and 5′-NMP. Specific activities of the enzyme are 2-fold higher for 2′:3′-cyclic NMP than for 3′:5′-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared −Pi culture medium as a source of 3′-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates. A2 - C1 - Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 2335 TI - Metal Stress Response and Tolerance of Cultured Cells from Silene vulgaris and Lycopersicon peruvianum: Role of Heat Stress Proteins JO - J. Plant Physiol. PY - 1999 SP - 547-553 AU - Wollgiehn, R. AU - Neumann, D. AU - VL - 154 UR - DO - 10.1016/S0176-1617(99)80296-X AB - The influence of the heavy metal ions Hg++, Cu++, Cd++ and Zn++ and of arsenite on growth, amino acid uptake, protein- and heat shock protein synthesis was investigated in cell cultures of a heavy metal tolerant Silene vulgaris and the sensitive Lycopersicon peruvianum.A distinct tolerance of Silene cell growth in comparison to tomato cells against Cu, Cd and Zn was observed. Synthesis of the small heat stress proteins was induced in both species, however, with quantitative differences depending on species and metal ion. While in tomato cells all metals induce HSP synthesis, in Silene the induction by Cu and Cd was found to be significantly lower in comparison to Hg and arsenite, and Zn did not induce HSP. Therefore, generally a lower tolerance of the cells against a metal is connected with a higher HSP synthesis. From comparison of cell growth and HSP accumulation in the presence of metal ions it was further concluded that HSP synthesis is a part of HM stress response of tolerant and non tolerant cells as under heat shock, but HSPs are not responsible for the heritable metal tolerance of Silene.In contrast to heat shock, metal stress does not inhibit the cell protein synthesis directly. In cultured tomato and Silene cells the inhibition of protein synthesis under metal stress was found to be a consequence of the inhibition of amino acid uptake. Zn has no effect on amino acid uptake of Silene cells. It is concluded that only Zn tolerance of Silene seems to be related with membrane stability. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2332 TI - Phenylpropanoids in mycorrhizas of the Pinaceae JO - Planta PY - 1999 SP - 491-502 AU - Weiss, M. AU - Schmidt, J. AU - Neumann, D. AU - Wray, V. AU - Christ, R. AU - Strack, D. AU - VL - 208 UR - DO - 10.1007/s004250050586 AB - Tissue-specific accumulation of phenylpropanoids was studied in mycorrhizas of the conifers, silver fir (Abies alba Mill.), Norway spruce [Picea abies (L.) Karst.], white pine (Pinus strobus L.), Scots pine (Pinus silvestris L.), and Douglas fir [Pseudotsuga menziesii (Mirbel) Franco], using high-performance liquid chromatography and histochemical methods. The compounds identified were soluble flavanols (catechin and epicatechin), proanthocyanidins (mainly dimeric catechins and/or epicatechins), stilbene glucosides (astringin and isorhapontin), one dihydroflavonol glucoside (taxifolin 3′-O-glucopyranoside), and a hydroxycinnamate derivative (unknown ferulate conjugate). In addition, a cell wall-bound hydroxycinnamate (ferulate) and a hydroxybenzaldehyde (vanillin) were analysed. Colonisation of the root by the fungal symbiont correlated with the distribution pattern of the above phenylpropanoids in mycorrhizas suggesting that these compounds play an essential role in restricting fungal growth. The levels of flavanols and cell wall-bound ferulate within the cortex were high in the apical part and decreased to the proximal side of the mycorrhizas. In both Douglas fir and silver fir, which allowed separation of inner and outer parts of the cortical tissues, a characteristic transversal distribution of these compounds was found: high levels in the inner non-colonised part of the cortex and low levels in the outer part where the Hartig net is formed. Restriction of fungal growth to the outer cortex may also be achieved by characteristic cell wall thickening of the inner cortex which exhibited flavanolic wall infusions in Douglas fir mycorrhizas. Long and short roots of conifers from natural stands showed similar distribution patterns of phenylpropanoids and cell wall thickening compared to the respective mycorrhizas. These results are discussed with respect to co-evolutionary adaptation of both symbiotic partners regarding root structure (anatomy) and root chemistry. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions; Cell and Metabolic Biology ER - TY - JOUR ID - 2322 TI - Cloning and Expression of a Potato cDNA Encoding Hydroxycinnamoyl-CoA:Tyramine N-(Hydroxycinnamoyl)transferase JO - J. Biol. Chem. PY - 1999 SP - 4273-4280 AU - Schmidt, A. AU - Grimm, R. AU - Schmidt, J. AU - Scheel, D. AU - Strack, D. AU - Rosahl, S. AU - VL - 274 UR - DO - 10.1074/jbc.274.7.4273 AB - Hydroxycinnamoyl-CoA:tyramineN-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) catalyzes the transfer of hydroxycinnamic acids from the respective CoA esters to tyramine and other amines in the formation ofN-(hydroxycinnamoyl)amines. Expression of THT is induced byPhytophthora infestans, the causative agent of late blight disease in potato. The amino acid sequences of nine endopeptidase LysC-liberated peptides from purified potato THT were determined. Using degenerate primers, a THT-specific fragment was obtained by reverse transcription-polymerase chain reaction, and THT cDNA clones were isolated from a library constructed from RNA of elicitor-treated potato cells. The open reading frame encoding a protein of 248 amino acids was expressed in Escherichia coli. Recombinant THT exhibited a broad substrate specificity, similar to that of native potato THT, accepting cinnamoyl-, 4-coumaroyl-, caffeoyl-, feruloyl- and sinapoyl-CoA as acyl donors and tyramine, octopamine, and noradrenalin as acceptors tested. Elicitor-induced THT transcript accumulation in cultured potato cells peaked 5 h after initiation of treatment, whereas enzyme activity was highest from 5 to 30 h after elicitation. In soil-grown potato plants, THT mRNA was most abundant in roots. Genomic Southern analyses indicate that, in potato, THT is encoded by a multigene family. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions; Cell and Metabolic Biology ER - TY - JOUR ID - 2315 TI - Accumulation of Silicon in the Monocotyledons Deschampsia caespitosa, Festuca lemanil and Schoenus nigricans JO - Plant Biol. PY - 1999 SP - 290-298 AU - Neumann, D. AU - Schwieger, W. AU - Lichtenberger, O. AU - VL - 1 UR - DO - 10.1111/j.1438-8677.1999.tb00255.x AB - Conventional and analytical electron microscopy (EDX, ESI, EELS) were used to investigate the silicon accumulation, the chemical nature of the Si deposits and their formation in three species of monocotyledons. In Deschampsia , in particular parts of the outer epidermal cell wall silicon is accumulated as silicic acid. Electron dense, needle‐shaped crystals in the vacuoles of epidermal cells and in the intercellular spaces were also identified as silicic acid. In xylem parenchyma cells, silicon is accumulated as SiO2, which is formed from Sn silicate. In Festuca , crystal‐like deposits of SiO2 occur on the epidermal surface, in the epidermal and parenchyma cell walls, and in vacuoles of bundle sheath cells. Often the deposits disturb the cell walls and penetrate the envelope of plastids and mitochondria. The crystal‐like SiO2 deposits originate from Sn silicate. In the pericarp of ripe nuts of Schoenus , no stainable cell wall components are detected. The inner part of the pericarp consists of silicic acid, while in the outer regions small clusters of silicic acid are embedded in a matrix of SiO2. The silicic acid deposits show an unusual, layered structure, typical for lepidoic silicic acids, which consist of two‐dimensional crystals lying one above the other. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2308 TI - Phytochelatins and heavy metal tolerance JO - Phytochemistry PY - 1999 SP - 1323-1328 AU - Leopold, I. AU - Günther, D. AU - Schmidt, J. AU - Neumann, D. AU - VL - 50 UR - DO - 10.1016/S0031-9422(98)00347-1 AB - The induction and heavy metal binding properties of phytochelatins in heavy metal tolerant (Silene vulgaris) and sensitive (tomato) cell cultures, in water cultures of these plants and in Silene vulgaris grown on a medieval copper mining dump were investigated. Application of heavy metals to cell suspension cultures and whole plants of Silene vulgaris and tomato induces the formation of heavy metal–phytochelatin-complexes with Cu and Cd and the binding of Zn and Pb to lower molecular weight substances. The binding of heavy metal ions to phytochelatins seems to play only a transient role in the heavy metal detoxification, because the Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Free heavy metal ions were not detectable in the extracts of all investigated plants and cell cultures. Silene vulgaris plants grown under natural conditions on a mining dump synthesize low molecular weight heavy metal binding compounds only and show no complexation of heavy metal ions to phytochelatins. The induction of phytochelatins is a general answer of higher plants to heavy metal exposition, but only some of the heavy metal ions are able to form stable complexes with phytochelatins. The investigation of tolerant plants from the copper mining dump shows that phytochelatins are not responsible for the development of the heavy metal tolerant phenotypes. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER - TY - JOUR ID - 2294 TI - Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast JO - EMBO J. PY - 1999 SP - 3325-3333 AU - Clemens, S. AU - Kim, E. J. AU - Neumann, D. AU - Schroeder, J. I. AU - VL - 18 UR - DO - 10.1093/emboj/18.12.3325 AB - Phytochelatins play major roles in metal detoxification in plants and fungi. However, genes encoding phytochelatin synthases have not yet been identified. By screening for plant genes mediating metal tolerance we identified a wheat cDNA, TaPCS1 , whose expression in Saccharomyces cerevisiae results in a dramatic increase in cadmium tolerance. TaPCS1 encodes a protein of ∼55 kDa with no similarity to proteins of known function. We identified homologs of this new gene family from Arabidopsis thaliana , Schizosaccharomyces pombe , and interestingly also Caenorhabditis elegans . The Arabidopsis and S.pombe genes were also demonstrated to confer substantial increases in metal tolerance in yeast. PCS‐expressing cells accumulate more Cd2+ than controls. PCS expression mediates Cd2+ tolerance even in yeast mutants that are either deficient in vacuolar acidification or impaired in vacuolar biogenesis. PCS‐induced metal resistance is lost upon exposure to an inhibitor of glutathione biosynthesis, a process necessary for phytochelatin formation. Schizosaccharomyces pombe cells disrupted in the PCS gene exhibit hypersensitivity to Cd2+ and Cu2+ and are unable to synthesize phytochelatins upon Cd2+ exposure as determined by HPLC analysis. Saccharomyces cerevisiae cells expressing PCS produce phytochelatins. Moreover, the recombinant purified S.pombe PCS protein displays phytochelatin synthase activity. These data demonstrate that PCS genes encode phytochelatin synthases and mediate metal detoxification in eukaryotes. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2292 TI - Heavy Metal Tolerance of Silene vulgaris JO - J. Plant Physiol. PY - 1999 SP - 536-546 AU - Bringezu, K. AU - Lichtenberger, O. AU - Leopold, I. AU - Neumann, D. AU - VL - 154 UR - DO - 10.1016/S0176-1617(99)80295-8 AB - Silene vulgaris ssp. humilis, a heavy metal tolerant plant growing on the polluted soil of a medieval copper mining dump, accumulates considerable amounts of heavy metals (HM) in its roots and shoots. The intracellular distribution of HMs in the leaves was investigated by conventional and analytical (EDX, ESI, EELS) electron microscopy. Part of the HMs, Fe, Cu, and Zn occur as crystalline compounds on the surface of the leaves. The epidermal cell walls accumulate Fe, Ni, Cu, AI, Sn, and Zn. Cu within the cell walls is tightly bound to a protein with oxalate oxidase activity, evidencing a high homology to germin. Zn and Sn are accumulated in the cell walls as silicate. Cytoplasm and organelles contain only traces of Cu and Sn, while in the vacuoles no HMs are detected. In the epidermal cell walls, intercellular spaces, and in vacuoles there are high concentrations of Si, forming crystal-like structures. EELS and quantum-chemical calculations reveal these structures as SiO2. The role of Si in the HM-tolerance of Silene is discussed. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2288 TI - Suppression of Inward-Rectifying K+ Channels KAT1 and AKT2 by Dominant Negative Point Mutations in the KAT1 α-Subunit JO - J. Membr. Biol. PY - 1999 SP - 119-125 AU - Baizabal-Aguirre, V. M. AU - Clemens, S. AU - Uozumi, N. AU - Schroeder, J. I. AU - VL - 167 UR - DO - 10.1007/s002329900476 AB - The Arabidopsis thaliana cDNA, KAT1 encodes a hyperpolarization-activated K+ (K+ in ) channel. In the present study, we identify and characterize dominant negative point mutations that suppress K+ in channel function. Effects of two mutations located in the H5 region of KAT1, at positions 256 (T256R) and 262 (G262K), were studied. The co-expression of either T256R or G262K mutants with KAT1 produced an inhibition of K+ currents upon membrane hyperpolarization. The magnitude of this inhibition was dependent upon the molar ratio of cRNA for wild-type to mutant channel subunits injected. Inhibition of KAT1 currents by the co-expression of T256R or G262K did not greatly affect the ion selectivity of residual currents for Rb+, Na+, Li+, or Cs+. When T256R or G262K were co-expressed with a different K+ channel, AKT2, an inhibition of the channel currents was also observed. Voltage-dependent Cs+ block experiments with co-expressed wild type, KAT1 and AKT2, channels further indicated that KAT1 and AKT2 formed heteromultimers. These data show that AKT2 and KAT1 are able to co-assemble and suggest that suppression of channel function can be pursued in vivo by the expression of the dominant negative K + in channel mutants described here. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2320 TI - Antisense-mediated depletion of a potato lipoxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pests JO - Proc. Natl. Acad. Sci. U.S.A. PY - 1999 SP - 1146-1151 AU - Royo, J. AU - León, J. AU - Vancanneyt, G. AU - Albar, J. P. AU - Rosahl, S. AU - Ortego, F. AU - Castañera, P. AU - Sánchez-Serrano, J. J. AU - VL - 96 UR - DO - 10.1073/pnas.96.3.1146 AB - De novo jasmonic acid (JA) synthesis is required for wound-induced expression of proteinase inhibitors and other defense genes in potato and tomato. The first step in JA biosynthesis involves lipoxygenase (LOX) introducing molecular oxygen at the C-13 position of linolenic acid. We previously have shown that, in potato, at least two gene families code for 13-LOX proteins. We have now produced transgenic potato plants devoid of one specific 13-LOX isoform (LOX-H3) through antisense-mediated depletion of its mRNA. LOX-H3 depletion largely abolishes accumulation of proteinase inhibitors on wounding, indicating that this specific LOX plays an instrumental role in the regulation of wound-induced gene expression. As a consequence, weight gain of Colorado potato beetles fed on antisense plants is significantly larger than those fed on wild-type plants. The poorer performance of LOX-H3-deficient plants toward herbivory is more evident with a polyphagous insect; larvae of beet armyworm reared on the antisense lines have up to 57% higher weight than those fed on nontransformed plants. LOX-H3 thus appears to regulate gene activation in response to pest attack, and this inducible response is likely to be a major determinant for reducing performance of nonspecialized herbivores. However, the regulatory role of LOX-H3 is not caused by its involvement in the wound-induced increase of JA, as wild-type and LOX-H3 deficient plants have similar jasmonate levels after wounding. LOX-H3-deficient plants have higher tuber yields. The apparent effect of suppressing the inducible defensive response on plant vigor suggests that it may pose a penalty in plant fitness under nonstress situations. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2368 TI - Characterization of a methyljasmonate-inducible lipoxygenase from barley (Hordeum vulgare cv. Salome) leaves JO - Eur. J. Biochem. PY - 1998 SP - 36-44 AU - Vörös, K. AU - Feussner, I. AU - Kühn, H. AU - Lee, J. AU - Graner, A. AU - Löbler, M. AU - Parthier, B. AU - Wasternack, C. AU - VL - 251 UR - DO - 10.1046/j.1432-1327.1998.2510036.x AB - We found three methyl jasmonate−induced lipoxygenases with molecular masses of 92 kDa, 98 kDa, and 100 kDa (LOX‐92, ‐98 and ‐100) [Feussner, I., Hause, B., Vörös, K., Parthier, B. & Wasternack, C. (1995) Plant J. 7 , 949−957]. At least two of them (LOX‐92 and LOX‐100), were shown to be localized within chloroplasts of barley leaves. Here, we describe the isolation of a cDNA (3073 bp) coding for LOX‐100, a protein of 936 amino acid residues and a molecular mass of 106 kDa. By sequence comparison this lipoxygenase could be identified as LOX2‐type lipoxygenase and was therefore designated LOX2 : Hv : 1 . The recombinant lipoxygenase was expressed in Escherichia coli and characterized as linoleate 13‐LOX and arachidonate 15‐LOX, respectively. The enzyme exhibited a pH optimum around pH 7.0 and a moderate substrate preference for linoleic acid. The gene was transiently expressed after exogenous application of jasmonic acid methyl ester with a maximum between 12 h and 18 h. Its expression was not affected by exogenous application of abscisic acid. Also a rise of endogenous jasmonic acid resulting from sorbitol stress did not induce LOX2 : Hv : 1 , suggesting a separate signalling pathway compared with other jasmonate‐induced proteins of barley. The properties of LOX2 : Hv : 1 are discussed in relation to its possible involvement in jasmonic acid biosynthesis and other LOX forms of barley identified so far. A2 - C1 - Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 2364 TI - Elicitor-stimulated biosynthesis of hydroxycinnamoyltyramines in cell suspension cultures of Solanum tuberosum JO - Planta PY - 1998 SP - 51-55 AU - Schmidt, A. AU - Scheel, D. AU - Strack, D. AU - VL - 205 UR - DO - 10.1007/s004250050295 AB - Treatment of suspension-cultured potato cells (Solanum tuberosum L. cv. Desirée) with an elicitor from Phytophthora infestans induced increased incorporation of 4-hydroxybenzaldehyde, 4-hydroxybenzoate, and N-4-coumaroyl- and N-feruloyltyramine into the cell␣wall and secretion of N-4-coumaroyl- and N-feruloyltyramine into the culture medium. Induced metabolite accumulation was preceded by rapid and transient increases in activities of phenylalanine ammonia-lyase (EC 4.3.1.5) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25), exhibiting maximal activities 5–10 h after initiation of elicitor treatment. Activities of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase (EC 2.3.1.110), catalyzing the formation of N-4-coumaroyl- and N-feruloyltyramine, increased later and remained at high levels. The phenolic defense compounds appear to be involved in cell wall reinforcement and may further directly affect fungal growth in the apoplastic space. A2 - C1 - Cell and Metabolic Biology; Biochemistry of Plant Interactions ER - TY - JOUR ID - 2359 TI - Resistance response physiology and signal transduction JO - Curr. Opin. Plant Biol. PY - 1998 SP - 305-310 AU - Scheel, D. AU - VL - 1 UR - DO - 10.1016/1369-5266(88)80051-7 AB - Plants defend themselves against pathogen attack by activating a multicomponent defense response. The activation of this response requires recognition of the pathogen and initiation of signal transduction processes that finally result in a spatially and temporally regulated expression of individual defense reactions. Several components involved in signaling resistance reactions have recently been identified and characterized. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2354 TI - Characterization and partial purification of an oligopeptide elicitor receptor from parsley (Petroselinum crispum) JO - FEBS Lett. PY - 1998 SP - 405-410 AU - Nennstiel, D. AU - Scheel, D. AU - Nürnberger, T. AU - VL - 431 UR - DO - 10.1016/S0014-5793(98)00800-X AB - Parsley cells recognize the fungal phytopathogen Phytophthora sojae through a plasma membrane receptor. A 13 amino acid oligopeptide fragment (Pep-13) of a 42 kDa fungal cell wall glycoprotein was shown to bind to the receptor and stimulate a complex defense response in cultured parsley cells. The Pep-13 binding site solubilized from parsley microsomal membranes by non-ionic detergents exhibited the same ligand affinity and ligand specificity as the membrane-bound receptor. Chemical crosslinking and photoaffinity labeling assays with [125I]Pep-13 revealed that a monomeric 100 kDa integral plasma membrane protein is sufficient for ligand binding and may thus constitute the ligand binding domain of the receptor. Ligand affinity chromatography of solubilized microsomal membrane protein on immobilized Pep-13 yielded a 5000-fold enrichment of specific receptor activity. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2341 TI - The plant cDNA LCT1 mediates the uptake of calcium and cadmium in yeast JO - Proc. Natl. Acad. Sci. U.S.A. PY - 1998 SP - 12043-12048 AU - Clemens, S. AU - Antosiewicz, D. M. AU - Ward, J. M. AU - Schachtman, D. P. AU - Schroeder, J. I. AU - VL - 95 UR - DO - 10.1073/pnas.95.20.12043 AB - Nonessential metal ions such as cadmium are most likely transported across plant membranes via transporters for essential cations. To identify possible pathways for Cd2+ transport we tested putative plant cation transporters for Cd2+ uptake activity by expressing cDNAs in Saccharomyces cerevisiae and found that expression of one clone, LCT1, renders the growth of yeast more sensitive to cadmium. Ion flux assays showed that Cd2+ sensitivity is correlated with an increase in Cd2+ uptake. LCT1-dependent Cd2+ uptake is saturable, lies in the high-affinity range (apparent KM for Cd2+ = 33 μM) and is sensitive to block by La3+ and Ca2+. Growth assays demonstrated a sensitivity of LCT1-expressing yeast cells to extracellular millimolar Ca2+ concentrations. LCT1-dependent increase in Ca2+ uptake correlated with the observed phenotype. Furthermore, LCT1 complements a yeast disruption mutant in the MID1 gene, a non-LCT1-homologous yeast gene encoding a membrane Ca2+ influx system required for recovery from the mating response. We conclude that LCT1 mediates the uptake of Ca2+ and Cd2+ in yeast and may therefore represent a first plant cDNA encoding a plant Ca2+ uptake or an organellar Ca2+ transport pathway in plants and may contribute to transport of the toxic metal Cd2+ across plant membranes. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2399 TI - Receptor-Mediated Activation of a MAP Kinase in Pathogen Defense of Plants JO - Science PY - 1997 SP - 2054-2057 AU - Ligterink, W. AU - Kroj, T. AU - zur Nieden, U. AU - Hirt, H. AU - Scheel, D. AU - VL - 276 UR - DO - 10.1126/science.276.5321.2054 AB - Parsley cells recognize the fungal plant pathogenPhytophthora sojae through a plasma membrane receptor. A pathogen-derived oligopeptide elicitor binds to this receptor and thereby stimulates a multicomponent defense response through sequential activation of ion channels and an oxidative burst. An elicitor-responsive mitogen-activated protein (MAP) kinase was identified that acts downstream of the ion channels but independently or upstream of the oxidative burst. Upon receptor-mediated activation, the MAP kinase is translocated to the nucleus where it might interact with transcription factors that induce expression of defense genes. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2393 TI - Elicitor-stimulated ion fluxes and O2- from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley JO - Proc. Natl. Acad. Sci. U.S.A. PY - 1997 SP - 4800-4805 AU - Jabs, T. AU - Tschöpe, M. AU - Colling, C. AU - Hahlbrock, K. AU - Scheel, D. AU - VL - 94 UR - DO - 10.1073/pnas.94.9.4800 AB - Fungal elicitor stimulates a multicomponent defense response in cultured parsley cells (Petroselinum crispum). Early elements of this receptor-mediated response are ion fluxes across the plasma membrane and the production of reactive oxygen species (ROS), sequentially followed by defense gene activation and phytoalexin accumulation. Omission of Ca2+ from the culture medium or inhibition of elicitor-stimulated ion fluxes by ion channel blockers prevented the latter three reactions, all of which were triggered in the absence of elicitor by amphotericin B-induced ion fluxes. Inhibition of elicitor-stimulated ROS production using diphenylene iodonium blocked defense gene activation and phytoalexin accumulation. O2− but not H2O2 stimulated phytoalexin accumulation, without inducing proton fluxes. These results demonstrate a causal relationship between early and late reactions of parsley cells to the elicitor and indicate a sequence of signaling events from receptor-mediated activation of ion channels via ROS production and defense gene activation to phytoalexin synthesis. Within this sequence, O2− rather than H2O2 appears to trigger the subsequent reactions. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2420 TI - Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense JO - Proc. Natl. Acad. Sci. U.S.A. PY - 1997 SP - 2751-2755 AU - Zimmermann, S. AU - Nürnberger, T. AU - Frachisse, J.-M. AU - Wirtz, W. AU - Guern, J. AU - Hedrich, R. AU - Scheel, D. AU - VL - 94 UR - DO - 10.1073/pnas.94.6.2751 AB - Pathogen recognition at the plant cell surface typically results in the initiation of a multicomponent defense response. Transient influx of Ca2+ across the plasma membrane is postulated to be part of the signaling chain leading to pathogen resistance. Patch-clamp analysis of parsley protoplasts revealed a novel Ca2+-permeable, La3+-sensitive plasma membrane ion channel of large conductance (309 pS in 240 mM CaCl2). At an extracellular Ca2+ concentration of 1 mM, which is representative of the plant cell apoplast, unitary channel conductance was determined to be 80 pS. This ion channel (LEAC, for large conductance elicitor-activated ion channel) is reversibly activated upon treatment of parsley protoplasts with an oligopeptide elicitor derived from a cell wall protein of Phytophthora sojae. Structural features of the elicitor found previously to be essential for receptor binding, induction of defense-related gene expression, and phytoalexin formation are identical to those required for activation of LEAC. Thus, receptor-mediated stimulation of this channel appears to be causally involved in the signaling cascade triggering pathogen defense in parsley. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2437 TI - Characterization of Three Potato Lipoxygenases with Distinct Enzymatic Activities and Different Organ-specific and Wound-regulated Expression Patterns JO - J. Biol. Chem. PY - 1996 SP - 21012-21019 AU - Royo, J. AU - Vancanneyt, G. AU - Pérez, A. G. AU - Sanz, C. AU - Störmann, K. AU - Rosahl, S. AU - Sánchez-Serrano, J. J. AU - VL - 271 UR - DO - 10.1074/jbc.271.35.21012 AB - Lipoxygenases are ubiquitous enzymes in eukaryotes. In plants, lipoxygenases are involved in the synthesis of the hormone jasmonic acid that regulates plant responses to wounding and, in addition, is an inducer of tuberization in potato.We have isolated potato lipoxygenase cDNA clones. From their deduced amino acid sequences, three distinct classes are defined (Lox1, Lox2, and Lox3). They are encoded in gene families that display organ-specific expression, lox1 being expressed mostly in tubers and roots, lox2 in leaves, and lox3 in leaves and roots.Consistent with their organ-specific expression pattern, Lox1 expressed in bacteria preferentially uses as substrate linoleic acid, abundant in membrane lipids of tubers, whereas linolenic acid, prevalent in leaves, is the preferred substrate for the other two classes of lipoxygenase. Analyses on reaction products of the enzymes expressed in bacteria reveal that Lox1 primarily produces 9- hydroperoxides. In contrast, the jasmonic acid precursor, 13-hydroperoxylinolenic acid, is the major product of the action of Lox2 and Lox3 on linolenic acid. Upon wounding, the levels of Lox2 and Lox3 transcripts rise markedly in leaves. While Lox3 mRNA accumulation peaks as early as 30 min after wounding, Lox2 shows a steady increase over a 24-h time course, suggesting different roles for these lipoxygenase isoforms in the synthesis of the plant hormone jasmonic acid. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2436 TI - Lipoxygenases in Plants - Their Role in Development and Stress Response JO - Z. Naturforsch. C PY - 1996 SP - 123-138 AU - Rosahl, S. AU - VL - 51 UR - DO - 10.1515/znc-1996-3-401 AB - Lipoxygenases catalyze the hydroperoxidation of polyunsaturated fatty acids and thus the first step in the synthesis of fatty acid metabolites in plants. Products of the LOX pathway have multiple functions as growth regulators, antimicrobial compounds, flavours and odours as well as signal molecules. Based on the effects of LOX products or on the correlation of increases in LOX protein and the onset of specific processes, a physiological function for LOXs has been proposed for growth and development and for the plant response to patho­gen infection and wound stress. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2430 TI - Purification of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase from cell-suspension cultures of Solanum tuberosum L. cv. Datura JO - Planta PY - 1996 SP - 166-168 AU - Hohlfeld, H. AU - Scheel, D. AU - Strack, D. AU - VL - 199 UR - DO - 10.1007/BF00196893 AB - A pathogen-elicitor-inducible acyltransferase [tyramine hydroxycinnamoyltransferase (THT); EC 2.3.1], which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was purified to apparent homogeneity from cell-suspension cultures of potato (Solanum tuberosum L. cv. Datura), with a 1400-fold enrichment, a 5% recovery and a final specific activity of 208 mkat·(kg protein)−1. Affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent was the decisive step in the purification sequence. The purified protein showed a native molecular mass of ca. 49 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and in the absence of a reducing agent (2-mercaptoethanol) indicated that THT is a heterodimer in which the protein subunits (ca. 25 kDa) are non-covalently associated. The enzyme was stimulated fivefold by 10 mM Ca2+. The apparent K m value for tyramine was dependent on the nature of the hydroxycinnamoyl-CoA present. Thus, the K m value for tyramine was about tenfold greater (174 μM) in the presence of 4-coumaroyl-CoA than in the presence of feruloyl-CoA (20 μM). A2 - C1 - Cell and Metabolic Biology; Biochemistry of Plant Interactions ER - TY - JOUR ID - 2459 TI - Molecular characterization of nucleotide sequences encoding the extracellular glycoprotein elicitor from Phytophthora megasperma JO - Molec. Gen. Genet. PY - 1995 SP - 45-55 AU - Sacks, W. AU - Nürnberger, T. AU - Hahlbrock, K. AU - Scheel, D. AU - VL - 246 UR - DO - 10.1007/BF00290132 AB - cDNA sequences encoding the 42 kDa glycoprotein elicitor from the oomycete, Phytophthora megasperma, that induces the defense response in parsley have been cloned and sequenced. The 5′ end of the mRNA matches a consensus derived from sequences surrounding the transcription initiation sites of seven other oomycete genes. The major transcript of 1802 nucleotides contains a 529-codon open reading frame, which was predicted to encode a 57 kDa precursor protein. On the basis of peptide sequencing, the N-terminus of the mature protein is at position 163, suggesting that proteolytic processing events, in addition to signal peptide cleavage, generate the protein purified from the fungal culture filtrate. Expression studies in Escherichia coli with the cDNA as well as smaller subfragments demonstrated that a region of 47 amino acids located in the C-terminal third of the protein was sufficient to confer elicitor activity. The gene encoding the elicitor was found to be a member of a multigene family in P. megasperma. Homologous families of differing sizes were found in all eight other Phytophthora species tested, but not in other filamentous fungi including other Oomycetes. No significant similarity of the elicitor preprotein to sequences present in the databases has yet been detected. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2458 TI - Covalent cross-linking of the Phytophthora megasperma oligopeptide elicitor to its receptor in parsley membranes. JO - Proc. Natl. Acad. Sci. U.S.A. PY - 1995 SP - 2338-2342 AU - Nürnberger, T. AU - Nennstiel, D. AU - Hahlbrock, K. AU - Scheel, D. AU - VL - 92 UR - DO - 10.1073/pnas.92.6.2338 AB - An oligopeptide elicitor from Phytophthora megasperma f.sp. glycinea (Pep-13) that induces phytoalexin accumulation in cultured parsley cells was radioiodinated and chemically cross-linked to its binding site in microsomal and plasma membrane preparations with each of three homobifunctional reagents. Analysis by SDS/PAGE and autoradiography of solubilized membrane proteins demonstrated labeling of a 91-kDa protein, regardless of which reagent was used. Cross-linking of this protein was prevented by addition of excess unlabeled Pep-13. The competitor concentration found to half-maximally reduce the intensity of the cross-linked band was 6 nM, which is in good agreement with the IC50 value of 4.7 nM, obtained from ligand binding assays. No crosslinking of 125I-labeled Pep-13 was observed by using microsomal membranes from three other plant species, indicating species-specific occurrence of the binding site. Coupling of 125I-Pep-13 to the parsley 91-kDa protein required the same structural elements within the ligand as was recently reported for binding of 125I-Pep-13 to parsley microsomes, elicitor-induced stimulation of ion fluxes across the plasma membrane, the oxidative burst, the expression of defense-related genes, and phytoalexin production. These findings suggest that the 91-kDa protein identified in parsley membranes is the oligopeptide elicitor receptor mediating activation of a multicomponent defense response. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2468 TI - High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses JO - Cell PY - 1994 SP - 449-460 AU - Nürnberger, T. AU - Nennstiel, D. AU - Jabs, T. AU - Sacks, W. R. AU - Hahlbrock, K. AU - Scheel, D. AU - VL - 78 UR - DO - 10.1016/0092-8674(94)90423-5 AB - An oligopeptide of 13 amino acids (Pep-13) identified within a 42 kDa glycoprotein elicitor from P. megasperma was shown to be necessary and sufficient to stimulate a complex defense response in parsley cells comprising H+/Ca2+ influxes, K+/Cl− effluxes, an oxidative burst, defense-related gene activation, and phytoalexin formation. Binding of radiolabeled Pep-13 to parsley microsomes and protoplasts was specific, reversible, and saturable. Identical structural features of Pep-13 were found to be responsible for specific binding and initiation of all plant responses analyzed. The high affinity binding site recognizing the peptide ligand (KD = 2.4 nM) may therefore represent a novel class of receptors in plants, and the rapidly induced ion fluxes may constitute elements of the signal transduction cascade triggering pathogen defense in plants. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2481 TI - Transcriptional repression of light-induced flavonoid synthesis by elicitor treatment of cultured parsley cells JO - Plant J. PY - 1991 SP - 227-234 AU - Lozoya, E. AU - Block, A. AU - Lois, R. AU - Hahlbrock, K. AU - Scheel, D. AU - VL - 1 UR - DO - 10.1111/j.1365-313X.1991.00227.x AB - Cultured parsley (Petroselinum crispum) cells respond differentially to UV‐containing white light and fungal elicitor. Both stimuli activate the transcription of genes encoding enzymes of partly overlapping phenylpropanoid biosynthetic pathways. Irradiation induces vacuolar accumulation of flavonoids, whereas elicitor treatment stimulates the secretion of furanocoumarins. Simultaneous treatment of parsley cells with UV light and elicitor results in quantitative changes in both responses. Irradiation reduces elicitor‐induced furanocoumarin synthesis, possibly by post‐transcriptional mechanisms, whereas elicitor treatment completely blocks the light‐induced accumulation of flavonoids by repressing the transcription of the chalcone synthase gene. We have identified elicitor‐sensitive regions in the chalcone synthase promoter by transient expression analysis of selected promoter constructs linked to the β‐D‐glucuronidase reporter gene in parsley protoplasts. These regions are identical to those that were found to be sufficient for light inducibility of the chalcone synthase promoter. A2 - C1 - Biochemistry of Plant Interactions ER -