TY - INPR ID - 2502 TI - CAMTA3 repressor destabilization triggers TIR domain protein TN2-mediated autoimmunity in the Arabidopsis exo70B1 mutant JO - Plant Cell PY - 2024 SP - AU - Liu, N. AU - Jiang, X. AU - Zhong, G. AU - Wang, W. AU - Hake, K. AU - Matschi, S. AU - Lederer, S. AU - Hoehenwarter, W. AU - Sun, Q. AU - Lee, J. AU - Romeis, T. AU - Tang, D. AU - VL - UR - https://doi.org/10.1093/plcell/koae036 DO - 10.1093/plcell/koae036 AB - Calcium-dependent protein kinases (CPKs) can decode and translate intracellular calcium signals to induce plant immunity. Mutation of the exocyst subunit gene EXO70B1 causes autoimmunity that depends on CPK5 and the Toll/interleukin-1 receptor (TIR) domain resistance protein TIR-NBS2 (TN2), where direct interaction with TN2 stabilizes CPK5 kinase activity. However, how the CPK5–TN2 interaction initiates downstream immune responses remains unclear. Here, we show that, besides CPK5 activity, the physical interaction between CPK5 and functional TN2 triggers immune activation in exo70B1 and may represent reciprocal regulation between CPK5 and the TIR domain functions of TN2 in Arabidopsis (Arabidopsis thaliana). Moreover, we detected differential phosphorylation of the calmodulin-binding transcription activator 3 (CAMTA3) in the cpk5 background. CPK5 directly phosphorylates CAMTA3 at S964, contributing to its destabilization. The gain-of-function CAMTA3A855V variant that resists CPK5-induced degradation rescues immunity activated through CPK5 overexpression or exo70B1 mutation. Thus, CPK5-mediated immunity is executed through CAMTA3 repressor degradation via phosphorylation-induced and/or calmodulin-regulated processes. Conversely, autoimmunity in camta3 also partially requires functional CPK5. While the TIR domain activity of TN2 remains to be tested, our study uncovers a TN2–CPK5–CAMTA3 signaling module for exo70B1-mediated autoimmunity, highlighting the direct embedding of a calcium-sensing decoder element within resistance signalosomes. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 205 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 - INPR ID - 2498 TI - Stromule geometry allows optimal spatial regulation of organelle interactions in the quasi-2D cytoplasm JO - Plant Cell Physiol. PY - 2023 SP - AU - Erickson, J. L. AU - Prautsch, J. AU - Reynvoet, F. AU - Niemeyer, F. AU - Hause, G. AU - Johnston, I. G. AU - Schattat, M. H. AU - VL - UR - https://doi.org/10.1093/pcp/pcad098 DO - 10.1093/pcp/pcad098 AB - In plant cells, plastids form elongated extensions called stromules, the regulation and purposes of which remain unclear. Here, we quantitatively explore how different stromule structures serve to enhance the ability of a plastid to interact with other organelles: increasing the effective space for interaction and biomolecular exchange between organelles. Interestingly, electron microscopy and confocal imaging showed that the cytoplasm in Arabidopsis thaliana and Nicotiana benthamiana epidermal cells is extremely thin (around 100 nm in regions without organelles), meaning that inter-organelle interactions effectively take place in 2D. We combine these imaging modalities with mathematical modeling and new in planta experiments to demonstrate how different stromule varieties (single or multiple, linear or branching) could be employed to optimize different aspects of inter-organelle interaction capacity in this 2D space. We found that stromule formation and branching provide a proportionally higher benefit to interaction capacity in 2D than in 3D. Additionally, this benefit depends on optimal plastid spacing. We hypothesize that cells can promote the formation of different stromule architectures in the quasi-2D cytoplasm to optimize their interaction interface to meet specific requirements. These results provide new insight into the mechanisms underlying the transition from low to high stromule numbers, the consequences for interaction with smaller organelles, how plastid access and plastid to nucleus signaling are balanced and the impact of plastid density on organelle interaction. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 271 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 - 217 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 - 282 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 - 360 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 - 352 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 - 350 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 - 330 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 - 414 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 - 450 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 - 442 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 - 472 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 - 508 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 - 554 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 - 553 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 - 546 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 - 620 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 - 616 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 - INPR ID - 2493 TI - Prioritization of abiotic and biotic stress responses by direct linkage of ABI1 phosphatase and CPK5 calcium-dependent protein kinase JO - bioRxiv PY - 2019 SP - AU - Seybold, H. AU - Bortlik, J. AU - Conrads, B. AU - Hoehenwarter, W. AU - Romeis, T. AU - VL - UR - DO - 10.1101/839662 AB - In nature plants are constantly challenged by simultaneous abiotic and biotic stresses, and under conflicting stress scenarios prioritization of stress responses is required for plant survival. Calcium-dependent protein kinase CPK5 is a central hub in local and distal immune signaling, required upstream of hormone salicylic acid (SA)-dependent systemic acquired resistance (SAR). Here we show that CPK5 signaling-dependent immune responses are effectively blocked and pathogen resistance is reverted either upon treatment of plants with abscisic acid (ABA) or in genetic mutant backgrounds lacking PP2C phosphatase activities including abi1-2. Consistently, enhanced immune responses occur upon co-expression of CPK5 kinase with active variants of ABI1 phosphatase ABI1G180S and ABI1G181A. Biochemical studies and mass spectrometry-based phosphosite analysis reveal a direct ABI1 phosphatase-catalyzed de-phosphorylation of CPK5 at T98, a CPK5 auto-phosphorylation site. CPK5T98A, mimicking continuous de-phosphorylation through ABI1, correlates with an increase in kinase activity and CPK5 function in ROS production. CPK5T98D, mimicking a CPK5 auto-phosphorylated status under ABA-induced phosphatase inhibition, leads to inactivated CPK5 causative to an immediate stop of immune responses.Our work reveals an elegant mechanism for plant stress prioritization, where the ABA-dependent phosphatase ABI1, negative regulator of abiotic responses, functions as positive regulator of biotic stress responses, stabilizing CPK5-dependent immune responses in the absence of ABA. This mechanism allows continuous immune signaling during pathogen survey in environmentally non-challenging conditions. Under severe abiotic stress, immune signaling is discontinued via a direct biochemical intersection through a phosphatase/kinase pair recruiting two key regulatory enzymes of these antagonistic signaling pathways. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1130 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 - 1263 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 - 1970 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 -