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Publikationen - Stress- und Entwicklungsbiologie

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Caillaud, M.-C.; Wirthmueller, L.; Fabro, G.; Piquerez, S. J. M.; Asai, S.; Ishaque, N.; Jones, J. D. G. Mechanisms of Nuclear Suppression of Host Immunity by Effectors from the Arabidopsis Downy Mildew Pathogen Hyaloperonospora arabidopsidis (Hpa) Cold Spring Harb Symp Quant Biol 77, 285-293, (2012) DOI: 10.1101/sqb.2012.77.015115

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.

Wirthmueller, L.; Roth, C.; Banfield, M. J.; Wiermer, M. Hop-on hop-off: importin-α-guided tours to the nucleus in innate immune signaling Front Plant Sci 4, 149, (2013) DOI: 10.3389/fpls.2013.00149

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.

Wirthmueller, L.; Maqbool, A.; Banfield, M. J. On the front line: structural insights into plant–pathogen interactions Nat Rev Microbiol 11, 761-776, (2013) DOI: 10.1038/nrmicro3118

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.

Heidrich, K.; Tsuda, K.; Blanvillain-Baufumé, S.; Wirthmueller, L.; Bautor, J.; Parker, J. E. Arabidopsis TNL-WRKY domain receptor RRS1 contributes to temperature-conditioned RPS4 auto-immunity Front Plant Sci 4, 403, (2013) DOI: 10.3389/fpls.2013.00403

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.

Zhou, X.; Graumann, K.; Wirthmueller, L.; Jones, J. D. G.; Meier, I. Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants J Cell Biol 205, 677-692, (2014) DOI: 10.1083/jcb.201401138

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.

Wirthmueller, L.; Roth, C.; Fabro, G.; Caillaud, M.-C.; Rallapalli, G.; Asai, S.; Sklenar, J.; Jones, A. M. E.; Wiermer, M.; Jones, J. D. G.; Banfield, M. J. Probing formation of cargo/importin-α transport complexes in plant cells using a pathogen effector Plant J 81, 40-52, (2015) DOI: 10.1111/tpj.12691

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.

Asai, S.; Rallapalli, G.; Piquerez, S. J. M.; Caillaud, M.-C.; Furzer, O. J.; Ishaque, N.; Wirthmueller, L.; Fabro, G.; Shirasu, K.; Jones, J. D. G. Expression Profiling during Arabidopsis/Downy Mildew Interaction Reveals a Highly-Expressed Effector That Attenuates Responses to Salicylic Acid PLOS Pathog 10, e1004443, (2014) DOI: 10.1371/journal.ppat.1004443

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.

Caillaud, M.-C.; Wirthmueller, L.; Sklenar, J.; Findlay, K.; Piquerez, S. J. M.; Jones, A. M. E.; Robatzek, S.; Jones, J. D. G.; Faulkner, C. The Plasmodesmal Protein PDLP1 Localises to Haustoria-Associated Membranes during Downy Mildew Infection and Regulates Callose Deposition PLOS Pathog 10, e1004496, (2014) DOI: 10.1371/journal.ppat.1004496

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.

Sarris, P. F.; Duxbury, Z.; Huh, S. U.; Ma, Y.; Segonzac, C.; Sklenar, J.; Derbyshire, P.; Cevik, V.; Rallapalli, G.; Saucet, S. B.; Wirthmueller, L.; Menke, F. L. H.; Sohn, K. H.; Jones, J. D. G. A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors Cell 161, 1089-1100, (2015) DOI: 10.1016/j.cell.2015.04.024

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.

Chen, S.; Wirthmueller, L.; Stauber, J.; Lory, N.; Holtkotte, X.; Leson, L.; Schenkel, C.; Ahmad, M.; Hoecker, U. The functional divergence between SPA1 and SPA2 in Arabidopsis photomorphogenesis maps primarily to the respective N-terminal kinase-like domain BMC Plant Biol 16, 165, (2016) DOI: 10.1186/s12870-016-0854-9

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.
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