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Plants evolve nucleotide-binding leucine-rich repeat receptors (NLRs) to induce immunity. Activated coiled-coil (CC) domain containing NLRs (CNLs) oligomerize and form apparent cation channels promoting calcium influx and cell death, with the alpha-1 helix of the individual CC domains penetrating the plasma membranes. Some CNLs are characterized by putative N-myristoylation and S-acylation sites in their CC domain, potentially mediating permanent membrane association. Whether activated Potentially Membrane Localized NLRs (PMLs) mediate cell death and calcium influx in a similar way is unknown. We uncovered the cell-death function at the vacuole of an atypical but conserved Arabidopsis PML, PML5, which has a significant deletion in its CC G10/GA domain. Active PML5 oligomers localize in Golgi membranes and the tonoplast, alter vacuolar morphology, and induce cell death, with the short N-terminus being sufficient. Mutant analysis supports a potential role of PMLs in plant immunity. PML5-like deletions are found in several Brassicales paralogs, pointing to the evolutionary importance of this innovation. PML5, with its minimal CC domain, represents the first identified CNL utilizing vacuolar-stored calcium for cell death induction., (© 2024. The Author(s).)

Intracellular plant immune receptors, termed NLRs (Nucleotide-binding Leucine-rich repeat Receptors), confer effector-triggered immunity. Sensor NLRs are responsible for pathogen effector recognition. Helper NLRs function downstream of sensor NLRs to transduce signaling and induce cell death and immunity. Activation of sensor NLRs that contain TIR (Toll/interleukin-1receptor) domains generates small molecules that induce an association between a downstream heterodimer signalosome of EDS1 (EnhancedDisease Susceptibility 1)/SAG101 (Senescence-AssociatedGene 101) and the helper NLR of NRG1 (NRequired Gene 1). Autoactive NRG1s oligomerize and form calcium signaling channels largely localized at the plasma membrane (PM). The molecular mechanisms of helper NLR PM association and effector-induced NRG1 oligomerization are not well characterized. We demonstrate that helper NLRs require positively charged residues in their N-terminal domains for phospholipid binding and PM association before and after activation, despite oligomerization and conformational changes that accompany activation. We demonstrate that effector activation of a TIR-containing sensor NLR induces NRG1 oligomerization at the PM and that the cytoplasmic pool of EDS1/SAG101 is critical for cell death function. EDS1/SAG101 cannot be detected in the oligomerized NRG1 resistosome, suggesting that additional unknown triggers might be required to induce the dissociation of EDS1/SAG101 from the previously described NRG1/EDS1/SAG101 heterotrimer before subsequent NRG1 oligomerization. Alternatively, the conformational changes resulting from NRG1 oligomerization abrogate the interface for EDS1/SAG101 association. Our data provide observations regarding dynamic PM association during helper NLR activation and underpin an updated model for effector-induced NRG1 resistosome formation.

The ever-growing world population, increasingly frequent extreme weather events and conditions, emergence of novel devastating crop pathogens and the social strive for quality food products represent a huge challenge for current and future agricultural production systems. To address these challenges and find realistic solutions, it is becoming more important by the day to understand the complex interactions between plants and the environment, mainly the associated organisms, but in particular pathogens. In the past several years, research in the fields of plant pathology and plant-microbe interactions has enabled tremendous progress in understanding how certain receptor-based plant innate immune systems function to successfully prevent infections and diseases. In this review, we highlight and discuss some of these new ground-breaking discoveries and point out strategies of how pathogens counteract the function of important core convergence hubs of the plant immune system. For practical reasons, we specifically place emphasis on potential applications that can be detracted by such discoveries and what challenges the future of agriculture has to face, but also how these challenges could be tackled., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Competing Interests: The authors have declared that no competing interests exist.

TIR domains are NAD-degrading enzymes that function during immune signaling in prokaryotes, plants, and animals. In plants, most TIR domains are incorporated into intracellular immune receptors termed TNLs. In Arabidopsis, TIR-derived small molecules bind and activate EDS1 heterodimers, which in turn activate RNLs, a class of cation channel-forming immune receptors. RNL activation drives cytoplasmic Ca 2+ influx, transcriptional reprogramming, pathogen resistance, and host cell death. We screened for mutants that suppress an RNL activation mimic allele and identified a TNL, SADR1. Despite being required for the function of an autoactivated RNL, SADR1 is not required for defense signaling triggered by other tested TNLs. SADR1 is required for defense signaling initiated by some transmembrane pattern recognition receptors and contributes to the unbridled spread of cell death in lesion simulating disease 1 . Together with RNLs, SADR1 regulates defense gene expression at infection site borders, likely in a non-cell autonomous manner. RNL mutants that cannot sustain this pattern of gene expression are unable to prevent disease spread beyond localized infection sites, suggesting that this pattern corresponds to a pathogen containment mechanism. SADR1 potentiates RNL-driven immune signaling not only through the activation of EDS1 but also partially independently of EDS1. We studied EDS1-independent TIR function using nicotinamide, an NADase inhibitor. Nicotinamide decreased defense induction from transmembrane pattern recognition receptors and decreased calcium influx, pathogen growth restriction, and host cell death following intracellular immune receptor activation. We demonstrate that TIR domains can potentiate calcium influx and defense and are thus broadly required for Arabidopsis immunity.

Arabidopsis BAK1/SERK3, a co-receptor of leucine-rich repeat pattern recognition receptors (PRRs), mediates pattern-triggered immunity (PTI). Genetic inactivation of BAK1 or BAK1-interacting receptor-like kinases (BIRs) causes cell death, but the direct mechanisms leading to such deregulation remains unclear. Here, we found that the TIR-NBS-LRR protein CONSTITUTIVE SHADE AVOIDANCE 1 (CSA1) physically interacts with BIR3, but not with BAK1. CSA1 mediates cell death in bak1-4 and bak1-4 bir3-2 mutants via components of effector-triggered immunity-(ETI) pathways. Effector HopB1-mediated perturbation of BAK1 also results in CSA1-dependent cell death. Likewise, microbial pattern pg23-induced cell death, but not PTI responses, requires CSA1. Thus, we show that CSA1 guards BIR3 BAK1 homeostasis and integrates pattern- and effector-mediated cell death pathways downstream of BAK1. De-repression of CSA1 in the absence of intact BAK1 and BIR3 triggers ETI cell death. This suggests that PTI and ETI pathways are activated downstream of BAK1 for efficient plant immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Activation of nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death. NLR cell death activity requires oligomerization and in some cases plasma membrane (PM) localization. The exact mechanisms underlying PM localization of NLRs lacking predicted transmembrane domains or recognizable lipidation motifs remain elusive. We used confocal microscopy, genetically encoded molecular tools and protein-lipid overlay assays to determine whether PM localization of members of the Arabidopsis HeLo-/RPW8-like domain 'helper' NLR (RNL) family is mediated by the interaction with negatively charged phospholipids of the PM. Our results show that PM localization and stability of some RNLs and one CC-type NLR (CNL) depend on the direct interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate from the PM led to a mis-localization of the analysed NLRs and consequently inhibited their cell death activity. We further demonstrate homo- and hetero-association of members of the RNL family. Our results provide new insights into the molecular mechanism of NLR localization and defines an important role of phospholipids for CNL and RNL PM localization and consequently, for their function. We propose that RNLs interact with anionic PM phospholipids and that RNL-mediated cell death and immune responses happen at the PM., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

Plants utilize cell-surface localized and intracellular leucine-rich repeat (LRR) immune receptors to detect pathogens and to activate defense responses, including transcriptional reprogramming and the initiation of a form of programmed cell death of infected cells. Cell death initiation is mainly associated with the activation of nucleotide-binding LRR receptors (NLRs). NLRs recognize the presence or cellular activity of pathogen-derived virulence proteins, so-called effectors. Effector-dependent NLR activation leads to the formation of higher order oligomeric complexes, termed resistosomes. Resistosomes can either form potential calcium-permeable cation channels at cellular membranes and initiate calcium influxes resulting in activation of immunity and cell death or function as NADases whose activity is needed for the activation of downstream immune signaling components, depending on the N-terminal domain of the NLR protein. In this mini-review, the current knowledge on the mechanisms of NLR-mediated cell death and resistance pathways during plant immunity is discussed., (© 2021 The Author(s).)

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) regulate immunity and cell death. In Arabidopsis , a subfamily of "helper" NLRs is required by many "sensor" NLRs. Active NRG1.1 oligomerized, was enriched in plasma membrane puncta, and conferred cytoplasmic calcium ion (Ca 2+ ) influx in plant and human cells. NRG1.1-dependent Ca 2+ influx and cell death were sensitive to Ca 2+ channel blockers and were suppressed by mutations affecting oligomerization or plasma membrane enrichment. Ca 2+ influx and cell death mediated by NRG1.1 and ACTIVATED DISEASE RESISTANCE 1 (ADR1), another helper NLR, required conserved negatively charged N-terminal residues. Whole-cell voltage-clamp recordings demonstrated that Arabidopsis helper NLRs form Ca 2+ -permeable cation channels to directly regulate cytoplasmic Ca 2+ levels and consequent cell death. Thus, helper NLRs transduce cell death signals directly., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Plant nucleotide-binding (NB) leucine-rich repeat (LRR) receptor (NLR) proteins function as intracellular immune receptors that perceive the presence of pathogen-derived virulence proteins (effectors) to induce immune responses. The 2 major types of plant NLRs that "sense" pathogen effectors differ in their N-terminal domains: these are Toll/interleukin-1 receptor resistance (TIR) domain-containing NLRs (TNLs) and coiled-coil (CC) domain-containing NLRs (CNLs). In many angiosperms, the RESISTANCE TO POWDERY MILDEW 8 (RPW8)-CC domain containing NLR (RNL) subclass of CNLs is encoded by 2 gene families, ACTIVATED DISEASE RESISTANCE 1 (ADR1) and N REQUIREMENT GENE 1 (NRG1), that act as "helper" NLRs during multiple sensor NLR-mediated immune responses. Despite their important role in sensor NLR-mediated immunity, knowledge of the specific, redundant, and synergistic functions of helper RNLs is limited. We demonstrate that the ADR1 and NRG1 families act in an unequally redundant manner in basal resistance, effector-triggered immunity (ETI) and regulation of defense gene expression. We define RNL redundancy in ETI conferred by some TNLs and in basal resistance against virulent pathogens. We demonstrate that, in Arabidopsis thaliana, the 2 RNL families contribute specific functions in ETI initiated by specific CNLs and TNLs. Time-resolved whole genome expression profiling revealed that RNLs and "classical" CNLs trigger similar transcriptome changes, suggesting that RNLs act like other CNLs to mediate ETI downstream of sensor NLR activation. Together, our genetic data confirm that RNLs contribute to basal resistance, are fully required for TNL signaling, and can also support defense activation during CNL-mediated ETI., Competing Interests: The authors have declared that no competing interests exist.

Plant nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins function as intracellular receptors in response to pathogens and activate effector-triggered immune responses (ETI). The activation of some sensor NLRs (sNLR) by their corresponding pathogen effector is well studied. However, the mechanisms by which the recently defined helper NLRs (hNLR) function to transduce sNLR activation into ETI-associated cell death and disease resistance remains poorly understood. We briefly summarize recent examples of sNLR activation and we then focus on hNLR requirements in sNLR-initiated immune responses. We further discuss how shared sequence homology with fungal self-incompatibility proteins and the mammalian mixed lineage kinase domain like pseudokinase (MLKL) proteins informs a plausible model for the structure and function of an ancient clade of plant hNLRs, called RNLs., (Copyright © 2019. Published by Elsevier Ltd.)

Phytopathogenic microbes multiply in the apoplast-a plant's intercellular spaces-of infected plants, and hence their success relies on the conditions in this habitat. Despite being extracellular parasites, most microbes translocate effectors into host cells that promote disease by acting inside cells. Initial studies suggested that effectors act predominantly as suppressors of plant immunity. These pioneering studies were trend-setting, causing a strong bias in the functional investigation of effectors. Yet, recent studies on bacterial model pathogens have identified effectors that promote disease by causing either increased sugar or water levels in the apoplast. These studies are likely to initiate a new era of effector research that will clarify the disease-promoting rather than defense-suppressing function of effectors, a molecular rather than genetic distinction., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Membrane vesicles delivered to the cell-division plane fuse with one another to form the partitioning membrane during plant cytokinesis, starting in the cell center. In Arabidopsis, this requires SNARE complexes involving the cytokinesis-specific Qa-SNARE KNOLLE. However, cytokinesis still occurs in knolle mutant embryos, suggesting contributions from KNOLLE-independent SNARE complexes. Here we show that Qa-SNARE SYP132, having counterparts in lower plants, functionally overlaps with the flowering plant-specific KNOLLE. SYP132 mutation causes cytokinesis defects, knolle syp132 double mutants consist of only one or a few multi-nucleate cells, and SYP132 has the same SNARE partners as KNOLLE. SYP132 and KNOLLE also have non-overlapping functions in secretion and in cellularization of the embryo-nourishing endosperm resulting from double fertilization unique to flowering plants. Evolutionarily ancient non-specialized SNARE complexes originating in algae were thus amended by the appearance of cytokinesis-specific SNARE complexes, meeting the high demand for membrane-fusion capacity during endosperm cellularization in angiosperms., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Intracellular membrane fusion is effected by SNARE proteins that reside on adjacent membranes and form bridging trans-SNARE complexes. Qa-SNARE members of the Arabidopsis SYP1 family are involved in membrane fusion at the plasma membrane or during cell plate formation. Three SYP1 family members have been classified as pollen-specific as inferred from gene expression profiling studies, and two of them, SYP124 and SYP125, are confined to angiosperms. The SYP124 gene appears genetically unstable, whereas its sister gene SYP125 shows essentially no variation among Arabidopsis accessions. The third pollen-specific member SYP131 is sister to SYP132, which appears evolutionarily conserved in the plant lineage. Although evolutionarily diverse, the three SYP1 proteins are functionally overlapping in that only the triple mutant syp124 syp125 syp131 shows a specific and severe male gametophytic defect. While pollen development and germination appear normal, pollen tube growth is arrested during passage through the style. Our results suggest that angiosperm pollen tubes employ a combination of ancient and modern Qa-SNARE proteins to sustain their growth-promoting membrane dynamics during the reproductive process., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Alleles that confer multiple disease resistance (MDR) are valuable in crop improvement, although the molecular mechanisms underlying their functions remain largely unknown. A quantitative trait locus, qMdr 9.02 , associated with resistance to three important foliar maize diseases-southern leaf blight, gray leaf spot and northern leaf blight-has been identified on maize chromosome 9. Through fine-mapping, association analysis, expression analysis, insertional mutagenesis and transgenic validation, we demonstrate that ZmCCoAOMT2, which encodes a caffeoyl-CoA O-methyltransferase associated with the phenylpropanoid pathway and lignin production, is the gene within qMdr 9.02 conferring quantitative resistance to both southern leaf blight and gray leaf spot. We suggest that resistance might be caused by allelic variation at the level of both gene expression and amino acid sequence, thus resulting in differences in levels of lignin and other metabolites of the phenylpropanoid pathway and regulation of programmed cell death.

Plants evolved intracellular immune receptors that belong to the NOD-like receptor (NLR) family to recognize the presence of pathogen-derived effector proteins. NLRs possess an N-terminal Toll-like/IL-1 receptor (TIR) or a non-TIR domain [some of which contain coiled coils (CCs)], a central nucleotide-binding (NB-ARC) domain, and a C-terminal leucine-rich repeat (LRR). Activation of NLR proteins results in a rapid and high-amplitude immune response, eventually leading to host cell death at the infection site, the so-called hypersensitive response. Despite their important contribution to immunity, the exact mechanisms of NLR activation and signaling remain unknown and are likely heterogenous. We undertook a detailed structure-function analysis of the plasma membrane (PM)-localized CC NLR Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1) using both stable transgenic Arabidopsis and transient expression in Nicotiana benthamiana We report that immune signaling is induced only by activated full-length PM-localized RPM1. Our interaction analyses demonstrate the importance of a functional P-loop for in planta interaction of RPM1 with the small host protein RPM1-interacting protein 4 (RIN4), for constitutive preactivation and postactivation self-association of RPM1 and for proper PM localization. Our results reveal an additive effect of hydrophobic conserved residues in the CC domain for RPM1 function and RPM1 self-association and their necessity for RPM1-RIN4 interaction. Thus, our findings considerably extend our understanding of the mechanisms regulating NLR activation at, and signaling from, the PM., Competing Interests: The authors declare no conflict of interest.

Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll-interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 ( RBA1 ), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the heme-binding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR-TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that "truncated" NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system.

Competing Interests: The authors declare no conflict of interest.

Nucleotide-binding leucine-rich repeat proteins (NLRs) serve as intracellular immune receptors in animals and plants. Sensor NLRs perceive pathogen-derived effector molecules and trigger robust host defense. Recent studies revealed the role of three coiled-coil-type NLRs (CNLs) of the ADR1 family - ADR1, ADR1-L1 and ADR1-L2 - as redundant helper NLRs, whose function is required for defense mediated by multiple sensor NLRs. From a mutant snc1-enhancing (MUSE) forward genetic screen in Arabidopsis targeted to identify negative regulators of snc1 that encodes a TIR-type NLR (TNL), we isolated two alleles of muse15, both carrying mutations in ADR1-L1. Interestingly, loss of ADR1-L1 also enhances immunity-related phenotypes in other autoimmune mutants including cpr1, bal and lsd1. This immunity-enhancing effect is not mediated by increased SNC1 protein stability, nor is it fully dependent on the accumulation of the defense hormone salicylic acid (SA). Transcriptional analysis revealed an upregulation of ADR1 and ADR1-L2 in the adr1-L1 background, which may overcompensate the loss of ADR1-L1, resulting in enhanced immunity. Interestingly, autoimmunity of snc1 and chs2, which encode typical TNLs, is fully suppressed by the adr1 triple mutant, suggesting that the ADRs are required for TNL downstream signaling. This study extends our knowledge on the interplay among ADRs and reveals their complexity in defense regulation., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

Membrane trafficking is required during plant immune responses, but its contribution to the hypersensitive response (HR), a form of programmed cell death (PCD) associated with effector-triggered immunity, is not well understood. HR is induced by nucleotide binding-leucine-rich repeat (NB-LRR) immune receptors and can involve vacuole-mediated processes, including autophagy. We previously isolated lazarus (laz) suppressors of autoimmunity-triggered PCD in the Arabidopsis thaliana mutant accelerated cell death11 (acd11) and demonstrated that the cell death phenotype is due to ectopic activation of the LAZ5 NB-LRR. We report here that laz4 is mutated in one of three VACUOLAR PROTEIN SORTING35 (VPS35) genes. We verify that LAZ4/VPS35B is part of the retromer complex, which functions in endosomal protein sorting and vacuolar trafficking. We show that VPS35B acts in an endosomal trafficking pathway and plays a role in LAZ5-dependent acd11 cell death. Furthermore, we find that VPS35 homologs contribute to certain forms of NB-LRR protein-mediated autoimmunity as well as pathogen-triggered HR. Finally, we demonstrate that retromer deficiency causes defects in late endocytic/lytic compartments and impairs autophagy-associated vacuolar processes. Our findings indicate important roles of retromer-mediated trafficking during the HR; these may include endosomal sorting of immune components and targeting of vacuolar cargo., (© 2015 American Society of Plant Biologists. All rights reserved.)

Cytokinesis separates the forming daughter cells. Higher plants have lost the ability to constrict the plasma membrane (PM) in the division plane. Instead, trans-Golgi network (TGN)-derived membrane vesicles are targeted to the centre of the division plane and generate, by homotypic fusion, the partitioning membrane named cell plate (CP). The CP expands in a centrifugal fashion until its margin fuses with the PM at the cortical division site. Mutant screens in Arabidopsis have identified a cytokinesis-specific syntaxin named KNOLLE and an interacting Sec1/Munc18 (SM) protein named KEULE both of which are required for vesicle fusion during cytokinesis. KNOLLE is only made during M-phase, targeted to the division plane and degraded in the vacuole at the end of cytokinesis. Here we address mechanisms of KNOLLE trafficking and interaction of KNOLLE with different soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) partners and with SM-protein KEULE, ensuring membrane fusion in cytokinesis.

Plants detect microbes via two functionally interconnected tiers of immune receptors. Immune detection is suppressed by equally complex pathogen mechanisms. The small plasma-membrane-tethered protein RIN4 negatively regulates microbe-associated molecular pattern (MAMP)-triggered responses, which are derepressed upon bacterial flagellin perception. We demonstrate that recognition of the flagellin peptide MAMP flg22 triggers accumulation of RIN4 phosphorylated at serine 141 (pS141) that mediates derepression of several immune outputs. RIN4 is targeted by four bacterial type III effector proteins, delivered temporally after flagellin perception. Of these, AvrB acts with a host kinase to increase levels of RIN4 phosphorylated at threonine 166 (pT166). RIN4 pT166 is epistatic to RIN4 pS141. Thus, AvrB contributes to virulence by enhancing "rerepression" of immune system outputs. Our results explain the evolution of independent effectors that antagonize accumulation of RIN4 pS141 and of a specific plant intracellular NLR protein, RPM1, which is activated by AvrB-mediated accumulation of RIN4 pT166., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. Although membrane fusion is required for separating daughter cells in eukaryotic cytokinesis, the SNARE complexes involved are not known. In plants, membrane vesicles targeted to the cell division plane fuse with one another to form the partitioning membrane, progressing from the center to the periphery of the cell. In Arabidopsis, the cytokinesis-specific Qa-SNARE KNOLLE interacts with two other Q-SNAREs, SNAP33 and novel plant-specific SNARE 11 (NPSN11), whose roles in cytokinesis are not clear. Here we show by coimmunoprecipitation that KNOLLE forms two SNARE complexes that differ in composition. One complex is modeled on the trimeric plasma membrane type of SNARE complex and includes, in addition to KNOLLE, the promiscuous Qb,c-SNARE SNAP33 and the R-SNARE vesicle-associated membrane protein (VAMP) 721,722, also involved in innate immunity. In contrast, the other KNOLLE-containing complex is tetrameric and includes Qb-SNARE NPSN11, Qc-SNARE SYP71, and VAMP721,722. Elimination of only one or the other type of KNOLLE complex by mutation, including the double mutant npsn11 syp71, causes a mild or no cytokinesis defect. In contrast, the two double mutants snap33 npsn11 and snap33 syp71 eliminate both types of KNOLLE complexes and display knolle-like cytokinesis defects. Thus the two distinct types of KNOLLE complexes appear to jointly mediate membrane fusion in Arabidopsis cytokinesis.

Syntaxins and interacting SNARE proteins enable membrane fusion in diverse trafficking pathways. The Arabidopsis SYP1 family of plasma membrane-localized syntaxins comprises nine members, of which KNOLLE and PEN1 play specific roles in cytokinesis and innate immunity, respectively. To identify mechanisms conferring specificity of action, we examined one member of each subfamily-KNOLLE/SYP111, PEN1/SYP121 and SYP132-in regard to subcellular localization, dynamic behavior and complementation of knolle and pen1 mutants when expressed from the same promoters. Our results suggest that cytokinesis-specific syntaxin requires high-level accumulation during cell-plate formation, which necessitates de novo synthesis rather than endocytosis of pre-made protein from the plasma membrane. In contrast, syntaxin in innate immunity does not need upregulation of expression but instead requires pathogen-induced and endocytosis-dependent retargeting to the infection site. This feature of PEN1 is not afforded by SYP132. Additionally, PEN1 could not substitute for KNOLLE because of SNARE domain differences, as revealed by protein chimeras. In contrast, SYP132 was able to rescue knolle as did KNOLLE-SYP132 chimeras. Unlike KNOLLE and PEN1, which appear to have evolved to perform specialized functions, SYP132 stably localized at the plasma membrane and thus might play a role in constitutive membrane fusion., (© 2011 John Wiley & Sons A/S.)

Membrane traffic contributes to plant growth and development. However, the functional significance of SNARE proteins involved in membrane fusion of the early secretory pathway has not been explored with respect to plant development. Here we analyze the Arabidopsis v-SNARE SEC22. Loss of SEC22 function impairs gametophyte development, as indicated by reciprocal crosses between wild-type plants and plants heterozygous for T-DNA insertions in the SEC22 gene. sec22 mutant pollen becomes abnormal during the bicellular stage, eventually giving rise to degenerated pollen grains. Most mutant embryo sacs fail to support embryogenesis and display unfused polar nuclei in their central cell. Immunolocalization by both light and electron microscopy revealed an association of mutant-complementing Myc-tagged SEC22 with the central and peripheral endoplasmic reticulum (ER). Ultrastructural analysis of developing sec22 mutant pollen demonstrated Golgi fragmentation and consumption. As a consequence, the plasma membrane-targeted syntaxin SYP124 was retained in the ER. Our results suggest that SEC22 plays an essential role in early secretory traffic between the ER and the Golgi., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Immunolabelling of (ultra)thin thawed cryosections according to Tokuyasu is one of the most reliable and efficient immunolocalisation techniques for cells and tissues. However, chemical fixation at ambient temperature, a prerequisite of this technique, can cause problems for samples, like plant tissue, because cell walls, hydrophobic surfaces and intercellular air slow down diffusion of fixative molecules into the sample. We show that a hybrid technique, based on a combination of cryofixation/freeze-substitution and Tokuyasu cryosection immunolabelling, circumvents the disadvantages associated with chemical fixation and results in an improved ultrastructure and antigenicity preservation of Tokuyasu cryosections used for light and electron microscopic immunolabelling (as shown for Myc- or mRFP-tagged proteins, KNOLLE and carbohydrate epitopes). In combination with the most sensitive particulate marker systems, like 1-nm gold or quantum dot markers, we were able to obtain a differentiated labelling pattern which allows a more detailed evaluation of plant Golgi, trans-Golgi network and multivesicular body/prevacuolar compartment markers (COPI-specific gammaCOP, the ADP-ribosylation factor GTPase ARF1, ARA7/RabF2b and the vacuolar sorting receptor VSR). We also discuss possibilities to improve membrane contrast, e.g., of transport vesicles like COPI, COPII and clathrin-coated vesicles, and of compartments of endosomal trafficking like the trans-Golgi network., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Cell-autonomous immunity is widespread in plant-fungus interactions and terminates fungal pathogenesis either at the cell surface or after pathogen entry. Although post-invasive resistance responses typically coincide with a self-contained cell death of plant cells undergoing attack by parasites, these cells survive pre-invasive defence. Mutational analysis in Arabidopsis identified PEN1 syntaxin as one component of two pre-invasive resistance pathways against ascomycete powdery mildew fungi. Here we show that plasma-membrane-resident PEN1 promiscuously forms SDS-resistant soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complexes together with the SNAP33 adaptor and a subset of vesicle-associated membrane proteins (VAMPs). PEN1-dependent disease resistance acts in vivo mainly through two functionally redundant VAMP72 subfamily members, VAMP721 and VAMP722. Unexpectedly, the same two VAMP proteins also operate redundantly in a default secretory pathway, suggesting dual functions in separate biological processes owing to evolutionary co-option of the default pathway for plant immunity. The disease resistance function of the secretory PEN1-SNAP33-VAMP721/722 complex and the pathogen-induced subcellular dynamics of its components are mechanistically reminiscent of immunological synapse formation in vertebrates, enabling execution of immune responses through focal secretion.

Plant cytokinesis starts in the center of the division plane, with vesicle fusion generating a new membrane compartment, the cell plate, that subsequently expands laterally by continuous fusion of newly arriving vesicles to its margin. Targeted delivery of vesicles is assisted by the dynamic reorganization of a plant-specific cytoskeletal array, the phragmoplast, from a solid cylinder into an expanding ring-shaped structure. This lateral translocation is brought about by depolymerization of microtubules in the center, giving way to the expanding cell plate, and polymerization of microtubules along the edge. Whereas several components are known to mediate cytokinetic vesicle fusion [8-10], no gene function involved in phragmoplast dynamics has been identified by mutation. Mutations in the Arabidopsis HINKEL gene cause cytokinesis defects, such as enlarged cells with incomplete cell walls and multiple nuclei. Proper targeting of the cytokinesis-specific syntaxin KNOLLE [8] and lateral expansion of the phragmoplast are not affected. However, the phragmoplast microtubules appear to persist in the center, where vesicle fusion should result in cell plate formation. Molecular analysis reveals that the HINKEL gene encodes a plant-specific kinesin-related protein with a putative N-terminal motor domain and is expressed in a cell cycle-dependent manner similar to the KNOLLE gene. Our results suggest that HINKEL plays a role in the reorganization of phragmoplast microtubules during cell plate formation.