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1.
Structure-function analysis of the coiled-coil and leucine-rich repeat domains of the RPS5 disease resistance protein 总被引:1,自引:0,他引:1
The Arabidopsis (Arabidopsis thaliana) RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) disease resistance protein mediates recognition of the Pseudomonas syringae effector protein AvrPphB. RPS5 belongs to the coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) family and is activated by AvrPphB-mediated cleavage of the protein kinase PBS1. Here, we present a structure-function analysis of the CC and LRR domains of RPS5 using transient expression assays in Nicotiana benthamiana. We found that substituting the CC domain of RPS2 for the RPS5 CC domain did not alter RPS5 specificity and only moderately reduced its ability to activate programmed cell death, suggesting that the CC domain does not play a direct role in the recognition of PBS1 cleavage. Analysis of an RPS5-super Yellow Fluorescent Protein fusion revealed that RPS5 localizes to the plasma membrane (PM). Alanine substitutions of predicted myristoylation (glycine-2) and palmitoylation (cysteine-4) residues affected RPS5 PM localization, protein stability, and function in an additive manner, indicating that PM localization is essential to RPS5 function. The first 20 amino acids of RPS5 were sufficient for directing super Yellow Fluorescent Protein to the PM. C-terminal truncations of RPS5 revealed that the first four LRR repeats are sufficient for inhibiting RPS5 autoactivation; however, the complete LRR domain was required for the recognition of PBS1 cleavage. Substitution of the RPS2 LRR domain resulted in the autoactivation of RPS5, indicating that the LRR domain must coevolve with the NBS domain. We conclude that the RPS5 LRR domain functions to suppress RPS5 activation in the absence of PBS1 cleavage and promotes RPS5 activation in its presence. 相似文献
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Dong Qi Ullrich Dubiella Sang Hee Kim D. Isaiah Sloss Robert H. Dowen Jack E. Dixon Roger W. Innes 《Plant physiology》2014,164(1):340-351
The recognition of pathogen effector proteins by plants is typically mediated by intracellular receptors belonging to the nucleotide-binding leucine-rich repeat (NLR) family. NLR proteins often detect pathogen effector proteins indirectly by detecting modification of their targets. How NLR proteins detect such modifications is poorly understood. To address these questions, we have been investigating the Arabidopsis (Arabidopsis thaliana) NLR protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5), which detects the Pseudomonas syringae effector protein Avirulence protein Pseudomonas phaseolicolaB (AvrPphB). AvrPphB is a cysteine protease that specifically targets a subfamily of receptor-like cytoplasmic kinases, including the Arabidopsis protein kinase AVRPPHB Susceptible1 (PBS1). RPS5 is activated by the cleavage of PBS1 at the apex of its activation loop. Here, we show that RPS5 activation requires that PBS1 be localized to the plasma membrane and that plasma membrane localization of PBS1 is mediated by amino-terminal S-acylation. We also describe the development of a high-throughput screen for mutations in PBS1 that block RPS5 activation, which uncovered four new pbs1 alleles, two of which blocked cleavage by AvrPphB. Lastly, we show that RPS5 distinguishes among closely related kinases by the amino acid sequence (SEMPH) within an exposed loop in the C-terminal one-third of PBS1. The SEMPH loop is located on the opposite side of PBS1 from the AvrPphB cleavage site, suggesting that RPS5 associates with the SEMPH loop while leaving the AvrPphB cleavage site exposed. These findings provide support for a model of NLR activation in which NLR proteins form a preactivation complex with effector targets and then sense a conformational change in the target induced by effector modification.Pathogen recognition by plants is mediated by both transmembrane cell surface receptors and intracellular receptors (Jones and Dangl, 2006). The latter receptors typically belong to the nucleotide-binding leucine-rich repeat (NLR) superfamily of proteins, which also play a central role in the innate immune systems of many animals, including humans (von Moltke et al., 2013). In plants, most NLR proteins detect pathogen “effector” proteins, which are proteins secreted by pathogens to promote virulence on susceptible hosts. The immune response activated by NLR proteins is thus referred to as effector-triggered immunity. In the majority of examples studied, effector-triggered immunity is accompanied by localized host cell death around the site of pathogen ingress, which is referred to as the hypersensitive response (HR; Goodman and Novacky, 1994).Several NLR proteins have been shown to detect pathogen effector proteins indirectly by detecting the modification of other host proteins mediated by the effectors (DeYoung and Innes, 2006). The best characterized examples of NLR proteins that employ indirect recognition mechanisms are the RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) and RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2) proteins of Arabidopsis (Arabidopsis thaliana), which detect modification to the RPM1 INTERACTING4 (RIN4) protein (Mackey et al., 2002; Axtell and Staskawicz, 2003), the RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) protein of Arabidopsis, which detects modification of the AVRPPHB SUSCEPTIBLE1 (PBS1) protein kinase (Ade et al., 2007), and the Pseudomonas resistance and fenthion sensitivity (Prf) protein of tomato (Solanum lycopersicum), which detects modification of the Pseudomonas syringae pv tomato resistance (Pto) protein kinase (Salmeron et al., 1996; Rathjen et al., 1999). Our group has focused on RPS5, which detects the effector protein Avirulence protein Pseudomonas phaseolicolaB (AvrPphB) from Pseudomonas syringae (Simonich and Innes, 1995). AvrPphB functions as a Cys protease (Zhu et al., 2004) and specifically targets a subclass of plant receptor-like cytoplasmic kinases that include PBS1 (Shao et al., 2003; Zhang et al., 2010). AvrPphB likely targets these kinases in order to suppress defense responses induced by cell surface-localized plant immune receptors such as FLAGELLIN SENSITIVE2 (FLS2; Zhang et al., 2010). PBS1 can be coimmunoprecipitated with FLS2, and mutation of PBS1 reduces FLS2-mediated production of hydrogen peroxide and callose deposits (Zhang et al., 2010), confirming that PBS1 functions in defense signaling.Cleavage of PBS1 by AvrPphB is both necessary and sufficient to activate RPS5 (Ade et al., 2007), and null mutations in PBS1 block RPS5 activation (Swiderski and Innes, 2001). Because AvrPphB can cleave multiple closely related kinases in Arabidopsis (Zhang et al., 2010), these observations indicate that RPS5 can distinguish among these kinases, with only PBS1 cleavage activating RPS5. The molecular basis for this specificity is unknown.One contributor to the specificity of RPS5 may be subcellular localization. RPS5 localizes to the plasma membrane (PM), and amino acid substitutions that displace RPS5 from the PM eliminate RPS5-mediated defense responses (Qi et al., 2012). PBS1 is also expected to localize to the PM, because fusion of the N-terminal 100 amino acids of PBS1 to GFP causes GFP to localize to the PM in both Arabidopsis and Nicotiana benthamiana (Takemoto et al., 2012). Consistent with this expectation, PBS1 and RPS5 can be coimmunoprecipitated when transiently overexpressed in N. benthamiana (Ade et al., 2007). Furthermore, AvrPphB is both myristoylated and palmitoylated upon entry into plant cells and localizes to the PM, with PM localization of AvrPphB being required for the activation of RPS5 (Dowen et al., 2009). Although these data all point to a PM localization for PBS1, full-length PBS1 protein has not yet been localized, nor has the functional significance of PBS1 localization been assessed relative to the activation of RPS5.In this study, we demonstrate that PBS1 is targeted to the PM via S-acylation at its N terminus and that PM localization is required for RPS5 activation. We also describe a high-throughput genetic screen for uncovering new mutations in PBS1 that block RPS5 activation, which uncovered four new pbs1 alleles. Lastly, we show that RPS5 distinguishes PBS1 from closely related kinases based on a specific loop in the C-terminal half of PBS1. 相似文献
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The Arabidopsis PBS1 resistance gene encodes a member of a novel protein kinase subfamily 总被引:1,自引:0,他引:1
Specific recognition of Pseudomonas syringae strains that express the avirulence gene avrPphB requires two genes in Arabidopsis, RPS5 and PBS1. Previous work has shown that RPS5 encodes a member of the nucleotide binding site-leucine rich repeat class of plant disease resistance genes. Here we report that PBS1 encodes a putative serine-threonine kinase. Southern blot analysis revealed that the pbs1-1 allele contained a deletion of the 3' end of the PBS1 open reading frame. DNA sequence analysis of the pbs1-2 allele showed it to be a missense mutation that caused a glycine to arginine substitution in the activation segment of PBS1, a region known to regulate substrate binding and catalytic activity in many protein kinases. The identity of PBS1 was confirmed using both transient transformation and stable transformation of mutant pbs1 plants. Comparison of the predicted PBS1 amino acid sequence with other plant protein kinases revealed that PBS1 belongs to a distinct subfamily of protein kinases that contains no other members of known function. The Pto kinase of tomato, which is required for specific resistance to P. syringae strains expressing avrPto, did not fall in the same subfamily as PBS1 and is only 42% identical in the kinase domain. These data suggest that PBS1 and Pto may fulfil different functions in the recognition of pathogen avirulence proteins. We discuss several possible models for the roles of PBS1 and RPS5 in AvrPphB recognition. 相似文献
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Runyao Bai Huanhuan Li Wenjia Du Niu Niu Wenxia Li Qican Gao Chongyang Yao Zikai Zhou Wenhua Bao Mingjia Che Yongxiu Zhao Bin Zhou Yaohui Wang Hada Wuriyanghan 《Molecular Plant Pathology》2022,23(6):901-908
Potato virus Y (PVY) is an important pathogen of potato (Solanum tuberosum). Although the PBS1–RPS5 immune system is well documented in Arabidopsis thaliana, it has not been reported in potato. In Arabidopsis, the bacterial effector AvrPphB cleaves AtPBS1 to trigger an immune response. Here, we show that the AvrPphB-triggered immune response is mediated by StPBS1, a close homologue of AtPBS1 in potato. However, downstream signalling of StPBS1 was mediated by unknown resistance (R) proteins other than potato orthologues of AtRPS5 and HvPBR1, which is important for HvPBS1 signalling in barley. Immune signalling of StPBS1 is mediated by the AvrPphB C-terminal cleavage domain and an STKPQ motif, in contrast to AtPBS1-mediated immunity in which both AvrPphB cleavage fragments and an SEMPH motif are essential. The cleavage sequence of AvrPphB in StPBS1 was replaced with that of the PVY NIa-Pro protease to obtain StPBS1NIa. StPBS1NIa overexpression potato displayed stronger immunity to PVY infection than did the StPBS1 transgenic lines. StPBS1NIa was cleaved at the expected target site by NIa-Pro protease from PVY. Thus, we characterized the function of StPBS1 in potato immunity and provide a biotechnology control method for PVY via transformation of decoy-engineered StPBS1NIa. 相似文献
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Huiren Cai Wei Wang Lu Rui Libo Han Mingyu Luo Na Liu Dingzhong Tang 《The Plant journal : for cell and molecular biology》2021,107(3):775-786
Nucleotide-binding site (NBS)–leucine-rich repeat (LRR) domain receptor (NLR) proteins play important roles in plant innate immunity by recognizing pathogen effectors. The Toll/interleukin-1 receptor (TIR)-NBS (TN) proteins belong to a subtype of the atypical NLRs, but their function in plant immunity is poorly understood. The well-characterized Arabidopsis thaliana typical coiled-coil (CC)-NBS-LRR (CNL) protein Resistance to Pseudomonas syringae 5 (RPS5) is activated after recognizing the Pseudomonas syringae type III effector AvrPphB. To explore whether the truncated TN proteins function in CNL-mediated immune signaling, we examined the interactions between the Arabidopsis TN proteins and RPS5, and found that TN13 and TN21 interacted with RPS5. However, only TN13, but not TN21, was involved in the resistance to P. syringae pv. tomato (Pto) strain DC3000 carrying avrPphB, encoding the cognate effector recognized by RPS5. Moreover, the regulation of Pto DC3000 avrPphB resistance by TN13 appeared to be specific, as loss of function of TN13 did not compromise resistance to Pto DC3000 hrcC− or Pto DC3000 avrRpt2. In addition, we demonstrated that the CC and NBS domains of RPS5 play essential roles in the interaction between TN13 and RPS5. Taken together, our results uncover a direct functional link between TN13 and RPS5, suggesting that TN13 acts as a partner in modulating RPS5-activated immune signaling, which constitutes a previously unknown mechanism for TN-mediated regulation of plant immunity. 相似文献
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Innes R 《Molecular microbiology》2003,50(2):363-365
The enzymatic activities and/or targets of four type III effector proteins from plant pathogens have been reported in a flurry of new papers. In this issue, XopD is shown to remove SUMO groups from host cell proteins, while in previous issues of Molecular Microbiology, HopPtoD2 was shown to function as a tyrosine phosphatase and AvrRpt2 as probably a cysteine protease that targets the host RIN4 protein. Finally, AvrPphB is revealed in a recent Science paper to function as a cysteine protease that targets the host PBS1 kinase. This work is providing some of the first insights into how plant pathogens subvert host cell signalling machinery to cause disease. 相似文献
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Arabidopsis (Arabidopsis thaliana) NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), a plasma membrane-localized protein, plays an essential role in resistance mediated by the coiled-coil-nucleotide-binding site-leucine-rich repeat class of resistance (R) proteins, which includes RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2), RESISTANCE TO PSEUDOMONAS SYRINGAE PV MACULICOLA1, and RPS5. Infection with Pseudomonas syringae pv tomato DC3000 expressing the bacterial effector proteins AvrRpt2, AvrB, and AvrPphB activates resistance by the aforementioned R proteins. Whereas the genetic requirement for NDR1 in plant disease resistance signaling has been detailed, our study focuses on determining a global, physiological role for NDR1. Through the use of homology modeling and structure threading, NDR1 was predicted to have a high degree of structural similarity to Arabidopsis LATE EMBRYOGENESIS ABUNDANT14, a protein implicated in abiotic stress responses. Specific protein motifs also point to a degree of homology with mammalian integrins, well-characterized proteins involved in adhesion and signaling. This structural homology led us to examine a physiological role for NDR1 in preventing fluid loss and maintaining cell integrity through plasma membrane-cell wall adhesions. Our results show a substantial alteration in induced (i.e. pathogen-inoculated) electrolyte leakage and a compromised pathogen-associated molecular pattern-triggered immune response in ndr1-1 mutant plants. As an extension of these analyses, using a combination of genetic and cell biology-based approaches, we have identified a role for NDR1 in mediating plasma membrane-cell wall adhesions. Taken together, our data point to a broad role for NDR1 both in mediating primary cellular functions in Arabidopsis through maintaining the integrity of the cell wall-plasma membrane connection and as a key signaling component of these responses during pathogen infection. 相似文献
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A Yersinia effector and a Pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis 总被引:7,自引:0,他引:7
A Yersinia effector known as YopT and a Pseudomonas avirulence protein known as AvrPphB define a family of 19 proteins involved in bacterial pathogenesis. We show that both YopT and AvrPphB are cysteine proteases, and their proteolytic activities are dependent upon the invariant C/H/D residues conserved in the entire YopT family. YopT cleaves the posttranslationally modified Rho GTPases near their carboxyl termini, releasing them from the membrane. This leads to the disruption of actin cytoskeleton in host cells. The proteolytic activity of AvrPphB is essential for autoproteolytic cleavage of an AvrPphB precursor as well as for eliciting the hypersensitive response in plants. These findings provide new insights into mechanisms of animal and plant pathogenesis. 相似文献
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Arabidopsis RPS2 is a typical disease resistance (R) protein with nucleotide-binding leucine-rich repeats (NB-LRR). Previously, we reported that RPS2 is physically associated with some Arabidopsis hypersensitive induced reaction (AtHIR) proteins, which are enriched in membrane microdomains. Biochemical and genetic analyses suggested that members of the AtHIR gene family have a function in RPS2-mediated immune signaling. Here, we provide evidence that the pattern recognition receptor (PRR) FLS2 is also physically associated with AtHIR2 in a N. benthamiana transient expression system. We thus speculate that PM microdomains provide a platform for both types of immune receptors, R proteins and PRRs, and that the activation of the receptors is facilitated by AtHIR proteins. 相似文献
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Arabidopsis RIN4 negatively regulates disease resistance mediated by RPS2 and RPM1 downstream or independent of the NDR1 signal modulator and is not required for the virulence functions of bacterial type III effectors AvrRpt2 or AvrRpm1 总被引:10,自引:0,他引:10
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Bacterial pathogens deliver type III effector proteins into the plant cell during infection. On susceptible (r) hosts, type III effectors can contribute to virulence. Some trigger the action of specific disease resistance (R) gene products. The activation of R proteins can occur indirectly via modification of a host target. Thus, at least some type III effectors are recognized at site(s) where they may act as virulence factors. These data indicate that a type III effector's host target might be required for both initiation of R function in resistant plants and pathogen virulence in susceptible plants. In Arabidopsis thaliana, RPM1-interacting protein 4 (RIN4) associates with both the Resistance to Pseudomonas syringae pv maculicola 1 (RPM1) and Resistance to P. syringae 2 (RPS2) disease resistance proteins. RIN4 is posttranslationally modified after delivery of the P. syringae type III effectors AvrRpm1, AvrB, or AvrRpt2 to plant cells. Thus, RIN4 may be a target for virulence functions of these type III effectors. We demonstrate that RIN4 is not the only host target for AvrRpm1 and AvrRpt2 in susceptible plants because its elimination does not diminish their virulence functions. In fact, RIN4 negatively regulates AvrRpt2 virulence function. RIN4 also negatively regulates inappropriate activation of both RPM1 and RPS2. Inappropriate activation of RPS2 is nonspecific disease resistance 1 (NDR1) independent, in contrast with the established requirement for NDR1 during AvrRpt2-dependent RPS2 activation. Thus, RIN4 acts either cooperatively, downstream, or independently of NDR1 to negatively regulate RPS2 in the absence of pathogen. We propose that many P. syringae type III effectors have more than one target in the host cell. We suggest that a limited set of these targets, perhaps only one, are associated with R proteins. Thus, whereas any pathogen virulence factor may have multiple targets, the perturbation of only one is necessary and sufficient for R activation. 相似文献
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Kim MG Geng X Lee SY Mackey D 《The Plant journal : for cell and molecular biology》2009,57(4):645-653
Plant disease resistance (R) proteins recognize potential pathogens expressing corresponding avirulence (Avr) proteins through 'gene-for-gene' interactions. RPM1 is an Arabidopsis R-protein that triggers a robust defense response upon recognizing the Pseudomonas syringae effector AvrRpm1. Avr-proteins of phytopathogenic bacteria include type III effector proteins that are often capable of enhancing virulence when not recognized by an R-protein. In rpm1 plants, AvrRpm1 suppresses basal defenses induced by microbe-associated molecular patterns. Here, we show that expression of AvrRpm1 in rpm1 plants induced PR-1, a classical defense marker, and symptoms including chlorosis and necrosis. PR-1 expression and symptoms were reduced in plants with mutations in defense signaling genes ( pad4 , sid2 , npr1 , rar1 , and ndr1 ) and were strongly reduced in rpm1 rps2 plants, indicating that AvrRpm1 elicits defense signaling through the Arabidopsis R-protein, RPS2. Bacteria expressing AvrRpm1 grew more on rpm1 rps2 than on rpm1 plants. Thus, independent of its classical 'gene-for-gene' activation of RPM1, AvrRpm1 also induces functionally relevant defenses that are dependent on RPS2. Finally, AvrRpm1 suppressed host defenses and promoted the growth of type III secretion mutant bacteria equally well in rps2 and RPS2 plants, indicating that virulence activity of over-expressed AvrRpm1 predominates over defenses induced by weak activation of RPS2. 相似文献
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Effector proteins injected by the pathogenic bacteria Pseudomonas syringae into plants can have profound effects on the pathogen-host interaction due to their efficient recognition by plants and the subsequent triggering of defenses. The AvrRpt2 effector triggers strong local and systemic defense (called systemic acquired resistance [SAR]) responses in Arabidopsis thaliana plants that harbor a functional RPS2 gene that encodes an R protein in the coiled-coil, nucleotide-binding domain, leucine-rich repeat class. The newly identified win3-T mutant shows greatly reduced resistance to P syringae carrying avrRpt2. In win3-T plants, RIN4 cleavage, an early AvrRpt2-induced event, is normal. However, salicylic acid accumulation is compromised, as is SAR induction and the local hypersensitive cell death response after infection by P syringae carrying avrRpt2. WIN3 encodes a member of the firefly luciferase protein superfamily. Expression of WIN3 at an infection site partially requires PAD4, a protein known to play a quantitative role in RPS2-mediated signaling. WIN3 expression in tissue distal to an infection site requires multiple salicylic acid regulatory genes. Finally, win3-T plants show modestly increased susceptibility to virulent P syringae and modestly reduced SAR in response to P. syringae carrying avrRpm1. Thus, WIN3 is a key element of the RPS2 defense response pathway and a basal and systemic defense component. 相似文献
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The Pseudomonas syringae type III effector tyrosine phosphatase HopAO1 suppresses innate immunity in Arabidopsis thaliana 总被引:2,自引:0,他引:2
The bacterial pathogen Pseudomonas syringae pv. tomato (Pst) strain DC3000 infects tomato and Arabidopsis plants, and is a model for studying the molecular basis of bacterial disease. Pst DC3000 secretes a battery of largely uncharacterized effector proteins into host cells via a type-III secretion system (TTSS). Little is currently known about the molecular mechanisms by which individual TTSS effectors promote virulence. The effector HopAO1 has similarity to protein tyrosine phosphatases, including a conserved catalytic site, and suppresses the hypersensitive response (HR) in some non-host plants. Whether HopAO1 has a similar effect in the host Arabidopsis is not clear. Here, we show that transgenic expression of HopAO1 in Arabidopsis suppresses callose deposition elicited by the Pst DC3000 hrpA mutant, and allows the normally non-pathogenic hrpA mutant to multiply within the leaf tissue. HopAO1 also suppresses resistance to Pst DC3000 induced by flg22, a pathogen-associated molecular pattern (PAMP). However, HopAO1 does not suppress the HR triggered by several classical avirulence genes. These results suggest that HopAO1 targets primarily PAMP-induced innate immunity in Arabidopsis. The virulence function of HopAO1 is dependent on an intact phosphatase catalytic site, as transgenic plants expressing a catalytically inactive derivative do not show these effects. Intriguingly, expression of the catalytically inactive HopAO1 has a dominant-negative effect on the function of the wild-type HopAO1. Analysis of mitogen-activated protein kinase (MAPK) activity suggests that HopAO1 targets a step downstream or independent of MAPK activation. Genome-wide expression analysis revealed that expression of several well-known defense genes was suppressed in hrpA mutant-infected HopAO1 transgenic plants. 相似文献
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Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4 总被引:38,自引:0,他引:38
Plants have evolved a sophisticated innate immune system to recognize invading pathogens and to induce a set of host defense mechanisms resulting in disease resistance. Pathogen recognition is often mediated by plant disease resistance (R) proteins that respond specifically to one or a few pathogen-derived molecules. This specificity has led to suggestions of a receptor-ligand mode of R protein function. Delivery of the bacterial effector protein AvrRpt2 by Pseudomonas syringae specifically induces disease resistance in Arabidopsis plants expressing the RPS2 R protein. We demonstrate that RPS2 physically interacts with Arabidopsis RIN4 and that AvrRpt2 causes the elimination of RIN4 during activation of the RPS2 pathway. AvrRpt2-mediated RIN4 elimination also occurs in the rps2, ndr1, and Atrar1 mutant backgrounds, demonstrating that this activity can be achieved independent of an RPS2-mediated signaling pathway. Therefore, we suggest that RPS2 initiates signaling based upon perception of RIN4 disappearance rather than direct recognition of AvrRpt2. 相似文献