Convergent evolution of disease resistance genes

The resistance genes Rpg1-b in soybean and RPM1 in Arabidopsis recognize the same bacterial avirulence protein (AvrB). Recent map-based cloning of Rpg1-b has provided the first opportunity to compare functionally analogous R genes in distantly related species. Rpg1-b and RPM1 are not orthologs. Rather, these genes descended from distinct evolutionary lineages in which recognition of AvrB has probably evolved independently. This result, together with new insights into RPM1-mediated recognition of AvrB, provides an exciting opportunity to reconsider classical views on the evolution of pathogen recognition specificity.     

Expression of the Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specific hypersensitive cell death.

The nonpathogenic bacteria Pseudomonas fluorescens and Escherichia coli can elicit a genotype-specific hypersensitive response (HR) in plants if they express both the HR and pathogenesis (Hrp) protein secretion system and the HrpZ harpin from P. syringae pv syringae 61 and a P. syringae avirulence (avr) gene whose presence is recognized by a corresponding disease resistance gene in the plant. We have found that the recognition event appears to require transfer of the Avr protein into the plant cell. Elicitation of a genotype-specific HR was observed with avrB+ P. fluorescens in soybean and Arabidopsis plants carrying resistance genes RPG1 and RPM1, respectively, and with avrPto+ E. coll in tomato plants carrying resistance gene PTO, but only if the Hrp secretion system, HrpZ, and the appropriate Avr proteins were produced in the same bacterial cell. The failure of avrB hyperexpression and exogenous AvrB or HrpZ to alleviate these requirements in soybean and Arabidopsis suggests that the site of AvrB action is not in the bacterial cell or plant apoplast. An Arabidopsis rps3 (rpm1) glabrous1 mutant was transformed with constructs expressing avrB and was crossed with an Arabidopsis ecotype Columbia (RPM1 GLABROUS1) plant. F1 seedlings (identified by their kanamycin-resistant, pubescent phenotype) exhibited extensive necrosis on cotyledon leaves 10 days postgermination. Ecotype Columbia and rps3-1 leaves biolistically cobombarded with plasmids expressing the beta-glucuronidase (GUS) gene and avrB failed to produce GUS activity (indicative of cell death) only when RPM1 and avrB were present in the leaf. Thus, both stable and transient expression of avrB in Arabidopsis resulted in RPM1-dependent necrosis, and the only demonstrable site of action for AvrB was inside plant cells.     

Convergent evolution of disease resistance gene specificity in two flowering plant families

Plant disease resistance (R) genes that mediate recognition of the same pathogen determinant sometimes can be found in distantly related plant families. This observation implies that some R gene alleles may have been conserved throughout the diversification of land plants. To address this question, we have compared R genes from Glycine max (soybean), Rpg1-b, and Arabidopsis thaliana, RPM1, that mediate recognition of the same type III effector protein from Pseudomonas syringae, AvrB. RPM1 has been cloned previously, and here, we describe the isolation of Rpg1-b. Although RPM1 and Rpg1-b both belong to the coiled-coil nucleotide binding site (NBS) Leu-rich repeat (LRR) class of R genes, they share only limited sequence similarity outside the conserved domains characteristic of this class. Phylogenetic analyses of A. thaliana and legume NBS-LRR sequences demonstrate that Rpg1-b and RPM1 are not orthologous. We conclude that convergent evolution, rather than the conservation of an ancient specificity, is responsible for the generation of these AvrB-specific genes.     

Pseudomonas syringae effector avrB confers soybean cultivar-specific avirulence on Soybean mosaic virus adapted for transgene expression but effector avrPto does not

Soybean mosaic virus (SMV) was adapted for transgene expression in soybean and used to examine the function of avirulence genes avrB and avrPto of Pseudomonas syringae pvs. glycinea and tomato, respectively. A cloning site was introduced between the P1 and HC-Pro genes in 35S-driven infectious cDNAs of strains SMV-N and SMV-G7. Insertion of the uidA gene or the green fluorescent protein gene into either modified cDNA and bombardment into primary leaves resulted in systemic expression that reflected the pattern of viral movement into uninoculated leaves. Insertion of avrB blocked symptom development and detectable viral movement in cv. Harosoy, which carries the Rpg1-b resistance gene corresponding to avrB, but not in cvs. Keburi or Hurrelbrink, which lack Rpg1-b. In Keburi and Hurrelbrink, symptoms caused by SMV carrying avrB appeared more quickly and were more severe than those caused by the virus without avrB. Insertion of avrPto enhanced symptoms in Harosoy, Hurrelbrink, and Keburi. This result was unexpected because avrPto was reported to confer avirulence on P. syringae pv. glycinea inoculated to Harosoy. We inoculated Harosoy with P syringae pv. glycinea expressing avrPto, but observed no hypersensitive reaction, avrPto-dependent induction of pathogenesis-related protein la, or limitation of bacterial population growth. In Hurrelbrink, avrPto enhanced bacterial multiplication and exacerbated symptoms. Our results establish SMV as an expression vector for soybean. They demonstrate that resistance triggered by avrB is effective against SMV, and that avrB and avrPto have general virulence effects in soybean. The results also led to a reevaluation of the reported avirulence activity of avrPto in this plant.     

A disease resistance gene in Arabidopsis with specificity for two different pathogen avirulence genes.

The RPS3 and RPM1 disease resistance loci of Arabidopsis confer resistance to Pseudomonas syringae strains that carry the avirulence genes avrB and avrRpm1, respectively. We have previously shown that RPS3 and RPM1 are closely linked genetically. Here, we show that RPS3 and RPM1 are in fact the same gene. We screened a mutagenized Arabidopsis population with a P. syringae strain carrying avrB and found 12 susceptible mutants. All 12 mutants were also susceptible to an isogenic strain carrying avrRpm1, indicating a loss of both RPS3 and RPM1 functions. No mutants were recovered that lost only RPS3 function. Genetic analysis of four independent mutants revealed that the lesions were in RPS3. Thus, a single gene in Arabidopsis confers resistance that is specific to two distinct pathogen avirulence genes--a gene-for-genes interaction. This observation suggests that the RPS3/RPM1 gene product can bind multiple pathogen ligands, or alternatively, that it does not function as a receptor.     

Type III effector activation via nucleotide binding, phosphorylation, and host target interaction

The Pseudomonas syringae type III effector protein avirulence protein B (AvrB) is delivered into plant cells, where it targets the Arabidopsis RIN4 protein (resistance to Pseudomonas maculicula protein 1 [RPM1]-interacting protein). RIN4 is a regulator of basal host defense responses. Targeting of RIN4 by AvrB is recognized by the host RPM1 nucleotide-binding leucine-rich repeat disease resistance protein, leading to accelerated defense responses, cessation of pathogen growth, and hypersensitive host cell death at the infection site. We determined the structure of AvrB complexed with an AvrB-binding fragment of RIN4 at 2.3 A resolution. We also determined the structure of AvrB in complex with adenosine diphosphate bound in a binding pocket adjacent to the RIN4 binding domain. AvrB residues important for RIN4 interaction are required for full RPM1 activation. AvrB residues that contact adenosine diphosphate are also required for initiation of RPM1 function. Nucleotide-binding residues of AvrB are also required for its phosphorylation by an unknown Arabidopsis protein(s). We conclude that AvrB is activated inside the host cell by nucleotide binding and subsequent phosphorylation and, independently, interacts with RIN4. Our data suggest that activated AvrB, bound to RIN4, is indirectly recognized by RPM1 to initiate plant immune system function.     

Crystal structure of the type III effector AvrB from Pseudomonas syringae

AvrB is a Pseudomonas syringae type III effector protein that is translocated into host plant cells during attempted pathogenesis. Arabidopsis harboring the corresponding resistance protein RPM1 can detect AvrB and mount a rapid host defense response, thus avoiding active infection. In the plant cell, AvrB induces phosphorylation of RIN4, a key component in AvrB/RPM1 recognition. Although the AvrB/RPM1 system is among the best characterized of the numerous bacterial effector/plant resistance protein systems involved in plant disease resistance and pathogenesis, the details of the molecular recognition mechanism are still unclear. To gain further insights, the crystal structure of AvrB was determined. The 2.2 A structure exhibits a novel mixed alpha/beta bilobal fold. Aided by the structural information, we demonstrate that one lobe is the determinant of AvrB/RPM1 recognition specificity. This structural information and preliminary structure-function studies provide a framework for the future understanding of AvrB function on the molecular level.     

Identification of a disease resistance locus in Arabidopsis that is functionally homologous to the RPG1 locus of soybean

A new disease resistance locus in Arabidopsis, RPS3 , was identified using a previously cloned avirulence gene from a non- Arabidopsis pathogen. The avrB avirulence gene from the soybean pathogen Pseudomonas syringae pv. glycinea was transferred into a P. syringae pv. tomato strain that is virulent on Arabidopsis , and conversion to avirulence was assayed on Arabidopsis plants. The avrB gene had avirulence activity on most, but not all, Arabidopsis ecotypes. Of 53 ecotypes examined, 45 were resistant to a P. syringae pv. tomato strain carrying avrB , and eight were susceptible. The inheritance of this resistance was examined using crosses between the resistant ecotype Col-0 and the susceptible ecotype Bla-2. In F₂ plants from this cross, the ratio of resistant:susceptible plants was approximately 3:1, indicating that resistance to P. syringae expressing avrB is determined by a single dominant locus in ecotype Col-0, which we have designated RPS3 . Using RFLP analysis, RPS3 was mapped to chromosome 3, adjacent to markers M583 and G4523, and ≤ 1 cM from another disease resistance locus, RPM1 . In soybean, resistance to P. syringae strains that carry avrB is controlled by the locus RPG1 . Thus, RPG1 and RPS3 both confer avrB -specific disease resistance, suggesting that these genes may be homologs.     

Soybean Resistance Genes Specific for Different Pseudomonas Syringae Avirulence Genes Are Allelic, or Closely Linked, at the Rpg1 Locus

RPG1 and RPM1 are disease resistance genes in soybean and Arabidopsis, respectively, that confer resistance to Pseudomonas syringae strains expressing the avirulence gene avrB. RPM1 has recently been demonstrated to have a second specificity, also conferring resistance to P. syringae strains expressing avrRpm1. Here we show that alleles, or closely linked genes, exist at the RPG1 locus in soybean that are specific for either avrB or avrRpm1 and thus can distinguish between these two avirulence genes.     

Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes

Alleles or tightly linked genes at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F. Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases. Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region. Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glycine. Members from different subgroups are physically intermixed and display a high level of polymorphism between soybean cultivars, suggesting that this region is rearranging at a high frequency. At least five CC-NBS-LRR-type genes cosegregate with Rpg1-b in our large mapping populations.     

RIN4-like proteins mediate resistance protein-derived soybean defense against Pseudomonas syringae

Resistance (R) protein mediated recognition of pathogen avirulence effectors triggers signaling that induces a very robust form of species-specific immunity in plants. The soybean Rpg1-b protein mediates this form of resistance against the bacterial blight pathogen, Pseudomonas syringae expressing AvrB_Pgyrace4. Likewise, the Arabidopsis RPM1 protein also mediates species-specific resistance against AvrB expressing bacteria. RPM1 and Rpg1-b are non-orthologous and differ in their requirements for downstream signaling components. We recently showed that the activation of Rpg1-b derived resistance signaling requires two host proteins that directly interact with AvrB. These proteins share high sequence similarity with the Arabidopsis RPM1 interacting protein 4 (RIN4), which is essential for RPM1-derived resistance. The two soybean RIN4-like proteins (GmRIN4a and b) differ in their abilities to interact with Rpg1-b as well as to complement the Arabidopsis rin4 mutation. Because the two GmRIN4 proteins interact with each other, we proposed that they might function as a heteromeric complex in mediating Rpg1-b-derived resistance. Absence of GmRIN4a or b enhanced basal resistance against bacterial and oomycete pathogens in soybean. Lack of GmRIN4a also enhanced the virulence of avrB bacteria in plants lacking Rpg1-b. Our studies suggest that multiple RIN4-like proteins proteins mediate R-mediated signaling, in soybean.Key words: AvrB, soybean defense, effector recognition, gaurdee, resistance protein, bacterial blight, gene silencingRecognition of pathogens in a species-specific manner results in the generation of a very robust mode of resistance in plants. This form of protection termed resistance (R) protein-mediated or effector-triggered immunity is induced when a plant encoded R protein “perceives” the presence of a pathogen-derived avirulence (Avr) effector. “Perception” occurs either via direct or indirect interactions between the R and Avr proteins.^1–7 One or more plant proteins, that themselves usually physically associate with the Avr and R proteins, mediate indirect R-Avr interactions. Such proteins have been termed “guardee” based upon the hypothesis that Avr-derived alterations of these proteins are guarded by R proteins.^5–7 First proposed to explain the perception of AvrPto_PtoJL1065 from Pseudomonas syringae in tomato,^8,9 the “guard” model has been extended to several other R-Avr interactions.^5,7,10 This mode of interaction is typified in the recognition of the Pseudomonas syringae AvrB effector by the Arabidopsis R protein, RPM1 (resistance to P. syringae pv. maculicola 1). RPM1 mediates resistance against bacteria expressing either AvrRpm1_PmaM6 or AvrB_Pgyrace4.¹¹ However, direct interactions between RPM1 and its cognate Avr proteins have not been detected. Rather, RPM1 associates with the host protein, RIN4 (RPM1-interacting 4), which in turn interacts with AvrRpm1 and AvrB. Consistent with its role as a “guardee” protein, RIN4 is required for RPM1-induced resistance and is phosphorylated by AvrRpm1/AvrB, albeit only in the presence of a plant-derived factor.¹² The phosphorylation status of RIN4 is likely monitored by RPM1 for the induction of resistance signaling. The “guard” model implies that unlike R proteins, “guardee” proteins are highly conserved. Indeed, RIN4-like proteins appear to be conserved in diverse plants including cowpea, lettuce, maize, potato, rice, tobacco and tomato.^13–16 Additionally, the tomato and lettuce RIN4 proteins are known to mediate defense against microbial pathogens.^14,15 However, due to the fact that “guardee” proteins have only been identified in the context of specific R-Avr pairs, their requirement in mediating responses to a common avirulence effector in diverse hosts has remained untested. We tested this corollary of the “guard” model in soybean since soybean too can induce resistance to AvrB expressing bacteria in an R gene-specific manner. We demonstrated that soybean does encode RIN4-like proteins and that these are important for mediating resistance to avrB P. syringae.¹⁷ This is an important finding since the soybean R protein Rpg1-b is non-orthologous to RPM1 and differs in its requirements for downstream signaling components.^18–21 Furthermore, unlike RPM1, Rpg1-b does not provide resistance against bacteria expressing the AvrRpm1 effector.²²Genome sequence search identified four genes encoding RIN4-like proteins in soybean, designated GmRIN4a-d. Both in planta bimolecular florescence complementation (BiFC) and in vitro “pull-down” assays detected binding between AvrB and all four GmRIN4 proteins, indicating that these interactions did not require additional plant-derived factors. Interactions were further confirmed by co-immunoprecipitation (Co-IP, Fig. 1). AvrB tagged with the FLAG (3X) epitope and the various GmRIN4 isoforms tagged with the MYC epitope were transiently expressed in Nicotiana benthamiana. Total protein extracts from leaves expressing AvrB-FLAG, GmRIN4a/b/c/d-MYC or co-expressing AvrB-FLAG with GmRIN4a/b/c/d-MYC proteins were used for immunoprecipitation with anti-FLAG antibodies. Immunoprecipitated proteins were visualized using anti-MYC antibodies in western blots (Fig. 1). Three of these (GmRIN4b, c and d) also interacted with Rpg1-b directly. However, GmRIN4a was unable to interact with Rpg1-b in planta or in vitro. Although GmRIN4a and b share very high amino acid identity (∼94%), only GmRIN4b interacted with Rpg1-b. However, silencing either GmRIN4a or b abrogated resistance to avrB bacteria in Rpg1-b plants, suggesting that both proteins were essential for the activation of Rpg1-b derived signaling.¹⁷ This raised the possibility that GmRIN4a and b might oligomerize to function in Rpg1-b-derived signaling. Indeed, GmRIN4b interacted with GmRIN4a as well as with GmRIN4c and d.¹⁷ GmRIN4b also interacted with itself. GmRIN4a, c and d neither interacted with each other, nor themselves. Together, these results suggest that the GmRIN4 isoforms might oligomerize. Whether the oligomer exists in the presence or absence of AvrB, and whether binding of one or the other isoform alters the dynamics of the complex to change affinities for Rpg1-b and/or AvrB, remains to be examined. The fact that the GmRIN4c and d isoforms also interact with Rpg1-b and that they associate with GmRIN4b raises the untested possibility that these too might function in Rpg1-b mediated resistance.Open in a separate windowFigure 1GmRIN4 proteins co-immunoprecipitate with AvrB. Agrobacterium cells expressing MYC-tagged GmRIN4a, b, c or d were expressed individually or together with FLAG (3X)-tagged AvrB in Nicotiana benthamiana. Proteins were immunoprecipitated (IP) from total extracts (T) using anti-FLAG antibodies, electrophoresed on SDS-PAGE and visualized using tagspecific antibodies (α-MYC for the various GmRIN4 proteins, α-FLAG for AvrB). Part showing AvrB is from the AvrB-GmRIN4a co-immunoprecipitation (Co-IP) experiment and is representative of Co-IPs with GmRIN4b, c and d.In Arabidopsis, RIN4 also associates with the RPS2 (resistance to P. syringae 2) protein, which mediates resistance against P. syringae expressing avrRpt2. RPS2-mediated signaling is activated when AvrRpt2_PtoJL1065, a cysteine protease, cleaves RIN4.^23–25 Since absence of RIN4 results in the ectopic induction of RPS2 activity and thereby lethality, the rin4 mutation can be generated only in plants lacking RPS2 (rps2). Absence of RIN4 also activates residual RPM1 activity.¹³ Therefore, rin4 rps2 plants exhibit increased PR-1 (pathogenesis related 1) gene expression and enhanced basal resistance to virulent bacteria. The residual RPM1 activity is not however sufficient to provide resistance against avrB or avrRpm1 expressing bacteria. Thus, rin4 rps2 plants are compromised in RPM1-derived resistance against the avrB/avrRpm1 bacterial strains. Interestingly, overexpression of GmRIN4b, but not GmRIN4a, was able to restore RPM1 function in the Arabidopsis rin4 rps2 mutant. Pathogen inoculation of transgenic Arabidopsis rin4 rps2 mutant plants constitutively expressing GmRIN4a showed that these plants were as susceptible to avrB or avrRpm1 P. syringae as the rin4 rps2 mutant. In contrast, the 35S-GmRIN4b transgenic plants accumulated similar avrB or avrRpm1 bacteria as wild-type (ecotype Col-0) plants. Likewise, transgenic overexpression of GmRIN4b, but not GmRIN4a was able to complement the ecotopic induction of defenses in the rin4 rps2 mutant. The failure of GmRIN4a to complement the rin4 was not related to interaction with the R protein; both GmRIN4a and b associated with RPM1 as well as AvrRpm1 in BiFC¹⁷ as well as Co-IP assays (Fig. 2). Deciphering the reason underlying inability of GmRIN4a to complement the rin4 mutation should provide important insights into the RIN4 dependent activation of RPM1 activity.Open in a separate windowFigure 2GmRIN4 proteins co-immunoprecipitate with RPM1 (A) and AvrRpm1 (B). Agrobacterium cells expressing MYC tagged GmRIN4a, b, c or d were expressed individually (GmRIN4) or together with 3XFLAG tagged AvrRpm1 (A) or RPM1 (B) in Nicotiana benthamiana. Proteins were immunoprecipitated (IP) from total extracts (T) using anti-FLAG antibodies. Proteins were visualized on western blots using tag-specific antibodies. Parts showing RPM1 and AvrRpm1 are from co-immunoprecipitation (Co-IP) experiments with GmRIN4a and are representative of Co-IPs with GmRIN4b, c and d.Interestingly, silencing either GmRIN4a or b enhanced resistance to virulent strains of P. syringae and the oomycete pathogen Phytophthora sojae in soybean. This suggested that both GmRIN4a and b contributed to basal defense in soybean. Increased basal defense in the GmRIN4a/b-silenced plants could not be attributed to residual Rpg1-b activity since the enhanced resistance phenotype was observed in the rpg1-b background (cv. Essex). Furthermore, the GmRIN4a- or b-silenced Rpg1-b plants (cv. Harosoy) accumulated similar levels of virulent bacteria as the control plants (Fig. 3). However, the possibility that loss of GmRIN4a or b activates other unidentified R proteins in the Essex cultivar cannot be ruled out. Assessing resistance in different genetic backgrounds lacking GmRIN4a and/or b will help clarify this. Inoculation of the GmRIN4a- or b-silenced rpg1-b plants with avrB bacteria showed that neither GmRIN4a nor b was required for the virulence function of AvrB; presence of AvrB enhanced bacterial growth on both GmRIN4a- and b-silenced rpg1-b plants. Interestingly, avrB bacteria were even more virulent on the GmRIN4a-silenced rpg1-b plants as compared to the control or GmRIN4b-silenced rpg1-b plants. These data suggest that GmRIN4a might negatively regulate the virulence function of AvrB. Analyzing the effects of GmRIN4a overexpression in the rpg1-b background will help clarify this.Open in a separate windowFigure 3Silencing GmRIN4a or b does not enhance resistance to virulent Pseudomonas syrinage in Rpg1-b (cv. Harosoy) plants. Bacterial counts in plants silenced for GmRIN4a (S_4a) or GmRIN4b (S_4b) as compared to vector-inoculated (V) plants. LOG values of colony forming units (CFU) per unit leaf area from infected leaves at 0 or 4 days post-inoculation (dpi) are presented. Error bars indicate standard deviation (n = 5).     

Large-scale structure-function analysis of the Arabidopsis RPM1 disease resistance protein

The Arabidopsis RPM1 gene confers resistance against Pseudomonas syringae expressing either the AvrRpm1 or the AvrB type III effector protein. We present an exhaustive genetic screen for mutants that no longer recognize avrRpm1. Using an inducible avrRpm1 expression system, we identified 110 independent mutations. These mutations represent six complementation groups. None discriminates between avrRpm1 and avrB recognition. We identified 95 rpm1 alleles and present a detailed structure--function analysis of the RPM1 protein. Several rpm1 mutants retain partial function, and we deduce that their residual activity is dependent on the level of avrRpm1 signal. In these mutants, the hypersensitive response remains activated if the signal goes above a certain threshold. Missense mutations in rpm1 are highly enriched in the nucleotide binding domain, suggesting that this region plays a key role either in the hypersensitive response associated with RPM1 activation or in RPM1 stability. Cluster analysis of rpm1 alleles defines functionally important residues that are highly conserved between nucleotide binding site leucine-rich repeat R proteins and those that are unique to RPM1. Regions of RPM1 to which no loss-of-function alleles map may represent domains in which variation is tolerated and may contribute to the evolution of new R gene specificities.     

Plant defense: one post,multiple guards?!

Arabidopsis RIN4 is a key bacterial virulence target that is guarded by the resistance (R) protein RPM1. Two recent studies suggest that another R protein, RPS2, also guards RIN4. Bacterial avirulence (Avr) effectors AvrB, AvrRpm1, and AvrRpt2 alter this key protein. R proteins RPM1 and RPS2 recognize the altered status and initiate a defense-signaling response. The guard hypothesis is in!     

Specific threonine phosphorylation of a host target by two unrelated type III effectors activates a host innate immune receptor in plants

The Arabidopsis NB-LRR immune receptor RPM1 recognizes the Pseudomonas syringae type III effectors AvrB or AvrRpm1 to mount an immune response. Although neither effector is itself a kinase, AvrRpm1 and AvrB are known to target Arabidopsis RIN4, a negative regulator of basal plant defense, for phosphorylation. We show that RIN4 phosphorylation activates RPM1. RIN4(142-176) is necessary and, with appropriate localization sequences, sufficient to support effector-triggered RPM1 activation, with the threonine residue at position 166 being critical. Phosphomimic substitutions at T166 cause effector-independent RPM1 activation. RIN4 T166 is phosphorylated in vivo in the presence of AvrB or AvrRpm1. RIN4 mutants that lose interaction with AvrB cannot be coimmunoprecipitated with RPM1. This defines a common interaction platform required for RPM1 activation by phosphorylated RIN4 in response to pathogenic effectors. Conservation of an analogous threonine across all RIN4-like proteins suggests a key function for this residue beyond the regulation of RPM1.     

A resistance gene product of the nucleotide binding site -- leucine rich repeats class can form a complex with bacterial avirulence proteins in vivo

Resistance (R) genes in plants mediate gene-for-gene disease resistance. The ligand-receptor model, which explains the gene-for-gene specificity, predicts a physical interaction between an elicitor, which is directly or indirectly encoded by an avirulence (avr) gene in the pathogen, and the corresponding R gene product. The nucleotide binding site (NBS) - leucine rich repeats (LRR) class of R genes is the largest known class of R genes. Here we report that an NBS-LRR R protein and its cognate Avr protein form a complex together in the plant cell. The Arabidopsis thaliana R genes RPS2 and RPM1 confer gene-for-gene disease resistance to strains of the phytopathogenic bacterium Pseudomonas syringae carrying the avr genes avrRpt2 and avrB, respectively. Using transient expression of these genes in Arabidopsis leaf mesophyll protoplasts, we first demonstrated that the protoplast system is appropriate for the investigation of the gene-for-gene recognition mechanism. Formation of an in vivo complex containing the RPS2 and AvrRpt2 proteins was demonstrated by co-immunoprecipitation of the proteins following expression of the genes in protoplasts. This complex contained at least one additional plant protein of approximately 75 kDa. Unexpectedly, RPS2 also formed a complex with AvrB. We speculate that complex formation between AvrRpt2 and RPS2 is productive and leads to the elicitation of the resistance response, whilst complex formation between AvrB and RPS2 is unproductive and possibly competes with complex formation between AvrRpt2 and RPS2.     

Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea.

A wide-host-range cosmid cloning vector, pLAFR3, was constructed and used to make cosmid libraries of partially digested Sau3A DNA from race 0 and race 1 of Pseudomonas syringae pv. glycinea. Two avirulence genes, avrB0 and avrC, cloned from race 0, elicited the hypersensitivity reaction (HR) on specific cultivars of soybean. Race 4 transconjugants containing avrB0 induced a dark brown necrotic HR within 24 h on the soybean cultivars Harosoy and Norchief, whereas race 4 transconjugants containing avrC induced a light brown necrotic HR within 48 h on the soybean cultivars Acme, Peking, Norchief, and Flambeau. An additional avirulence gene, avrB1, cloned from race 1, appeared to be identical to avrB0 from race 0. The avrB0 and avrC genes from race 0 were characterized by restriction enzyme mapping, Southern blot analysis, Tn5 transposon mutagenesis, and site-directed gene replacements. The effects of these three genes on the in planta bacterial growth of race 4 transconjugants have also been examined. The identification and cloning of avrB1 provides genetic evidence for a gene-for-gene interaction in the bacterial blight disease of soybean, as avrB1 from race 1 interacts with the soybean disease resistance locus, Rpg1.     

Determining the GmRIN4 Requirements of the Soybean Disease Resistance Proteins Rpg1b and Rpg1r Using a Nicotiana glutinosa-Based Agroinfiltration System

Rpg1b and Rpg1r are soybean disease resistance (R) genes responsible for conferring resistance to Pseudomonas syringae strains expressing the effectors AvrB and AvrRpm1, respectively. The study of these cloned genes would be greatly facilitated by the availability of a suitable transient expression system. The commonly used Niciotiana benthamiana-based system is not suitable for studying Rpg1b and Rpg1r function, however, because expression of AvrB or AvrRpm1 alone induces a hypersensitive response (HR), indicating that N. benthamiana contains endogenous R genes that recognize these effectors. To identify a suitable alternative host for transient expression assays, we screened 13 species of Nicotiana along with 11 accessions of N. tabacum for lack of response to transient expression of AvrB and AvrRpm1. We found that N. glutinosa did not respond to either effector and was readily transformable as determined by transient expression of β-glucuronidase. Using this system, we determined that Rpg1b-mediated HR in N. glutinosa required co-expression of avrB and a soybean ortholog of the Arabidopsis RIN4 gene. All four soybean RIN4 orthologs tested worked in the assay. In contrast, Rpg1r did not require co-expression of a soybean RIN4 ortholog to recognize AvrRpm1, but recognition was suppressed by co-expression with AvrRpt2. These observations suggest that an endogenous RIN4 gene in N. glutinosa can substitute for the soybean RIN4 ortholog in the recognition of AvrRpm1 by Rpg1r.     

RPS2, an Arabidopsis disease resistance locus specifying recognition of Pseudomonas syringae strains expressing the avirulence gene avrRpt2.

A molecular genetic approach was used to identify and characterize plant genes that control bacterial disease resistance in Arabidopsis. A screen for mutants with altered resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) expressing the avirulence gene avrRpt2 resulted in the isolation of four susceptible rps (resistance to P. syringae) mutants. The rps mutants lost resistance specifically to bacterial strains expressing avrRpt2 as they retained resistance to Pst strains expressing the avirulence genes avrB or avrRpm1. Genetic analysis indicated that in each of the four rps mutants, susceptibility was due to a single mutation mapping to the same locus on chromosome 4. Identification of a resistance locus with specificity for a single bacterial avirulence gene suggests that this locus, designated RPS2, controls specific recognition of bacteria expressing the avirulence gene avrRpt2. Ecotype Wü-0, a naturally occurring line that is susceptible to Pst strains expressing avrRpt2, appears to lack a functional allele at RPS2, demonstrating that there is natural variation at the RPS2 locus among wild populations of Arabidopsis.     

Phosphorylation of the Plant Immune Regulator RPM1-INTERACTING PROTEIN4 Enhances Plant Plasma Membrane H+-ATPase Activity and Inhibits Flagellin-Triggered Immune Responses in Arabidopsis

The Pseudomonas syringae effector AvrB targets multiple host proteins during infection, including the plant immune regulator RPM1-INTERACTING PROTEIN4 (RIN4) and RPM1-INDUCED PROTEIN KINASE (RIPK). In the presence of AvrB, RIPK phosphorylates RIN4 at Thr-21, Ser-160, and Thr-166, leading to activation of the immune receptor RPM1. Here, we investigated the role of RIN4 phosphorylation in susceptible Arabidopsis thaliana genotypes. Using circular dichroism spectroscopy, we show that RIN4 is a disordered protein and phosphorylation affects protein flexibility. RIN4 T21D/S160D/T166D phosphomimetic mutants exhibited enhanced disease susceptibility upon surface inoculation with P. syringae, wider stomatal apertures, and enhanced plasma membrane H⁺-ATPase activity. The plasma membrane H⁺-ATPase AHA1 is highly expressed in guard cells, and its activation can induce stomatal opening. The ripk knockout also exhibited a strong defect in pathogen-induced stomatal opening. The basal level of RIN4 Thr-166 phosphorylation decreased in response to immune perception of bacterial flagellin. RIN4 Thr166D lines exhibited reduced flagellin-triggered immune responses. Flagellin perception did not lower RIN4 Thr-166 phosphorylation in the presence of strong ectopic expression of AvrB. Taken together, these results indicate that the AvrB effector targets RIN4 in order to enhance pathogen entry on the leaf surface as well as dampen responses to conserved microbial features.