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 receptor-like cytoplasmic kinase phosphorylates the host target RIN4, leading to the activation of a plant innate immune receptor

Plants have evolved sophisticated surveillance systems to recognize pathogen effectors delivered into host cells. RPM1 is an NB-LRR immune receptor that recognizes the Pseudomonas syringae effectors AvrB and AvrRpm1. Both effectors associate with and affect the phosphorylation of RIN4, an immune regulator. Although the kinase and the specific mechanisms involved are unclear, it has been hypothesized that RPM1 recognizes phosphorylated RIN4. Here, we identify RIPK as a RIN4-interacting receptor-like protein kinase that phosphorylates RIN4. In response to bacterial effectors, RIPK phosphorylates RIN4 at amino acid residues T21, S160, and T166. RIN4 phosphomimetic mutants display constitutive activation of RPM1-mediated defense responses and RIN4 phosphorylation is induced by AvrB and AvrRpm1 during P. syringae infection. RIPK knockout lines exhibit reduced RIN4 phosphorylation and blunted RPM1-mediated defense responses. Taken together, our results demonstrate that the RIPK kinase associates with and modifies an effector-targeted protein complex to initiate host immunity.     

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.     

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

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.     

A duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and RPS2-mediated hypersensitive response

The Arabidopsis RPM1 protein confers resistance to disease caused by Pseudomonas syringae strains delivering either the AvrRpm1 or AvrB type III effector proteins into host cells. We characterized two closely related RPM1-interacting proteins, RIN2 and RIN3. RIN2 and RIN3 encode RING-finger type ubiquitin ligases with six apparent transmembrane domains and an ubiquitin-binding CUE domain. RIN2 and RIN3 are orthologs of the mammalian autocrine motility factor receptor, a cytokine receptor localized in both plasma membrane caveolae and the endoplasmic reticulum. RIN2 is predominantly localized to the plasma membrane, as are RPM1 and RPS2. The C-terminal regions of RIN2 and RIN3, including the CUE domain, interact strongly with an RPM1 N-terminal fragment and weakly with a similar domain from the Arabidopsis RPS2 protein. RIN2 and RIN3 can dimerize through their C-terminal regions. The RING-finger domains of RIN2 and RIN3 encode ubiquitin ligases. Inoculation with P. syringae DC3000(avrRpm1) or P. syringae DC3000(avrRpt2) induces differential decreases of RIN2 mobility in SDS-PAGE and disappearance of the majority of RIN2. A rin2 rin3 double mutant expresses diminished RPM1- and RPS2-dependent hypersensitive response (HR), but no alteration of pathogen growth. Thus, the RIN2/RIN3 RING E3 ligases apparently act on a substrate that regulates RPM1- and RPS2-dependent HR.     

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.     

Two Pseudomonas syringae type III effectors inhibit RIN4-regulated basal defense in Arabidopsis

Plant cells have two defense systems that detect bacterial pathogens. One is a basal defense system that recognizes complex pathogen-associated molecular patterns (PAMPs). A second system uses disease-resistance (R) proteins to recognize type lll effector proteins that are delivered into the plant cell by the pathogen's type III secretion system. Here we show that these two pathways are linked. We find that two Pseudomonas syringae type III effectors, AvrRpt2 and AvrRpm1, inhibit PAMP-induced signaling and thus compromise the host's basal defense system. RIN4 is an Arabidopsis protein targeted by AvrRpt2 and AvrRpm1 for degradation and phosphorylation, respectively. We find that RIN4 is itself a regulator of PAMP signaling. The R proteins, RPS2 and RPM1, sense type III effector-induced perturbations of RIN4. Thus, R proteins guard the plant against type III effectors that inhibit PAMP signaling and provide a mechanistic link between the two plant defense systems.     

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!     

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.     

AvrB mutants lose both virulence and avirulence activities on soybean and Arabidopsis

The Pseudomonas syringae pv. glycinea effector protein AvrB induces resistance responses in soybean varieties that contain the resistance gene Rpg1-b and Arabidopsis varieties that carry RPM1. In addition to this avirulence activity, AvrB also enhances bacterial virulence on soybean plants that lack Rpg1-b and induces a chlorotic phenotype on Arabidopsis plants that lack RPM1. We screened a library of avrB mutants for loss of avirulence on soybean and Arabidopsis, and assayed selected avirulence mutants for loss of virulence on both plants. All mutants screened were recognized similarly on both plant species. Nine single-site avrB mutations that affected avirulence localized to a solvent-accessible pocket in the protein structure. Seven of these mutated residues are absolutely conserved between AvrB and its nine homologues. Avirulence mutants generally lost virulence enhancement on susceptible soybean varieties and lost the ability to induce a chlorotic response on the rpm1 null Arabidopsis variety Mt-0. Three of four avirulence mutants tested failed to interact with RIN4, an Arabidopsis protein previously shown to be required for RPM1 function. Our results suggest that soybean and Arabidopsis recognize AvrB in the same manner, and that AvrB enzymatic activity is required for its function as an avirulence and virulence effector on two different plant species.     

RPG1-B-Derived Resistance to AvrB-Expressing Pseudomonas syringae Requires RIN4-Like Proteins in Soybean

Soybean (Glycine max) RPG1-B (for resistance to Pseudomonas syringae pv glycinea) mediates species-specific resistance to P. syringae expressing the avirulence protein AvrB, similar to the nonorthologous RPM1 in Arabidopsis (Arabidopsis thaliana). RPM1-derived signaling is presumably induced upon AvrB-derived modification of the RPM1-interacting protein, RIN4 (for RPM1-interacting 4). We show that, similar to RPM1, RPG1-B does not directly interact with AvrB but associates with RIN4-like proteins from soybean. Unlike Arabidopsis, soybean contains at least four RIN4-like proteins (GmRIN4a to GmRIN4d). GmRIN4b, but not GmRIN4a, complements the Arabidopsis rin4 mutation. Both GmRIN4a and GmRIN4b bind AvrB, but only GmRIN4b binds RPG1-B. Silencing either GmRIN4a or GmRIN4b abrogates RPG1-B-derived resistance to P. syringae expressing AvrB. Binding studies show that GmRIN4b interacts with GmRIN4a as well as with two other AvrB/RPG1-B-interacting isoforms, GmRIN4c and GmRIN4d. The lack of functional redundancy among GmRIN4a and GmRIN4b and their abilities to interact with each other suggest that the two proteins might function as a heteromeric complex in mediating RPG1-B-derived resistance. Silencing GmRIN4a or GmRIN4b in rpg1-b plants enhances basal resistance to virulent strains of P. syringae and the oomycete Phytophthora sojae. Interestingly, GmRIN4a- or GmRIN4b-silenced rpg1-b plants respond differently to AvrB-expressing bacteria. Although both GmRIN4a and GmRIN4b function to monitor AvrB in the presence of RPG1-B, GmRIN4a, but not GmRIN4b, negatively regulates AvrB virulence activity in the absence of RPG1-B.One of the myriad plant defense responses activated upon pathogen invasion is signaling induced via the activation of resistance (R) proteins. R gene-mediated resistance is generally activated in response to race-specific pathogen effectors, termed avirulence proteins (Avr), and often results in the development of a hypersensitive reaction at the site of pathogen entry (Dangl et al., 1996). The hypersensitive reaction is a form of programmed cell death that results in the formation of necrotic lesions around the site of pathogen entry and is thought to help prevent pathogen spread by confining it to the dead cells.A majority of the known R proteins contain conserved structural domains, including N-terminal coiled coil (CC) or Toll-interleukin 1 receptor (TIR)-like domains, central nucleotide-binding site (NBS), and C-terminal Leu-rich repeat (LRR) domains (Martin et al., 2003). While some R proteins “perceive” pathogen presence via direct physical interactions with the cognate Avr proteins (Scofield et al., 1996; Jia et al., 2000; Leister and Katagiri, 2000; Deslandes et al., 2003), several others likely do so indirectly. This led to the suggestion that R proteins monitor the presence of Avr proteins by “guarding” other host proteins targeted by the pathogen effector (Van der Biezen and Jones, 1998; Innes, 2004; Jones and Dangl, 2006). Avr proteins enhance pathogen virulence in genetic backgrounds lacking cognate R proteins by targeting components of the host basal defense machinery, including “guardee” proteins (Chang et al., 2000; Guttman and Greenberg, 2001; Chen et al., 2004, Kim et al., 2005b; Ong and Innes, 2006; van Esse et al., 2007; Shan et al., 2008; Xiang et al., 2008). However, some Avr proteins were found to also target host proteins that do not contribute to the virulence function of the effector (Shang et al., 2006; Shabab et al., 2008; Zhou and Chai, 2008; Zipfel and Rathjen, 2008). This led to the proposition that plants express “decoy” proteins that mimic Avr-guardee recognition in the presence of the R protein. This decoy model suggests that, unlike guardees, decoy proteins do not directly contribute to host basal immunity, such that Avr-derived alterations of decoys do not enhance pathogen virulence in plants lacking the R protein (van der Hoorn and Kamoun, 2008).A well-studied example of an indirect mode of effector recognition is that of the Arabidopsis (Arabidopsis thaliana) R protein, RPM1 (for resistance to Pseudomonas syringae pv maculicola 1). RPM1 mediates resistance against bacteria expressing two different Avr proteins, AvrRpm1 (AvrRpm1_PmaM6) and AvrB (AvrB1_Pgyrace4). Although RPM1 does not directly interact with either AvrRpm1 or AvrB, it does associate with RIN4 (for RPM1-interacting 4), which interacts with AvrRpm1 and AvrB. RIN4 is required for RPM1-induced resistance to AvrRpm1/AvrB-expressing P. syringae (Mackey et al., 2002). Both AvrRpm1 and AvrB induce the phosphorylation of RIN4, which is thought to induce RPM1-mediated resistance signaling. RIN4 also associates with a second Arabidopsis R protein, RPS2 (for resistance to P. syringae), which mediates resistance against P. syringae expressing AvrRpt2. RPS2-mediated signaling is activated when AvrRpt2 (AvrRpt2_PtoJL1065), a Cys protease, cleaves RIN4 (Axtell and Staskawicz, 2003; Mackey et al., 2003; Kim et al., 2005a). The AvrRpt2-triggered loss of RIN4 compromises RPM1-mediated resistance, because RIN4 is not available for phosphorylation (Ritter and Dangl, 1996; Axtell and Staskawicz, 2003; Mackey et al., 2003).The avirulence effector AvrB was first isolated from a P. syringae strain colonizing soybean (Glycine max) and used to identify the cognate resistance locus RPG1 in soybean (Staskawicz et al., 1987; Keen and Buzzell, 1991). This locus contains the RPG1-B (for resistance to P. syringae pv glycinea) gene, which encodes a CC-NBS-LRR protein conferring resistance to AvrB-expressing P. syringae in soybean (Bisgrove et al., 1994; Ashfield et al., 2004). Unlike RPM1, RPG1-B does not confer specificity to AvrRpm1 (Ashfield et al., 1995). However, as in Arabidopsis, the soybean RPG1-B-derived hypersensitive reaction to AvrB-expressing bacteria is inhibited by the presence of AvrRpt2-expressing bacteria (Axtell and Staskawicz, 2003, Mackey et al., 2003; Ashfield et al., 2004). This suggests that RPG1-B and RPM1 might utilize common signaling components even though they share very limited sequence identity. Therefore, we investigated the possible involvement of RIN4-like proteins in RPG1-B-mediated resistance signaling. In addition to Arabidopsis, RIN4-like proteins have also been identified in tomato (Solanum lycopersicum) and lettuce (Lactuca sativa; Jeuken et al., 2009; Luo et al., 2009). In tomato, the NBS-LRR protein, Prf (for Pseudomonas resistance and fenthion sensitivity), and its interacting protein kinase, Pto, mediate resistance to the AvrPto (AvrPto1_PtoJL1065)-expressing strain of P. syringae (Scofield et al., 1996; Tang et al., 1996; Kim et al., 2002; Mucyn et al., 2006). AvrPto binds RIN4 proteins from both Arabidopsis (AtRIN4) and tomato (SlRIN4). Similar to AvrRpt2, AvrPto induces the proteolysis of RIN4, albeit only in the presence of Pto and Prf (Luo et al., 2009). However, in the case of AvrPto, degradation of RIN4 is the result of induced proteolytic activity in the plant, rather than that of AvrPto itself. In Lactuca (lettuce) species, the L. saligna RIN4 allele was recently shown to be essential for resistance to an avirulent strain of the downy mildew pathogen, Bremia lactucae (Jeuken et al., 2009).Here, we report that two functionally nonredundant isoforms of soybean RIN4 (GmRIN4) function in RPG1-B-derived resistance as well as in the virulence activity of AvrB in the absence of RPG1-B.     

NDR1 interaction with RIN4 mediates the differential activation of multiple disease resistance pathways in Arabidopsis

Recognition of pathogens by plants involves the coordinated efforts of molecular chaperones, disease resistance (R) proteins, and components of disease resistance signaling pathways. Characterization of events associated with pathogen perception in Arabidopsis thaliana has advanced understanding of molecular genetic mechanisms associated with disease resistance and protein interactions critical for the activation of resistance signaling. Regulation of R protein-mediated signaling in response to the bacterial pathogen Pseudomonas syringae in Arabidopsis involves the physical association of at least two R proteins with the negative regulator RPM1 INTERACTING PROTEIN4 (RIN4). While the RIN4-RPS2 (for RESISTANCE TO P. SYRINGAE2) and RIN4-RPM1 (for RESISTANCE TO P. SYRINGAE PV MACULICOLA1) signaling pathways exhibit differential mechanisms of activation in terms of effector action, the requirement for NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) is shared. Using a yeast two-hybrid screen, followed by a series of coimmunoprecipitation experiments, we demonstrate that the RIN4-NDR1 interaction occurs on the cytoplasmically localized N-terminal portion of NDR1 and that this interaction is required for the activation of resistance signaling following infection by P. syringae expressing the Cys protease Type III effector protein AvrRpt2. We demonstrate that like RPS2 and RPM1, NDR1 also associates with RIN4 in planta. We suggest that this interaction serves to further regulate activation of disease resistance signaling following recognition of P. syringae DC3000-AvrRpt2 by Arabidopsis.     

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.     

Rpa1 mediates an immune response to avrRpm1Psa and confers resistance against Pseudomonas syringae pv. actinidiae

The type three effector AvrRpm1_Pma from Pseudomonas syringae pv. maculicola (Pma) triggers an RPM1‐mediated immune response linked to phosphorylation of RIN4 (RPM1‐interacting protein 4) in Arabidopsis. However, the effector–resistance (R) gene interaction is not well established with different AvrRpm1 effectors from other pathovars. We investigated the AvrRpm1‐triggered immune responses in Nicotiana species and isolated Rpa1 (R esistance to P seudomonas syringae pv. a ctinidiae 1) via a reverse genetic screen in Nicotiana tabacum. Transient expression and gene silencing were performed in combination with co‐immunoprecipitation and growth assays to investigate the specificity of interactions that lead to inhibition of pathogen growth. Two closely related AvrRpm1 effectors derived from Pseudomonas syringae pv. actinidiae biovar 3 (AvrRpm1_Psa) and Pseudomonas syringae pv. syringae strain B728a (AvrRpm1_Psy) trigger immune responses mediated by RPA1, a nucleotide‐binding leucine‐rich repeat protein with an N‐terminal coiled‐coil domain. In a display of contrasting specificities, RPA1 does not respond to AvrRpm1_Pma, and correspondingly AvrRpm1_Psa and AvrRpm1_Psy do not trigger the RPM1‐mediated response, demonstrating that separate R genes mediate specific immune responses to different AvrRpm1 effectors. AvrRpm1_Psa co‐immunoprecipitates with RPA1, and both proteins co‐immunoprecipitate with RIN4. In contrast with RPM1, however, RPA1 was not activated by the phosphomimic RIN4^T166D and silencing of RIN4 did not affect the RPA1 activity. Delivery of AvrRpm1_Psa by Pseudomonas syringae pv. tomato (Pto) in combination with transient expression of Rpa1 resulted in inhibition of the pathogen growth in N. benthamiana. Psa growth was also inhibited by RPA1 in N. tabacum.     

Membrane release and destabilization of Arabidopsis RIN4 following cleavage by Pseudomonas syringae AvrRpt2

The Arabidopsis RIN4 protein mediates interaction between the Pseudomonas syringae type III effector proteins AvrB, AvrRpm1, and AvrRpt2 and the Arabidopsis disease-resistance proteins RPM1 and RPS2. Confocal laser-scanning fluorescence microscopy following particle bombardment of tobacco leaf epidermal cells was used to examine the subcellular localization of fusions between GFP and RIN4 or several of its homologs and to examine the effects of cobombardment with AvrRpt2 or AvrRpml. This study showed that RIN4 was attached to the plasma membrane at its carboxyl terminus and that a carboxyl-terminal CCCFxFxxx prenylation or acylation (typically palmitoylation) motif, or both, was essential for this attachment. RIN4 was cleaved by AvrRpt2 at two PxFGxW motifs, one releasing a large portion of RIN4 from the plasma membrane and both exposing amino-terminal residues that destabilized the carboxyl-terminal cleavage products by targeting them for N-end ubiquitylation and proteasomal degradation. Plasma-membrane localization of RIN4 was not affected by AvrRpml. RIN4 was found to be part of a protein family comprising two full-length homologs and at least 11 short carboxyl-terminal homologs. Representatives of this family, comprising a full-length RIN4 homolog and two short carboxyl-terminal RIN4 homologs, were also attached to the plasma membrane and cleaved near their amino termini by AvrRpt2, but in contrast to RIN4, the major portions of these proteins remained on the plasma membrane. N-end degradation may play a minor role in RIN4 degradation but probably plays a major role in the degradation of RIN4 homologs and is, therefore, a major pathogenic consequence of AvrRpt2 cleavage.     

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).     

The Pseudomonas syringae type III effector AvrRpm1 induces significant defenses by activating the Arabidopsis nucleotide-binding leucine-rich repeat protein RPS2

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.