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.     

RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis

In Arabidopsis, RPM1 confers resistance against Pseudomonas syringae expressing either of two sequence unrelated type III effectors, AvrRpm1 or AvrB. An RPM1-interacting protein (RIN4) coimmunoprecipitates from plant cell extracts with AvrB, AvrRpm1, or RPM1. Reduction of RIN4 protein levels inhibits both the hypersensitive response and the restriction of pathogen growth controlled by RPM1. RIN4 reduction causes diminution of RPM1. RIN4 reduction results in heightened resistance to virulent Peronospora parasitica and P. syringae, and ectopic defense gene expression. Thus, RIN4 positively regulates RPM1-mediated resistance yet is, formally, a negative regulator of basal defense responses. AvrRpm1 and AvrB induce RIN4 phosphorylation. This may enhance RIN4 activity as a negative regulator of plant defense, facilitating pathogen growth. RPM1 may "guard" against pathogens that use AvrRpm1 and AvrB to manipulate RIN4 activity.     

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.     

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!     

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.     

Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4

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.     

Proteolysis of a Negative Regulator of Innate Immunity Is Dependent on Resistance Genes in Tomato and Nicotiana benthamiana and Induced by Multiple Bacterial Effectors

RPM1-interacting protein 4 (RIN4), a negative regulator of the basal defense response in plants, is targeted by multiple bacterial virulence effectors. We show that RIN4 degradation is induced by the effector AvrPto from Pseudomonas syringae and that this degradation in Solanaceous plants is dependent on the resistance protein, Pto, a protein kinase, and Prf, a nucleotide binding site–leucine-rich repeat protein. Our data demonstrate overlap between two of the best-characterized pathways for recognition of pathogen virulence effectors in plants. RIN4 interacts with multiple plant signaling components and bacterial effectors in yeast and in planta. AvrPto induces an endogenous proteolytic activity in both tomato (Solanum lycopersicum) and Nicotiana benthamiana that degrades RIN4 and requires the consensus site cleaved by the protease effector AvrRpt2. The interaction between AvrPto and Pto, but not the kinase activity of Pto, is required for proteolysis of RIN4. Analysis of many of the effectors comprising the secretome of P. syringae pv tomato DC3000 led to the identification of two additional sequence-unrelated effectors that can also induce degradation of RIN4. Therefore, multiple bacterial effectors besides AvrRpt2 elicit proteolysis of RIN4 in planta.     

Layered basal defenses underlie non-host resistance of Arabidopsis to Pseudomonas syringae pv. phaseolicola

Arabidopsis is a non-host for Pseudomonas syringae pv. phaseolicola NPS3121 (Pph), a bacterial pathogen of bean. Pph does not induce a hypersensitive response in Arabidopsis. Here we show that Arabidopsis instead resists Pph with multi-layered basal defense. Our approach was: (i) to identify defense readouts induced by Pph; (ii) to determine whether mutations in known Arabidopsis defense genes disrupt Pph-induced defense signaling; (iii) to determine whether heterologous type III effectors from pathogens of Arabidopsis suppress Pph-induced defense signaling, and (iv) to ascertain how basal defenses contribute to resistance against Pph by individually or multiply disrupting defense signaling pathways with mutations and heterologous type III effectors. We demonstrate that Pph elicits a minimum of three basal defense-signaling pathways in Arabidopsis. These pathways have unique readouts, including PR-1 protein accumulation and morphologically distinct types of callose deposition. Further, they require distinct defense genes, including PMR4, RAR1, SID2, NPR1, and PAD4 . Finally, they are suppressed differentially by heterologous type III effectors, including AvrRpm1 and HopM1. Pph growth is enhanced only when multiple defense pathways are disrupted. For example, mutation of NPR1 or SID2 combined with the action of AvrRpm1 and HopM1 renders Arabidopsis highly susceptible to Pph. Thus, non-host resistance of Arabidopsis to Pph is based on multiple, individually effective layers of basal defense.     

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.     

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.     

Disruption of PAMP-induced MAP kinase cascade by a Pseudomonas syringae effector activates plant immunity mediated by the NB-LRR protein SUMM2

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) serves as a primary plant defense response against microbial pathogens, with MEKK1, MKK1/MKK2, and MPK4 functioning as a MAP kinase cascade downstream of PAMP receptors. Plant Resistance (R) proteins sense specific pathogen effectors to initiate a second defense mechanism, termed effector-triggered immunity (ETI). In a screen for suppressors of the mkk1 mkk2 autoimmune phenotype, we identify the nucleotide-binding leucine-rich repeat (NB-LRR) protein SUMM2 and find that the MEKK1-MKK1/MKK2-MPK4 cascade negatively regulates SUMM2-mediated immunity. Further, the MEKK1-MKK1/MKK2-MPK4 cascade positively regulates basal defense targeted by the Pseudomonas syringae pathogenic effector HopAI1, which inhibits MPK4 kinase activity. Inactivation of MPK4 by HopAI1 results in activation of SUMM2-mediated defense responses. Our data suggest that SUMM2 is an R protein that becomes active when the MEKK1-MKK1/MKK2-MPK4 cascade is disrupted by pathogens, supporting the hypothesis that R proteins evolved to protect plants when microbial effectors suppress basal resistance.     

Separable fragments and membrane tethering of Arabidopsis RIN4 regulate its suppression of PAMP-triggered immunity

RPM1-interacting protein 4 (RIN4) is a multifunctional Arabidopsis thaliana protein that regulates plant immune responses to pathogen-associated molecular patterns (PAMPs) and bacterial type III effector proteins (T3Es). RIN4, which is targeted by multiple defense-suppressing T3Es, provides a mechanistic link between PAMP-triggered immunity (PTI) and effector-triggered immunity and effector suppression of plant defense. Here we report on a structure-function analysis of RIN4-mediated suppression of PTI. Separable fragments of RIN4, including those produced when the T3E AvrRpt2 cleaves RIN4 and each containing a plant-specific nitrate-induced (NOI) domain, suppress PTI. The N-terminal and C-terminal NOIs each contribute to PTI suppression and are evolutionarily conserved. Native RIN4 is anchored to the plasma membrane by C-terminal acylation. Nonmembrane-tethered derivatives of RIN4 activate a cell death response in wild-type Arabidopsis and are hyperactive PTI suppressors in a mutant background that lacks the cell death response. Our results indicate that RIN4 is a multifunctional suppressor of PTI and that a virulence function of AvrRpt2 may include cleaving RIN4 into active defense-suppressing fragments.     

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.     

Phospholipase-dependent signalling during the AvrRpm1- and AvrRpt2-induced disease resistance responses in Arabidopsis thaliana

Bacterial pathogens deliver type III effector proteins into plant cells during infection. On susceptible host plants, type III effectors contribute to virulence, but on resistant hosts they betray the pathogen to the plant's immune system and are functionally termed avirulence (Avr) proteins. Recognition induces a complex suite of cellular and molecular events comprising the plant's inducible defence response. As recognition of type III effector proteins occurs inside host cells, defence responses can be elicited by in planta expression of bacterial type III effectors. We demonstrate that recognition of either of two type III effectors, AvrRpm1 or AvrRpt2 from Pseudomonas syringae , induced biphasic accumulation of phosphatidic acid (PA). The first wave of PA accumulation correlated with disappearance of monophosphatidylinosotol (PIP) and is thus tentatively attributed to activation of a PIP specific phospholipase C (PLC) in concert with diacylglycerol kinase (DAGK) activity. Subsequent activation of phospholipase D (PLD) produced large amounts of PA from structural phospholipids. This later wave of PA accumulation was several orders of magnitude higher than the PLC-dependent first wave. Inhibition of phospholipases blocked the response, and feeding PA directly to leaf tissue caused cell death and defence-gene activation. Inhibitor studies ordered these events relative to other known signalling events during the plant defence response. Influx of extracellular Ca²⁺ occurred downstream of PIP-degradation, but upstream of PLD activation. Production of reactive oxygen species occurred downstream of the phospholipases. The data presented indicate that PA is a positive regulator of RPM1- or RPS2-mediated disease resistance signalling, and that the biphasic PA production may be a conserved feature of signalling induced by the coiled-coil nucleotide binding domain leucine-rich repeat class of resistance proteins.     

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.     

Functional analysis of the type III effectors AvrRpt2 and AvrRpm1 of Pseudomonas syringae with the use of a single-copy genomic integration system

Gram-negative phytopathogenic bacteria require a type III secretion apparatus for pathogenesis, presumably to deliver Avr effector proteins directly into plant cells. To extend previous studies of Avr effectors that employed plasmids encoding Avr proteins, we developed a system that permits the integration of any gene into the Pseudomonas syringae genome in single copy. With this system, we confirmed earlier findings showing that P. syringae pv. maculicola strain PsmES4326 expressing the AvrRpt2 effector induces a resistance response in plants with the cognate R gene, RPS2. Chromosomally located avrRpt2, however, provoked a stronger resistance response than that observed with plasmid-expressed AvrRpt2 in RPS2+ plants. Additionally, chromosomal expression of AvrRpt2 conferred a fitness advantage on P. syringae grown in rps2- plants, aiding in growth within leaves and escape to leaf surfaces that was difficult to detect with plasmid-borne avrRpt2. Finally, with the use of the genomic integration system, we found that a chimeric protein composed of the N terminus of the heterologous AvrRpml effector and the C-terminal effector region of AvrRpt2 was delivered to plant cells. Because the C terminus of AvrRpt2 cannot translocate into plant cells on its own, this indicates that the N-terminal region can direct secretion and translocation during an infection, which supports the view that Avr proteins have a modular design. This work establishes a readily manipulatable system to study type III effectors in a biologically realistic context.     

Direct delivery of bacterial avirulence proteins into resistant Arabidopsis protoplasts leads to hypersensitive cell death

Many bacterial avirulence (Avr) proteins, including the Pseudomonas syringae proteins, AvrRpt2 and AvrB, appear to be recognized inside the host plant cell by resistance mechanisms mediated by the cognate resistance (R) genes. It is thought that Avr proteins are either delivered directly into the host cell via the bacterial type III secretion system (TTSS) or taken up by the plant cell following secretion into the apoplast through the TTSS. Recently, it was shown that the Xanthomonas campestris AvrBs2 protein can be delivered directly into the host plant cell by the TTSS. However, it is not known whether other type III effectors of phytopathogens behave similarly. Here, using a novel protein transfection method, we demonstrate that AvrRpt2 and AvrB must enter the plant cell to be recognized by R gene-mediated mechanisms. First, we established a hypersensitive cell death assay for protoplasts using the membrane-impermeable, nuclear-staining dye, YO-PRO-1, and transgenic Arabidopsis plants that carry an inducible avrRpt2 gene. Second, we transfected E. coli-produced AvrRpt2 or AvrB proteins into Arabidopsis protoplasts using a protein transfection kit based on the carrier peptide Pep-1, and demonstrated that hypersensitive cell death occurs in a gene-for-gene-specific manner. In contrast, these Avr proteins failed to elicit hypersensitive cell death when they were applied to protoplasts without the carrier peptide. We conclude that our preparations of E. coli-produced AvrRpt2 and AvrB are active, that AvrRpt2 and AvrB must be delivered into the plant cell to be recognized, and that a method based on a carrier peptide can be used to introduce proteins into plant cells.     

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.