Prf immune complexes of tomato are oligomeric and contain multiple Pto‐like kinases that diversify effector recognition

Cytoplasmic recognition of pathogen virulence effectors by plant NB‐LRR proteins leads to strong induction of defence responses termed effector triggered immunity (ETI). In tomato, a protein complex containing the NB‐LRR protein Prf and the protein kinase Pto confers recognition of the Pseudomonas syringae effectors AvrPto and AvrPtoB. Although structurally unrelated, AvrPto and AvrPtoB interact with similar residues in the Pto catalytic cleft to activate ETI via an unknown mechanism. Here we show that the Prf complex is oligomeric, containing at least two molecules of Prf. Within the complex, Prf can associate with Pto or one of several Pto family members including Fen, Pth2, Pth3, or Pth5. The dimerization surface for Prf is the novel N‐terminal domain, which also coordinates an intramolecular interaction with the remainder of the molecule, and binds Pto kinase or a family member. Thus, association of two Prf N‐terminal domains brings the associated kinases into close promixity. Tomato lines containing Prf complexed with Pth proteins but not Pto possessed greater immunity against P. syringae than tomatoes lacking Prf. This demonstrates that incorporation of non‐Pto kinases into the Prf complex extends the number of effector proteins that can be recognized.     

Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases

Plants use receptor kinases, such as FLS2 and EFR, to perceive bacterial pathogens and initiate innate immunity. This immunity is often suppressed by bacterial effectors, allowing pathogen propagation. To counteract, plants have evolved disease resistance genes that detect the bacterial effectors and reinstate resistance. The Pseudomonas syringae effector AvrPto promotes infection in susceptible plants but triggers resistance in plants carrying the protein kinase Pto and the associated resistance protein Prf. Here we show that AvrPto binds receptor kinases, including Arabidopsis FLS2 and EFR and tomato LeFLS2, to block plant immune responses in the plant cell. The ability to target receptor kinases is required for the virulence function of AvrPto in plants. The FLS2-AvrPto interaction and Pto-AvrPto interaction appear to share similar sequence requirements, and Pto competes with FLS2 for AvrPto binding. The results suggest that the mechanism by which AvrPto recognizes virulence targets is linked to the evolution of Pto, which, in association with Prf, recognizes the bacterium and triggers strong resistance.     

The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate specific plant immunity

Immunity in tomato (Solanum lycopersicum) to Pseudomonas syringae bacteria expressing the effector proteins AvrPto and AvrPtoB requires both Pto kinase and the NBARC-LRR (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4 fused to C-terminal leucine-rich repeats) protein Prf. Pto plays a direct role in effector recognition within the host cytoplasm, but the role of Prf is unknown. We show that Pto and Prf are coincident in the signal transduction pathway that controls ligand-independent signaling. Pto and Prf associate in a coregulatory interaction that requires Pto kinase activity and N-myristoylation for signaling. Pto interacts with a unique Prf N-terminal domain outside of the NBARC-LRR domain and resides in a high molecular weight recognition complex dependent on the presence of Prf. In this complex, both Pto and Prf contribute to specific recognition of AvrPtoB. The data suggest that the role of Pto is confined to the regulation of Prf and that the bacterial effectors have evolved to target this coregulatory molecular switch.     

The solution structure of type III effector protein AvrPto reveals conformational and dynamic features important for plant pathogenesis

Pseudomonas syringae pv. tomato, the causative agent of bacterial speck disease of tomato, uses a type III secretion system (TTSS) to deliver effector proteins into the host cell. In resistant plants, the bacterial effector protein AvrPto physically interacts with the host Pto kinase and elicits antibacterial defense responses. In susceptible plants, which lack the Pto kinase, AvrPto acts as a virulence factor to promote bacterial growth. The solution structure of AvrPto reveals a functional core consisting of a three-helix bundle motif flanked by disordered N- and C-terminal tails. Residues required for Pto binding lie in a 19 residue Omega loop. Modeling suggests a hydrophobic patch involving the activation loop of Pto forms a contact surface with the AvrPto Omega loop and that helix packing mediates interactions between AvrPto and putative virulence targets Api2 and Api3. The AvrPto structure has a low stability that may facilitate chaperone-independent secretion by the TTSS.     

Crystal Structure of the Complex between Pseudomonas Effector AvrPtoB and the Tomato Pto Kinase Reveals Both a Shared and a Unique Interface Compared with AvrPto-Pto

Resistance to bacterial speck disease in tomato (Solanum lycopersicum) is activated upon recognition by the host Pto kinase of either one of two sequence-unrelated effector proteins, AvrPto or AvrPtoB, from Pseudomonas syringae pv tomato (Pst). Pto induces Pst immunity by acting in concert with the Prf protein. The recently reported structure of the AvrPto-Pto complex revealed that interaction of AvrPto with Pto appears to relieve an inhibitory effect of Pto, allowing Pto to activate Prf. Here, we present the crystal structure of the Pto binding domain of AvrPtoB (residues 121 to 205) at a resolution of 1.9Å and of the AvrPtoB_121-205–Pto complex at a resolution of 3.3 Å. AvrPtoB_121-205 exhibits a tertiary fold that is completely different from that of AvrPto, and its conformation remains largely unchanged upon binding to Pto. In common with AvrPto-Pto, the AvrPtoB-Pto complex relies on two interfaces. One of these interfaces is similar in both complexes, although the primary amino acid sequences from the two effector proteins are very different. Amino acid substitutions in Pto at the other interface disrupt the interaction of AvrPtoB-Pto but not that of AvrPto-Pto. Interestingly, substitutions in Pto affecting this unique interface also cause Pto to induce Prf-dependent host cell death independently of either effector protein.     

The N-Terminal Domain of the Tomato Immune Protein Prf Contains Multiple Homotypic and Pto Kinase Interaction Sites

Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats).     

Lessons learned from type III effector transgenic plants

The Gram negative bacterial phytopathogen Pseudomonas syringae employs a molecular syringe termed the type III secretion system (TTSS) to deliver an array of type III secreted effector (TTSE) proteins into plant cells. The major function ascribed to type III effectors of P. syringae is their ability to suppress plant immunity. Because individual pathovars of P. syringae can possess over 30 TTSEs, functional redundancy can provide a hurdle to ascribing functions by TTSE-deletion or -overexpression in such TTSE-rich backgrounds. Approaches to overcome functional redundancy have included the deletion of multiple TTSEs from individual pathovars as well as engineering the plant commensal P. fluorescens strain to express the P. syringae TTSS and deliver P. syringae TTSEs. As we describe here, transgenic Arabidopsis plants expressing individual TTSEs have also been used to overcome problems of functional redundancy and provide invaluable insights into TTSE virulence functions.Key words: pathogen, virulence, effector, plant immunity, HopF2_Pto, RIN4     

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.     

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.     

The Pseudomonas syringae effector HopF2 suppresses Arabidopsis immunity by targeting BAK1

Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage immune responses and modulate physiology to favor infection. The P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple microbe‐associated molecular patterns (MAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms for suppression of two branches of MAMP‐activated MAP kinase (MAPK) cascades. In addition to blocking MKK5 (MAPK kinase 5) activation in the MEKK1 (MAPK kinase kinase 1)/MEKKs–MKK4/5–MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in the MEKK1–MKK1/2–MPK4 cascade and the plasma membrane‐localized receptor‐like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane‐localized receptor‐like kinase that is involved in multiple MAMP signaling. The interaction between BAK1 and HopF2 and between two other P. syringae effectors, AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of AvrPto, AvrPtoB and HopF2, the strong growth defects or lethality associated with ectopic expression of these effectors in wild‐type Arabidopsis transgenic plants were largely alleviated in bak1 mutant plants. Thus, our results provide genetic evidence to show that BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2. Identification of BAK1 as an additional target of HopF2 virulence not only explains HopF2 suppression of multiple MAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors act through multiple targets in host cells.     

Phosphorylation of the Pseudomonas syringae effector AvrPto is required for FLS2/BAK1-independent virulence activity and recognition by tobacco

The type III effector protein AvrPto from Pseudomonas syringae pv. tomato is secreted into plant cells where it promotes bacterial growth and enhances symptoms of speck disease on susceptible tomato plants. The virulence activity of AvrPto is due, in part, to its interaction with components of host pattern recognition receptor complexes, which disrupts pathogen-associated molecular pattern-triggered immunity. This disruption mechanism requires a structural element of the AvrPto protein, the CD loop, which is also required for triggering Pto/Prf-mediated resistance in tomato. We have shown previously that the carboxyl-terminal domain (CTD) of AvrPto is phosphorylated and also contributes to bacterial virulence. Here we report that phosphorylation of the CTD on S147 and S149 promotes bacterial virulence in an FLS2/BAK1-independent manner, which is mechanistically distinct from the CD loop. In a striking corollary with Pto recognition of the CD loop in tomato, the tobacco species Nicotiana sylvestris and Nicotiana tabacum have a recognition mechanism that specifically detects the phosphorylation status of the CTD. Thus different species in the Solanaceae family have evolved distinct recognition mechanisms to monitor the same type III effector.     

Pseudomonas syringae effector HopZ3 suppresses the bacterial AvrPto1–tomato PTO immune complex via acetylation

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1_Psy-triggered immunity. HopZ3 acetylates AvrPto1_Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1_Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1_Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3’s targets such as AvrPto1_Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3’s unusual capacity to modify histidine in addition to serine, threonine and lysine residues.     

AvrE1 and HopR1 from Pseudomonas syringae pv. actinidiae are additively required for full virulence on kiwifruit

Pseudomonas syringae pv. actinidiae ICMP 18884 biovar 3 (Psa3) produces necrotic lesions during infection of its kiwifruit host. Bacterial growth in planta and lesion formation are dependent upon a functional type III secretion system (T3S), which translocates multiple effector proteins into host cells. Associated with the T3S locus is the conserved effector locus (CEL), which has been characterized and shown to be essential for the full virulence in other P. syringae pathovars. Two effectors at the CEL, hopM1 and avrE1, as well as an avrE1-related non-CEL effector, hopR1, have been shown to be redundant in the model pathogen P. syringae pv. tomato DC3000 (Pto), a close relative of Psa. However, it is not known whether CEL-related effectors are required for Psa pathogenicity. The Psa3 allele of hopM1, and its associated chaperone, shcM, have diverged significantly from their orthologs in Pto. Furthermore, the CEL effector hopAA1-1, as well as a related non-CEL effector, hopAA1-2, have both been pseudogenized. We have shown that HopM1 does not contribute to Psa3 virulence due to a truncation in shcM, a truncation conserved in the Psa lineage, probably due to the need to evade HopM1-triggered immunity in kiwifruit. We characterized the virulence contribution of CEL and related effectors in Psa3 and found that only avrE1 and hopR1, additively, are required for in planta growth and lesion production. This is unlike the redundancy described for these effectors in Pto and indicates that these two Psa3 genes are key determinants essential for kiwifruit bacterial canker disease.     

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.     

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.     

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.     

The T-loop Extension of the Tomato Protein Kinase AvrPto-dependent Pto-interacting Protein 3 (Adi3) Directs Nuclear Localization for Suppression of Plant Cell Death

In tomato (Solanum lycopersicum), resistance to Pseudomonas syringae pv. tomato is elicited by the interaction of the host Pto kinase with the pathogen effector protein AvrPto, which leads to various immune responses including localized cell death termed the hypersensitive response. The AGC kinase Adi3 functions to suppress host cell death and interacts with Pto only in the presence of AvrPto. The cell death suppression (CDS) activity of Adi3 requires phosphorylation by 3-phosphoinositide-dependent protein kinase 1 (Pdk1) and loss of Adi3 function is associated with the hypersensitive response cell death initiated by the Pto/AvrPto interaction. Here we studied the relationship between Adi3 cellular localization and its CDS activity. Adi3 is a nuclear-localized protein, and this localization is dictated by a nuclear localization signal found in the Adi3 T-loop extension, an ∼80 amino acid insertion into the T-loop, or activation loop, which is phosphorylated for kinase activation. Nuclear localization of Adi3 is required for its CDS activity and loss of nuclear localization causes elimination of Adi3 CDS activity and induction of cell death. This nuclear localization of Adi3 is dependent on Ser-539 phosphorylation by Pdk1 and non-nuclear Adi3 is found in punctate structures throughout the cell. Our data support a model in which Pdk1 phosphorylation of Adi3 directs nuclear localization for CDS and that disruption of Adi3 nuclear localization may be a mechanism for induction of cell death such as that during the Pto/AvrPto interaction.