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The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding oligomerization domain-like receptors (NLRs) L6 from flax and disease resistance protein RPS4 from Arabidopsis Here we show that the crystal structure of the TIR domain from the Arabidopsis NLR suppressor of npr1-1, constitutive 1 (SNC1) contains both an L6-like interface involving helices αD and αE (DE interface) and an RPS4-like interface involving helices αA and αE (AE interface). Mutations in either the AE- or DE-interface region disrupt cell-death signaling activity of SNC1, L6, and RPS4 TIR domains and full-length L6 and RPS4. Self-association of L6 and RPS4 TIR domains is affected by mutations in either region, whereas only AE-interface mutations affect SNC1 TIR-domain self-association. We further show two similar interfaces in the crystal structure of the TIR domain from the Arabidopsis NLR recognition of Peronospora parasitica 1 (RPP1). These data demonstrate that both the AE and DE self-association interfaces are simultaneously required for self-association and cell-death signaling in diverse plant NLRs.

SUMMARY: Arabidopsis thaliana WRKY proteins are potential targets of pathogen‐secreted effectors. RESISTANT TO RALSTONIA SOLANACEARUM 1 (RRS1; AtWRKY52) is a well‐studied Arabidopsis nucleotide‐binding and leucine‐rich repeat (NLR) immune receptor carrying a C‐terminal WRKY domain that functions as an integrated decoy. RRS1‐R recognizes the effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia pseudosolanacearum by direct interaction through its WRKY domain. AvrRps4 and PopP2 were previously shown to interact with several AtWRKYs. However, how these effectors selectively interact with their virulence targets remains unknown. Here, we show that several members of subgroup IIIb of the AtWRKY family are targeted by AvrRps4 and PopP2. We demonstrate that several AtWRKYs induce cell death when transiently expressed in Nicotiana benthamiana, indicating the activation of immune responses. AtWRKY54 was the only cell death‐inducing AtWRKY that interacted with both AvrRps4 and PopP2. We found that AvrRps4 and PopP2 specifically suppress AtWRKY54‐induced cell death. We also demonstrate that the amino acid residues required for the avirulence function of AvrRps4 and PopP2 are critical for suppressing AtWRKY54‐induced cell death. AtWRKY54 residues predicted to form a binding interface with AvrRps4 were predominantly located in the DNA binding domain and necessary for inducing cell death. Notably, one AtWRKY54 residue, E164, contributes to affinity with AvrRps4 and is exclusively present among subgroup IIIb AtWRKYs, yet is located outside of the DNA‐binding domain. Surprisingly, AtWRKY54 mutated at E164 evaded AvrRps4‐mediated cell death suppression. Taking our observations together, we propose that AvrRp4 and PopP2 specifically target AtWRKY54 to suppress plant immune responses. Significance Statement: RESISTANT TO RALSTONIA SOLANACEARUM 1 (RRS1) is an Arabidopsis disease resistance protein that recognizes two sequence‐unrelated bacterial effectors, AvrRps4 and PopP2, through its WRKY domain. To better understand the functions of AvrRps4 and PopP2, we explored Arabidopsis WRKY proteins (AtWRKY) and found that AvrRps4 and PopP2 selectively associate with AtWRKY54 and suppress AtWRKY54‐induced immune response. [ABSTRACT FROM AUTHOR]

The bacterial wilt disease caused by soilborne bacteria of the Ralstonia solanacearum species complex (RSSC) threatens important crops worldwide. Only a few immune receptors conferring resistance to this devastating disease are known so far. Individual RSSC strains deliver around 70 different type III secretion system effectors into host cells to manipulate the plant physiology. RipE1 is an effector conserved across the RSSC and triggers immune responses in the model solanaceous plant Nicotiana benthamiana. Here, we used multiplexed virus‐induced gene silencing of the nucleotide‐binding and leucine‐rich repeat receptor family to identify the genetic basis of RipE1 recognition. Specific silencing of the N. benthamiana homologue of Solanum lycopersicoides Ptr1 (confers resistance to Pseudomonas syringae pv. tomato race 1) gene (NbPtr1) completely abolished RipE1‐induced hypersensitive response and immunity to Ralstonia pseudosolanacearum. The expression of the native NbPtr1 coding sequence was sufficient to restore RipE1 recognition in Nb‐ptr1 knockout plants. Interestingly, RipE1 association with the host cell plasma membrane was necessary for NbPtr1‐dependent recognition. Furthermore, NbPtr1‐dependent recognition of RipE1 natural variants is polymorphic, providing additional evidence for the indirect mode of activation of NbPtr1. Altogether, this work supports NbPtr1 relevance for resistance to bacterial wilt disease in Solanaceae. [ABSTRACT FROM AUTHOR]

Summary: Some nucleotide‐binding and leucine‐rich repeat receptors (NLRs) indirectly detect pathogen effectors by monitoring their host targets. In Arabidopsis thaliana, RIN4 is targeted by multiple sequence‐unrelated effectors and activates immune responses mediated by RPM1 and RPS2. These effectors trigger cell death in Nicotiana benthamiana, but the corresponding NLRs have yet not been identified. To identify N. benthamiana NLRs (NbNLRs) that recognize Arabidopsis RIN4‐targeting effectors, we conducted a rapid reverse genetic screen using an NbNLR VIGS library.We identified that the N. benthamiana homolog of Ptr1 (Pseudomonas tomato race 1) recognizes the Pseudomonas effectors AvrRpt2, AvrRpm1, and AvrB.We demonstrated that recognition of the Xanthomonas effector AvrBsT and the Pseudomonas effector HopZ5 is conferred independently by the N. benthamiana homolog of Ptr1 and ZAR1. Interestingly, the recognition of HopZ5 and AvrBsT is contributed unequally by Ptr1 and ZAR1 in N. benthamiana and Capsicum annuum. In addition, we showed that the RLCK XII family protein JIM2 is required for the NbZAR1‐dependent recognition of AvrBsT and HopZ5.The recognition of sequence‐unrelated effectors by NbPtr1 and NbZAR1 provides an additional example of convergently evolved effector recognition. Identification of key components involved in Ptr1 and ZAR1‐mediated immunity could reveal unique mechanisms of expanded effector recognition. [ABSTRACT FROM AUTHOR]

Clavibacter michiganensis , a Gram-positive plant-pathogenic bacterium, utilizes apoplastic effectors for disease development in host plants. Here, we determine the roles of Pat-1Cm (a putative serine protease) in pathogenicity and plant immunity. Pat-1Cm was found to be a genuine secreted protein, and the secreted mature form did not carry the first 33 amino acids predicted to be a signal peptide (SP). The pat-1Cm mutant impaired to cause wilting, but still caused canker symptom in tomato. Moreover, this mutant failed to trigger the hypersensitive response (HR) in a nonhost Nicotiana tabacum. Among orthologs and paralogs of pat-1Cm , only chp-7Cs from Clavibacter sepedonicus , a potato pathogen, successfully complemented pat-1Cm function in pathogenicity in tomato, whereas all failed to complement pat-1Cm function in HR induction in N. tabacum. Based on the structural prediction, Pat-1Cm carried a catalytic triad for putative serine protease, and alanine substitution of any amino acids in the triad abolished both pathogenicity and HR-inducing activities of Pat-1Cm in C. michiganensis. Ectopic expression of pat-1Cm with an SP from tobacco secreted protein triggered HR in N. tabacum , but not in tomato, whereas a catalytic triad mutant failed to induce HR. Inoculation of the pat-1Cm mutant mixed with the mutant of another apoplastic effector CelA (cellulase) caused severe wilting in tomato, indicating that these two apoplastic effectors can functionally cooperate in pathogenicity. Overall, these results indicate that Pat-1Cm is a distinct secreted protein carrying a functional catalytic triad for serine protease and this enzymatic activity might be critical for both pathogenicity and HR-eliciting activities of Pat-1Cm in plants. [ABSTRACT FROM AUTHOR]