Arabidopsis CRK28 is synthesized upon immune perception, associates with the FLS2 receptor complex, and silencing multiple related CRKs enhances disease suscept

Nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins function as sensors that perceive pathogen molecules and activate immunity. In plants, the accumulation and activation of NLRs is regulated by SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1). In this work, we found that an effector protein named RipAC, secreted by the plant pathogen Ralstonia solanacearum, associates with SGT1 to suppress NLR-mediated SGT1-dependent immune responses, including those triggered by another R. solanacearum effector, RipE1. RipAC does not affect the accumulation of SGT1 or NLRs, or their interaction. However, RipAC inhibits the interaction between SGT1 and MAP kinases, and the phosphorylation of a MAPK target motif in the C-terminal domain of SGT1. Such phosphorylation is enhanced upon activation of immune signaling and contributes to the activation of immune responses mediated by the NLR RPS2. Additionally, SGT1 phosphorylation contributes to resistance against R. solanacearum. Our results shed light onto the mechanism of activation of NLR-mediated immunity, and suggest a positive feedback loop between MAPK activation and SGT1-dependent NLR activation. Plant immune receptors are subjected to a tight regulation in order to avoid auto-immune responses, but must be promptly activated upon perception of pathogen threats. Plant intracellular immune receptors can perceive effector proteins injected inside plant cells; for most of these receptors, the SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1) constitutes an essential regulator that controls their stability and activation. In this work, we found that phosphorylation mediated by MAPKs regulates SGT1, and is required for the activation of intracellular immune receptors. Supporting the biological relevance of this finding, we found that a bacterial pathogen, Ralstonia solanacearum, injects an effector protein that inhibits the interaction between MAPKs and SGT1, thereby suppressing SGT1 phosphorylation and the associated effector-triggered immune responses. These results suggest the existence of a phosphorylation-mediated feedback loop between MAPKs and SGT1, which leads to the activation of immune responses, and can be targeted by bacterial virulence activities.

In both animals and plants, the perception of bacterial flagella by immune receptors elicits the activation of defence responses. Most plants are able to perceive the highly conserved epitope flg22 from flagellin, the main flagellar protein, from most bacterial species. However, flagellin from Ralstonia solanacearum, the causal agent of the bacterial wilt disease, presents a polymorphic flg22 sequence (flg22^Rso) that avoids perception by all plants studied to date. In this work, we show that soybean has developed polymorphic versions of the flg22 receptors that are able to perceive flg22^Rso. Furthermore, we identify key residues responsible for both the evasion of perception by flg22^Rso in Arabidopsis and the gain of perception by the soybean receptors. Heterologous expression of the soybean flg22 receptors in susceptible plant species, such as tomato, enhances resistance to bacterial wilt disease, demonstrating the potential of these receptors to enhance disease resistance in crop plants.

InPseudomonas syringae, the type III secretion system (T3SS) is essential for disease in compatible hosts and for eliciting the hypersensitive response in incompatible hosts.P. syringaepathovars secrete a variable number of type III effectors that form their secretomes. The secretome ofPseudomonas syringaepv. phaseolicola 1448a (Pph1448a) currently includes 22 experimentally validated effectors, one HrpL-regulated candidate for which translocation results have been inconsistent, two translocated candidates for whichin plantaexpression has not been established, one bioinformatically identified candidate, and six candidates that have been experimentally discarded. We analyzed the translocation and/or expression of these and other candidates to complete thePph1448a effector inventory, bringing this inventory to 27 bona fide effectors, including a new one that does not belong to any of the previously described effector families. We developed a simple process for rapidly making single and double knockout mutants and apply it to the generation of an effector mutant collection that includes single knockouts for the majority of thePph1448a effector inventory. We also generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the single and double mutant strains as well as strains expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to virulence in bean plants, thus validating this approach for dissecting the contribution of thePph1448a type III effector inventory to virulence. We also analyzed the effect that the expression of these four effectors fromPseudomonas syringaepv. tomato DC3000 (PtoDC3000) has during its interaction withArabidopsis thaliana, establishing that AvrB4-1, but not the others, determines a restriction of bacterial growth that takes place mostly independently of the salicylic acid (SA)-signaling pathway.