Characterization of suppressor of slh1 immunity (sushi) mutations in Arabidopsis thaliana

Abstract

Plants have evolved the innate immune systems against various kind of pathogens, represented by PTI (pattern-triggered immunity) using PRRs (Pattern recognition receptors) and ETI (effector-triggered immunity) by NLRs (NB-LRR receptors). RRS1 and RPS4 are atypical paired TNL (TIR-NB-ARC-LRR) class R (resistance) proteins. RRS1 carries a WRKY transcription factor (TF) in C-terminus as a decoy to recognize effectors, such as PopP2 from Ralstonia solanacearum and AvrRps4 from Pseudomonas syringae. However, the detailed mechanism of recognition is yet unclear. In order to illustrate the RRS1/RPS4-mediated immune signaling in detail, suppressor mutants of slh1, an autoactive mutant form of RRS1 were identified using forward genetic mutant screening approach in previous studies. Here in this study, suppressor of slh1 mutants, so called ‘sushi’ mutations are categorized and characterized. The sushi mutations in RPS4 was previously characterized (Sohn et al., 2014). RPS4sushi mutation study revealed that heterodimerization of TIR domain in RPS4 and RRS1 is important in slh1 autoimmunity and the P-loop of RPS4 is necessary for autoimmune signaling while that of RRS1 is not (Sohn et al., 2014). In this study, 5 causal sushi mutations in RRS1 (RRS1sushi) were characterized. Interestingly, C15Y and P68L mutations were both in the TIR domain abolishing autoimmunity of slh1 but only P68L mutation could recognize effectors in transgenic Arabidopsis, demonstrating these two residues in TIR domain were differentially required for triggering autoimmune signaling and effector recognition. Furthermore, G176E mutation in NB-ARC domain and C607Y mutation in LRR domain could recognize effector, on the other hand, L816F mutations in LRR domain failed to recognize effectors. These results indicate that there are two different ways of signaling between effector recognition and triggering autoimmunity, which can be differentially interfered with RRS1sushi mutations. There is another type of sushi mutants which has no causal mutation in RRS1 or RPS4, therefore are categorized in ‘unknown’ sushi mutants. It was demonstrated here that one of the unknown sushi mutations, sushi63, was mapped approximately on the lower arm of chromosome 5 by bulked segregant analysis using F2 progenies between sushi63 and rrs1-3. Two SUSHI63 candidate genes were found by bioinformatics analysis after NGS (Next generation sequencing). These two candidates, At5g42250, an alcohol dehydrogenase (ADH) family member, and At5g45510, of the CC-LRR like family, will be confirmed whether they are SUSHI63 or not by introducing each gene in original sushi63. In this study, it was characterized that autoimmune signaling and effector recognition signaling have different mechanisms. Moreover, finding candidates of a new gene involving paired NLR defense signaling will give us a new insight of R gene signaling pathways.