P-081 - Structural and mutational analysis of a diversified amino acid motif across CC-NLR groups

Plant pathogens pose a risk to global food security. Resistance genes confer strain-specific resistance to pathogens, almost all of which encode for plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that induce immunity. The wheat coiled-coil (CC) domain containing NLR (CC-NLR) Sr35 forms a pentameric resistosome in complex with its pathogen effector, which binds to the leucin-rich repeat (LRR) domain, triggering a hypersensitive response (HR). An arginine cluster in the LRR-domain forms intramolecular salt bridges to the 'EDVID' motif in the CC-domain, which is crucial for resistosome function. Despite considerable progress in understanding of NLR protein structures and activation mechanisms (e.g. NRC2/4, ZAR1, MLA13) the function of the 'EDVID' motif in NLRs other than Sr35 and its closer homologs remains unclear. In this study, we analyse the variable function of this diversified amino acid motif in Arabidopsis thaliana CC-NLR across different phylogenetic groups, which occurs in reduced and extended variants, using structural and mutational analysis in HR-assays in Nicotiana benthamiana and place it into an evolutionary context. While mutations of the acidic residues in the extended motif have a clear effect, mutations in the reduced motif show varied results. Our data supports the hypothesis of divergent functions of this motif, suggesting different protein structures and currently unknown activation mechanisms.