Dissection of the FLS2/flagellin interaction in Arabidopsis thaliana

Recognition of pathogen-associated molecular patterns (PAMPs) by the plant innate immune system activates plant defense responses. Bacterial flagellin, a potent PAMP, is recognized by the Arabidopsis transmembrane receptor kinase FLS2. FLS2 binds flagellin, an interaction that is believed to occur within the FLS2 extracellular leucine-rich repeat (LRR) domain. However, methods to identify active sites within LRR domains have not been perfected. We identified flagellin and FLS2 residues involved in flagellin perception and studied the role of this interaction in bacterial virulence. Site-directed mutagenesis libraries of beta-stand/beta-turn residues within the FLS2 LRR domain were created and alleles were screened in Arabidopsis for their ability to perceive flg22, the elicitation-active domain of flagellin. Alanine scanning across the FLS2 LRR domain identified LRRs 9-15 as being involved in flg22 perception, a result supported by Conserved Functional Group analysis of FLS2 LRR domain homologues from diverse Brassicaceae. Saturation mutagenesis of beta-stand/beta-turn residues in LRRs 9-15 identified certain residues in LRRs 9-15 that are important for flagellin perception. A separate study investigating flagellins from diverse Xanthomonas campestris pv. campestris ( Xcc) strains revealed within-species, within-pathovar variation in flagellin defense-eliciting activity, a phenomenon that had not previously been described. A third study examined Ralstonia solanacearum (Rs), which requires flagellin-mediated motility for full virulence. While Rs extracts did contain a strong elicitor of defense-associated responses, we determined that the elicitor was not flagellin. The presence of flagellin or FLS2 had no effect on Rs-induced disease and a Rs flg22 peptide did not elicit defense responses. We conclude that Rs contains an unrecognized flagellin, and also a second elicitor that purifies at 5-10 kDa and also at higher molecular masses, possibly because of aggregation. From these and other studies, we conclude that plant immune systems do not uniformly recognize flagellins from a particular pathogen species and that Xcc and Rs have evolved to evade FLS2-mediated defense responses. These studies also have begun to identify determinants of specificity in the FLS2/flagellin interaction, and offer a conceptual framework for future study of other LRR-containing proteins.