flg22 is an elicitor of plant defense responses that binds to the LRRs of the receptor complex FLS2 and BAK1. This interaction triggers the recruitment of the intracellular kinases PBS1 and BIK1, and subsequently the activation of MAP kinase pathways, leading to cell death and disease resistance.
Previous targeted mutagenesis studies have shown that polymorphisms in flg22 abolish FLS2/BAK1 recognition of this pathogen peptide1,2. We here use a high-throughput sequencing approach to identify additional flg22 interacting partners and to determine the specific functional residues required for flg22 perception. Our data show that the C-terminal domain of flg22 (residues 30 to 51) is critical for its elicitor activity. flg22-interacting proteins include the kinases PIK3 and PIK5, the cytosolic protein PINK1, and the protein-protein complexes SPIK1 and PINK1. Moreover, we found that a single residue change in flg22Psy (I21 to A) abolished its ability to elicit a ROS burst in GmFLS2b transiently expressed in N. benthamiana, as well as its ability to trigger a detectable interaction between GmFLS2b and BAK1 assessed by coIP and Forster resonance energy transfer-fluorescence lifetime imaging.
Heterologous expression of GmFLS2/GmBAK1 in tomato plants conferred responsiveness to flg22Psy and induced resistance to R. solanacearum upon soil-drenching inoculation. We further showed that a gain-of-perception mutation in the flg22Psy I21 residue reduced binding energy with PIK3, suggesting that this site of polymorphism is responsible for avoiding perception by the PRRs GmFLS2b and BAK1. We also identified structural adaptions in the interaction of GmFLS2b with flg22, identifying several key amino acid residues that form hydrophobic interactions. These are likely to be involved in complex formation, and the flg22Psy-associated GmFLS2b polymorphisms can potentially function as a tool for engineering a gain-of-perception mutant of this important plant virulence factor.