A sunflower FLS2 guides reconstruction of recognition of a Xanthomonas flg22 escape variant across divergent FLS2 receptors

Abstract

FLS2-mediated perception of the bacterial flagellin epitope flg22 is frequently compromised by pathogen sequence variation, and Val14-containing Xanthomonas flg22 variants remain unrecognized by multiple characterized expanded-recognition FLS2 homologues. Using AlphaFold 3-based structural screening of 256 natural FLS2 homologues against the flg22 epitope from Xanthomonas campestris pv. campestris (flg22Xcc), we identified sunflower HaFLS2-1 as the top structural candidate and experimentally validated its recognition through biochemical and immune-response assays. We then used ligand-proximal HaFLS2-1 residue states as a donor reference for iterative engineering of divergent FLS2 homologues from two distinct plant families, PsFLS2 and TjFLS2. Distinct recipient-specific residue solutions emerged in the two backgrounds, and representative functional variants carrying ten or fewer amino-acid substitutions acquired flg22Xcc recognition. Together, these results show that, for this defined flg22Xcc escape challenge, AF3-guided discovery and iterative local sequence exploration can reconstruct recognition across divergent FLS2 scaffolds through a limited number of receptor substitutions.