Structural and biochemical basis of FLS2-mediated signal activation and transduction in rice

The receptor-like kinase FLAGELLIN-SENSITIVE 2 (FLS2) functions as a bacterial flagellin receptor localized on the cell membrane of plants. In Arabidopsis, the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) cooperates with FLS2 to detect the flagellin epitope flg22, resulting in the formation of a signaling complex that triggers plant defense responses. However, the co-receptor responsible for recognizing and signaling the flg22 epitope in rice remains undetermined, and the precise structural mechanism underlying FLS2-mediated signal activation and transduction has not been elucidated. This study presents the structural characterization of a kinase-dead mutant of the intracellular kinase domain of OsFLS2 (OsFLS2-KDD1013A) in complex with ATP or ADP, resolved at resolutions of 1.98 Å and 2.09 Å, respectively. Structural analysis reveals that OsFLS2 can adopt an active conformation in the absence of phosphorylation, albeit exhibiting significantly weak basal catalytic activity for autophosphorylation. Subsequent investigations demonstrate that OsSERK2 effectively phosphorylates OsFLS2, which reciprocally phosphorylates OsSERK2, leading to the complete activation of OsSERK2 and the rapid phosphorylation of the downstream substrate receptor-like cytoplasmic kinases OsRLCK176 and OsRLCK185. Through mass spectrometry experiments, we successfully identified critical autophosphorylation sites on OsSERK2, as well as sites transphosphorylated by OsFLS2. Furthermore, we demonstrated the interaction between OsSERK2 and OsFLS2, which is enhanced in the presence of flg22. Additionally, our genetic evidence suggests that OsRLCK176 and OsRLCK185 may function downstream of the OsFLS2-mediated signaling pathway. Our study elucidates the molecular mechanism of OsFLS2-mediated signal transduction pathways in rice and provides a valuable example for comprehending RLK-mediated signal pathways in plants.