Summary Phosphorylation of receptor-like kinases (RLKs) plays an important role in the regulation of pattern-triggered immunity (PTI). Arabidopsis thaliana FLAGELLIN-SENSITIVE2 (FLS2) is a typical RLK that can sense a conserved 22 amino acid sequence in the N-terminal region of flagellin (flg22) to initiate plant defense pathways. However, the mechanisms underlying the regulation of FLS2 phosphorylation activity at the plasma membrane in response to flg22 remain largely enigmatic. Here, by single-particle tracking, we demonstrated that Ser-938 phosphorylation site affected flg22-induced FLS2 spatiotemporal dynamics and dwell time. Furthermore, using Förster resonance energy transfer-fluorescence lifetime (FRET-FLIM) imaging microscopy coupled with protein proximity indexes (PPI), we revealed that the degree of co-localization of FLS2/FLS2 S938D -GFP with AtRem1.3-mCherry increased in response to flg22, whereas FLS2 S938A -GFP did not show significant changes, indicating that Ser-938 phosphorylation site facilitates efficient sorting of FLS2 into nanodomains. Importantly, we found that the Ser-938 phosphorylation of FLS2 significantly increased flg22-induced internalization and immune responses. Taken together, these results illustrate that the phosphorylated site of FLS2 regulates the partitioning of FLS2 into functional membrane nanodomains to activate flg22-induced plant immunity.

Single-molecule analysis unveils the phosphorylation of FLS2 regulates its spatiotemporal dynamics and immunity