Single-molecule analysis reveals the phosphorylation of FLS2 governs its spatiotemporal dynamics and immunity

TheArabidopsis thalianaFLAGELLIN-SENSITIVE2 (FLS2), a typical receptor kinase, recognizes the conserved 22 amino acid sequence in the N-terminal region of flagellin (flg22) to initiate plant defense pathways, which was intensively studied in the past decades. However, the dynamic regulation of FLS2 phosphorylation at the plasma membrane after flg22 recognition needs further elucidation. Through single-particle tracking, we demonstrated that upon flg22 treatment, the phosphorylation of Ser-938 in FLS2 impacts its spatiotemporal dynamics and lifetime. Following förster resonance energy transfer-fluorescence lifetime (FRET-FLIM) imaging microscopy and protein proximity indexes (PPI) assays revealed that flg22 treatment increased the co-localization of GFP-tagged FLS2/FLS2^S938Dbut not FLS2^S938Awith AtRem1.3-mCherry, a sterol-rich lipid marker, indicating that the phosphorylation of FLS2^S938affects FLS2 sorting efficiency to AtRem1.3-associated nanodomains. Importantly, we found that the phosphorylation of Ser-938 enhanced flg22-induced FLS2 internalization and immune responses, demonstrating that the phosphorylation may activate flg22-triggered immunity through partitioning FLS2 into functionalAtRem1.3-associated nanodomains, which fills the gap between the FLS2^S938phosphorylation and FLS2-mediated immunity.