Gymnosperm FLS2 orthologues mediate conserved flg22 perception and divergent responses to flg22 variants

O_LIWhether gymnosperm FLAGELLIN-SENSING 2 (FLS2) orthologues are functional receptors and whether their flg22-recognition spectra have already diversified remain unclear, despite the central role of FLS2 in flagellin perception in angiosperms. C_LIO_LIHere, we identified two gymnosperm FLS2 orthologues, GbFLS2 from Ginkgo biloba and PtFLS2 from Pinus tabuliformis, and analysed their function using transient and stable expression in the Nicotiana benthamiana fls2 mutant, in planta cross-linking assays, AlphaFold3 modelling and structure-guided mutagenesis. C_LIO_LIBoth receptors restored flg22Pst-triggered reactive oxygen species production and MAPK activation, physically associated with flg22Pst, and required conserved residues for flg22Pst recognition. In stable transgenic plants, both receptors mediated flg22Pst-triggered PTI outputs and flg22Pst-induced resistance to Pseudomonas syringae. PtFLS2 additionally mediated responsiveness to flg22Rso and enhanced resistance to Ralstonia solanacearum, whereas GbFLS2 retained a typical flg22Pst-recognition profile. C_LIO_LIThese findings provide direct evidence that gymnosperm FLS2 orthologues can function as bona fide flagellin receptors in a heterologous angiosperm background, and further suggest that diversification in flg22 recognition had already emerged within gymnosperms. C_LI