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

Whether 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.

Here, 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.

Both receptors restored flg22 ^Pst -triggered reactive oxygen species production and MAPK activation, physically associated with flg22 ^Pst , and required conserved residues for flg22 ^Pst recognition. In stable transgenic plants, both receptors mediated flg22 ^Pst -triggered PTI outputs and flg22 ^Pst -induced resistance to Pseudomonas syringae . PtFLS2 additionally mediated responsiveness to flg22 ^Rso and enhanced resistance to Ralstonia solanacearum , whereas GbFLS2 retained a typical flg22 ^Pst -recognition profile.

These 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.