Disulfide bond sculpts a peptide fold that mediates phytocytokine recognition

Precise ligand recognition by closely related leucine-rich repeat receptor kinases (LRR-RKs) is essential for plants to coordinate immunity, development, and environmental adaptation. Here, we show how HAESA-LIKE 3 (HSL3) specifically recognizes the folded, disulfide-stabilized CTNIP/SCREW phytocytokines in Arabidopsis. Quantitative binding assays define a minimal CTNIP4 region required for high-affinity HSL3 interaction and signaling activation. A 2.12 Å crystal structure of the HSL3-CTNIP4 complex reveals a unique C-terminal receptor pocket that accommodates the peptide’s cyclic architecture through a combination of hydrophobic and polar contacts, a feature absent in the closely related HAE/HSL LRR-RKs. The cyclic CTNIP4 fold further establishes a largely hydrophobic interface that bridges HSL3 to the SERK co-receptor, forming a distinct activation surface. Together, these structural, biochemical and physiological insights uncover a previously unrecognised mechanism of peptide perception and receptor activation, highlighting how subtle architectural variations enable precise ligand selectivity among highly conserved plant receptor kinases.