Fig. 5: Structure-guided neofunctionalization of orphan CNLs and MLA receptor hybrids.

a, Illustration of Sr35 domain structure and hybrid receptors made from Sr35 homologues (Sh) in bread wheat (Triticum aestivum; TaSh1) and barley (Hordeum vulgare; HvSh1). Sr35^LRR (red) substitutes TaSH1^LRR and HvSH1^LRR (TaSH1^Sr35LRR and HvSH1^Sr35LRR). GOF receptor variants (TaSh1^GOF and HvSh1^GOF) were derived from sequence polymorphisms between Sr35, TaSH1 and HvSH1. b, Wheat protoplast transfections of TaSh1^Sr35LRR, HvSh1^Sr35LRR and controls co-expressed with AvrSr35. EV treatment defined the relative baseline (mean ± s.e.m.; n = 6). Test statistics derived from ANOVA and Tukey post hoc tests (P < 0.05). Exact P values for all protoplast plots are provided in Supplementary Table 3. c, Tobacco cell death of TaSh1^Sr35LRR and HvSh1^Sr35LRR. Representative data shown from seven replicates and scored for cell death. d, Western blot of hybrid receptors tested in tobacco. Pooled three replicates. Ponceau S staining as loading control. Composite image of same blot. e, Cryo-EM structure of Sr35 and structural predictions of TaSH1^LRR and HvSH1^LRR (ref. ²⁸). Polymorphisms between TaSH1 or HvSH1, and Sr35 are shown (orange). Residues mutated are shown red. f, Wheat protoplast transfections of TaSh1^GOF, HvSh1^GOF and controls co-expressed with AvrSr35. Experiment and statistics as in b. g, Tobacco cell death of TaSh1^GOF and HvSh1^GOF. Replicates and scoring as in c. h, Western blot of GOF experiment in tobacco. Replicates and loading control as in d. i, Graphical illustration of MLA hybrid receptors; design as in a (HvMla10^Sr35LRR and HvMla13^Sr35LRR). j, Wheat protoplast transfections of HvMla10^Sr35LRR, HvMla13^Sr35LRR and controls co-expressed with AvrSr35. Experiment and statistics as in b. k, Tobacco cell death of HvMla10^Sr35LRR and HvMla13^Sr35LRR. Replicates and scoring as in c. l, Western blot of MLA hybrid receptors in tobacco. Replicates and loading control as in d. Composite image of two independent blots.