Fig. 4: A 22 amino acid fragment in the N terminus of SGP1 is sufficient to mediate cell death in N. benthamiana.

a–c Regions of SGP1 examined for cell death activity. Plus symbol (+), cell death phenotype; minus symbol (−), no clear cell death phenotype. Cell death phenotypes were recorded at 5 dpi. a Regions of SGP1 were examined for cell death activity by agrobacterium-mediated transient expression. Similar results were obtained from four independent experiments, with 10 plants per strain in each experiment (Supplementary Data 3). b Western blot analysis of SGP1 and truncated mutant variants transiently expressed in N. benthamiana leaves. The red asterisk (*) indicates the predicted protein sizes. c Regions of SGP1 examined for cell death activity in N. benthamiana by infiltration with 1 μM purified SGP1 variants from E. coli. Similar results were obtained from three independent experiments (Supplementary Data 3). d Sequence logos indicating the level of conservation for each residue of SNP22 region in GPI-anchored proteins that trigger cell death from plant-associated fungi. Sequence logos were constructed with Geneious R11 (Supplementary Data 6). The character and size of each logo represent the proportion of homologs with the amino acid at that specific site. e, f Cell death triggered by expression of recombinant SNP22 homolog proteins from other fungi and alanine-substituted versions of SNP22rec. e Sequence alignment and cell death-inducing activity of the SNP22 region of SGP1 homologs that can induce cell death. The homologous sequences of SNP22 from other fungi, including four amino acids flanking either terminus (Supplementary Data 7), were expressed in E. coli. Vm, KUI59516 (Valsa mali); Bc, Bcin01p05310.2 (Botrytis cinerea); Ss, EDO01950 (Sclerotinia sclerotiorum); Cp, CCE31100 (Claviceps purpurea); Fv, FVEG_00876T0 (Fusarium verticillioides); Fs, NechaP98847 (Fusarium solani); Vd, VDAG_JR2_Chr3g03370a-00001 (Verticillium dahliae); Mo, MGG_04599T0 (Magnaporthe oryzae). Three independent experiments were performed, using 10 plants for each SNP22 homolog (Supplementary Data 3) in each experiment. f Representative photos of cell death triggered by SNP22rec variants with single alanine substitutions at the indicated residues. Similar results were obtained from three independent experiments, with five plants per protein in each experiment (Supplementary Data 3).