Fig. 4

Nonsynonymous SNP sites of 319 and 326 in TIR1-TIR2 caused differences in HR strength between the two haplotypes, and the minimum functional region is TIR1-TIR2. a,b HR phenotypes associated with chimeras or site-directed mutants of TIR1-TIR2_R and TIR1-TIR2_S alleles. RS and SR are artificial constructs of a combination of R-TIR1-S-TIR2 and S-TIR1-R-TIR2, respectively. Site-directed mutants were converted to S alleles at the nonsynonymous SNP sites in a TIR1-TIR2_R background. c Evaluation of protein expression using a Flag antibody, and immunoblotting of plant actin with α-actin was used as a loading control. d,e LUC activity within the region co-transformed with a LUC plasmid and TIR1-TIR2 variants. Fluorescence intensity was captured and data represent means ± SE of three independent experiments. Different letters indicate significant difference at α = 0.05 level via one-way ANOVA analysis. f,g Cell death phenotype in cotton protoplast for cell viability assays. Protoplast preparation and construct transformation used 14-day-old plant leaves. Fluorescence intensity was captured. Three independent experiments are represented. h Schematic diagram of the GhRVD1 domain structure. Individual domains are presented in a colored box, and the boundaries of truncated sequences are marked on the top. The numbers and names of truncated sequences are marked on the right and left, respectively. i HR phenotypes of truncated derivatives of GhRVD1_R. j Evaluation of protein expression using a Flag antibody, immunoblotting of plant actin with α-actin was used as a loading control. k LUC activity within the region co-transformed with a LUC plasmid and a series of truncated derivatives of GhRVD1. Numbers on the top represent corresponding truncated sequences