Fig. 5
Self-association of TIR1-TIR2 and physical interaction between TIR1 and TIR2 depend on the DE interface and cause cell death. a HR phenotypes associated with site-directed mutants of TIR1-TIR2_R, T20A/Y21A, and N188A/H189A disrupting the AE interface of TIR1 and TIR2, respectively; G142R and G312 disrupting the DE interface of TIR1 and TIR2, respectively. AEM disrupts the AE interface of TIR1 and TIR2, and DEM disrupts the DE interface of TIR1 and TIR2. Autoactivity of TIR1-TIR2_R is disrupted by mutations in the predicted DE interface. b,c Evaluation of protein expression using a Flag antibody, immunoblotting of plant actin with α-actin was used as a loading control. LUC activity within the region is co-transformed with a LUC plasmid and TIR1-TIR2_R variants. Different letters indicate significant difference at α = 0.05 level via one-way ANOVA analysis. d Coimmunoprecipitation analysis of self-association of TIR1-TIR2_R and its variants. The indicated proteins were transiently expressed in N. benthamiana leaf cells by agroinfiltration. Total protein extracts were used for coimmunoprecipitation and immunoblot analyses. e Split luciferase complementation shows an interaction between TIR1-TIR2_R (left) and the interaction strength between TIR1-TIR2_R variants (right). Fluorescence signal intensity is indicated. f HR phenotypes associated with co-inoculation of TIR1L_R-YFP-HA and TIR2L_R-YFP-HA and that of their variants (1AEM and 2AEM disrupts the AE interface of TIR1L and TIR2L, respectively; 1DEM and 2DEM disrupts the DE interface of TIR1L and TIR2L, respectively). TIR1-TIR2-YFP-HA was used as positive control (top). Evaluation of protein expression using a HA antibody, and immunoblotting of plant actin with α-actin was used as a loading control (bottom). g Split luciferase complementation indicates the interaction between TIR1L_R and TIR2L _R (left), and the interaction strength between their variants (right). Fluorescence signal intensity is indicated. h Growth of yeast cells co-expressing AD-TIR1L_R-YFP and BD-TIR2L_R-YFP fusions, and their variants, on synthetic media lacking leucine and tryptophan (− LT) or selective media additionally lacking histidine and adenine (− LTHA). A series of dilutions are shown at the top of the figure. Autoactivation and toxicity of the fusion protein were first excluded, and AD-YFP and BD-YFP were used as a negative control for self-association activity. i Coimmunoprecipitation analysis of the interaction between TIR1L_R and TIR2L_R and that of their variants