Fig. 6
GhTIRP1 binds to TIR1-TIR2_R in vivo and inhibits autoactivity and self-association of TIR1-TIR2_R by competing for binding to it. a Growth of yeast cells co-expressing AD-TIRP1 and BD-TIR fusions on synthetic media; TIR domains of L6 (residues 29–248), TIR domains of RPP1_NdA (residues 1–254), and TIR domains of RPS4 (residues 1–183). Yeast cells co-expressing AD-TIRP1 and BD-TIR1-TIR2 can grow on the SD/ − LTHA plates. b Split luciferase complementation indicates an interaction between TIRP1 and TIR1-TIR2_R. c Heterodimerization of TIRP1 and TIR1-TIR2_R detected by Co-IP in N. benthamiana leaves; GFP was used as a negative control and actin was used as a loading control. d Interaction between nYFP-TIR1-TIR2 and TIRP1-cYFP and between nYFP-GhRVD1 and TIRP1-cYFP induced fluorescence in N. benthamiana leaves. EV, empty vector. Defense cells are marked by red arrows. e, f HR phenotypes associated with co-inoculation of TIRP1 and plant TNLTIR; increasing concentrations of TIRP1 (OD600 = 0, 0.5, 1.0, and 1.5) gradually alleviated the cell death phenotype induced by TIR1-TIR2; GFP was used as a negative control. Evaluation of protein expression using α-Flag (for TIR1-TIR2, RPP1_NdATIR, and RPS2) and α-GFP (for TIRP1). Immunoblotting of plant actin with α-actin was used as a loading control. g, h TIRP1 outcompetes TIR1-TIR2_R for binding to TIR1-TIR2_R in a concentration-dependent manner in N. benthamiana leaves, as determined by coimmunoprecipitation. A series of Agrobacterium concentrations of TIRP1-GFP are shown (OD600 = 0, 0.5, 1.0, and 1.5) and co-inoculated with a constant concentration of TIR1-TIR2 (OD600 = 1.5) into leaf tissues; GFP was used as a negative control. Blotting of IP groups from different lanes were measured by ImageJ, and the ratio between the maximum values (relative intensity) is represented in the histogram. Three independent experiments were analyzed, and between-group comparisons were made using Student’s t test