Residues within the 120–144 CC region of Sr33 are crucial for self-association and autoactivity. (A) Alignment showing the 120–160 fragment of MLA10, Sr33, and Sr50. Six mutations (numbered 1–6) were generated in which conserved hydrophobic residues (red) were replaced by glutamate, or a hydrophilic stretch (blue) was replaced by alanines. (B) WT and mutated constructs of the Sr33 CC1–160 domain or full-length protein fused to HA or CFP were transiently expressed in N. benthamiana, and cell death was visualized after 4 d. Equivalent results were obtained in three independent experiments. (C) The indicated mutants of the Sr33 CC1–160 domain fused to CFP or HA tags were coexpressed in N. benthamiana and analyzed by coimmunoprecipitation as described in Fig. 2A. RGA41–171:CFP was used as a control for specificity, and WT Sr331–160:HA and Sr331–160:CFP were used as positive controls. Three independent experiments gave equivalent results.