Fig. 4.

Pi02860-mediated degradation of StSWAP70 requires dimerization of StNRL1. (A) IP of protein extracts from N. benthamiana leaves transiently expressing GFP-StSWAP70 with either cMYC-StNRL1 or cMYC-StNRL1^NQ using GFP-Trap confirmed that cMYC-StNRL1 associates with GFP-StSWAP70 but the dimerization mutant cMYC-StNRL1^NQ does not. IP was analyzed by immunoblotting using an anti-GFP antibody showing protein fusion of GFP-StSWAP70 and the anti-cMYC antibody showing protein fusions of cMYC-StNRL1 and cMYC-StNRL1^NQ of the expected size. (B) The dimerization mutant StNRL1^NQ prevents degradation of StSWAP70. Transient coexpression of GFP-StSWAP70 and cMYC-Pi02860 in the presence of either wild-type FLAG-StNRL1 or mutant FLAG-StNRL1^NQ in N. benthamiana leaves, with or without MG132 treatment. Immunoblots with anti-GFP show stable protein fusion of GFP-StSWAP70, the anti-cMYC antibody shows protein fusion of cMYC-Pi02860 and anti-FLAG antibody shows stable protein fusions of FLAG-NRL1 and FLAG-StNRL1 of the expected size. (C) Expression of wild-type GFP-StNRL1 significantly enhances P. infestans infection compared with the control GFP-EV, whereas the dimerization mutant GFP-StNRL1^NQ reduces P. infestans colonization. The results shown are combinations of at least five individual biological replicates (ANOVA, P < 0.05; n = 82 per construct) and error bars show SE. Letters on the graph denote statistically significant differences. Representative leaf image showing P. infestans lesions following overexpression of each construct, as indicated, in N. benthamiana.