Fig. 5.

A Pi02860 mutant fails to interact with NRL1 or promote StSWAP70 degradation. (A) IP of protein extracts from N. benthamiana leaves transiently expressing cMYC-StNRL1 with either GFP-EV, GFP-Pi02860, or GFP-Pi02860^Δ126–135 using GFP-Trap confirmed that cMYC-StNRL1 associates with GFP-Pi02860 but not with GFP-Pi02860^Δ126–135. IP was analyzed by immunoblotting using an anti-GFP antibody showing protein fusions GFP-Pi02860, GFP-Pi02860^Δ126–135, GFP-EV, and the anti-cMYC antibody showing protein fusions of cMYC-StNRL1 of the expected size. (B) Expression of wild-type GFP-Pi02860 significantly enhances P. infestans infection compared with the control GFP-EV. However, the Pi02860^Δ126–135 fails to enhance P. infestans colonization. The results shown are combinations of three individual biological replicates (ANOVA, P < 0.001; n = 75 per construct) and error bars show SE. Letters on the graph denote statistically significant differences. (C) Suppression of ICD by Pi02860^Δ126–135 is significantly reduced (P < 0.001, n = 45). The graph shows the mean percentage of infiltration sites forming ICD by transient expression of INF1 with GFP-EV, GFP-Pi02860, GFP-PiAvr3a, or GFP-Pi02860^Δ126–135. The graph represents the combined data from three biological replicates. Letters on the graph denote statistically significant differences and error bars show SE. (D) The Pi02860^Δ126–135 mutant fails to degrade StSWAP70 by StNRL1. GFP-StSWAP70 and cMYC-StNRL1 were transiently coexpressed with either wild-type cMYC-Pi02860 or mutant cMYC-Pi02860^Δ126–135 in N. benthamiana leaves. Immunoblots with anti-GFP show the protein fusion of GFP-StSWAP70; the anti-cMYC antibody shows protein fusion of cMYC-Pi02860 and cMYC-Pi02860^Δ126–135 of the expected size.