Figure 5.
WPP2 and HSC70-1 decrease aggregation of WIT11-660 in N. benthamiana, and WPP2 promotes its NE-targeting. A, GFP-WIT11-660 was either expressed alone or coexpressed by coagroinfiltration in N. benthamiana leaves with one of the HA-tagged constructs: GUS, WPP2, and HSC70-1 in the presence of p19. A, Aggregation. Bars = 10 μm. B, Anti-HA immunoblot of protein extracts from infiltrated leaves expressing GFP-WIT11-660 with the constructs as explained in A (top). Anti-GFP immunoblot showing GFP-WIT11-660 expression levels in the same protein extracts (bottom). Ponceau S-stained membranes are shown below each blot to indicate equal loading. C and D, Quantification of aggregation (C) and NE targeting (D) in the coinfiltration experiment shown in A. The graphs depict the mean and sd values obtained from two independent experiments. A minimum of 100 cells total were scored for each agroinfiltrated combination. E, HA-WPP2 was coexpressed with either GFP, GFP-WIT1, or GFP-WIT11-660 (left); HA-HSC70-1 was coexpressed with either GFP, GFP-WIT1, or GFP-WIT11-660 (right) in N. benthamiana in the presence of p19. Immunoprecipitation was performed using the anti-HA antibody, and coimmunoprecipitated proteins were detected with the anti-GFP antibody. IP, Immunoprecipitate. [See online article for color version of this figure.]