Cytoplasmic localization of GFP-Pi02860 is important for P. infestans virulence and INF1 HR suppression phenotypes. A, Confocal images showing that GFP-Pi02860 is localized in the cytoplasm and nucleus, NESGFP-Pi02860 is greatly reduced in nuclear fluorescence, and NLSGFP-Pi02860 is concentrated in the nucleus and reduced in the cytoplasm. The top row shows stacked projections of single cells, while the bottom row shows single slice images of the nuclei. Bars =10 μm. B, GFP-Pi02860 and NESGFP-Pi02860 expression leads to statistically significant increases (P < 0.001, n = 68) in P. infestans lesion diameter compared with free GFP, whereas NLSGFP-Pi02860 shows an intermediate phenotype. C, Representative leaf image showing P. infestans lesions following overexpression of each construct, as indicated, in N. benthamiana. D, GFP-Pi02860 and NESGFP-Pi02860 coexpression with INF1 leads to a statistically significant decrease (P < 0.001, n = 41) in HR compared with free GFP, whereas NLSGFP-Pi02860 shows an intermediate phenotype. E, Representative leaf image showing INF1 HR following coexpression with each construct, as indicated, in N. benthamiana. Lowercase letters on graphs denote statistically significant differences by one-way ANOVA, with pairwise comparisons performed with the Holm-Sidak method. Results shown are combinations of three independent biological replicates, each with 18 infiltration zones. Error bars show se.