Figure 10.

Virulence of PiLLP-silenced transformants restored by DPI treatment. (A) Disease lesions induced by different isolates. Detached leaves of potato cultivar Impala were injected with H₂O, 0.05% DMSO and 0.5 μM DPI. The treated leaves were inoculated on injection sites with sporangia [~100 in 10 μL for WT, CK, and PiLLP-overexpressed sporangia, whereas 200 for PiLLP-silenced transformant sporangia]. Pictures were taken 5 day after inoculation. (B) Microscopic observations of invasive hyphae in potato leave. The inoculated leaves were boiled for ~2 min in lactophenol-trypan blue and decolorized in 2.5 g/ml chloral hydrate for 30–40 min. (C) Detached leaves of N. benthamiana were injected with H₂O, 0.05% DMSO and 0.5 μM DPI and then inoculated with sporangia of WT, CK, and PiLLP transformants. Twelve hours after inoculation, the leaves were incubated in 1 mg/mL DAB solution (pH 3.8), at room temperature for 8 h and destained with ethanol, and observed with a microscope. The bars indicate 10 μm. (D) Lesion diameter on DPI-untreated and -treated leaves induced by different isolates. The diameter of each lesion was measured at 5-day after inoculation. Three replicates were used for each treatment and the whole experiment was repeated once. Bars represent the standard deviation of three replicates. Statistical significance was analyzed using Student's t-test. The asterisk indicates the significant difference (^*P < 0.05, ^**P < 0.01). Two independent infection assays were performed with similar results.