FIG 5.

The HR-like response correlates with the silencing suppression activity of P25. (A) Leaves of N. benthamiana were infiltrated with GUS alone or combinations of Agrobacterium cultures containing binary constructs expressing a free GFP reporter gene plus T7-tagged versions of either P25wt or P25 A104V, T117A, K124E mutants or P25stop mutant, as indicated. Photographs were taken with long-wavelength UV light at 3 days postagroinoculation (dpa) (upper panels). Total RNA (10 μg) extracted from infiltrated tissues was hybridized with a probe complementary to GFP (lower panel). Ethidium bromide staining of 25S rRNA is shown as a loading control. (B) Western blot analysis of extracts derived from leaf patches infiltrated with combinations of PPV HCwt plus either P25 wt, A104V, T117A, K124E, P25stop, or GUS at 6 dpa, using antibodies against the T7 epitope. The lower panel shows the Ponceau S-stained membrane after blotting, as a loading control. (C) Leaves were infiltrated with GUS alone or combinations of Agrobacterium cultures containing PPV HCwt plus either T7-tagged versions of P25 wt, A104V, T117A, K124E, P25stop, or GUS. Leaves were stained with DAB solution at 9 dpa. (D) Electrolyte leakage from leaf disks infiltrated with T7-tagged versions of P25 wt, A104V, T117A, K124E, or P25stop, either alone or in combination with PPV HCwt at 9 dpa. GUS alone was infiltrated as a control. Data represent the means ± standard errors for 18 replicates, each consisting of four plants that received the same treatment in three independent experiments. Statistically significant differences between means were determined by employing Scheffé's multiple-range test. Different letters (a, b, c, d, e) indicate significant differences at P values of <0.05.