FIG 10.

Downregulation of RDR6 rescues PVX-induced SN of ago2 N. benthamiana. (A) VIGS-mediated downregulation of RDR6 activity was verified by a functional gene silencing assay. The assay is based on the observation that the inverted repeat (IR)-initiated posttranscriptional gene silencing is significantly enhanced by RDR6-dependent secondary siRNAs. Wild-type N. benthamiana plants were infected with either PVX-RDR6-VIGS or PVX-RDR1-VIGS recombinant PVX viruses (three plants each). Infections were initiated by agroinfiltration of a binary vector expressing the appropriate virus. At 14 dpi, systemically infected leaves of the plants were infiltrated with a 35S CaMV-GFP binary vector either alone (left side of leaves) or together with a GFP-IR binary construct (right side of leaves). Three days later, samples were collected from the leaves, and total protein lysates were prepared. GFP levels were analyzed by quantitative Western blotting using GFP antibody. GFP-IR-induced repression (plotted in the chart at the bottom) was obtained by dividing the GFP band intensities measured in the absence of GFP-IR by the intensities detected in its presence (lane pairs are indicated on the x axis). GFP band intensities were normalized for actin signals. Errors are given as standard deviations. Note that in PVX-RDR6-VIGS-infected plants, efficiency of IR posttranscriptional gene silencing decreased by approximately 1 order of magnitude compared to that of the control PVX-RDR1-VIGS-infected plants, indicating efficient downregulation of RDR6 activity. (B) PVX-RDR6-VIGS and PVX-RDR1-VIGS (negative control) virus infections were initiated in wild-type and ago2 N. benthamiana plants using agroinfiltration. Pictures of infected plants were taken at 21 dpi. Virus levels were monitored by Northern and Western blotting in systemically infected leaves (bottom left panels). The Northern blot was hybridized with radioactively labeled PVX-CP probe, while the Western blot was probed with PVX-CP antibody. As loading controls for Northern and Western blots, the corresponding ethidium bromide-stained gel and Ponceau-stained filter are shown, respectively. Necrotic marker gene expression was analyzed by Northern blotting using radioactively labeled PR-1a-specific probe (bottom right panels). The ethidium bromide-stained gel is shown as a loading control. The experiment was repeated three times with the same outcomes. w, week.