FIG 8.

Effects of knockdown of DRB proteins on PVX infection. (A) Wild-type and ago2 N. benthamiana plants were agroinfiltrated with the indicated PVX-DRB-VIGS recombinant constructs. Pictures of mock- and virus-infected plants were taken at 21 dpi. The cartoon at the top depicts the structure of the VIGS constructs. DRB fr., DRB VIGS fragment. (B) Downregulation of DRB2 rescues PVX-induced systemic necrosis of ago2 N. benthamiana. Pictures of PVX-DRB2-VIGS-infected wild-type and ago2 N. benthamiana plants were taken at 21 dpi (top panels). Downregulation of endogenous DRB2 mRNA level was verified by semiquantitative reverse transcription-PCR (bottom left panels). As a control, a fragment of the actin mRNA was amplified. Cleavage-independent translational repression can also contribute to the effects of VIGS. To take this into account, the ability of PVX-DRB2-VIGS to knock down the DRB2 protein level was also assessed (bottom right panels). N. benthamiana plants were infected with PVX-DRB2-VIGS and with empty PVX (as control). At 14 dpi, apical systemically infected leaves of the plants were coinfiltrated with suspensions of agrobacteria expressing epitope-tagged DRB2 and GFP (as negative control). Three days later, the expression levels of HA-DRB2 and GFP were monitored in the infiltrated leaves using Western blotting. The Ponceau-stained filter is shown as a loading control. M, marker lane. (C) Replication of PVX-DRB2-VIGS was verified by Northern blotting (using radioactive PVX-CP probe) and Western blotting (using PVX-CP specific antibody) in systemically infected leaves (left panels). The PR-1a necrotic marker gene expression level was analyzed by Northern blotting (right panel). As loading controls for Northern and Western blots, the corresponding ethidium bromide (EtBr)-stained gel and Ponceau-stained filter are shown, respectively. Each experiment was repeated at least three times with similar results. w, week.