FIG 4.

Inhibition of RNAi by DRB2 does not depend on RDR6. (A) Inverted-repeat-initiated posttranscriptional gene silencing is inhibited by DRB2. The indicated binary expression constructs were agroinfiltrated into leaves of wild-type N. benthamiana. At 5 dpi, samples were collected and pooled from three identically infiltrated leaves. From the collected samples protein lysates and total RNA were prepared. Viral protein synthesis and DRB2 protein levels were monitored by Western blotting using GFP and HA antibodies, respectively. Note that the HA antibody detects nonspecific bands in the first and third lanes at positions comparable to those of DRB2. The PVXΔTGB-GFP virus level was followed in Northern blotting using a radioactively labeled GFP probe. (B) DRB2 inhibits RNAi in rdr6 mutant N. benthamiana. Leaves of wild-type (wt) and rdr6 N. benthamiana were agroinfiltrated with a PVXΔTGB-GFP reporter either alone (left side of leaves) or combined with a DRB2 expression vector (right side of leaves). At 5 dpi, samples were collected and pooled from three identically infiltrated leaves. From the collected samples protein lysates and total RNA were prepared. Viral protein synthesis and DRB2 protein levels were monitored by Western blotting using PVX-CP and HA antibodies, respectively. PVXΔTGB-GFP virus level was followed by Northern blotting using a radioactively labeled GFP probe. The corresponding Ponceau-stained protein filters and ethidium bromide (EtBr)-stained RNA gels are shown as loading controls for the Western and Northern blots, respectively.