FIG 5.

DRB2 inhibits the activity of all DCLs involved in antiviral RNAi. (A) The RNA samples analyzed in the experiment shown in Fig. 1 were used for small RNA Northern blots to examine the vsiRNA production from different regions of PVXΔTGB-GFP in the presence or absence of DRB proteins. Two different probes were used to monitor vsiRNA levels. The positions of the probes are indicated at the top of the cartoon. The filter was first probed with a radioactively labeled PVX-RdRp fragment and subsequently with a PVX-CP fragment (after stripping). The ethidium bromide (EtBr)-stained gel is shown as a loading control. SGP, subgenomic promoter; (A)n, poly(A) tail. (B) DRB2 overexpression inhibits the activity of all DCLs involved in antiviral RNAi in rdr6 N. benthamiana. Leaves of rdr6 N. benthamiana were agroinfiltrated with PVX:ΔTGB-GFP either alone (left side of leaves) or together with a DRB2 binary expression construct (right side of leaves). At 5 dpi samples were collected and pooled from three infiltrated leaves, and total RNAs were prepared. RNAs were analyzed on a small RNA blot as described above. The filter was probed with radioactively labeled PVX-CP probe. The EtBr-stained gel is shown as a loading control.