FIG 2.

Analysis of the effects of DRB proteins on PVX replication. (A) An agroinfiltration-based transient virus replication assay was used to evaluate the effects of ectopic expression of DRB proteins on PVX replication. Suspensions of agrobacteria carrying either PVX-GFP- or PVXΔTGB-GFP-encoding binary vectors were infiltrated into leaves of wild-type (wt) or ago2 N. benthamiana. As a control a CaMV 35S promoter-driven GFP expression vector was used. Along with the reporter constructs, agrobacteria carrying binary expression vectors for DRB proteins were also codelivered. As a negative control, a coinfiltrated suspension of empty agrobacteria (∅) was employed. The bacterial suspensions (optical density at 600 nm of 1) were mixed at a 1:1 ratio. GFP expression in the infiltrated leaf patches was monitored using an appropriate UV light source at 3 and 7 dpi. Pictures of representative leaves are shown. At 7 dpi, PVX- and DRB2-coinfiltrated leaf patches frequently exhibited necrosis, which resulted in the apparent fading of the GFP signal. These necrotic areas are circled in red. Experiments were repeated three times. (B) Expression of PVXΔTGB-GFP-encoded GFP was monitored by Western blotting (WB). Leaves of wild-type N. benthamiana were agroinfiltrated with a PVXΔTGB-GFP reporter either alone (right side of leaves) or combined with an expression vector for one of the indicated DRB proteins (left side of leaves). At 5 dpi samples were collected and pooled from three identically infiltrated leaves. Protein lysates were prepared from the pooled samples and analyzed by GFP antibody. To verify the expression of the DRB proteins, the same filter was probed with HA antibody. As a loading control, the Ponceau-stained filter is shown. From the above-described infiltrated leaves, total RNA samples were also prepared, and viral RNA levels were monitored by Northern blotting (right panel). The Northern blot was probed with a radioactively labeled GFP DNA fragment. The ethidium bromide (EtBr)-stained gel is shown as a loading control. Protein lysate or total RNA prepared from PVXΔTGB-GFP and empty agrobacteria (∅)-infiltrated leaves were used as negative controls in the Western and Northern blots.