Figure 4

Bacterially expressed dsRNA interferes with PPV infection. (A) Production of PPV dsRNAs in E. coli. HT115 cells were separately transformed with the L4440 double-T7 vector containing either the HC or the CP genes of PPV. Bacterial cultures were induced with IPTG and processed for total nucleic acid. Samples were resolved by electrophoresis on 1% agarose gel before (lanes 1 to 4) or after treatment with RNase A (lanes 5 to 8), and nucleic acid was visualized by staining with ethidium bromide. Markers, λEcoRI-HindIII molecular weight markers. The positions of bacterially expressed 1492-bp HC and 1081-bp CP dsRNAs are indicated in the margin. (B) Detection of PPV in total RNA extracted from systemic leaves of N. benthamiana by RT-PCR at 14 dpi. Plants were mock inoculated or were inoculated with PPV (0.3 μg/ml) alone (-), or with mixtures of PPV plus French Press preparations derived from HT115 harboring either PPV HC dsRNA, PMMoV IR 54 or the empty vector, as indicated. Markers, λEcoRI-HindIII molecular weight markers. RT-PCR was performed with 1 μg of total RNA using primers corresponding to the CP coding sequence of PPV. The position of the 510-bp amplified fragment is indicated in the margin.