FIG 5.

CP-mediated inhibition of PVA gene expression is dependent on translation of the CP cistron. We studied the effects of CP^wt on the transcription and translation of viral and nonviral RNAs. Agrobacterium containing CP^wt was introduced to plants by infiltration 24 h before the viral and nonviral constructs were introduced. Leaf samples were collected at 3 and 10 dpi for RLuc activity measurements as well as for RNA quantification via qRT-PCR. Firefly luciferase (FLuc) was used as an internal control to normalize RLuc activity. For qRT-PCR, six biological replicates from each sample set were pooled, and eight technical replicates were produced from them. The protein phosphatase 2A gene (a housekeeping gene) was used as a reference gene, and RLuc was used as the target during quantitative PCR. Normalized gene expression was calculated according to equation 2 of Q-Gene (32). **, P < 0.01; *, P < 0.05. (A) Expression of CP^wt and GUS with RLuc-CP-3′UTR (middle panel) and RLuc^STOP-CP-3′UTR (right panel). The left panel shows the expression of bicistronic constructs RLuc-CP-3′UTR and RLuc^STOP-CP-3′UTR in Nicotiana benthamiana plants at 3 dpi, with no significant difference in the translation of both constructs. (B) Expression of CP^wt and GUS with PVA^wt (left and middle panels). The right panel shows the fold difference in RNA expression from plants expressing PVA^wt and GUS and those expressing PVA^wt and CP^wt, collected at 10 dpi. (C) Expression of CP^wt and GUS with PVA^STOPCP (left and middle panels). The right panel shows the fold difference in RNA expression from plants expressing PVA^STOPCP and GUS and those expressing PVA^STOPCP and CP^wt, collected at 10 dpi. **, P < 0.01; *, P < 0.05.