RNAi induced by dsPns10 knockdown the expression of Pns10 without significantly inhibiting virus multiplication in VCMs.

(A) Effects of the treatment of dsRNAs on multiplication of cell-associated RDV in VCMs. Viral titers were determined in duplicate by the fluorescent focus assay (see text for details). Error bars indicate standard deviations from three independent experiments. (B) Transfection of dsPns10 in VCMs results in a significant reduction in level of plus-strand RNA of Pns10 gene, without greatly inhibiting synthesis of plus-strand RNA of P8 gene, as revealed by northern blot. VCMs were transfected with transfection reagent (control), dsYFP or dsPns10, inoculated with RDV at an MOI of 10, then harvested 72 h later. Approximately 5 µg of total RNAs were probed with DIG-labeled negative-sense RNA transcripts of Pns10 or P8 genes. Lower panel: detection of 5.8S rRNA as a control to confirm loading of equal amounts of RNA in each lane. Image is representative of multiple experiments with multiple preparations. (C) Transfection of dsPns10 in VCMs caused about 80% reduction in level of plus-strand RNA of Pns10 gene, without greatly inhibiting synthesis of plus-strand RNA of P8 gene, as revealed by RT-qPCR assay. VCMs were transfected with transfection reagent (control), dsYFP or dsPns10, inoculated with RDV at an MOI of 10, then harvested 72 h later. Approximately 5 µg of total RNAs was extracted with TRIzol Reagent. The results of RT-qPCRs were normalized to the level of leafhopper actin gene. Error bars indicate standard deviations from three independent PCRs.