FIG. 3.

Mutations within TCV CP differentially affect its functions in silencing suppression, virion assembly, and viral systemic spread. (A) Evaluation of the silencing suppression capability of different TCV CP mutants using the agro-infiltration assay. TBSV P19 (leaf no. 4) was used as an additional positive control. The leaves were photographed at 5 days after infiltration. (B) Detection of GFP mRNA and siRNAs in the agro-infiltrated leaves with RNA blot hybridizations. (C) dsRNA-binding properties of CPA, CPB, CPC, and CPF mutants. Top, 5% nondenaturing polyacrylamide gel containing protein extracts preinoculated with ³²P-labeled dsRNA. Bottom, 12% SDS-PAGE gel serving showing the relative concentrations of mutant proteins used in the binding assay. (D) Systemic symptoms on Col-0 and dcl2 dcl3 dcl4 plants infected with various TCV mutants. Mock, buffer inoculated. Photographs were taken at 18 dpi. (E) Close-up images of dcl2 dcl3 dcl4 plants not inoculated (top left), inoculated with TCV-CPstop (bottom left), and inoculated with CPC (bottom right). Top right: systemically infected leaves taken from these plants. A relatively healthy class II vein on CPC-infected leaves is highlighted with an arrow. (F) Viral RNA, virion, and CP accumulations in plants infected with various TCV mutants as determined with RNA blot and Western blot hybridizations. Total RNA, protein, and virions were extracted from pooled systemic leaves (see Materials and Methods) at 11 to 12 dpi. Note that in the bottom two panels, lanes 5 and 6 were dilutions of the lane 4 sample. The panel at the very bottom is the stained gel serving as the control for protein load, with the TCV CP band easily visible.