The turnip crinkle virus-based vector TCV–GFPDCP had been devised previously to study cell-to-cell and long-distance spread of virus-induced RNA silencing. TCV–GFPDCP, which had been constructed by replacing the coat protein (CP) gene with a green fluorescent protein (GFP) coding sequence, was able to induce RNA silencing in single epidermal cells, from which RNA silencing spread from cell-to-cell. Using this unique local silencing assay together with mutagenesis analysis, two TCV genes, p8 and p9, which were involved in the intercellular spread of virus-induced RNA silencing, were identified. TCV–GFPDCP and its p8- or p9-mutated derivatives, TCVmp8–GFPDCP and TCVmp9–GFPDCP, replicated efficiently but were restricted to single Nicotiana benthamiana epidermal cells. TCV–GFPDCP, TCVmp8–GFPDCP, or TCVmp9–GFPDCP was able to initiate RNA silencing that targeted and degraded recombinant viral RNAs in inoculated leaves of the GFP-expressing N. benthamiana line 16c. However, cell-to-cell spread of silencing to form silencing foci was triggered only by TCV–GFPDCP. Non-replicating TCVmp88–GFPDCP and TCVmp28mp88–GFPDCP with dysfunctional replicase genes, and single-stranded gfp RNA did not induce RNA silencing. Transient expression of the TCV p9 protein could effectively complement TCVmp9–GFPDCP to facilitate intercellular spread of silencing. These data suggest that the plant cellular trafficking machinery could hijack functional viral proteins to permit cell-to-cell movement of RNA silencing
