Abstract

The turnip crinkle virus-based vector TCV-GFP Delta CP had been devised previously to study cell-to-cell and long-distance spread of virus-induced RNA silencing. TCV-GFP Delta CP, 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-GFP Delta CP and its p8- or p9-mutated derivatives, TCVmp8-GFP Delta CP and TCVmp9-GFP Delta CP, replicated efficiently but were restricted to single Nicotiana benthamiana epidermal cells. TCV-GFP Delta CP, TCVmp8-GFP Delta CP, or TCVmp9-GFP Delta CP 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-GFP Delta CP. Non-replicating TCVmp88-GFP Delta CP and TCVmp28mp88-GFP Delta CP 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-GFP Delta CP 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.