A begomovirus DNAβ-encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus

Our previous results demonstrated that the DNAP satellite (Y10 beta) associated with Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) is essential for induction of leaf curl symptoms in plants and that transgenic expression of its beta C1 gene in Nicotiana plants induces virus-like symptoms. In the present study, in vitro DNA binding activity of the beta C1 proteins of Y10 beta and DNA beta (Y35 beta) found in the Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) were studied following their expression as six-His fusion proteins in Escherichia coli. Electrophoretic mobility shift assays and UV cross-linking experiments revealed that beta C1 proteins could bind both single-stranded and double-stranded DNA without size or sequence specificity. Suppression of green fluorescent protein (GFP) transgene silencing was observed with the new leaves of GFP-expressing Nicotiana benthamiana plants coinoculated by TYLCCNV-Y10 plus Y10 beta or by TbCSV-Y35 plus Y35 beta. In a patch agroinfiltration assay, the transiently expressed beta C1 gene of Y10 beta or Y35 beta was able to suppress host RNA silencing activities and permitted the accumulation of high levels of GFP mRNA in the infiltrated leaf patches of GFP transgenic N. benthamiana plants. The beta C1 protein of Y10 beta accumulated primarily in the nuclei of plant and insect cells when fused with beta-glucuronidase or GFP and immunogold labeling showed that the beta C1 protein is present in the nuclei of infected N. benthamiana plants. A mutant version of Y10 beta carrying the mutations within the putative nuclear localization sequence of the Y10 beta C1 protein failed to induce disease symptoms, suppress RNA silencing, or accumulate in the nucleus, suggesting that nuclear localization of the beta C1 protein is a key requirement for symptom induction and silencing suppression.