A Begomovirus DNAβ-Encoded Protein Binds DNA, Functions as a Suppressor of RNA Silencing, and Targets the Cell Nucleus

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Abstract
Our previous results demonstrated that the DNAβ satellite (Y10β) 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 βC1 gene in Nicotiana plants induces virus-like symptoms. In the present study, in vitro DNA binding activity of the βC1 proteins of Y10β and DNAβ (Y35β) 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 βC1 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 Y10β or by TbCSV-Y35 plus Y35β. In a patch agroinfiltration assay, the transiently expressed βC1 gene of Y10β or Y35β 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 βC1 protein of Y10β accumulated primarily in the nuclei of plant and insect cells when fused with β-glucuronidase or GFP and immunogold labeling showed that the βC1 protein is present in the nuclei of infected N. benthamiana plants. A mutant version of Y10β carrying the mutations within the putative nuclear localization sequence of the Y10 βC1 protein failed to induce disease symptoms, suppress RNA silencing, or accumulate in the nucleus, suggesting that nuclear localization of the βC1 protein is a key requirement for symptom induction and silencing suppression.