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Journal of Virology, August 2005, p. 10764-10775, Vol. 79, No. 16
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.16.10764-10775.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Xiaofeng Cui,1 Guixin Li,1 Daowen Wang,2 Dongwei Hu,1 and Xueping Zhou1*

State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310029,1 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China2

Received 2 February 2005/ Accepted 8 May 2005

Our previous results demonstrated that the DNAß satellite (Y10ß) 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 ßC1 gene in Nicotiana plants induces virus-like symptoms. In the present study, in vitro DNA binding activity of the ßC1 proteins of Y10ß and DNAß (Y35ß) 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 ßC1 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 Y10ß or by TbCSV-Y35 plus Y35ß. In a patch agroinfiltration assay, the transiently expressed ßC1 gene of Y10ß or Y35ß 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 ßC1 protein of Y10ß accumulated primarily in the nuclei of plant and insect cells when fused with ß-glucuronidase or GFP and immunogold labeling showed that the ßC1 protein is present in the nuclei of infected N. benthamiana plants. A mutant version of Y10ß carrying the mutations within the putative nuclear localization sequence of the Y10 ßC1 protein failed to induce disease symptoms, suppress RNA silencing, or accumulate in the nucleus, suggesting that nuclear localization of the ßC1 protein is a key requirement for symptom induction and silencing suppression.

* Corresponding author. Mailing address: Institute of Biotechnology, Zhejiang University, Hangzhou 310029, People's Republic of China. Phone: 0086 571 86971680. Fax: 0086 571 86971498. E-mail: zzhou{at}zju.edu.cn.

Journal of Virology, August 2005, p. 10764-10775, Vol. 79, No. 16
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.16.10764-10775.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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