本项目拟以我国发生面最广、危害最重的单组份双生病毒番茄黄化曲叶病毒（TYLCV）为材料，在明确TYLCV具有回复转录水平基因沉默（TGS）的基础上，构建PVX重组表达载体并浸润16-TGS植株以鉴定TYLCV编码的TGS基因沉默抑制子（VSR）。构建VSR突变的病毒突变体的侵染性克隆，分析病毒突变体的生物学性状及其病毒基因组DNA甲基化水平的变化。将VSR遗传转化拟南芥，分析抑制子的表达对拟南芥已发生TGS沉默的内源基因甲基化的影响、从头甲基化（de novo methylation）的影响以及HEN1甲基转移酶活性的影响。利用酵母双杂交、BiFC、Co-IP筛选甲基化途径中与TYLCV基因沉默抑制子互作的寄主因子。研究结果将有助于我们了解单组份双生病毒抑制TGS和寄主DNA甲基化的作用机理，进而阐明病毒致病及逃逸寄主植物防御的分子机制，为TYLCV病毒病的防治提供新的策略和思路。

Tomato yellow leaf curl virus (TYLCV) is a monopartite begomovirus and has caused devastating damage in China. Our preliminary results showed TYLCV could reverse transcriptional gene silencing (TGS) on transgenic 16-TGS plant. In this project, we will use PVX vector to further characterize the viral suppressor of RNA silencing (VSR) encoded by TYLCV. The infectious clone of TYLCV mutant which introduce VSR mutation will be constructed to test the biological character induced by TYLCV mutant and DNA methylation level of viral mutant genome.In order to evaluate the methylation status at endogenous TGS-silenced loci and the impact of the de novo methylation and HEN1 MTase activity following VSR expression, a transgenic Arabidopsis line expressing VSR will be constructed, in addition to that, RT-PCR, ChIP, methylation sensitive restriction-PCR, bisulfite sequencing and methylation sensitive extension assay will be performed. Yeast two-hybrid analysis, Bimolecular fluorescence complementation and co-immunoprecipitation assay will be carried out to screen the interaction factor in methyl pathway. Based on this research work, it will expand our knowledge of how monopartite TYLCV encodes suppressor to repress TGS and DNA methylation to escape host defense.