植物选择性甲基化调控双生病毒DNA复制分子机理的研究

Epigenetic machinery involves RNA-directed DNA methylation (RdDM), DNA demethylation and chromatin remodeling. In plants, it is well-established that epigenetic control, in particular, RdDM and methylation maintenance, is an active form of cellular antiviral defense mechanisms. To counterattack such an epi-antiviral defense, geminiviruses, a large family of DNA viruses, have evolved to encode proteins capable of interfering with host cellular epigenetic mechanisms. Indeed, plants defective in RdDM and methylation maintenance pathways become highly susceptible to geminiviral infection. However it remains to be elucidated how cellular epigenetic machinery recognizes invading viral DNA for RdDM. We have established a unique transgenic pOri2 system to investigate geminiviral DNA replication and geminivirus-plant interactions. We unravel an unprecedented mechanism that plant epigenetic machinery can differentiate identical DNA sequences for selective DNA methylation (SDM). Only autonomous episomal DNAs were selectively methylated whilst identical viral sequences, if integrated into the nuclear genome, were not methylated. We propose that plant cells may have evolved specific machinery for SDM. This machinery consists of cellular SDM factors that can recruit geminiviral components including viral replication protein (Rep) and viral small interfering RNA (vsiRNA) so that they select and methylate replicative episomal DNA molecules to inhibit their replication. In this proposal, we aim to characterize and dissect the genetic/epigenetic requirements and the biochemical components for SDM through a combination of genetic crossing and a wide range of modern technologies. A successful outcome of this project will not only advance current theories of plant epigenetics and deepen our understanding of virus-plant epigenetic interactions and host defence systems, but also shed new light on how to develop novel antiviral strategies for the control of devastating geminivirus diseases in food, vegetable and economic crops in China and worldwide.

植物表观遗传涉及DNA甲基化、去甲基化和染色质重塑，影响DNA复制和基因转录，是植物防御双生病毒（gv）的一种有效机制。但植物细胞靶标甲基化gvDNA的机理并不清楚。我们建立了一个研究gvDNA复制的pOri2转基因烟草体系，首次发现植物表观遗传机制能选择性地靶标复制型游离的环状gvDNA分子，导致其甲基化。我们推测植物细胞甲基化因子和病毒因子vsiRNA、Rep等共同作用，以选择性地识别并甲基化gvDNA。本项目重点研究小RNA生物合成及甲基化途径关键基因RNAi对植物细胞选择性地靶标并甲基化gvDNA的影响，分析鉴定导致选择性甲基化的甲基化因子，探究其生物学功能，揭示表观遗传机器如何选择性地靶标gvDNA、对其进行甲基化修饰、从而影响gvDNA复制的分子机制，进而阐明表观遗传介导的防御双生病毒侵染的分子遗传基础，拓展植物表观遗传学理论，为建立新型病毒病害防御方案提供理论指导。

植物表观遗传涉及DNA甲基化、去甲基化和染色质重塑，影响DNA复制和基因转录，是植物防御双生病毒（gv）的一种有效机制。但植物细胞靶标甲基化gvDNA的机理并不清楚。我们建立了一个研究gvDNA复制的pOri2转基因烟草体系，首次发现植物表观遗传机制能选择性地靶标复制型游离的环状gvDNA分子，导致其甲基化。我们推测植物细胞甲基化因子和病毒因子vsiRNA、Rep等共同作用，来选择性地识别并甲基化gvDNA。本项目重点研究了小RNA生物合成及甲基化途径关键基因对植物细胞选择性地靶标并甲基化gvDNA的影响，我们发现不同的DCLs对gvDNA特异的同源小RNA的产生、gvDNA复制有不同的影响，但对gvDNA甲基化影响不显著；但是DRM2过表达导致gvDNA选择性甲基化增加、gvDNA复制下降、而对gvDNA特异的同源小RNA的产生没有影响；我们还分析鉴定了导致选择性gvDNA甲基化的甲基化因子“DNA-RNA-蛋白质”复合物DrPC，DrPC中存在gvDNA、gvDNA特异的同源siRNAs、选择性gvDNA甲基化关联的microRNAs、双生病毒DNA复制蛋白和细胞中与DNA甲基化相关的酶；并進一步探究了甲基化因子生物学功能。我们的研究揭示了植物细胞中表观遗传机器选择性靶标和甲基化gvDNA、并影响gvDNA复制的分子机制，进一步阐明了表观遗传介导的防御双生病毒侵染的分子遗传基础，拓展植物表观遗传学理论，为建立新型病毒病害防御方案和表观育种提供了理论基础。

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