一种调节植物细胞周期与病毒DNA复制的新机制

Whitefly-transmitted geminiviruses (WTGs) incited diseases result in reduced growth and significant reductions in yield and quality of important crop plants. WTGs have recently been spread from south to north in China, and become an even greater threat to agriculture due to climate changing and the appearance of a new and more aggressive biotype-B whitefly, The small (2.5-3.0 kb) genome of WTGs consists of one or two circular single-stranded DNA (ssDNA) molecules (DNA-A and DNA-B). The WTGs do not encode polymerases and they depend on host systems for replication and transcription.WTGs mainly infect terminally differentiated tissues and cells in plants. They need to reprogram host cellular machinery from a quiescent state to S-phase so that viral replication can occur. This process is thought to be mediated by inactivation of cell-cycle repressor proteins and by induction of host DNA synthesis protein expression through actions of the geminviral replication initiator protein (Rep). Rep protein interference with the G1/S transition control by interacts with plant regulatory proteins retinoblastoma-related (RBR). Although mechanistic studies on geminiviral DNA replication are emerging, more detailed biochemical studies on replication are necessary. One system in transgenic Nicotiana benthamiana pOri2 was generated to study WTG viral replication and interactions between viral proteins and host factors during rolling cycle replication. The latest results shown Rep protein alone cannot reprogram a "ceased" replication machinery through its interaction with RBR. Moreover, prior to Rep-mediated deregulation of cell-cycle control to provide an environment that is able to accommodate replication, the Rep gene must be transcribed from double-stranded (ds) DNA intermediates to express mRNA from which the Rep protein can then be translated. It remains an open question how a geminivirus generates dsDNA from its ssDNA genomes in cells where the replication machinery is inactive. We assume that some uncharacterized plant gene or viral factor may participate in reactivating the host cellular machinery for viral DNA replication in terminally differentiated cells.In this proposal, we aim to identify the unknown plant or viral factors that may affect regulation of cell cycle and viral DNA replication.

粉虱传双生病毒在我国蔓延迅速，来势凶猛，灾情严重，该类病毒病的控害减灾已成为当前生产中亟待解决的突出问题。粉虱传双生病毒极易产生重组变异，因此其复制机理研究是深入揭示该类病毒病灾变规律的关键。前期工作利用已构建的双生病毒Rep复制蛋白与植物内源相关因子互作研究实验体系，发现现有粉虱传双生病毒DNA复制理论仍然存在暗区:在已分化细胞中，双生病毒可以通过一种未知因子通过未知途径调节植物细胞周期与病毒DNA复制的新机制。本项申请拟通过对制备样本表达差异的精细分析，从转录和转译水平上，阐明这一未知的植物或病毒因子及其作用机制，探明可能存在的粉虱传双生病毒复制新机制。这无疑是对现有理论的拓展，是本项申请重要的科学创新，具有较高的学术价值。

粉虱传双生病毒病害在我国蔓延迅速，灾情严重，其控害减灾已成为当前生产中亟待解决的突出问题。而病毒的复制和重组是导致双生病毒变异和病害流行的重要原因，是减灾防病的核心问题。之前的研究发现粉虱传双生病毒在侵染成熟的寄主植株过程中，双生病毒的复制酶蛋白Rep通过调节一种未知因子，完成了细胞周期转变，起始了病毒DNA复制。本项目针对这一现象，构建了双生病毒复制相关蛋白基因Rep:: pOri2的转基因本氏烟实验体系，通过这一实验体系可以检测和分析双生病毒DNA复制起始周期和复制量。利用该实验体系，我们进一步发现在双生病毒侵染分裂期的植物细胞时，ACMV Rep也不能单独起始环状DNA分子的复制，需要未知因子的协助来完成双生病毒DNA在植物体内的积累。进一步的实验分析发现，植物表观遗传系统能够识别并甲基化外源 DNA的复制起始IR区序列，抑制其复制和表达，这是首次发现植物表观遗传机制的选择性识别作用机制。对于本氏烟RdDM途径中5个重要基因分别下调和上调它们的表达量，对双生病毒的复制产生了明显的影响， 影响了病毒DNA的积累。上述3个主要发现是本项研究的阶段性成果。