NbRFP1调控燕麦矮缩病毒 RepA诱导HR的分子机制研究

Ubiquitination and proteasomal degradation plays important roles in plant defense, including the hypersensitive response (HR). ODV RepA protein has been identified to trigger a HR-typed cell death. A previous yeast two-hybrid (Y2H) screen of a tobacco cDNA library using RepA as the bait revealed that a RING FINGER-like protein named as NbRFP1 interacts with RepA. Here, the interaction between them will be further confirmed by the bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation assays (Co-IP) in N. benthamiana cells. Further, the truncated mutants of NbRFP1 gene will be constructs to determine the domains involved in interactions with RepA and RepA-induced HR. Additional, the effects of RepA on the expression and subcellular localization of NbRFP1 will be identified. On the other hand, in vitro ubiquitination assays will be carried out to reveal that NbRFP1 is a functional E3 ubiquitin ligase. In addition, stabilization of NbRFP1 following inoculation with 35S-RepA will be determined in cell-free degradation, whereas the levels of ubiquitinated NbRFP1 be assessed. The effects of loss of NbRFP1 gene function or overexpression of NbRFP1 on RepA-induced HR will be investigated in these transgenic N. benthamiana plants. To determine the ubiquitinated substrate of NbRFP1, the partners that interact with NbRFP1 will be screened, following the analysis of gene functions involving in RepA-induced HR. The research works mentioned above will expanding our understanding of the molecular basis of E3 ubiquitin ligase involved in ODV RepA-induced HR and plant defense.

前期研究发现ODV RepA是一个HR反应的效应子，并且和潜在的E3泛素连接酶NbRFP1在体外互作。在此基础上，本项目拟利用Y2H、BiFC及Co-IP等进一步验证NbRFP1和RepA的互作并鉴定其互作结构域；利用qRT-PCR、western blot、激光共聚焦观察等分析RepA表达对NbRFP1蛋白水平和亚细胞定位等的影响；通过体外泛素化反应确认NbRFP1的E3泛素连接酶活性，并解析NbRFP1和RepA交互作用中RepA是否能抑制NbRFP1的自身泛素化进而阻止其被植物体内26S-蛋白酶体降解，从而提高NbRFP1的蛋白稳定性；构建NbRFP1过表达或抑制的转基因植株等，明确NbRFP1的表达变化是否影响ODV RepA诱导HR；筛选NbRFP1调控的下游互作分子并解析其对ODV RepA诱导HR的影响。项目的实施对于揭示ODV RepA诱导HR的分子机制有重要意义。

前期研究发现ODV RepA是一个激发HR反应的效应子，并且和潜在的E3泛素连接酶NbRFP1在体外互作。在此基础上，本项目利用Y2H、BiFC及Co-IP等进一步证实NbRFP1和RepA互作，并明确NbRFP1的RING结构域对于NbRFP1与RepA互作是必须的；利用western blot、激光共聚焦观察等明确RepA和NbRFP1共表达对于二者的蛋白表达水平及其亚细胞定位没有显著影响，进一步通过沉默植物内源的RPN14发现26S蛋白酶体降解系统并未参与NbRFP1蛋白的降解过程，而体内降解实验也证实NbRFP1蛋白在植物体内具有较好的稳定性；为了探讨NbRFP1和RepA互作的生物学意义，利用基因沉默和植物遗传转化抑制或者过表达NbRFP1，证实NbRFP1对RepA诱导HR有调控作用，而通过构建NbRFP1的缺失突变体确认其RING结构域是参与调控RepA诱导HR的关键元件；通过基因表达谱测序并结合免疫沉淀NbRFP1蛋白进行质谱检测，筛选NbRFP1调控的下游蛋白，揭示NbRFP1-RPM1-NbHSP90模块可能参与调控RepA诱导HR，而过氧化氢酶等也可能是NbRFP1作用的靶标蛋白。