茎尖脱毒过程中黄瓜花叶病毒和寄主植物互作的分子机制

The process that virus exclusion from shoot apexes of infected host plants is a classical biological phenomenon, and has been applied to producing virus-free seedlings. Nonetheless, the molecular mechanisms behind this process are largely unknown. Cucumber mosaic virus has a broaden host range of 1200 plants, and causes serious diseases world-widely. Therefore, we employ CMV and its host plants as materials to study the mechanisms of virus exclusion from shoot apexes. Our preliminary data has shown that wild-type CMV could invade the apical meristems of infected plants at 7 days post inoculation (dpi), and disappeared from shoot apexes after 14 dpi. Unexpectedly, the virus mutant of nucleolus signal of CMV CP region could continuously invade the meristem, thereby leading to stunting and deficient apical dominance. These results suggest that CMV CP regulate the viral invasion of apical meristems. Here, this proposal is consisted of three aims that (i) analyze the difference of tissues infection by wild-type CMV and mutant, (ii) detect the changes of plant hormones and transcriptomes upon virus challenge to explore plant genes involved in apical development and virus infection, (iii) screen and identify the functional genes responsible for the virus exclusion from shoot apexes by yeast two-hybrid systems and virus infection assay on lost function mutants and over-expressed plants. These results will provide new evidences in understanding the interaction of CMV and host plants during the virus exclusion of shoot apexes.

植物茎尖脱毒是一个经典的生物学现象，指植物茎尖分生组织能够有效抵抗植物病毒的侵染，已经应用于生产植物脱毒苗，但其中的分子机理尚不清楚。黄瓜花叶病毒（CMV）能侵染1200多种植物，在世界范围引起了严重危害。因此，我们用CMV和植物寄主为材料来研究茎尖脱毒的分子机制。前期初步结果表明CMV接种7天后能够瞬时侵染植物顶端分生组织，而14天后从顶端消失；而外壳蛋白 (CP) 的核仁信号突变体CMV-NM1能够持续侵染顶端分生组织，并造成植物矮化，顶端优势丧失等现象，说明CMV CP参与了病毒侵入顶端分生组织的调控过程。本申请课题拟利用原位杂交技术分析CMV和突变体系统侵染植物组织差异；检测侵染前后激素和转录组变化，分析顶端发育相关基因受病毒侵染的表达调控；通过酵母双杂交、缺失突变和超表达植株接种实验等筛选鉴定茎尖脱毒相关的寄主因子，为理解茎尖脱毒过程中CMV和寄主植物互作机制提供新的科学依据。

大多数植物病毒都不能侵染植物的顶端分生组织，利用此生物学现象，实际生产中常常利用茎尖离体培养获得无毒苗，然而其中的分子机制还不清楚。黄瓜花叶病毒 (Cucumber mosaic virus, CMV) 能侵染1200多种植物, 是造成多种经济作物减产的主要植物病毒之一，本项目就是探究病毒不能侵染茎尖的分子机理。我们发现野生型CMV (CMVWT) 在侵染初期进入寄主顶端分生组织，随后新生叶出现“茎尖脱毒”的现象，而一个突变体病毒CMVRA (CP的N端R13RRRPRR19突变为连续的A) 会持续地侵入茎尖并在顶端组织中积累，导致寄主顶端优势的丧失。深入研究发现，CPWT主要是促进依赖于寄主RDR6/SGS3的次级siRNA的产生，诱导更强的抗病毒RNA沉默，从而负调控病毒的茎尖侵染。同时，我们发现在黄瓜花叶病毒属其它病毒在病毒CP 也具有类似功能。第二方面的研究中，CMV Fny株系2b转基因拟南芥发育异常，主要是因为2b与AGO1互作并影响内源miRNA途径。在2b超表达植株中转入CP，发育异常会变弱甚至消失，并且一些受2b诱导的内源基因积累水平也恢复正常，说明CP能够影响2b对于miRNA途径的调控，深入研究表明CP与2b发生互作，减弱2b与AGO1的互作，释放AGO1行使体内miRNA通路。以上两种都是病毒自我弱化的新机制，保证了寄主相对正常的生长状态，有利于病毒的长期侵染。