免疫亲合素在玉米受病毒侵染表现矮花叶病状过程中的作用

The development of viral diseases and symptom exhibition is a result of interactions between viruses and their host plants. It is important to identify host interactors of viurses and subsequently elucidate their roles in the infection process. Our previous work showed that the CI protein of sugarcane mosaic virus (SCMV) which causes maize dwarf mosaic could interact with immunophilin FKBP12 from maize. Immunophilins are involved in protein-folding, regulation of cell cycle and influence pathogenecity of some plant-pathogenic fungi. However, it remains unknown what biological functions immunophilins perform in plants. Our previous results suggest that maize FKBP12 interact with CI of SCMV not only in the yeast, but also in the epidermal cells of tobacco leaves. In the proposed project, we would utilize various methods (such as bimolecular fluorescence complementation) to determine specific interactive regions along the polypepetide chains and subcellular localization of interaction using transgenic maize plants and viral vectors; The roles of interaction between FKBP12 and CI in pathogenesis and its effects on infection of maize will be investigated and the interactive network be dissected in order to reveal the molecular mechanism underlying the development of maize dwarf mosaic disease and ultimately seek effective strategies for controlling this disease by employing sustainable measures and/or varieties with durable resistance.

病毒病的发生以及症状表现是病毒与寄主相互作用的结果。鉴定与病毒互作的寄主因子并解析其在病毒侵染过程中的作用具有重要的意义。我们前期的研究表明玉米矮花叶病主要毒原甘蔗花叶病毒（SCMV）CI蛋白可与玉米免疫亲合素（immunophilin） FKBP12发生特异性互作。免疫亲合素参与蛋白质的折叠，调控细胞周期，还可以影响病原真菌对植物的致病性；但FKBP12在植物中的生物学功能尚不清楚。我们的结果表明玉米FKBP12与病毒CI的互作不仅存在于酵母中，而且也发生于烟草表皮细胞中。本课题将利用已有的转基因玉米和病毒载体等材料通过多种方法明确FKBP12与CI的互作的多肽区域及亚细胞定位;同时对互作在SCMV致病过程中的作用及其对侵染玉米的影响进行系统研究，解析SCMV与玉米蛋白的互作网络，以揭示玉米矮花叶病发生的分子机制，为采用基因调控措施创制具有持久抗病性的玉米种质提供理论依据。

甘蔗花叶病毒（SCMV）是我国玉米矮花叶病的主要毒原。 我们通过酵母双杂交系统筛选到与SCMV细胞质内含体蛋白CI互作的玉米免疫亲合素FKBP12蛋白。 为研究ZmFKBP12在SCMV侵染过程中的作用，在mRNA水平检测了SCMV侵染对ZmFKBP12基因表达的影响。在玉米接种SCMV后的早期ZmFKBP12的mRNA积累量下降60%；而在第14天时则上调1.5倍。高粱在SCMV侵染及玉米在同属的白草花叶病毒（PenMV）侵染后FKBP12 mRNA表达量均随侵染时间而变化，结果与SCMV侵染玉米的结果一致；因此，病毒的侵染能够在一定程度上影响FKBP12基因的表达。利用马铃薯X病毒(PVX)侵染性克隆构建了瞬时过量表达载体研究ZmFKBP12的瞬时表达对PVX侵染的影响, 结果PVX的积累量减少33%；在玉米原生质体瞬时过表达ZmFKBP12后SCMV RNA的积累显著下调，约为对照表达量的30%，说明ZmFKBP12可能参与植物抗病毒防卫反应。此外，我们以SCMV VPg为诱饵利用酵母双杂交筛选玉米cDNA文库得到了与之互作的玉米蛋白Elongin C。 双分子荧光互补实验（BiFC）证实了SCMV VPg与ZmElc在玉米原生质体中发生互作。ZmElc蛋白同时定位于玉米细胞质和细胞核中。在玉米原生质体中SCMV侵染对ZmElc的亚细胞定位没有影响。实时荧光定量 RT-PCR结果显示，接种SCMV 4天和6天后， ZmELC基因在接种叶和第一片系统侵染叶的表达上调，而接种10天后其在第一片系统侵染叶的表达保持稳定。在玉米原生质体中瞬时过量表达ZmELC导致SCMV RNA积累量的增加。 用BMV介导的基因沉默（VIGS）体系瞬时沉默ZmELC基因，未影响玉米植株的正常生长和发育，但是显著抑制了两种SCMV分离物的积累。因此，VPg与玉米Elc的互作可促进SCMV的复制与侵染，玉米Elc是该病毒的感病因子。通过基因编辑有可能获得对该病毒有抗性的玉米材料。