蛋白激发子Hrip1诱导番茄抗黄化曲叶病毒的互作蛋白鉴定与调控机制研究

Tomato yellow leaf curl virus (TYLCV) causes tremendous losses of tomato crops worldwide. Protein elicitor Hrip1, which was isolated from Alternaria tenuissima, had a significant effect on prevention and control of Tomato yellow leaf curl virus disease and the prevention rate was up to 78.62%. However, the mechanism of antiviral disease remains to be fully understood. In our previous study, a mutant, which had obtained from tomato EMS populations, was significantly decreased in the resistance to TYLCV under Hrip1 treatment. In this project, immunohistochemical method was applied for measuring the difference of location and expression of proteins coded by virus genome in tomato wild-type and mutant. The mutation was detected by mapping-by-sequencing, and the protein interacted with Hrip1 was identified by protein-protein interaction analysis, such as yeast-two-hybrid, GST pull-down and Co-IP. And then, the deletion mutant and overexpression were constructed by CRISP/Cas9 system for illuminating the biological functions of interacting protein. Furthermore, the signal transduction pathway, which interacting protein involved in, was explored during Hrip1 induced resistance to Tomato yellow leaf curl virus. This research will elucidate the mechanism of elicitor Hrip1-induced antiviral disease in tomato.

番茄黄化曲叶病毒引起的病毒病是番茄生产中重要的病害之一。Hrip1是申请人所在的课题组从极细链格孢菌（Alternaria tenuissima）中分离的一种蛋白激发子。它对番茄黄化曲叶病毒病的防治率高达78.62%，但是其抗病毒的分子机制尚不明确。前期研究中我们已经获得了一个突变体植株，突变体抗番茄黄化曲叶病毒的能力显著减弱。本项目拟采用免疫组化技术研究突变基因在Hrip1抑制番茄黄化曲叶病毒复制和侵染过程中的作用；通过Mapping-By-Sequencing技术实现对基因突变位点的定位，结合蛋白互作分析和共定位分析确定番茄中Hrip1的互作蛋白。通过构建互作蛋白的突变体和过表达植株研究互作蛋白在Hrip1诱导的番茄抗黄化曲叶病毒过程中的生物学功能；进一步通过分析互作蛋白参与番茄抗病毒相关的信号通路，阐明蛋白激发子Hrip1互作蛋白诱导番茄抗黄化曲叶病毒的分子机制。

由番茄黄化曲叶病毒引起的病毒病是番茄生产中重要的病害之一。Hrip1是本实验室从极细链格孢菌中分离的一种新型病原相关模式分子，它能够激活免疫防御反应，提高植物对病毒病的抗性，但是其抗病毒的分子机制尚不明确。在本基金项目的资助下，完成人首次明确了Hrip1作用的位点并且发现Hrip1可能干扰了细胞内TYLCV的复制过程。通过酵母双杂交和GST Pull-down实验证实了番茄中COP9复合体亚基SlCSN5B能够与Hrip1互作，并且发现在细胞内Hrip1也能够与TYLCV-AC2蛋白互作，表明Hrip1在细胞内可能通过与AC2互作影响了TYLCV的侵染。亚细胞定位观察结果表明SlCSN5B定位于细胞核内。通过构建SlCSN5B的突变体植株发现SlCSN5B可能参与Hrip1对番茄SA途径和苯丙氨酸解氨酶活性的激活。基于label-free差异蛋白质组分析我们发现Hrip1通过调控病程相关蛋白的表达参与了番茄抗病毒的过程。转录组测序的结果表明TYLCV侵染与Hrip1处理共有的差异表达基因3458个；其中表达变化相反的基因共1621个，这些基因主要富集到“苯丙素生物合成”和“植物激素信号传导”等信号通路；在Hrip1处理与TYLCV侵染的样品中发现91共有的差异表达miRNA。Hrip1处理后上调、TYLCV侵染下调表达的miRNA共有9个；Hrip1处理后下调、TYLCV侵染上调表达的miRNA共有23个。差异表达miRNA靶基因通路富集结果表明Hrip1通过调控“苯丙素代谢”、“植物激素信号传导”和“过氧化物酶体”等信号通路参与了番茄抗病毒过程。关联分析的结果表明sly-miR166c-5p、sly-miR172a、sly-miR172b、sly-miR172d和sly-miR1917能够与RNA-seq的数据建立相互关系。这些miRNA调控的靶基因参与了植物应答生物胁迫、苯丙素的生物合成、赖氨酸代谢途径以及乙烯信号通路，表明Hrip1通过调控这些miRNA参与番茄抗黄化曲叶病毒的过程。通过本项目的执行，我们明确了Hrip1在番茄抗黄化曲叶病毒中的作用方式；鉴定了Hrip1的互作蛋白SlCSN5B，初步阐明了SlCSN5B的功能；最后从蛋白水平、转录水平和转录后调控三个层面揭示了Hrip1诱导番茄抗黄化曲叶病毒的分子机制。