中国番茄黄化曲叶病毒卫星编码的βC1干扰植物MAPK信号途径机制研究

Eukaryotic mitogen-activated protein kinase (MAPK) cascade have evolved to transduce environmental and developmental signals into intracellular responses, and MAPK cascade has been confirmed to play important roles in defense against infection of microbial pathogens. While the studies on how plant utilize MAPK signal pathway to defense plant viruses is limited, the mechanisms of the early detection and quick response to invading virus and antiviral signaling transduction in plants are still unknown. Our previous data showed that the infection of Tomato yellow leaf curl China virus associated with Tomato yellow leaf curl China betasattite (TYLCCNV/TYLCCNB complex) could induce the activation of host MAPK cascade, while βC1 encoded by TYLCCNB suppressed the MAPK signals, and the Arabidopsis MKK2 protein was found to interact with βC1 in the immunoprecipitation-mass spectrometry analysis. In this project, the in planta interaction between MKK2 and βC1 will be confirmed using Y2H, BiFC and LCI assays. In order to test whether MKK2 is involved in basal defense against viral infection, the virus-challenging assay on mkk2 mutant Arabidopsis and NbSIPKK knock out Nicotiana benthamiana by CRISPR/Cas9 will be performed, and the viral accumulation and MAPK activity will be compared between the mutant and the wild type plants. The transgenic MKK2 overexpression Arabidopsis plants will be generated to analyze the biological relationship between βC1 and mkk2 by in vitro kinase assay. The transgenic βC1 overexpression in mkk2 mutant Arabidopsis plants will be produced to assess the MAPK activity, transcripts of downstream genes, and the transcripts level, protein level and kinase activity of MPK4 between the transgenic plants and that of the mkk2 mutant Arabidopsis. With these research work above, it will expand our knowledge of the biological mechanism of the rapid response to invading virus and βC1 counter defense against MAPK signal transduction.

国际上植物病毒参与寄主的MAPK信号级联途径的研究很少，植物识别病毒及响应病毒侵染的信号传导还不得而知。本项目以严重危害我国农业生产的中国番茄黄化曲叶病毒为材料，发现病毒侵染能激活植物MAPK信号通路，而病毒βC1蛋白影响MAPK信号传导，利用免疫沉淀-质谱分析筛选到与βC1互作的MKK2蛋白。在此基础上，本项目拟用Y2H、BiFC及LCI等验证βC1与MAPK途径相关蛋白的互作；病毒接种mkk2缺失拟南芥或本氏烟，检测其MAPK活性及病毒积累量的变化，明确MKK2是否参与寄主防御病毒的防卫反应；构建MKK2过表达转基因拟南芥，体外磷酸化分析βC1与MKK2的关系；βC1转化拟南芥mkk2缺失突变体，以mkk2缺失突变体为对照，比较两者MAPK活性与下游相关基因的转录水平、MPK4蛋白水平与转录水平及其激酶活性的变化，揭示寄主响应病毒侵染及βC1对寄主MAPK信号传导的反向调控的分子机制。

MAPK（mitogen-activated protein kinase, MAPK）级联途径是真核生物中保守的信号传导方式。国际上植物病毒参与寄主的MAPK信号级联途径的研究很少，植物识别病毒及响应病毒侵染的信号传导还不得而知。本课题以严重危害我国农业生产的中国番茄黄化曲叶病毒（TYLCCNV/TYLCNB）为材料，通过遗传学、生物化学和分子生物学等对其编码的βC1蛋白干扰寄主MAPK信号途径的作用进行研究。.发现TYLCCNV侵染能诱导本氏烟MAPK途径中SIPK的激活，三种不同双生病毒粒子粗提液激活拟南芥MAPK途径中MPK3和MPK6的磷酸化，说明MAPK途径参与对双生病毒侵染的防御。利用免疫沉淀的方法富集βC1蛋白并进行质谱分析，检测到7条匹配本氏烟MAPK激酶NbSIPKK的肽段。以NbSIPKK在拟南芥中的同源物MKK2进行分析，通过酵母双杂交、双分子荧光互补、免疫共沉淀和互补荧光素酶的方法验证βC1与MKK2的互作，明确互作区域为MKK2的激酶功能域。体外磷酸化实验发现MKK2不能磷酸化βC1，而βC1抑制MKK2的激酶活性。利用flg22处理过表达βC1转基因拟南芥，发现βC1抑制flg22诱导的MAPK激活、胼胝质积累和下游报告基因的转录。转录组结果显示，βC1负调控flg22诱导的上调和下调的基因转录。mkk2突变体拟南芥中βC1不再影响flg22诱导的MAPK激活和下游报告基因的转录，说明靶标MKK2蛋白对于βC1抑制flg22诱导的MAPK激活是必须的。同样发现βC1能和MKK2下游的MPK4蛋白互作，并抑制MPK4的激酶活性。.TYLCCNV/TYLCCNB侵染野生型和mkk2突变体拟南芥后发现mkk2突变体拟南芥中病毒侵染率要高于野生型。Crispr/cas9敲除本氏烟NBSIPKK和NBMPK4基因的nbsipkk-crispr、nbmpk4-crispr突变体植株对TYLCCNV/TYLCCNB侵染均表现出更高的病毒积累量和更明显的病毒表型，说明MKK2和MPK4参与双生病毒的防御。本工作揭示MAPK途径参与植物抗病毒而双生病毒βC1干扰寄主MAPK途径成功侵染植物的新机制。

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