病原物效应子及与其互作番茄靶标基因的发掘和功能分析

Tomato powdery mildew, late blight and verticillium wilt are important cosmopolitan plant disease caused by Oidium neolycopersici, Phytophthora infestans, Verticillium dahliae in biotrophic, semibiotrophic and nectrophic manners, respectively. To investigate the molecular mechanisms of their interactions with tomato will benefit long-lasting and broad-spectrum resistance breeding of tomato. In this project, yeast two hybrid (Y2H) AD-cDNA libraries of tomato induced by water, O. neolycopersici, P. infestans and V. dahliae will be constructed for mining tomato genes targeted by pathogen effectors, while the Y2H libraries of V. dahliae will be constructed for mining new effectors. Many pathogen effectors were predicted by using bioinfomatics methods with the completion of whole genome sequencing. Based on the sequences of the predicted effectors, the genes can be cloned and constructed into the Y2H inducer vectors for screening the induced tomato library, to mine their target genes in tomato. On the other hand, the known and newly found target genes of tomato will be constructed into Y2H inducer vectors for screening pathogen Y2H libraries, to obtain their interacting effectors. Their function in tomato interaction with three pathogens of newly found effectors and tomato effector target genes will be analyzed by using virus induced gene silencing and transient super-expression methods. Pathogen effectors will be analyzed for their role in interaction between tomato and pathogens by using transient super-expression in tomato. With the functional analysis, tomato key genes targeted by effectors can be selected for stable transformation in tomato or tobacco for further study of their roles in signal transduction. With the results, the molecular mechanism of tomato interactions with O. neolycopersici, P. infestan and V. dahliae will be deciphered, while new effectors and effector target tomato genes will be mined. It will be helpful for long-lasting and broad-spectrum resistance breeding of tomato.

由番茄白粉菌、致病疫霉和黄萎菌引起的白粉病、晚疫病和黄萎病是重要的番茄病害，三种病原物分属于活体寄生、半活体寄生和腐生类型。番茄与不同类型病原物互作分子机制的分析，有助于番茄持久和广谱抗性育种。本研究拟构建番茄、病原诱导番茄及不同病原物的酵母双杂交(Y2H)文库；利用已预测的病原物效应子为诱饵，筛选番茄Y2H文库获得与其互作的番茄靶标基因；利用番茄靶标基因为诱饵，筛番茄白粉菌、致病疫霉和黄萎菌的Y2H文库，获得新的病原物效应子基因；利用沉默和超表达靶标基因植株，分析其在效应子激发免疫反应(ETI)和病原相关分子模式激发免疫反应(PTI)的作用；利用超表达效应子基因植株，分析其在番茄与病原物互作中的作用；利用超表达和RNAi具有功能的番茄靶标基因番茄，分析其可能参与的信号途径。通过发掘不同病原物的效应子及与其互作的靶标基因，分析其作用机理，可为番茄持久和广谱抗病育种提供基因资源和策略。

由番茄白粉菌、致病疫霉和黄萎菌引起的白粉病、晚疫病和黄萎病是重要的番茄病害，三种病原物分属于活体寄生、半活体寄生和腐生类型。番茄与不同类型病原物互作分子机制的分析和互作参与基因的挖掘，有助于番茄持久和广谱抗性育种。1）本项目成功的建立了多个番茄病原菌诱导的酵母双杂交的AD-cDNA文库，构建了番茄的多个抗病基因和白粉菌效应子BEC1019、致病疫霉菌AVR3a、黄萎菌VdNEP的效应子基因的诱饵载体，采用自创的多诱饵筛库方法获得多个番茄抗病反应的候选基因，正在进行下一步的基因功能验证；2）ShoRR1-16是番茄抗病反应必需基因ShoRR1的互作蛋白，它们以同源二聚体的形式发挥作用，参与抗病反应的多个通路；3）完成了番茄白粉菌、致病疫霉菌和黄萎菌与番茄亲和互作的比较转录组分析，发现活体寄生真菌易于抑制番茄的差异表达基因（DEGs）的表达，腐生真菌易于诱导番茄DEGs的表达，对DEGs进行GO、KEGG、COG的功能注释和分析，最终获得522个番茄抗病反应的重要候选基因，为番茄抗病育种提供了丰富的基因资源；4）建立了多种稳定的番茄遗传转化体系，在酵母双杂交文库筛选和转录组测序获得的关键候选基因中，挑选了19个基因，构建其过表达载体和RNAi载体，目前部分基因已获得T2代转基因植株，正在对其接种各种病原菌进行功能验证；5）建立了番茄白粉菌、致病疫霉菌和黄萎菌等病原菌的遗传转化体系；6）利用改进的定点突变方法对CRISPR/cas9基因组编辑体系进行优化，提高载体构建的效率；7）首次将TRV-VIGS系统运用到单子叶植物中，成功沉默了靶标基因；8）建立了快速的真菌鉴定体系。本项目初步阐释了番茄与不同病原菌互作的分子机制，为挖掘持久、广谱抗性的番茄基因提供基因资源。基于以上研究成果，本项目共申请专利5项，获授权专利2项，发表SCI论文11篇，中文核心论文9篇。