葡萄霜霉菌效应因子筛选及中国野生葡萄抗霜霉病基因鉴定

Downy mildew, caused by the Oomycete Plasmopara viticola , is a serious disease in the cultivated European grapevines (Vitis vinifera L.). Pathogen effectors are known as Avr factors when they are recognized by plants. Effectors of the Oomycete P. viticola play an important role in the process of pathogenesis. At present study, three Chinese wild grapevines (V. pseudoreticulata Baihe-35-1, V. davidii var. cyanocarpa Langao-5 and V. piasezkii Liuba-8) and a European cultivated variety (V. vinifera Piont Nior) were inoculated by P. vticola and histological survey was undertaken. In conclusion, V. piasezkii Liuba-8 was classified as highly resistant to downy mildew, V. pseudoreculata Baihe-35-1 and V. davidii var. cyanocarpa Langao-5 were resistant to downy mildew, while V. vinifera Piont Nior was susceptible to downy mildew; and we performed in-depth transcriptome analysis by high-through sequencing of germinated zoospores cDNAs from P. viticola. A substantial proportion of transcripts coding for predicted secreted proteins comprises effector candidates. The objective of this study is to clone target effector genes from germinated zoospores of P. viticola in vitro. To identify whether these target effectors play a role in regulating plant immunity, predicted effectors are functionally assayed by using a PVX-based high-through transient plant expression system in Nicotiana benthamiana. Our research involves analysis of function of target effctors, which can block the programmed cell death induced by mouse pro-apoptosis protein BAX in N. benthamiana and can trigger cell death in N. benthamiana, identifying that P. viticola effectors maybe play an important role in pathogehicity. In the following study, a cDNA library, constructed using diseased leaf tissues of V. piasezkii Liuba-8 infected with P. viticola, were screened for proteins interacting with the target effector protein using a yeast two-hybrid approach. A candidate protein was obtained by screening cDNA library clones and was confirmed by co-transformation to be effector-interacting. These target genes from host plant were sub-cloned into the plant expression vector. The function of these resistant genes were further analyzed in the transferred European grapevine plants inoculated by P. viticola. The goals are to identify key effectors of the Oomycete P. viticola and resistant genes of host plant interacting with these effectors, and to investigate on the interaction between oomycete pathogens and their host plants and the disease resistance mechanisms at the molecular level. This study facilitates further investigation on translocation of effector and its virulence mechanisms in host plant cells and will facilitate the development of novel disease control strategies.

葡萄霜霉病是危害最严重的葡萄病害之一，霜霉菌孢子致病效应因子在抑制葡萄防御反应中的起关键作用。本研究是在鉴定不同葡萄材料抗霜霉病反应基础之上；从感染霜霉菌的欧洲葡萄叶片上，收集霜霉菌孢子，诱导游动孢子萌发，进行转录组测序，克隆候选的效应因子基因。以小鼠促凋亡蛋白BAX为诱导因子，采用PVX病毒表达载体在本氏烟中表达目的候选效应因子，分析这些效应因子对BAX诱导的过敏性细胞死亡的抑制作用和诱导烟草细胞死亡的功能，鉴定致病效应因子。以致病效应因子为诱饵，利用酵母双杂交方法筛选与效应因子互作的蛋白。克隆与效应因子互作蛋白基因，转入感霜霉病的欧洲葡萄无核白中，接种霜霉病鉴定转基因植株的抗病反应。确定抑制植物防御反应关键的效应因子和与效应因子互作的靶标蛋白基因，揭示寄主细胞抗病蛋白与霜霉菌效应因子互作的抗病分子机理，推动认识葡萄-病原卵菌互作的分子机制。

完成了葡萄霜霉菌侵染寄主生活史观察，开展了葡萄霜霉菌与4种抗、感葡萄种质互作超微结构观察，分析了抗感葡萄与霜霉菌互作关系，鉴定了复叶葡萄‘留坝-8’属于高抗类型，华东葡萄‘白河-35-1’和秋葡萄‘岚皋-5’属于抗病类型，欧洲葡萄‘黑比诺’属于感病类型。完成了欧洲葡萄‘黑比诺’（PN），中国野生华东葡萄‘白河-35-1’（BH），复叶葡萄‘留坝-8’（LB）接种葡萄霜霉菌后0，12，24，48，96，120 h后转录组测序，克隆了野生葡萄NAC72转录因子和乙二醛酶I-4（GLYI-4）基因，揭示了葡萄NAC72转录因子负调控乙二醛酶I-4（GLYI-4）功能；克隆了葡萄VDAC基因家族VvVDAC1.1、VvVDAC1.2、VvVDAC2、VvVDAC3、VvVDAC4、VvVDAC5，VvVDAC6.1与VvVDAC6.2基因。接种葡萄霜霉菌后中国野生复叶葡萄‘留坝-8’ VpVDA1.1与VpVDAC3在72hpi、48hpi有上调表达，其中VpVDAC1.1、 VpVDAC3过表达能不同程度的引起本氏烟草叶片内ROS积累和细胞凋亡。获得VpVDAC3转基因拟南芥株系VpVDAC3-OE-1、VpVDAC3-OE-8接种丁香假单胞菌5d后，两个转基因株系相对于野生型Col-0较为抗病，解析了葡萄VpVDAC能过激发植物细胞活性氧代谢，产生过氧化氢，诱导侵染位点细胞死亡，抑制病原菌生长的抗病功能。完成了葡萄霜霉菌基因组测序，共预测102个RxLR效应因子蛋白基因，已经克隆了80个葡萄霜霉菌RxLR效应因子。分析了42个RxLR效应因子的功能，完成了部分葡萄霜霉菌效应因子引起寄主致死反应和抑制寄主细胞致死反应鉴定。建立了鉴定霜霉菌效应因子蛋白功能的葡萄叶片瞬时转化体系，为后续筛选能激发抗病葡萄免疫反应的效应因子蛋白提供了依据。证明了效应因子PvAvh74可以诱导烟草细胞的死亡，致死功能与效应因子糖基化位点和核定位有关，揭示了PvAvh74引起的烟草细胞死亡依赖于SGT1， HSP90，RAR1，EDS1，NDR1，MEK2，WIPK，SIPK途径。分别以霜霉菌效应因子RxLR53和RxLR31154为诱饵蛋白，筛选获得RxLR53与葡萄GATA26转录因子互作蛋白，RxLR31154与中国野生葡萄OEE2互作的蛋白。