青枯菌效应蛋白RipP1诱导茄科植物抗病反应的分子机理

The occurrence of a resistant or incompatible interaction depends on both the presence of a gene for resistance (R) in the host and the corresponding gene for avirulence (Avr) in the pathogen. The investigation of the interaction between R gene and Avr gene is a hot spot in pathogenic bacteria study. The model pathogen Ralstonia solanacearum GMI1000 is the causal agent of the bacterial wilt disease that attacks many solanaceous plants and other hosts but not tobacco (Nicotiana spp.). Two type III secretion system effector genes, ripP1 and avrA, are limiting the host range of strain GMI1000 on tobacco species, and both effectors elicit the hypersensitive response (HR) on these tobacco species. Only the double inactivation of the ripP1 and avrA genes allowed GMI1000 to wilt tobacco plants, thus showing that the interaction between GMI1000 and tobacco is supposed to have a gene-for-gene basis. But the molecular mechanisms in association with those phenotypes remain unclear. In present project, the Y2H experiment was employed to identify RipP1 interacting proteins from cDNA library of Nicotiana benthamiana. The exact target protein of RipP1 was confirmed by VIGS-mediated gene silencing. Accordingly, the proposal here will mainly focus on the proteins that RipP1 interacts with to induce the defense response in host plant, and try to explain why the strain contained RipP1 elicits HR on tobacco but not on other Solanaceae crops, such as tomato. This project aims to identify the resistance gene from plants and provide a fundamental research for breeding solanaceous plants conferring resistance against bacterial wilt disease.

植物病原细菌无毒基因诱导的抗病反应决定了病原菌在寄主上的非亲和性互作，寄主植物抗性基因对无毒基因的识别是非亲和性互作的分子基础，是病原细菌学研究的热点科学问题。茄科作物青枯菌模式菌株GMI1000为害多种茄科作物，却不能侵染烟草；所含有的效应蛋白RipP1在烟草叶片上诱导抗病坏死反应，并且与效应蛋白RipAA一起共同决定了GMI1000在烟草植物上的非亲和性互作，但作用机理还不清楚。本项目利用蛋白-蛋白互作技术筛选茄科植物中RipP1的互作蛋白，用VIGS介导的基因沉默筛选识别RipP1的寄主靶蛋白，揭示RipP1诱导寄主产生抗病反应的分子机制；以靶蛋白与RipP1的互作识别为基础，发掘寄主植物来源的抗性基因，为培育抗青枯菌茄科作物新品种提供科学思路。

青枯菌GMI1000效应蛋白RipP1与RipAA共同决定了青枯菌与烟草之间的亲和性，而关于此效应子的转录调控机制以及其诱发烟草过敏性坏死反应的机制未知。为了探究这些问题，本项目通过分析ripP1上游两段启动子序列，并构建启动子缺失突变体菌株，证明ripP1的转录不完全受HrpB调控。另外，本项目根据基因对基因假说，利用酵母双杂交技术从本氏烟cDNA文库中筛选到了翻译起始因子eIF-5，且证明了该蛋白与RipP1在体内体外均互作。通过TRV介导的VIGS系统，证明了eIF-5参与了RipP1引发的抗病反应。同时，对其它茄科作物的eIF-5同源蛋白的序列分析，发现N端同源性高，虽然N端是RipP1与eIF-5互作的关键区域，但是番茄eIF-5依然不能识别RipP1。最后，通过致病力试验发现，eIF-5减弱了本氏烟的抗病性。本项目的研究结果揭示了RipP1引起抗病反应的机理，为茄科作物的抗病育种提供了理论依据。