番茄与青枯菌效应子PopP2互作蛋白的鉴定与功能研究

Bacterial wilt is an important obstacle restricting the development of tomato industry in south China. However, the molecular mechanism of the interaction between tomato and Ralstonia solanacearum is not clear. Previously, the oxidative stress phenotypes were observed in PopP2-overexpression lines of Arabidopsis. And the resistance-related proteins were obtained by CoIP (Co-Immunoprecipitation) and mass spectrometry analysis. In addition, the PopP2-overexpression lines of tomatos were obtained. In this study, we will focus on the functions of the target proteins of PopP2 in tomato resistant to bacterial wilt, We will carry out experiments to isolate and identify the target proteins in tomato resistance to bacterial wilt with CoIP combine with mass spectrometry analysis and Y2H (yeast two hybrid). Further more, we will conduct research on the functional analysis of genes encoding target proteins through reverse genetics methods to elucidate the molecular mechanism of the target proteins participated in tomato resistance to bacterial wilt. This study will provide insights into epigenetic regulation in plant defense of bacterial wilt.

青枯病是制约我国南方番茄产业发展的重要障碍，然而番茄与青枯菌互作的分子机制尚不明确。前期发现青枯菌效应子蛋白PopP2编码基因异源表达的拟南芥株系具有氧化胁迫表型，通过免疫共沉淀及质谱分析获得抗病相关蛋白，此为番茄中PopP2靶标蛋白的分离鉴定提供技术及理论基础；同时获得了PopP2异源过表达的番茄抗（LS89）、感（Micro-Tom）青枯病自交系的转基因植株；在此基础上，本项目针对青枯菌效应子PopP2在番茄中靶标蛋白的分离鉴定，及其在番茄抗青枯病过程中扮演何种角色，以PopP2为切入点，通过免疫共沉淀与质谱联用、酵母双杂交筛选抗病材料cDNA文库，分离和鉴定PopP2在番茄抗青枯病材料中的靶标（互作）蛋白；并利用反向遗传学方法研究靶标蛋白编码基因参与番茄抗青枯病的分子调控途径。以期揭示PopP2在番茄中靶标蛋白参与抗青枯病的分子机制，将为番茄青枯病抗性的遗传改良奠定坚实的基础。

番茄是世界上重要的经济作物和模式植物之一，由茄科雷尔氏菌侵染引起的番茄青枯病是严重制约我国华南地区番茄生产的细菌性病害。本项目通过免疫共沉淀与质谱联用、酵母双杂交筛选抗病材料cDNA文库，结合双荧光素酶验证PopP2互作蛋白，揭示了具有乙酰化活性的青枯菌效应子蛋白PopP2通过与JA合成通路蛋白JAZ1、3、7、10和MSI1互作，发挥抗病作用；通过病毒诱导的基因沉默技术研究了番茄组蛋白去乙酰化酶参与番茄抗青枯病机制，结果表明，SlSRT2(54.17%)和SlHDA6(50.00%)基因沉默后显著降低了感病品种发病率(100%)，采用QPCR和ChIP-QPCR分析了抗病通路基因的表达和乙酰化修饰，结果表明SlHDACs基因沉默提高了抗病相关基因的转录和乙酰化活性水平，参与增强番茄青枯病抗性；以PopP2为诱饵蛋白筛选抗病材料LS89的cDNA文库获得的120个阳性克隆中包含ATP合成酶α亚基蛋白，我们前期研究表明其参与提高番茄灰霉病抗性，在此基础上本项目研究发现atpA基因过量表达可以增强番茄叶霉病、青枯病抗性。本研究结果为进一步揭示宿主植物和青枯菌间的互作机制提供了有益线索，但组蛋白乙酰化与具有乙酰化活性的青枯菌效应子蛋白PopP2的关系及二者参与调控青枯病抗性的作用机制还有待于进一步研究。