黄龙病菌Tol-Pal操纵子影响菌体增殖及触发柑橘防卫反应分子机制研究

黄龙病是柑橘生产上毁灭性病害，迄今因病菌极难获得纯培养而限制了其功能基因组学和抗病育种工作的开展。我们前期研究发现Tol-Pal操纵子影响菌体增殖且触发柑橘防卫反应。本课题以S.meliloti为替代菌株，通过构建突变体和功能互补验证Tol-Pal各元件的作用及互作关系。同时构建Pal突变体并诱导融合蛋白表达，验证触发防卫反应差异及特定功能氨基酸位点，结合PR蛋白表达差异、ROS、TMV抗性变化及转录组、蛋白质组等阐明触发柑橘防卫反应信号通路。运用分裂泛素酵母双杂交结合CoIP技术从柑橘文库中调取模式识别受体Rc-Pal，通过BiFC和Pull-down验证蛋白之间的互作Motif及亚细胞定位特点，进而构建Rc-Pal超表达和CRISPR-Cas9敲除载体，明确靶基因有无对Pal的应答特点。最终明确黄龙病菌Tol-Pal操纵子影响菌体增殖及触发柑橘防卫反应机制，为抗黄龙病育种提供新的策略。

Huanglongbing is a destructive disease of citrus. Due to the pathogens' resistance of in vitro culture, research on functional genome and plant resist breeding is greatly hindered. Our previous work showed Tol-Pal operon affected cell proliferation and triggered citrus defense response. In this project, S. meliloti will be used as substitute strain for functional analysis of Tol-Pal operon by creating mutants and carrying out complementary experiments. Series of Pal mutants will be studied by characterizing its difference in triggering defense response with the corresponding fusion protein. Functional single amino acid triggering defense response will be determined. Signal transduction pathway will be figured out by quantifying the expression of PR proteins, ROS, the resistance variation to TMV, transcriptional and proteomics analysis. Split-ubiquitin based membrane yeast two-hybrid (MYTH) system and Co-Immunoprecipitation (CoIP) will be performed to screen the receptor protein RcPal from citrus library. Protein interaction motif between PalCalas and Rc-PalCalas will be confirmed by bimolecular fluorescence complementation (BiFC) and Pull-down. Subcellular localization of PalCalas and Rc-PalCalas will be verified using confocal laser scanning microscopy and BiFC. Furthermore, overexpression of Rc-PalCalas and knocking out by CRISPR-Cas9 in citrus will be studied, the response to PalCalas will be reconfirmed. The final goal is to figure out the function of Tol-Pal operon affecting cell proliferation and the molecular mechanism of triggering citrus defense response. It will provide new strategies for citrus resist breeding which play vital important in health development of citrus industry.