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

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.

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。

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。本项目重点围绕黄龙病菌Tol-Pal操纵子的功能进行了系统研究，结果发现：Tol-Pal操纵子的重要构成元件PalCLas具有多重功能，可影响菌体增殖速率和形态建成，敲除突变体生长速率明显变慢，回补菌株可在一定程度上恢复生长速率。带有完整信号肽的PalCLas可触发HR反应，具有PAMP特性，信号肽区域关键氨基酸缺失或突变后ΔPalCLas不能引起HR反应，其中ΔPal6th(aa7-161)-103、ΔPal7th(aa8-161) -103和ΔPal7th (Phe-Gly)-103能诱导本氏烟草产生HR反应，其他缺失突变体和定点突变体不能引起烟草HR反应，证实第八位异亮氨酸（IIe）是引起本氏烟草HR反应的关键氨基酸。Tol-Pal各元件存在复杂的互作关系：通过酵母双杂交点对点验证发现PalCLas与TolACLas和TolBCLas蛋白均存在直接互作关系，PalCLas与TolQ和TolR不存在直接互作。TolA与TolQ存在直接互作但与TolR不存在直接互作，TolQ与TolR也不存在直接互作。运用酵母双杂交技术从柑橘文库中调取互作受体蛋白，结合点对点验证、BiFC和Pull-down试验证实，PalCLas与DRM/ARP(XP_006467758.1)蛋白、纤维素酶（GAY35888.1）以及HSP90 (KDO58427.1)蛋白存在直接互作，同时PalCLas能触发烟草防卫相关基因表达，诱导氧爆发和胼胝体沉积，证实PalCLas具有PAMP特性。进而构建了PalCLas及受体靶蛋白的植物超表达载体，甜橙中超表达PalCLas没有明显的症状表型，超表达植株防卫相关基因表达增强，提高了对黄龙病菌抗性。本项目明确了黄龙病菌Tol-Pal操纵子影响黄龙病菌菌体增殖的机理，阐明了Tol-Pal系统各元件之间的互作关系，以及PalCLas的PAMP特性及其与受体靶蛋白之间的互作关系。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的分子机制，为抗黄龙病育种提供新的靶点和策略。