CaLRR51与CaCML15互作在辣椒抗青枯病中的作用及其分子机制解析

Bacterial wilt of pepper, caused by Ralstonia solanacearum, is one of devastating diseases worldwide. Revealing the underlying mechanism of disease resistance can benefit genetic improvement of pepper against the pathogen. We previously found that a novel leucine-rich repeat protein, CaLRR51, plays as a positive regulator in pepper’s response to R. solanacearum infection. We also identified a calcium binding protein CaCML15 by yeast two-hybrid screening and Co-immunoprecipitation confirmation, which can interact with CaLRR51, and was transcriptionally upregulated by R. solanacearum infection. But the underlying mechanism of defense reaction triggered by CaLRR51/CaCML15 interaction remains unknown. In the present project, the functions of CaCML15 in plant immunity will be identified using comprehensive approaches including transient over-expression and virus-induced gene silencing (VIGS) in pepper plants as well as ectopic stable over-expression in tobacco plants. The subcellular localization and intensity change of the interaction between CaLRR51 and CaCML15 will be assayed by both Bimolecular fluorescence complementation and Split-LUC analysis during R. solanacearum infection, and the roles of defense reaction triggered by CaLRR51/CaCML15 interaction will be assayed using comprehensive approaches including transient over-expression and VIGS of CaLRR51 and CaCML15 singly or in combination in pepper plants. Furthermore, some other candidate proteins which can interact with CaLRR51 or CaCML15 will be identified as well as the cascades upstream or downstream of CaLRR51/CaCML15 interaction will also be elucidated. The results of this project will help to reveal the underlying mechanism of the defense reaction triggered by CaLRR51/CaCML15 interaction and benefit the genetic improvement of pepper disease resistance.

青枯病等病害严重威胁辣椒的农业生产，剖析抗病机制是辣椒抗病遗传改良的重要基础。我们前期研究发现CaLRR51是辣椒抗青枯病的正调节因子，并分离了一个与CaLRR51互作的类钙调蛋白CaCML15，且该基因在青枯菌接种后表达增强，但其作用机制仍不清楚。本项目拟通过病毒诱导基因沉默（VIGS）和在辣椒和烟草中超表达分析CaCML15表达减弱或增强对应答青枯菌和免疫反应的影响；通过双分子荧光互作和Split-LUC等分析CaLRR51与CaCML15互作在青枯菌接种前后的亚细胞定位及其互作强度的变化；通过VIGS和瞬间超表达分析CaLRR51和CaCML15沉默对彼此超表达激活免疫反应的影响；最后进一步分离和验证CaLRR51和CaCML15的其他互作蛋白以分析其可能的上、下游信号联结。研究结果有利于进一步阐明CaLRR51/CaCML15介导的辣椒抗青枯病分子机制，为其抗病遗传改良提供依据。

青枯病严重威胁辣椒的农业生产，剖析辣椒抗病分子机制是其遗传改良的重要基础。我们前期研究发现CaLRR51是辣椒抗青枯病的正调节因子，并分离了一个与CaLRR51互作的类钙调蛋白CaCML15，但它们作用机制还不清楚。本研究表明：CaCML15在细胞核和细胞膜质中均有表达，且在细胞膜的一些部位聚集分布。CaCML15的表达受青枯病菌及SA、JA、ET和ABA等外源激素诱导上调表达。通过病毒诱导的基因沉默、稳定超表达和病原菌接种等分析，表明CaCML15作为一个类钙调蛋白家族成员，可能存在功能冗余的现象；而CaLRR51转基因拟南芥植株的抗青枯病能力显著增强。通过双分子荧光互补分析，表明CaLRR51与CaCML15在细胞膜上互作。利用瞬间超表达分析，表明在辣椒叶片中单独表达CaLRR51可以产生细胞坏死反应和H2O2积累，而单独表达CaCML15却不能产生，但是当两者共同表达时，产生的细胞坏死斑和H2O2的积累比CaLRR51单独表达时要强，这说明在CaLRR51诱导产生过敏反应的过程中，CaCML15可能起着一种正向调控的促进或信号放大作用。研究结果有利于阐明CaLRR51/CaCML15介导的辣椒抗青枯病分子机制，为其抗病遗传改良提供依据。