南方根结线虫效应蛋白Mi-V86抑制植物免疫分子机制研究

南方根结线虫是严重危害农业生产的一种植物病原生物，效应蛋白在线虫成功寄生植物过程中具有重要的作用。Mi-V86是从南方根结线虫中新发现的一种类毒液过敏原蛋白，前期工作已证实该效应蛋白能够抑制ETI反应且与烟草中的E3泛素连接酶蛋白NtRNF14互作，本项目拟采用原位杂交和定量PCR技术检测Mi-V86的时空表达模式；利用病毒介导的基因沉默和转基因过表达研究其在线虫寄生过程中的作用；借助Pull-down和Co-IP技术验证Mi-V86与NtRNF14在离体和活体条件下的互作；明确NtRNF14在植物免疫调控中所具有的功能，测定NtRNF14的E3泛素连接酶活性并验证其参与Mi-V86的泛素化修饰及代谢过程，研究结果将有望证实南方根结线虫能够利用自身分泌的效应蛋白，通过与E3泛素连接酶互作从而调控泛素/26S蛋白酶体介导的植物免疫，为深入揭示线虫与植物互作的分子机制提供理论依据。

Root-knot nematode Meloidogyne incognita is one of the most important plant parasitic nematodes and causes serious damage to agricultural industry in the world. The nematode effectors secreted into the host plants during its infection are proven to play key role in successful parasitism. The effector Mi-V86 from M. incognita is a novel candidate member of the venom allergen-like protein family. Our preliminary results demonstrated that Mi-V86 can suppress the effector-triggered immunity (ETI), and interact with Nicotiana tabacum NtRNF14 which encodes an E3 ubiquitin-protein ligase. In order to elucidate clearly the function of Mi-V86 during the nematode infection process, the spatial and temporal transcription patterns of Mi-V86 will be analyzed using in situ hybridization and RT-qPCR, then the virulence of Mi-V86 during M. incognita infection will be examined by the virus-induced gene silencing and the overexpression in transgenic plants. Meanwhile the interaction between Mi-V86 and NtRNF14 will be confirmed in vitro and in vivo using the Pull-down and Co-IP technique, respectively. In order to clarify the function of NtRNF14 in manipulating the plant immunity, the E3 ligase activity of NtRNF14 will be evaluated and its involvement in ubiquitinating Mi-V8 through the ubiquitin-proteasome pathway will be confirmed. The results will demonstrate that the cytoplasmic effector Mi-V86 of M. incognita may manipulate the plant immunity by interacting with the host E3 ligase that involved in ubiquitin-26S proteasome pathway, so as to improve our understanding of the interactions between nematodes and host plants, and benefit to the designing of new control strategies for nematode diseases.