南方根结线虫激活JAZ效应子MiISE23对茉莉酸通路负调控机制研究

Root-knot nematodes (RKNs) are highly specialized parasites that cause vast losses annually, and the secreted effectors play important roles during the interactions of RKNs and their host plants. Based on the screening and functional analysis of immunity suppressing effectors from Meloidogyne incognita, we found a new type of effectors which can negatively regulate jasmonic defense responses by activating JAZ reppressors. In this research, the target genes in tomato interacting with MiISE23 are isolated and cloned by yeast two hybrid system, and their function are identified with bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP). Moreover, the function of tomato JAZ genes and MiISE23 targeting gene in nematode resistances was verified using in Planta RNAi and tomato transgenic technology. Our experimental results would provide evidence to decipher the molecular mechanisms underlying the manipulation of host immune defense responses by plant and provide a source of potential targets for RKNs control.

根结线虫每年在生产上造成严重危害。效应子在根结线虫与寄主互作过程中发挥重要作用。本研究基于前期南方根结线虫免疫抑制效应子的筛选和功能鉴定，发现一类能够通过激活JAZ基因来抑制茉莉酸防卫反应的效应子，但是其具体作用机理尚不清楚。本研究以这类效应子中的MiISE23为代表，拟采用酵母双杂交技术获得其在番茄中的互作靶标，并通过双分子荧光互补（BiFC）及Co-IP对靶标基因进行验证，解析MiISE23对茉莉酸通路调控的分子机制；同时，通过in Planta RNAi及番茄转基因的方法验证JAZ基因及效应子直接互作靶标基因在根结线虫防治中的作用。这些研究结果对于揭示南方根结线虫的致病机理，防治根结线虫危害具有重要意义。

根结线虫(RKNs, Meloidogyne spp.)为专性植物内寄生线虫，寄主范围十分广泛。根结线虫侵染寄主时能够通过口针分泌一些效应子来帮助自身侵染。本研究基于前期转录组测序及谷枯菌系统的筛选，发现MiISE23能够抑制谷枯菌在烟草上形成的免疫坏死反应。原位杂交及实时荧光定量PCR结果表明其定位于线虫的亚腹食道腺中，且在侵染初期上调表达。为了进一步探究MiISE23在线虫侵染中的作用机制，后续通过免疫沉淀和质谱技术发现MiISE23和JAZ6能够互作，双分子荧光互补及亚细胞定位试验证明MiISE23和JAZ6在植物细胞核中存在互作。实时荧光定量PCR结果表明，MiISE23在植物中的过表达能够使茉莉酸防卫基因的表达量下调，且MiISE23具有稳定JAZ6的作用。番茄外施JA-Ile后能够增加番茄对线虫的抗性，且JAZ6基因在番茄中的过表达能够促进线虫侵染。该研究表明，茉莉酸通路是作物抗线虫作用的关键通路，MiISE23在线虫侵染过程中通过增加JAZ6的稳定性，抑制茉莉酸通路来促进线虫的侵染。该项目有助于我们理解茉莉酸通路在根结线虫和寄主互作中的关系，有助于阐明根结线虫入侵的分子机理。