LysM蛋白激酶受体调节香蕉枯萎病抗性的分子机理研究

Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. cubense race (Fox) is known as a destructive disease of banana (Musa spp.). Currently, the molecular mechanism of resistance to Fox still focuses on the isolation and function analysis of defense genes, which can not be used to well explain the reason of resistance. Here, our previous study present the first evidence that the banana LysM receptor like kinase 1 (BLysM1) can induce the plant immune responses against Fox4 by perceiving Fox4 or chitin effectors, which is a main component of the fungal cell wall. However, the molecular mechanism of the chitin recognition by BLysM1 still remains unknown. Therefore, to further investigate the recognition mechanism between LysM domains of BLysM1 and pathogen associated molecular patterns (PAMPs), the BLysM1 expressed in vivo will be used to discuss the binding activities of extracelluar domains to soluble glycol chitin or high concentration of chitooligosaccharides having 1-8 residues of N-acetyl glucosamine (GlcNAc). By comparing the characteristics of LysM domains from LysM receptor kinases of the different resistant varieties to Fox, the roles of diverse LysM units of BLysM1 will be clarified in perceiving the chitin effectors. In addition, the effects of chitin treatment on the intracellular domain phosphorylation of BLysM1 in vivo and in vitro and the roles of posttranslational modification in plant innate immunity are also discussed in this study. Finally, we will provide evidence how Banana LysM receptor kinase perceives chitin signal and activates the downstream pathway of defense response. In theory, the novel research content and pattern will be provided to illustrate the activity mechanism between plant pattern recognition receptors and PAMPs. In practice, this project will lay on a theoretical foundation for the wilt resistance breeding.

香蕉枯萎病（Fox）是一种毁灭性的病害，当前对于其分子抗性机制的研究仍停留在防御基因分离和功能分析上，不能很好地解析抗性机理。我们前期研究发现香蕉LysM蛋白激酶受体1（BLysM1）可识别Fox 4效应因子或几丁质信号，诱导植株对Fox的抗性。为进一步探讨LysM激酶受体与病原菌分子模式（PAMPs）的识别机制，以已分离的BLysM1基因为研究对象，通过分析胞外LysM结构域对几丁质或几丁质寡居糖的绑定活性，比较不同抗性品种LysM胞外结构域的特征，明确不同LysM结构域单元在识别几丁质信号中的作用；并用体内和体外表达的受体激酶蛋白分析几丁质诱导对胞内激酶区磷酸化位点修饰的影响，最终探明香蕉LysM蛋白激酶受体在识别几丁质信号激活下游防御信号通路中的作用。在理论上，为阐明植物细胞表面识别受体与PAMPs互作激活机制提供新的研究内容与模式。在实践上，为香蕉枯萎病抗性育种提供理论基础。

香蕉LysM蛋白激酶受体1（MaLYK1）可提高植株对枯萎病（Foc4）的抗性，然而，该受体如何识别配体激活防御信号通路仍不清楚。本项目运用生物信息学、蛋白质生物化学、细胞生物学等方法与技术，进一步探讨LysM激酶受体与病原菌分子模式（PAMPs）的互作机制。研究结果表明，MaLYK1在体外可直接结合几丁质寡聚糖，最低长度要求含有五个寡聚糖残基；利用蛋白质三维重建技术对比不同抗性香蕉品种LysM胞外结构域的特征，预测MaLYK1胞外LysM2结构域单元在识别配体中发挥重要作用。后将不同的LysM结构单元分别在烟草叶片进行瞬时表达和纯化，通过体外几丁质或几丁质寡聚糖的绑定活性分析，进一步证明了LysM2结构域对配体识别的重要性。而体内和体外分析受体蛋白激酶磷酸化位点，发现结构域中YAQ氨基酸位点对于激活下游防御信号较为重要。除此之外，我们还发现几丁质可诱导MaLYK1胞外结构域形成二聚体或多聚体，继而识别几丁质信号，引起胞内结构域的构象变化，激活下游防御信号通路。这一结果不仅为阐明植物细胞表面识别受体与PAMPs互作激活机制提供新的研究内容与模式，而且为香蕉枯萎病抗性育种提供理论基础。