Analysis of COI1-dependent signalling processes in Arabidopsis roots upon infection with the fungal pathogen Verticillium longisporum

子囊菌长孢黄萎病（verticillium longisporum）是一种土壤传播的维管病原菌，对油菜造成重大产量损失。我们之前对V. longisporum/Arabidopsis thaliana相互作用的分析揭示了两个意想不到的发现：(1)植物防御激素茉莉酸异亮氨酸（JA-Ile）的受体COI1是根部真菌在茎部有效增殖所必需的。(2)新的COI1功能即使在没有植物来源的JA-Ile或真菌来源的JA-Ile模拟物的情况下也能起作用。本研究的长期目标是识别促进疾病的根到茎信号，并阐明coi1依赖但ja - ile不依赖的信号转导途径，从而导致其合成。在拟议的资助期内，下一代测序（NGS）将产生在受感染的aos （JA生物合成突变体）和coi1 （JA受体突变体）根中差异表达的植物和/或真菌基因。将选定的植物和真菌基因与荧光蛋白的翻译融合体分别转化到拟南芥和V. longisporum中，并通过共聚焦激光显微镜同时监测它们的表达。将整合表达模式（时间点、细胞类型特异性）和编码区域的信息，以选择可能编码敏感性增强信号的候选植物或真菌基因。这些基因将通过分析相应的突变体来进行功能表征。为了证实COI1可以独立于其配体发挥作用的假设，我们将用编码不能结合JA-Ile的突变COI1蛋白的转基因来补充COI1突变体。此外，在根特异性启动子的控制下，利用表达显性负JAZ蛋白的植物来分析JAZ蛋白的参与。为了鉴定新的COI1相互作用伙伴，将在高通量筛选系统中进行几种酵母双杂交筛选。转基因植物或突变体的分析将通过监测疾病表型和NGS鉴定的coi1依赖性但ja - ile非依赖性基因的表达来完成。

The ascomyceteVerticillium longisporum is a soil-borne vascular pathogen that causes major yield losses on rapeseed (Brassica napus). Our previous analysis of the V. longisporum/Arabidopsis thaliana interaction has unraveled two unexpected findings: (1) COI1, the receptor of the plant defense hormone jasmonic acid-isoleucine (JA-Ile), is required in roots for efficient proliferation of the fungus in the shoot. (2) The novel COI1 function operates even in the absence of plant-derived JA-Ile or fungus-derived JA-Ile mimics. The long-term goals of this proposal are to identify the disease-promoting root-to-shoot signal and to elucidate the COI1-dependent but JA-Ile-independent signal transduction pathway leading to its synthesis. During the proposed funding period, next generation sequencing (NGS) shall yield plant and/or fungal genes that are differentially expressed in infected aos (JA biosynthesis mutant) and coi1 (JA receptor mutant) roots. Translational fusions of selected plant and fungal genes to fluorescent proteins will be transformed into Arabidopsis and V. longisporum, respectively, and their expression will be monitored simultaneously by confocal laser microscopy. Information on the expression pattern (time point, cell-type-specificity) and coding region will be integrated to select candidate plant or fungal genes that might encode the susceptibility-enhancing signal. These genes will functionally characterized by analysis of corresponding mutants. To substantiate our hypothesis that COI1 can function independently from its ligand, we will complement the coi1 mutant with transgenes encoding mutant COI1 proteins that cannot bind JA-Ile. Moreover, the involvement of JAZ proteins will be analyzed using plants expressing a dominant negative JAZ protein under the control of a root-specific promoter. In order to identify novel COI1 interaction partners, several variants of the yeast two hybrid screen will be performed in a hi-throughput screening system. Analysis of transgenic plants or mutants will be done by monitoring the disease phenotype and the expression of the COI1-dependent but JA-Ile-independent genes identified by NGS.