菊花CmWRKYs响应黑斑病链格孢菌侵染的分子机制

Chrysanthemum (Chrysanthemum morifolium), one of the ten traditional famous flower and one of four important cut flowers over the world, is of high ornamental and economic values. The black leaf spot disease caused by the fungi Alternaria spp. is one of the most severe diseases during chrysanthemum production. Broad spectrum fungicide is currently less effective to alleviate the disease, which not only cause serious environmental pollution, but also have the high cost and energy consumption. We have previously screened candidate CmWRKY genes from full transcripts generated from chrysanthemum infected by Alternaria spp. The present proposal aimed to identify its function by analyzing the expression pattern of the relevant CmWRKY, its subcellular localization and transcriptional activation activity; to obtain the CmWRKY overexpressed and silenced gene function transgenic chrysanthemum lines, thereby, to screen and verify genes regulated downstream by CmWRKY by bioinformatics analysis; finally to obtain a clear post-translational regulatory mechanism of CmWRKY, using the yeast two-hybrid to screen the proteins of interaction with CmWRKY, the interaction would be validated both in vitro and in vivo by biochemical and molecular methods. Taking all these experimental evidence, we will identify the function and clarify the mechanism of CmWRKY involved in the response of chrysanthemum to Alternaria fungus infection. Our proposal can enrich molecular mechanisms of chrysanthemum in response to pathogen infection, which meanwhile will provide a theoretical basis for the control of Alternaria leaf spot disease.

菊花是我国十大传统名花和世界四大切花之一，有很高的观赏和经济价值。链格孢菌为死体营养型致病真菌，适应性强，其引发的黑斑病严重危害菊花生产，广谱杀菌剂防治效果差，消耗大量人力、物力，环境污染严重。因此，本申请项目在先前获得链格孢菌侵染显著诱导的CmWRKY候选基因基础上，拟分析其表达特性、亚细胞定位、转录激活活性，初步阐述其功能；利用超表达和融合抑制技术获得CmWRKY转基因菊花，通过生物信息学筛选CmWRKY下游调控基因，阐述CmWRKY响应菊花链格孢菌侵染的分子调控路径；利用酵母双杂交技术获得CmWRKY翻译调控及行使功能相关的互作蛋白，并进行体内体外验证，进一步运用生化及分子实验阐明CmWRKY翻译后调控机制。本申请项目的实施可鉴定CmWRKY的功能，揭示CmWRKY响应菊花链格孢菌侵染的分子机制，丰富菊花抗病分子机理，并为菊花黑斑病防治提供理论指导。

链格孢菌引发的黑斑病严重危害菊花生产。项目组成功克隆了菊花链格孢菌病原侵染响应基因CmWRKY15、CmWRKY11，发现其受链格孢菌诱导表达，均定位于细胞核中，其中CmWRKY15不具备转录激活活性，CmWRKY11具有转录激活活性；CmWRKY15、CmWRKY11均介导菊花对黑斑病的敏感性。进一步对CmWRKY15转基因植株进行了转录组分析，发现其调控ABA、SA、JA信号路径以及MYB、WRKY等转录因子的表达而介导菊花对链格孢菌病原侵染的转录调控分子网络；筛选出CmWRKY15互作蛋白CmWRYK4，发现其定位于细胞核，并具有转录激活活性，初步从蛋白翻译后水平揭示了CmWRKY15介导菊花对链格孢菌病原侵染响应的分子机制；此外，CmWRKY11可调控ABA激素的合成或信号路径从而介导菊花对病原侵染的敏感性。CmWRKY15、CmWRKY11含有多个磷酸化位点，病原侵染下的磷酸化修饰可能是其介导菊花应答链格孢菌侵染的翻译后修饰调控机制。本研究丰富了 WRKY 转录因子参与死体营养型病原菌防卫的分子机制。