百里香酚靶向西瓜枯萎病菌Noxs的抑菌机制研究

Fungal cell death mediated by over-generated reactive oxygen species (ROS) is the general mode of agricultural fungicides. However, the molecular mechanism of ROS generation induced by fungicides is rarely reported, which limits the targeted screening of effective fungicides. In our previous work, we had identified a plant-derived fungicide, thymol, which showed great potential in controlling watermelon fusarium wilt. In addition, thymol remarkably induced the growth inhibition of Fusarium oxyporum f. sp. niveum (FON) by triggering rapid ROS burst. NADPH oxidases (Noxs), located on the cell membrane, have been demonstrated to be a mainly stable source of ROS generation in sensing external stimuli. However, whether Nox could be a specific target of fungicide remains unclear. Therefore, this project would focus on the following studies. (1) analyzing the correlation among thymol-induced cell death, ROS burst, and the expression of Nox genes in FON; (2) cloning the full-length DNA of a series of Noxs and constructing Noxs knock-out mutants; (3) detecting the sensitivity of the mutants to thymol and the changes in ROS generation in mutants under thymol treatment. The above work will help us figure out the exact Nox responsible for ROS burst during thymol-induced cell death of FON, which would provide the scientific basis for the screening and development of biopesticides targeting Noxs and the field application of such formulations.

过量活性氧（ROS）介导的真菌细胞死亡是多数农用杀菌剂普遍的作用方式，但杀菌剂诱导ROS产生的分子机理鲜有报道，从而限制了农用杀菌剂的靶向筛选与研发。申请者前期筛选到植物源杀菌剂百里香酚，对目前难以防治的西瓜枯萎病具有良好防效，且诱导西瓜枯萎病菌ROS迅速爆发是其表现显著抑菌活性的重要作用方式。位于细胞膜上的NADPH氧化酶（Nox）是产生ROS并响应外界胁迫的重要稳定来源，鉴于Nox作为杀菌剂药靶的作用尚不明晰，本项目拟通过：1）分析百里香酚诱导的西瓜枯萎病菌死亡与ROS产生以及Noxs基因表达的相关性；2）克隆西瓜枯萎病菌一系列Noxs基因全长序列，构建Noxs缺失突变体；3）分析Noxs突变体对百里香酚敏感性及菌体内ROS代谢变化，明确具体Nox基因在百里香酚通过诱导西瓜枯萎病菌ROS爆发这一高效抑菌方式中的决定性作用，为该类制剂以及靶向Nox的生物农药研发及田间应用提供理论依据。

枯萎病是西瓜的一种毁灭性病害，在世界各西瓜产区分布广泛，造成严重的经济损失，限制了西瓜生产。本研究发现植物源化合物百里香酚在室内和田间对西瓜枯萎病具有良好的防效。进一步研究发现百里香酚对西瓜枯萎病菌生长、产孢、孢子萌发，以及蛋白酶、纤维素酶、淀粉酶、镰刀菌毒等致病因子有显著的抑制作用。同时，百里香酚能够诱导西瓜枯萎病菌Nox1基因迅速表达产生大量活性氧物质，引起丙二醛含量上升，细胞膜透性增加、甘油含量增加，麦角甾醇含量显著下降，从而抑制细胞膜物质的合成，造成膜脂过氧化损伤，破坏细胞膜完成性，导致细胞渗透压失衡菌体死亡。转录组测序结果表明，百里香酚对西瓜枯萎病菌参与多种生物过程基因表达水平具有较大影响。其中发现差异表达基因5057个， 2440个基因表达上调，2617个基因表达下调。根据现有枯萎病菌基因组信息对其中4869个差异表达基因进行功能注释。通过注释，发现大量的差异表达基因（1348个）富集在氧化还原代谢过程，这表明百里香酚诱导西瓜枯萎病菌快速死亡是一个氧化还原代谢占主导地位过程，在此间Noxs基因在应答“百里香酚”这个外界胁迫产生大量活性氧物质起到了关键作用。结合抑菌实验与转录组测序探明百里香酚抑菌机制包括：1）百里香酚抑制了细胞壁的构建，并促进了细胞壁的降解；2）通过抑制糖鞘糖脂和鞘脂代谢破坏了膜的生物合成；3）可能会影响TCA循环并积累活性氧。以上研究为百里香酚在田间防治西瓜枯萎病害应用提供了一定的理论依据。