核盘菌病毒性低毒菌株在油菜中内生性生长及其与油菜的分子互作机制

Previously, we reported Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) has a potential to control stem rot of rapeseed (Brassica napus) caused by Sclerotinia sclerotiorum. Recently, we found that the SsHADV-1-mediated hypovirulent strain could grow endophytically in rapeseed, and this hypovirulent strain could increase seed oil content by up to 3.75 % when plants were treated with hypovirulent strain. Our finding showed that S. sclerotiorum is converted to an endophyte from a typical necrotrophic pathogen, and the endophytic growth is likely to give strong impact on its host. Based on the endophytic property of the hypovirulent strain of S. sclerotiorum, we propose to study the endophytism of SsHADV-1-mediated hypovirulent strain of S. sclerotiorum and molecular interaction with rapeseed, and four aspects listed below will be considered.① Probing if there is any possible mutualistic symbiosis among mycovirus, fungal and rapeseed. The distribution and life cycle of the endophyte growing in rapeseed will be studied, and its impact on the growth, development and stress-resistance of rapeseed and on the transmission of mycovirus will be examined. ② Unravelling the endophytism of S. sclerotiorum. The key signal transduction pathways and metabolism pathways of endophytic growth which are significantly different from those pathways of necrotrophic growth and saprobiotic growth will be identified, and the genes whose expressions are significantly regulated will be analyzed functionally. ③ Understanding the response of rapeseed to the endophytic growth of S. sclerotiorum. The key metabolism pathways and signal transduction pathways of rapeseed which are significantly different from S. sclerotiorum-free plants will be studied, and the metabolites and secondary metabolites responding to those changed pathways will be detected and key genes whose expressions are significantly regulated will be also founctionally analysed. ④Understanding the mechanism for promoting seed oil content by endophytic growth of S. sclerotiorum. The lipids metabolism pathways and the key genes which are regulated by the endophytic growth of S. sclerotiorum will also be studied during bloom stage of rapeseed. Hopefully, our study will shed light up on the endophytism of a typical necrotrophic pathogen which may help us to understand the potential contribution of endophytic growth of fungal pathogens on the development and prevalence of crop diseases; our study may also be used to establish a model for triple interaction among mycovirus, fungal pathogen and plant host which may improve our knowledge on mycovirus transmission and help us to explore hypovirulence-associated mycoviruses as biological control agents; finally, our study also will help us to develop a biological agent to control stem rot of rapeseed and enhance seed oil content simultaneously which may be used to impove the productivity effect of rapeseed in China.

前期研究发现感染低毒相关DNA病毒（SsHADV-1）的核盘菌低毒菌株可在油菜上进行内生性生长，用其处理油菜可显著提高油菜籽含油量，最高可提高3.75 %。针对这一特性，本项目拟研究低毒菌株在油菜体内的分布和生活史，研究低毒菌株内生性生长对油菜生长、发育和抗逆及对SsHADV-1传播的影响；利用RNA-Seq、基因超量表达和基因沉默等技术及KEGG PATHWAY数据库等，研究核盘菌从死体营养型病原菌转变为油菜内生真菌后关键信号通路和代谢通路的变化以及油菜应答核盘菌内生性生长的分子机制，研究核盘菌内生性生长对油菜脂肪代谢通路及关键基因表达的影响以之解析刺激油菜籽含油量提高的分子机制。项目的完成将对解析病原真菌转变为内生真菌的机制有显著促进作用；将建立“真菌病毒－病原真菌－寄主”交互作用模式，丰富真菌病毒学理论和实践；也将为创建一种显著提高油菜生产效益的新型防病、增油生物菌剂提供理论依据。

在该项目的资助下，我们致力于“病毒SsHADV-1－核盘菌－油菜”交互作用分析、核盘菌由死体营养型病菌转变为内生真菌的分子机制、油菜应答内生性核盘菌进入和生长的分子机理和内生性核盘菌提高油菜籽含油量的分子基础等四个方面的研究。发现在感染SsHADV-1的核盘菌DT-8中，植物细胞壁降解酶类基因和类似效应子蛋白基因等致病相关基因的表达受到显著抑制、激发油菜抗病反应的角质酶基因的表达显著上调。发现一种新的效应子类似蛋白SsCP1，它与油菜中的PR１蛋白互作，抑制油菜的抗病反应，但SsCP1的表达在DT-8中受到抑制。DT-8的这种基因表达模式可能是核盘菌向内生真菌转变的机制。DT-8对油菜的生长和抗病都有显著的促进作用。对经DT-8处理的油菜顶端进行RNA_Seq分析，发现油菜中植物与病原物互作、MAPK信号、植物激素信号转导、角质/亚角质和蜡质的生物合成及生物节律等均发生变化。鉴定和克隆出对油菜的抗病、油菜籽产量和含油量有显著影响的基因（BNcx9c），并通过将BNCX9C导入拟南芥中证明了该基因的功能。DT-8在油菜中内生可以向核盘菌传染SsHADV-1，也可以营腐生生活。基于这些发现，建立了利用DT-8作为植物疫苗控制油菜菌核病、促进植物生长的技术；该技术操作简单，可以实现8%以上的增产。研究还发现了一种噬真菌的昆虫厉眼蕈蚊与SsHADV-1存在互惠性互作，该昆虫可以作为传播介体传播病毒。研究还发现核盘菌也是禾本科植物的有益内生真菌，可以促进禾本科植物生长和抗病。而且对油菜致病的和不致病的菌株均可以进入禾本科植物体内。利用核盘菌处理小麦和水稻可以增强小麦和水稻的抗病能力。基于上述的发现，我们认为SsHADV-1、历眼蕈蚊、核盘菌和多种科属的植物之间存在交互作用；利用这种交互作用可以建立高效、绿色的病害防控技术，如利用DT-8控制菌核病和小麦赤霉病和锈病等。