小麦条锈菌效应子鉴定及其调控机理

Puccinia striiformis Westend. f. sp. tritici Eriks (PST), as an obligate parasitism fungi, causes devastating stripe rust disease in wheat. Resistance in wheat cultivars is usually overcome quickly by the rapid emergence of new virulent PST races, which frequently leads to the epidemic of the disease. PST can not be cultivated in vitro and lack the stable transformant system, which hinder the intensive researches on the pathogenetic mechanism of PST via the traditional genetic techniques. Thus it is difficult to effectively control the disease. To speed up the deciphering the mechanism of PST pathogenicity and the rapid virulence variation, we completed the genome sequencing of PST CYR32 and deep sequencing of five PST races with variant virulence, which provide us abundant gene resources for the screening of PST effectors. Thus, in this project, we will screen the PST effectors in the whole PST genome scale by transient expression on the tobacco, combining with the analysis of the intra-/inter-species polymorphism of the effectors, the key effectors in PST will be determined for further studies. And then analyse their spatial and temporal characteristics during wheat-Pst interaction, identify their host targets, analyse the roles of the target genes, reveal their biochemical function via sets of technology and methods and different research systems. Collectively, the results will reveal the roles of PST effectors in regulating host defense and metabolism, and provide strong theoretical basis for deeply exploring the mechanism of PST pathogenicity and virulence variation, which will then lay the foundation for establishing brand strategies for durable control the stripe rust disease.

小麦条锈菌为专性寄生真菌，在小麦上引起严重的病害。病菌频繁变异常导致小麦品种抗性不断“丧失”，引发病害流行。由于条锈菌不能培养，缺乏遗传转化体系，难以通过遗传学方法深入研究条锈菌致病机理，病害防治始终处于被动状态。为加快深入解析条锈菌致病及其变异机理的研究步伐，本项目拟在前期完成了条锈菌CYR32全基因组测序及5个不同毒性菌系重测序的基础上，大规模筛选条锈菌效应蛋白，解析条锈菌效应蛋白种间和种内变异特征；挖掘条锈菌关键效应蛋白基因，并利用瞬时表达、免疫荧光定位、酵母双杂交、寄主诱导的基因沉默（HIGS）、RNAi等技术方法，开展效应蛋白在条锈菌侵染小麦过程中的毒性功能、条锈菌效应蛋白作用的时空特征、效应蛋白互作靶标及其功能、效应蛋白生化功能等方面研究内容，以期揭示条锈菌效应蛋白调控寄主的作用机理，为深入揭示条锈菌致病及其变异机理提供理论依据，进而为开辟持久有效的条锈病防控策略奠定了基础。

小麦条锈菌为专性寄生真菌，在小麦上引起严重的病害。病菌的频繁变异导致小麦品种抗性不断“丧失”，常常引发病害流行。由于条锈菌不能培养，缺乏遗传转化体系，难以通过遗传学方法深入研究条锈菌致病机理，病害防治始终处于盲目被动状态。为了深入解析条锈菌致病及其变异机理，本项目从基因组水平对条锈菌效应蛋白进行了大规模筛选鉴定，系统深入解析了关键效应蛋白的功能及作用机理，取得了重要研究进展。①获得了19个在烟草中能够抑制Bax诱导的细胞坏死的效应蛋白，qRT-PCR分析表明其中15个在条锈菌侵染过程诱导表达，4个下调表达。②利用细菌三型分泌系统在小麦中瞬时表达，发现其中16个能够明显抑制小麦PTI相关胼胝质的积累，14个抑制小麦ETI中活性氧（ROS）与过敏性坏死反应（HR）发生。HIGS （Host induced gene silencing）沉默19个效应蛋白基因鉴定到6个能够显著影响条锈菌致病性的关键效应蛋白。③通过酵母双杂交筛选获得了5个关键效应蛋白在寄主细胞内的互作蛋白，揭示了效应蛋白Pst18363靶向并稳定小麦防卫反应的负调控子—Nudix水解酶23（TaNUDX23），抑制小麦ROS产生，促进病菌侵染；富含甘氨酸和丝氨酸的效应蛋白PstGSRE1与LSD转录因子TaLOL2互作，通过阻止TaLOL2的细胞核定位，干扰其诱导细胞坏死功能，抑制寄主防御反应；叶绿体效应蛋白Pst_12806与小麦叶绿体细胞色素b6f复合体元件TaISP互作，通过干扰叶绿体中ROS的产生，影响叶绿体代谢以抑制植物免疫；初步阐明效应蛋白PsKE1和PsKE2能够与小麦抗条锈病负调控因子硫氧还蛋白、H3K4去乙酰化酶和脱水素互作，调控寄主的防御反应。④鉴定到5个潜在无毒基因，能够在小麦不同近等基因系中激发HR。⑤创制了效应蛋白基因PstGSRE1-RNAi小麦转基因材料，增强了小麦对条锈病的抗性。研究结果加深了对条锈菌致病机理的认识，为小麦抗条锈病遗传改良奠定基础，为绿色、持久控制条锈病提供理论依据和新材料。在本项目资助下，在Nature Communications、Molecular Plant、New Phytologists和Environmental Microbiology等期刊发表SCI论文8篇，出版英文专著《Stripe Rust》，获陕西省科学技术一等奖1项。