小麦叶锈菌效应蛋白Pt-2567对小麦抗叶锈病NILTcLr28无毒效应研究

The effector triggered immunity (ETI) is an important part in plant disease resistance. Therefore, studies on the interaction between Puccinia triticina avirulence (Avr) gene and wheat leaf rust resistance (Lr) gene are very important for discovering the immune mechanism of wheat against wheat leaf rust, and utilizing of avirulence gene and resistance gene in the future. In our previous study, by transient expression of Pt effectors in wheat leaves using Agrobacterium, one effector Pt-2567 was identified to specifically trigger HR on wheat near isogenic line (NIL) TcLr28. Therefore, in this proposal, we plan to further validate the avirulence role of Pt-2567 on TcLr28 by transient expressing it in wheat NILs carrying different Lr genes using Agrobacterium, and silencing it by host induced gene silencing (HIGS). Meanwhile, targets of Pt-2567 and their associative functions will be identified and analyzed by yeast two-hybrid and bimolecular fluorescence complementation (BiFC),Co-Immunoprecipitation (Co-IP), mass spectrometry and HIGS. Furthermore, co-expressing of the targets of Pt-2567 and Pt-2567 gene in Nicotiana benthamiana and expression analysis of the Pt-2567 on wheat NILs carrying different Lr genes will be conducted. The current project will reveal the mechanism of Pt-2567 in triggering cell death in TcLr28, and is of great significance in moving forward our understanding of the interaction between Pt and wheat.It provides foundation for the identification of avirlence genes of obligate parasitic pathogene.

病原菌效应蛋白激发的免疫反应是植物的重要抗病表现。开展小麦叶锈菌无毒基因（Avr）与小麦抗叶锈病基因（Lr）的互作研究，有助于深入揭示小麦抗病反应的分子机制。对于小麦叶锈菌Avr和小麦R的利用具有重要意义。本课题组前期在小麦中瞬时表达效应蛋白，获得了1个能够在TcLr28中诱导过敏性坏死反应的效应蛋白Pt-2567，为探明该基因如何激发TcLr28的免疫反应机理，本研究拟利用小麦抗叶锈病近等基因系，结合酵母双杂交、BiFC、共表达分析、HIGS分析等多种技术，明确Pt-2567对TcLr28无毒效应的特异性，Pt-2567的结构、表达特性和互作靶标，阐明Pt-2567介导的对小麦NILTcLr28的无毒效应。研究结果对于深入理解小麦叶锈菌与小麦的互作机制具有重要意义。

开展效应蛋白的研究对于明确其致病机制，培育持久抗病本品种具有重要意义。本研究通过分析小麦叶锈菌生理小种感病寄主Thatcher互作的孢子休眠、萌发、侵染时期的转录组数据，利用生物信息学手段筛选到了635个候选效应蛋白，选择Pt2567进行研究。通过软件SignalP v4.1, TargetP v1.1, TMHMM v2.0 和 EffectorP v2.0发现Pt2567含有信号肽、定位于分泌途径中、不含有跨膜结构域，EffectorP预测其有94%的概率是一个候选效应蛋白。利用异源表达系统明确了Pt2567可抑制由BAX诱导的PCD；实时荧光定量 技术分析发现该基因在互作后36h和60h表达量最高，与转录组测序的表达水平表现一致；利用9个毒力不同的叶锈菌菌株开展对效应蛋白Pt2567的种内多态性分析，发现该序列共有4个氨基酸位点突变，现低多态性水平；分别在烟草细胞和小麦原生质体中对Pt2567进行亚细胞定位，以pRG107:GFP为对照，发现该融合表达蛋白在寄主细胞内发挥作用。利用农杆菌瞬时注射法将Pt2567递送至37个近等(单)基因系中，发现该基因能在TcLr28中特异引起过敏性坏死反应，表明Pt2567可能为Lr28的候选无毒基因。通过细菌三型分泌系统对Pt2567在不同小麦品种中进行过表达，通过胼胝质的积累量确定该基因能够在Thatcher中抑制PTI，在TcLr19中抑制ETI，发挥毒性功能；能够在近等基因系TcLr28中增强PTI和ETI，发挥无毒功能；利用HIGS在TcLr28中沉默Pt2567，对TcLr28表现低毒力的菌株(KHHT)在TcLr28上的反应型由“0”变为“4”。120 h时，与对照组BSMV:00相比，沉默Pt2567的TcLr28中出现了密集的菌丝，沉默Pt2567有效地抑制了TcLr28的抗病性。说明Pt2567在小麦叶锈菌侵染TcLr28过程中发挥无毒性功能。本研究表明小麦叶锈菌在寄主病原物互作的侵染阶段分泌大量效应蛋白。效应蛋白Pt2567在寄主细胞内发挥作用并兼具毒性和无毒性两中特性，在TcLr28上表现无毒效应。