小麦叶锈菌侵染小麦过程中效应因子的鉴定及功能分析

Wheat leaf rust is one of very importance disease, and can cause 15%~40% or more yield losses under high disease pressure. Resistant cultivars are the most efficient and environmentally friendly method for reducing damage caused by leaf rust. With the rapid variation of Puccinia triticina(Pt) and deversity of the pathoptyes, the resistance was overcome rapidly when released. Therefore, it has become urgent to speed up the understanding of the mechanisms of virulence variations of the pathogen for having effective protection the wheat production from rust. Effector proteins were secreted into host tissues to promote infection through the manipulation of host processes, and play very important role in pathogenicity. It is also the importance research focus. Therefore, in this project we intended to identify Pt effectors through large-scale screening based on the available Pt transcriptome data, screen the protein candidate effect can be suppressed by BAX induced cell necrosis in tobacco; analysis expression characteristics of effector proteinsby qRT-PC; study on the effect of protein toxic and non-toxic function and the functional domains of function by combining with the method of gradual loss of amino acid and the tobacco transient expression technology; clear the subcellular localization of effetor by tobacco transient expression and locate the place in host by immunofluorescence and electron microscopy; study the translocate effector proteins and domains responsible for transport by using tobacco transient expression technique; analyze the virulence and avirulence functions of effectors and identify the motifs responsible for virulence and translocation; analyze the function by Bacterial type III secretion system and planta induced transient gene silencing (PITGS) assay in wheat; reveal the variation of effect protein genes in wheat leaf rust in different toxicity between pathogenic types.This research might provide new insights in revealing the pathogenicity mechanisms of Pt, in revealing the interaction mechanism between pathogen and host, reveal the mechanism of host resistance, and offer a new approach to engineer durable resistance through modulation of the pathogen genes,which is of great theoretical and practical significance in the sustainable control of wheat leaf rust.

小麦叶锈病是小麦生产的主要病害之一，抗病品种是防治该病害最安全、经济、有效的方法，但小麦叶锈菌致病性多样化及其频繁变异，导致小麦抗叶锈性不断丧失。效应因子在致病中发挥重要作用，因此，研究叶锈菌效应因子对探明叶锈菌致病分子机理及控制叶锈病尤为重要。本研究在已构建吸器阶段转录组文库基础上利用生物信息学技术筛选候选分泌蛋白；利用YSST和烟草瞬时表达技术筛选候选分泌效应蛋白；用qRT-PCR 分析效应因子表达特征；用烟草瞬时表达结合氨基酸逐步缺失法，分析效应蛋白功能及结构域；利用烟草瞬时表达和免疫电镜进行效应因子定位；研究效应因子转运及其转运结构域；利用细菌III型分泌系统和PITGS在小麦中验证候选效应因子的功能；揭示叶锈菌不同致病类型间序列差异。研究对揭示病菌致病机理具有重要意义，同时，对深入揭示寄主抗病及病原物致病专化性具有重要指导意义, 为发掘持久控制病害新策略提供重要理论依据与支撑。

小麦叶锈病是小麦生产的主要病害之一，抗病品种是防治该病害最安全、经济、有效的方法，但小麦叶锈菌致病性多样化及其频繁变异，导致小麦抗叶锈性不断丧失。效应因子在致病中发挥重要作用，因此，研究叶锈菌效应因子对探明叶锈菌致病分子机理及控制叶锈病尤为重要。本研究在已构建吸器阶段转录组文库基础上利用生物信息学技术筛选到635个候选效应蛋白；克隆得到候选效应蛋白58个，对其进行了结构分析；筛选到51个能够抑制由BAX诱导的细胞坏死的效应蛋白，而Pt31812能诱导产生细胞坏死；用烟草瞬时表达结合氨基酸逐步缺失法，对其中6个效应蛋白进行功能结构域分析；分析了10个与发育和侵染过程有关基因的转录表达模式；利用YSST技术验证候选分泌效应蛋白；利用烟草瞬时表达对9个效应因子的作用位置进行验证；利用细菌III型分泌系统和瞬时表达技术在小麦中验证候选效应因子的功能；明确8个效应因子在不同抗病小麦中的作用及在不同小麦叶锈菌中的序列差异；明确效应蛋白Pt31812和Pt3372的毒性功能。以上研究结果对揭示叶锈菌不同致病类型间序列差异、病菌致病机理具有重要意义，同时，对深入揭示寄主抗病及病原物致病专化性具有重要指导意义, 为发掘持久控制病害新策略提供重要理论依据与支撑。