剑麻斑马纹病原菌效应蛋白PnRXLR1的致病机理研究

Zebra disease in Agave sisalana caused by Phytophthora nicotianae is one of the most serious diseases in sisal production. RXLR effector proteins are important cytoplasmic effectors of oomycete and more than 300 RXLR effector genes were identified in the genomes of P. nicotianae. Based on our preliminary results, a RXLR effector candidate named PnRXLR1 was obtained through comparative RNA-seq analysis between the disease-resistant sisal and the susceptible sisal which were inoculated P. nicotianae. We previously have shown that PnRXLR1 can cause leaf to decay. However, it is unclear how PnRXLR1 manipulates plant innate immunity to promote infection. In this research, we propose a serial of experiments including gene expression analysis under different inductions, signal peptide validation, subcellular localization, mutations assay to define the basic features of PnRXLR1. To further understand the mechanism on how PnRXLR1 functions in virulence by interfering host programmed cell death (PCD), reactive oxygen species (ROS) production and callose deposits, by overexpressing of PnRXLR1 gene in Nicotiana benthamiana. Meanwhile, we perform yeast two hybrid (Y2H) assay to indentify the host factors directly interaction with PnRXLR1. In conclusion, this project will provide clues regarding the role of PnRXLR1 in the pathogenicity and lay the theory foundations for the future prevention of zebra disease, advancing selection and breeding of new varieties.

烟草疫霉引起的斑马纹病是剑麻生产上的毁灭性病害之一。疫霉菌能分泌RXLR效应蛋白进入寄主促进侵染。前期分析接种烟草疫霉的抗病、感病品种转录组数据，获得7个差异表达的RXLR效应蛋白基因，在剑麻感病品种叶片中瞬时表达，发现4926号基因引起的症状最严重（命名为PnRXLR1），提示PnRXLR1在烟草疫霉侵染剑麻的过程中发挥重要作用，但不清楚PnRXLR1的致性作用机制。本项目拟采用qRT-PCR和亚细胞定位明确PnRXLR1致病时间和致病部位；通过在酵母和植物中表达PnRXLR1突变基因明确信号肽的分泌作用、C端致病区段及关键氨基酸位点；过表达分析PnRXLR1对植物胼胝沉淀、ROS产生和细胞死亡等防卫反应的影响；筛选剑麻中与PnRXLR1互作的靶蛋白。明确PnRXLR1介导的烟草疫霉对剑麻的致病作用，从而促进烟草疫霉对剑麻致病机制的认识，为培育抗斑马纹病剑麻新品种提供理论依据。

烟草疫霉引起的斑马纹病是剑麻生产上的毁灭性病害之一。疫霉菌能分泌RXLR效应蛋白进入寄主促进侵染。本项目以烟草疫霉RXLR效应蛋白作为切入点，探讨效应蛋白在烟草疫霉对剑麻致病中的作用机理，旨在通过本项目的开展促进对烟草疫霉对剑麻致病机制的认识，为培育抗斑马纹病剑麻新品种提供理论依据。经过三年的努力，取得如下重要研究进展：1）明确了烟草疫霉中308个编码RXLR效应蛋白基因中表达量前35个基因表达模式的分析，11个基因呈现先增加后减少的表达模式，4个基因的表达量从侵染开始逐渐增加的表达模式，1个基因在侵染后的表达量逐渐降低，而这些基因中PnRXLR1的表达量最高、变化量最大；2）完成了35个RXLR效应蛋白的结构分析，其中20个拥有信号肽和RxLR基序，同时发现一些蛋白中含有W和Y保守结构域，包括PnRXLR1在内11个蛋白的信号肽能将转化酶分泌到酵母细胞外；3）PnRXLR1能诱导细胞死亡。4）鉴定了PnRXLR1在剑麻中的互作蛋白，获得17个靶蛋白。5）在原计划之外，我们对全部45个CRN效应蛋白进行了生物信息分析， 3个蛋白含有信号肽，13个蛋白为非典型分泌蛋白，功能分析发现仅CRN0793能诱导细胞死亡，8个CRN效应蛋白能抑制BAX诱导的细胞死亡，CRN0793的信号肽能将转化酶分泌到酵母细胞外。在项目的资助下，我们完成了烟草疫霉中两类主要效应蛋白RXLR和CRN的筛查工作，发现了引起细胞死亡和抑制细胞死亡的效应蛋白，结果为进一步深入研究这些效应蛋白的致病机制提功能重要研究基础。发表论文1篇，培养研究生3名，本科生5名。