水稻细菌性褐条病菌VI型分泌系统的组成、互作和功能研究

In recent years, bacterial brown stripe is gradually becoming the main rice disease in China. In order to prevent and control the disease efficiently, it is necessary to understand the pathogenic mechanism of the pathogen. The applicant has sequenced the whole genome of brown stripe pathogen and identified six different types of secretion systems based on bioinformatics analysis. Furthermore, knockout of dotU, icmF, evpB, hcp and vgrG gene make this pathogen loss pathogenicity to rice, which revealed that T6SS play an important role in pathogenicity of brown stripe pathogen. In addition, knockout of dotU, icmF and vgrG gene influences the expression of other T6SS genes while the interaction between dotU and icmF was identified by a bacterial two-hybrid system, indicating the correlation between different T6SS genes. This proposed project intends to further knock out the genes encoding the 7 other core components of T6SS, and compare the difference in growth, pathogenicity, and T6SS expression between the T6SS mutants with the wild type strain, and further identified the interaction between the other core components of T6SS. Results from this study will not only help to understand the composition, interaction and function of T6SS in pathogenicity of brown stripe pathogen, but also provide a basis for controlling brown stripe by the modulation of T6SS expression.

水稻细菌性褐条病这一传统次要病害近年来正逐渐上升为我国水稻主要细菌病害之一。为有效预防和控制该病害，有必要搞清楚该病菌的致病机理。申请者前期已测定完成该病菌的全基因组，生物信息学分析发现我国病菌存在Ⅰ-Ⅵ型分泌系统；敲除VI型分泌系统（T6SS）的dotU, icmF, evpB, hcp和5个vgrG基因均基本丧失了对水稻的致病性，揭示了T6SS对病菌的致病性至关重要；而且dotU, icmF和vgrG基因被敲除影响T6SS其它基因表达，加上细菌双杂交鉴定的dotU和icmF蛋白互作，揭示了T6SS各组分间的紧密联系。本申请项目拟在前期基础上，继续敲除编码T6SS其它7个核心成分的基因，并比较各突变体与野生型的细菌学特性及T6SS各基因的表达；继续鉴定T6SS各组分间的关系，以全面阐明水稻褐条病菌T6SS的组成，互作和功能特别是其水稻致病功能，为通过调控T6SS来控制水稻褐条病提供基础。

鉴于水稻细菌性褐条病这一传统次要病害近年来正逐渐上升为我国水稻主要细菌病害之一,为搞清楚该病菌的致病机理以有效预防和控制该病害，本项目在前期已测定完成水稻细菌性褐条病菌的全基因组，生物信息学分析发现我国病菌存在Ⅰ-Ⅵ型分泌系统（T1SS-T6SS）且活体感染转录组发现T6SS可能与该病菌的致病性密切相关的基础上，以菌株RS-1和RS-2为对象，首先通过同源重组的方法逐一构建了T6SS的突变体和回补体，表型生物学测定发现这些T6SS基因突变体影响细菌的生长、生物膜形成、游动性、胞外多糖产生、水稻幼苗致病性等一系列致病相关表型，揭示了T6SS对该病菌的致病性至关重要；然后利用细菌双杂交系统、双分子荧光互补技术、pull down和ELISA等鉴定出水稻细菌性褐条病菌T6SS组分间的蛋白互作；最后分别通过qRT-PCR和ELISA鉴定出T6SS单个基因的突变影响其它T6SS的表达以及效应蛋白Hcp的分泌，基本明确了T6SS致病功能的作用机制。总之，本项目已顺利完成了各项研究内容和目标，基本阐明了水稻褐条病菌T6SS的组成、互作和功能特别是其水稻致病功能，为通过调控T6SS来控制水稻细菌性褐条病提供了坚实的基础。