Molecular Basis of Phytopathogenicity of Pseudomonas solanacearum

假单胞菌（Pseudomonas solanacearum）是一种具有重要生态和经济意义的植物病原菌，可引起世界上200多种茄属植物的致命枯萎病。拟从主要的细胞外大分子出发，探讨其致病性的生化和遗传基础。在P. solanacearum基因组中，有一个大于10kb的片段（I区），其中的插入同时导致毒力大幅下降，产生细胞外多糖黏液（EPS），并合成两种主要的细胞外蛋白（EXPs）。拟议研究的一个目的是表征I区基因产物，并研究它们在毒力和细胞外毒力分子合成中的作用。这将通过大肠杆菌I区亚克隆的大细胞分析、TnphoA诱变、I区非极性分离突变体的体外生化和植物研究来完成。编码28-kDa EXP的基因（其水平与EPS水平相关）将被克隆并进行类似的分析。对于生化研究，我们将首先开发检测EPS合成中中间体积累的方法。区域I的表达依赖于编码输出（膜）蛋白的两个不同的非连锁位点，以及位于区域I下游6kb的位点；其他研究表明，所有这些位点都控制着与I区无关的其他毒力基因。其他项目目标是通过DNA序列和基因产物分析来表征调控蛋白，通过分析多突变背景下I区表达来探索它们之间的串扰，以及研究影响毒力基因调控的信号。

Pseudomonas solanacearum is an ecologically and economically important phytopathogen causing a lethal wilt disease of over 200 solanaceous plants worldwide. The research proposed investigates biochemical and genetic basis of its pathogenicity, focusing on major extracellular macromolecules. A greater than 10-kb segment (Region I) of the P. solanacearum genome in which insertions simultaneously cause a major decrease in virulence, production of extracellular polysaccharide slime (EPS), and synthesis of two major extracellular proteins (EXPs) has been identified. One objective of the proposed research is to characterize Region I gene products and investigate their involvement in virulence and synthesis of extracellular virulence molecules. This will be accomplished by E. coli maxicell analysis of Region I subclones, TnphoA mutagenesis, and in vitro biochemical and in planta studies of nonpolar isertion mutants of Region I. The gene encoding a major 28-kDa EXP, whose level correlates with EPS levels, will be cloned and similarly analyzed. For the biochemical studies we will first develop methods to detect accumulation of intermediates in EPS synthesis. Expression of Region I depends on two distinct, unlinked loci that encode exported (membrane) proteins, and a locus located 6 kb downstream of Region I; other studies suggest that all these loci control other virulence genes unlinked to Region I. Additional project objectives are characterization of the regulatory proteins by DNA sequence and gene product analysis, exploring crosstalk between them by analysis of Region I expression in multiple mutant background, and investigation of signals affecting virulence gene regulation.