Novel Multichannel Environmental Monitoring Network Controlling Virulence of Ralstonia solanacearum

假单胞菌（Ralstonia, Pseudomonas） solanacearum)是一种致命的萎蔫病，在世界范围内对200多种不同的植物造成危害。分泌的植物细胞壁降解外酶和EPS I外多糖都有助于其在根入侵后成功定植植物维管系统。因为EPS I阻断了木质部的水流，它也是导致枯萎和死亡的主要原因。EPS I是一种由3个氨基糖组成的长链无支链聚合物，其生物合成和输出由16 kb的EPS操纵子编码。eps操纵子的转录，以及编码一些外泌酶和其他毒力基因的基因，是由一个由超过12种蛋白质组成的大的、相互作用的调控网络控制的，这些蛋白质对多种环境信号有反应。该网络由两个不同的双组分调控系统（VsrAD和VsrBC）、独特的信号整合蛋白XpsR和不寻常的Phc信号转导系统组成。初步数据表明，Phc模块采用非典型磷接力级联反应3-羟基铝酸甲酯（3-OH PAME），这是一种新型挥发性，群体指示分子。Phc和3-OH PAME依次调节phcA，编码一个全局的lysr型转录调节因子，控制两种截然不同的生理状态之间的可逆转换，一种适应植物的毒力，另一种适应腐生生存。该项目的主要目标是在分子水平上了解Phc模块如何响应3-OH PAME，以及Phc组件随后如何控制phcA功能。利用两种不同的方法，一种是遗传方法（转座子诱变），另一种是生化方法（在eps调控区共价结合的柱上对茄茄提取物进行dna亲和层析），寻找、克隆和表征了XpsR和VsrC介导eps调控的缺失基因。这些对番茄红霉毒力调控网络的研究非常重要，因为成功致病的最关键因素之一是根据环境信号协调产生毒力和致病性因子的能力，而且番茄红霉网络处理和整合多个信号输入的能力在很大程度上是无与伦比的。这些研究应该能让我们深入了解病原体处理多种环境信号以调节毒力基因表达的方式。最后，通过克隆和分析几种vsrd调控外蛋白的编码基因，寻找R. solanacearum在植物中快速、有效定植所需的基因。同时，体内表达技术也将适应并应用于龙舌兰。该技术鉴定和分离高表达的基因。只有在寄主的定植过程中才会被病原体感染。这些方法中的任何一种或两种都将提供一种获取新的重要基因的方法，并为病原体成功定植植物的策略提供见解。

Ralstonia (Pseudomonas) solanacearum causes a lethal wilting disease of over 200 different plants worldwide. Secreted plant cell-wall-degrading exoenzymes and the EPS I exopolysaccharide, both contribute to its successful colonization of a plant's vascular system after root invasion. Because EPS I blocks water flow in the xylem, it is also a primary cause of wilting and killing. Biosynthesis and export of EPS I, a long unbranched polymer of 3 amino sugars, are encoded by the 16-kb eps operon. Transcription of the eps operon, as well as genes encoding some exoenzymes and other virulence genes, is controlled by a large, interactive regulatory network of over 12 proteins that is responsive to multiple environmental signals. This network is comprised of two distinct two-component regulatory systems (VsrAD and VsrBC), the unique signal integrator protein XpsR and the unusual Phc signal transduction system. Preliminary data suggest that the Phc module employs an atypical phosphorelay cascade that responds to 3-hydroxypalmitic acid methyl ester (3-OH PAME), a new type of volatile, quorum-indicating molecule. Phc and 3-OH PAME in turn regulate phcA, encoding a global, LysR-type transcriptional regulator that controls reversible switching between two very different physiological states, one adapted for virulence in plants, the other for saprophytic survival. A major goal of this project is to understand at the molecular level how the Phc module responds to 3-OH PAME, and how the Phc components subsequently control phcA function. Using two different approaches, a genetic one (transposon mutagenesis) or a biochemical one (DNA-affinity chromatography of R. solanacearum extracts on columns with the eps regulatory region covalently bound), missing genes that mediate regulation of eps by XpsR and VsrC), will be searched for, cloned, and characterized. These studies of the R. solanacearum virulence regulatory network are important because one of the most critical factors for successful pathogenesis is the ability to coordinate production of virulence and pathogenicity factors in response to environmental signals, and because the ability of the R. solanacearum network to process and integrate multiple signal inputs is largely unparalleled. These studies should give insight into the ways multiple environmental cues are processed by pathogens to adjust virulence gene expression. Finally, genes required by R. solanacearum for rapid, efficient colonization of plants by cloning and analysis of genes encoding several vsrD-regulated exoproteins will be searched for. In parallel, in vivo expression technology will be adapted and applied to R. solanacearum. This technology identifies and isolates genes that are highly expressed. by a pathogen only during colonization of a host. Either or both of these approaches will provide a way to access new, important genes, and provide insight into a pathogen's strategies for successful colonization of plants.