Interactions Between Agrobacterium and Host Plants

农杆菌与宿主植物之间的相互作用

• 批准号: 6702311
• 负责人: Stephen C. Winans
• 金额: $ 34.38万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6702311
• 关键词: Agrobacterium tumefaciens; Arabidopsis; Burkholderia; Escherichia coli; Yersinia; bacterial genetics; bacterial proteins; cell cell interaction; gene expression; host organism interaction; immunoprecipitation; laboratory rabbit; microorganism growth; molecular cloning; pheromone; receptor; receptor binding

DESCRIPTION (provided by applicant): The long-range goal of this lab is to continue developing the Agrobacterium-plant pathosystem as a model for studying molecular interactions between pathogenic bacteria and their hosts. After the successful oncogenic transformation of host plants, A. tumefaciens colonizes the transformed plant and uses tumor released compounds (opines) as nutrients. Under these conditions, the bacterium uses a quorum-sensing system composed of Tral, which synthesizes the bacterial pheromone N-3-oxooctanoylhomoserine lactone (OOHL) and TraR, which is an OOHL receptor and OOHL-dependent transcriptional regulator. We have purified both proteins and reconstituted their activities in vitro. We recently collaborated in solving the crystal structure of TraR, complexed with OOHL and DNA. We will take advantage of this structure to do structural studies of TraR, by doing alanine-scanning mutagenesis of the TraR surface, its OOHL binding determinants, and its DNA binding determinants. We will attempt to isolate positive control mutants, and will do a selection for constitutively active TraR mutants and for mutants able to detect heterologous autoinducers. We have shown that TraR synthesized in the absence of OOHL is rapidly targeted for proteolysis, probably by the CIp and Lon proteases. We will disrupt the Ion and the three clpP genes of Agrobacterium and measure the half life of the protein, and whether it still requires OOHL for stability and activity. We will overproduce the N-terminal domain of TraR in the present of C13 and N15 isotopes for collaborative studies of TraR folding. We will express the C-terminal domain in vivo in a form that dimerizes to check for DNA binding and for transcription activation. We have been to compare the biochemical properties of TraR to those of two other homologous proteins: CepR of Burkholderia cepacia, and YenR of Yersinia enterocolitica. CepR, requires its cognate pheromone for solubility, while YenR does not. In this respect, YenR is especially interesting, as we can purify this protein as an apo-protein or as an AHL complex and compare their properties. YenR binds to its binding site near the YenR promoter only in the apoprotein form, suggesting that the pheromone antagonizes protein function. We will do standard biochemical analysis of YenR will use several approaches to identify target genes of the YenR-Yenl regulon. In a final project, we will screen for AHL synthase genes from DNA that is purified from environmental samples and cloned into cosmids in E. coli.

描述（由申请人提供）：该实验室的远程目标是继续开发农业植物的病原系统，作为研究致病细菌与其宿主之间分子相互作用的模型。在成功的宿主植物致癌转化之后，曲霉曲霉的植物定居于转化的植物，并使用肿瘤释放的化合物（Pipines）作为营养。在这些条件下，细菌使用由TRAL组成的法定感应系统，该系统合成了细菌信息素N-3-oxOctanoylhomoserine Lactone（OOHL）和Trar，这是OOHL受体和OOHL依赖性转录器调节剂。我们已经纯化了蛋白质，并在体外重构了其活性。我们最近合作解决了与OOHL和DNA复合的Trar的晶体结构。我们将利用这种结构来进行TRAR的结构研究，通过对Trar表面，其OOHL结合决定簇及其DNA结合决定簇进行Trar表面的丙氨酸扫描诱变。我们将尝试分离阳性对照突变体，并为组成型活性的Trar突变体和能够检测异源诱导剂的突变体做出选择。我们已经表明，在没有OOHL的情况下合成的Trar迅速针对蛋白水解，可能是由CIP和LON蛋白酶靶向。我们将破坏农杆菌的离子和三个CLPP基因，并测量蛋白质的半寿命，以及它是否仍然需要OOHL才能稳定和活性。在C13和N15同位素的当下，我们将过量生产TRAR的N末端结构域，以进行TRAR折叠的协作研究。我们将以二聚体以检查DNA结合和转录激活的形式表达C末端结构域。我们已经将TRAR的生化特性与另外两种同源蛋白的生化特性进行了比较：伯克霍尔德氏菌的CEPR和Yersinia Enterocolitica的Yenr。 CEPR，需要其同源信息素的溶解度，而Yenr则不需要。在这方面，Yenr特别有趣，因为我们可以将这种蛋白质纯化为Apo蛋白质或AHL复合物并比较其特性。 YENR仅以载脂蛋白形式与Yenr启动子附近的结合位点结合，表明信息素拮抗蛋白质功能。我们将对YENR进行标准的生化分析，将使用几种方法来识别Yenr-Yenl调节的靶基因。在最后一个项目中，我们将从DNA中筛选AHL合酶基因，该基因从环境样品中纯化并克隆到大肠杆菌中的宇宙中。