Interactions Between Agrobacterium and Host Plants

描述(申请人提供)：本实验室的长期目标是继续开发农杆菌-植物病理系统作为研究病原菌与其宿主之间的分子相互作用的模型。在寄主植物成功致癌转化后，根癌农杆菌在转化植物上定植，并利用肿瘤释放的化合物(OPINS)作为营养物质。在这些条件下，细菌使用由Tral组成的群体感应系统，该系统合成细菌信息素N-3-氧辛酰高丝氨酸内酯(Oohl)和TraR，TraR是Oohl受体和Oohl依赖的转录调节因子。我们已经纯化了这两种蛋白，并在体外重组了它们的活性。我们最近合作解决了TRAR的晶体结构，它与Oohl和DNA络合。我们将利用这种结构来研究TRAR的结构，方法是对TRAR表面、其OOHL结合决定簇和DNA结合决定簇进行丙氨酸扫描突变。我们将尝试分离阳性对照突变体，并对成分活性TRAR突变体和能够检测到异源自体诱导子的突变体进行选择。我们已经证明，在没有OOHL的情况下合成的TRIR迅速被蛋白质降解所靶向，可能是通过CIP和Lon蛋白酶。我们将破坏农杆菌的离子和三个ClpP基因，并测量蛋白质的半衰期，以及它是否仍然需要Oohl来保持稳定性和活性。我们将在C13和N15同位素的存在下过度生产TRAR的N-末端结构域，以用于TRAR折叠的合作研究。我们将在体内以二聚体的形式表达C末端结构域，以检查DNA结合和转录激活。我们已经比较了TRAR与另外两个同源蛋白的生化性质：洋葱伯克霍尔德氏菌的CEPR和小肠结肠炎耶尔森氏菌的YenR。CEPR，需要它的同源信息素来溶解，而YenR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脱辅基蛋白或AHL复合体，并比较它们的性质。YenR仅以载脂蛋白的形式与其靠近YenR启动子的结合部位结合，表明信息素拮抗蛋白质的功能。我们将对YenR进行标准的生化分析，将使用几种方法来鉴定YenR-Yen1调节子的靶基因。在最后一个项目中，我们将从从环境样本中纯化的DNA中筛选AHL合成酶基因，并将其克隆到大肠杆菌的粘粒中。

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.