Phenolic/Receptor Interactions in Agrobacterium

Mcb-94-19252摘要这是一项研究致病基因(VIR)表达中信号识别步骤的建议，这些基因是将DNA从根癌农杆菌转移到敏感的、受伤的植物细胞中所必需的。VIR基因系统编码在Ti质粒上，部分由转运体/受体复合体(VirA和VIRG)的基因组成。VIR A/VIR G系统对广泛的化合物作出反应的能力，但仍提供足够的相互作用能量来传递信息，可能是通过催化酚质子转移到受体，从而激活信号转导级联反应实现的。这个模型做出了几个机械性的预测，这些预测已经导致了第一批已知的病毒表达抑制物的合成。以前的研究是在体内进行的；在这项研究中，这些预测的体外测试是通过检查重组系统中VirA的自动磷酸化来完成的。合成所需的诱导剂类似物和对病毒进行分子遗传操作的方法已经到位。基于抑制剂的亲和标记试剂表明，两个苯酚结合蛋白p10和p21可能是苯酚受体的候选者。这些蛋白质，以及在必要的化学反应筛选中确定的其他蛋白质，将被提纯、测序，并用于分离各自的基因，以测试它们在病毒表达中的相关性。获得用于基础研究和提高我国食品供应的生物技术的转基因植物的能力在很大程度上是通过植物感染转化的根癌农杆菌而实现的，根癌农杆菌是一种在自然界中导致冠胆疾病的细菌。目前尚不清楚DNA转移的机制以及细菌如何“感觉”到合适的转移部位。这项建议涉及到后一点，并将分离和鉴定能够感知植物提供的适当“信号”信号的蛋白质。这些线索是植物酚类物质，在大多数情况下，它们充当抗生素。根癌农杆菌对这些酚很敏感，并通过一种有趣的信号机制来感知它们，这可能为植物识别激素或细菌识别小分子提供一个模型。这项工作描述了苯酚结合蛋白以及将结合信息传递给细胞所涉及的蛋白质。要做到这一点，部分信号通路的体外重建将使用参与这一信号级联的纯化的“稳定的”蛋白质来完成。通过基因工程合成具有活性但被截断的蛋白质，从而使蛋白质稳定下来。这些实验的结果将阐明这一在生物技术上重要但又根本性的信号事件。***

MCB-94-19252 Abstract This is a proposal to examine the signal recognition steps in the expression of the virulence (vir) genes necessary for the transfer of DNA from Agrobacterium tumefaciens into susceptible, wounded plant cells. The vir gene system is encoded on the Ti plasmid and is in part comprised of the genes for the transmitter/receiver complex (virA and vir G). The ability of the vir A/vir G system to respond to a broad range of compounds, and yet provide sufficient interaction enrgy to transmit information, might be achieved by the catalysis of the transfer of a phenolic proton to the receptor, thereby activating the signal transduction cascade. This model makes several mechanistic predictions which have already given rise to the synthesis of the first known inhibitors of vir expression. The previous studies have been in vivo; in this study in vitro testing of these predictions is accomplished by examination of the autophosphorylation of VirA in a reconstituted system. Methods for the synthesis of the required inducer analogs and molecular genetic manipulation of VirA are in place. Affintiy labeling reagents based on the inhibitors have implicated two phenol-binding proteins, p10 and P21, as likely candidates for the phenol receptors. These, as well as the other proteins identified in screens for the requisite chemical reactivity, will be purified, sequenced and the sequence used to isolate the respective genes to test their relevance in vir expression. %%% The ability to obtain transgenic plants for basic research and for biotechnological enhancement of our nation's food supply has in large part been made possible through plant infection with transformed Agrobacterium tumifaciens, a bacterium which causes Crown Gall disease in nature. The mechanism of DNA transfer and how the bacterium can "sense" an appropriate site for transfer is unknown. This proposal deals with the latter point and will isolate and characterize the proteins which perceive the appropriate "signalling" cues provided by the plant. These cues are plant phenolics which, in most instances, serve as antibiotics. A. tumifaciens is sensitive to these phenols and "hones in" on them, perceiving them through an interesting signalling mechanism which may provide a model for plant recognition of hormones or bacterial recognition of small molecules. The work characterizes the phenol binding proteins and the proteins involved in relaying binding information to the cell. To do that, in vitro reconstitution of part of the signalling pathway will be done using purified "stabilized" proteins involved in this signaling cascade. The proteins are stabilized by synthesizing an active, but truncated version of the protein through genetic engineering. The outcome of the experiments will be to shed light on this biotechnologically important, yet fundamental signalling event. ***