Phenolic/Receptor Interactions in Agrobacterium

摘要本研究旨在研究农杆菌DNA向易感植物损伤细胞转移所必需的毒力（vir）基因表达过程中的信号识别步骤。vir基因系统编码在Ti质粒上，部分由传递/接收复合物（virA和vir G）的基因组成。vir A/vir G系统能够对多种化合物作出反应，并提供足够的相互作用能量来传递信息，这可能是通过催化酚类质子向受体的转移来实现的，从而激活信号转导级联。该模型做出了几个机制预测，这些预测已经引起了第一个已知的vir表达抑制剂的合成。之前的研究都是在体内进行的；在这项研究中，这些预测的体外测试是通过在重组系统中检测病毒的自磷酸化来完成的。所需的诱导剂类似物的合成方法和病毒的分子遗传操作已经到位。基于抑制剂的亲和标记试剂暗示了两种酚结合蛋白p10和P21可能是酚受体的候选者。这些蛋白以及在筛选中鉴定出的其他蛋白的必要化学反应性，将被纯化、测序，并将序列用于分离各自的基因，以测试它们在vir表达中的相关性。获得用于基础研究和加强我国粮食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转化的农杆菌感染植物而成为可能的，农杆菌是一种在自然界中引起冠瘿病的细菌。DNA转移的机制以及细菌如何“感知”合适的转移位点尚不清楚。该建议涉及后一点，并将分离和表征感知植物提供的适当“信号”线索的蛋白质。这些线索是植物酚类物质，在大多数情况下，它们起到抗生素的作用。a . tumifaciens对这些酚类物质很敏感，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了一种模式。这项工作表征了苯酚结合蛋白和参与将结合信息传递给细胞的蛋白。为了做到这一点，在体外重建部分信号通路将使用纯化的“稳定的”蛋白质参与这个信号级联。通过基因工程合成一种活性的，但被截断的蛋白质来稳定蛋白质。这些实验的结果将揭示这一生物技术上重要而又基本的信号事件。＊＊＊

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. ***