Phenolic/Receptor Interactions in Agrobacterium

MCB-94-19252摘要 这是一个建议，以检查的毒力（vir）基因的表达所需的DNA从根癌农杆菌转移到敏感的，受伤的植物细胞的信号识别步骤。 vir基因系统在Ti质粒上编码，并且部分由用于发射器/接收器复合物的基因（virA和vir G）组成。 vir A/vir G系统对广泛的化合物作出反应并提供足够的相互作用能量来传递信息的能力，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来实现的。 这个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。 以前的研究已经在体内，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的自磷酸化完成的。 合成所需诱导物类似物和VirA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已经暗示了两种酚结合蛋白，p10和P21，作为酚受体的可能候选物。 这些以及在筛选必需的化学反应性中鉴定的其他蛋白质将被纯化、测序，并将序列用于分离相应的基因以测试它们在vir表达中的相关性。 获得转基因植物用于基础研究和生物技术提高我国粮食供应的能力在很大程度上是通过用转化的根癌农杆菌感染植物而实现的，根癌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。 这项建议涉及后一点，并将分离和表征的蛋白质，感知适当的“信号”线索提供的植物。 这些线索是植物酚，在大多数情况下，作为抗生素。 A. tumifaciens对这些酚类敏感，并通过一种有趣的信号传导机制对它们进行感知，这可能为植物识别激素或细菌识别小分子提供了一种模型。这项工作的特点是苯酚结合蛋白质和蛋白质参与中继绑定信息的细胞。 为此，将使用参与该信号级联的纯化的“稳定化”蛋白质来进行部分信号通路的体外重建。 通过基因工程合成活性但截短形式的蛋白质来稳定蛋白质。 实验的结果将揭示这一生物技术上重要的，但基本的信号事件。 ***

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. ***