Identification and Characterization of Agrobacterium and Plant Proteins Interacting with Proteins of the Agrobacterium T-complex

农杆菌与植物之间的相互作用是自然界中唯一有记录的基因工程案例。农杆菌与多种高等植物相互作用和遗传转化的分子机制的几个方面已经得到了很好的了解。农杆菌大肿瘤诱导质粒（Ti）的毒力（vir）调控被受伤植物合成的许多信号分子激活，导致核蛋白复合物（t复合物）从细菌到植物细胞的产生和转移。t复合物由T-DNA （Ti质粒的单链DNA片段）、VirD2（与T-DNA 5'端共价结合的先导蛋白）和VirE2（包裹T-DNA的单链DNA结合蛋白）组成。一旦转移到植物细胞中，t复合物就会进入植物细胞核，在那里T-DNA被整合和表达，导致植物生长调节剂的过量产生，最终形成肿瘤。然而，t复合物如何在细菌包膜中转移，t复合物如何在植物细胞内运输，以及T-DNA如何通过非同源重组整合到植物染色体中仍然知之甚少。该项目启动了一项研究计划，有两个目标：了解农杆菌中t复合物及其运输装置之间的相互作用，并确定参与T-DNA在植物细胞内转移和整合的植物因子。由于T-DNA转移过程的特异性是由t复合体的蛋白质组分决定的，因此将重点研究农杆菌以及与t复合体的两种蛋白质组分VirD2和VirE2相互作用的植物蛋白。研究蛋白质-蛋白质相互作用的一些分子工具将用于鉴定和研究农杆菌蛋白，这些农杆菌蛋白识别VirD2或VirE2并与之相互作用，从而促进t复合物从细菌转移到植物细胞。这些包括酵母双杂交系统、交联和共免疫沉淀。本研究还将确定与细菌转运装置相互作用的VirD2和VirE2的信号域。这些研究有望为农杆菌与其寄主之间的分子相互作用的未开发领域提供见解，例如细菌和寄主植物细胞中t复合物转移的顺序事件。这将使农杆菌-植物冠瘿系统不仅成为研究微生物-真核生物相互作用的模型系统，而且成为研究和探索细菌和高等植物中核蛋白转运和易位的模型系统。对参与T-DNA转移和整合过程的寄主因素的了解也可能导致对抗性植物物种进行基因工程的改进方法。

Nester 9723735 The interaction between Agrobacterium tumefaciens and plants is the only documented case of genetic engineering in nature. Several aspects of the molecular mechanism by which Agrobacterium tumefaciens interacts with and genetically transforms a wide variety of higher plants are well understood. The virulence (vir) regulon of a large tumor-inducing (Ti) plasmid of Agrobacterium is activated by a number of signal molecules synthesized by wounded plants, resulting in the production and transfer of a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of the T-DNA (a single-stranded DNA segment of the Ti plasmid), VirD2 (the pilot protein covalently bonded to the 5' end of the T-DNA), and VirE2 (the single-stranded DNA binding protein which envelops the T-DNA). Once transferred into the plant cell, the T-complex finds its way into the plant nucleus where the T-DNA is integrated and expressed, leading to the overproduction of plant growth regulators and ultimately tumor formation. However, how the T-complex is transferred across the bacterial envelope, how the T-complex is trafficked once within the plant cell, and how the T-DNA is integrated into plant chromosomes through non-homologous recombination remain poorly understood. This project initiates a research program that has two objectives: to understand the interaction between the T-complex and its transport apparatus within Agrobacterium, and to identify plant factors involved in the transfer and integration of the T-DNA inside the plant cell. Since the specificity of the T-DNA transfer process is determined by the protein components of the T-complex, efforts will focus on identifying the Agrobacterium as well as the plant proteins interacting with the two protein components of the T-complex, VirD2 and VirE2. Several molecular tools for studying protein-protein interactions will be used to identify and study Agrobacterium proteins which recognize and interact with either VirD2 or VirE2 or both and thereby facilitate the transfer of the T-complex from the bacterium to the plant cell. These include the yeast two-hybrid system, cross linking, and co-immunoprecipitation. This study will also identify the signal domains of VirD2 and VirE2 which interact with the bacterial transport apparatus. These studies are expected to provide insights into unexplored areas of the molecular interaction between Agrobacterium and its host, such as the sequential events of the T-complex transfer in both the bacterium and the host plant cell. This will make the Agrobacterium-plant crown gall system a model system not only for studying microorganism-eukaryote interactions but also for studying and probing nucleoprotein trafficking and translocation in bacteria as well as higher plants. The understanding of the host factors involved in the T-DNA transfer and integration process will also likely lead to improved methods for genetically engineering recalcitrant plant species.