VirB Mediated DNA and Protein Transfer from A.tumefaciens

小行星9513662 DNA从根癌农杆菌向植物细胞中的移动取决于Ti质粒的毒力（vir）基因的活性。 几种Vir蛋白是产生单链DNA结合蛋白VirE 2所必需的。 这种“T-复合物”被认为是转移的中间体。 VirB蛋白被提出形成负责T复合物转移的膜定位装置。 与这一假设相一致，VirB蛋白是膜相关的，并且是毒力所必需的。 令人惊讶的是，其他人已经表明，如果在植物感染之前，将含有突变体virE 2（负）菌株的T-DNA与携带野生型vir基因（VirE 2供体）但缺乏T-DNA的菌株混合，则含有突变体virE 2（负）菌株的T-DNA可以起到将其T-DNA递送到植物细胞的作用。 这种“胞外互补”表明VirE 2可以独立于T-DNA转移进入植物细胞。 由于VirB基因是细菌作为VirE 2供体所必需的，因此似乎VirB复合物也可以起到转移蛋白质的作用。 在这个项目中，三个目标的设计，以进一步表征VirB蛋白之间的相互作用，并与运输基板。 首先，需要对virB操纵子进行持续的遗传分析，以了解各种VirB蛋白如何相互作用以形成能够进行大分子转移的膜结合复合物。 这些研究将集中在virB 5、7、8、9和10上。 数据表明，virB 7 -10相互作用的方式，这是一个转移复合物的建设是至关重要的，这种相互作用的遗传基础将被定义; virB 5是感兴趣的，因为它已被提出编码一种蛋白质，与运输底物相互作用，但还没有得到广泛的特点。 第二，胞外互补现象表明农杆菌可能能够不依赖于T-DNA而将蛋白质转运到植物细胞中。 尽管这一有趣的观察结果，以及virB操纵子与编码某些细菌的蛋白质分泌装置的基因的相似性，但实际上没有该过程的遗传或细胞表征。 将在单个植物细胞水平上进行细胞外互补的定量分析，并将表征拟议的VirE 2移动到植物细胞中的遗传和细胞基础。 将开发监测植物细胞中VirE 2存在的方法，并用于表征植物-细菌相互作用开始后的VirE 2转移，并对VirE 2运输所需区域进行遗传分析。 第三，分析incQ RSF 1010衍生质粒pjW 323对大分子转移的抑制。 这些实验将增加我们对DNA从根癌农杆菌特异性转移到植物细胞的机制的理解。 农杆菌介导的DNA转移是农业生物技术中一种重要的基因转移方法。 更好地理解这一过程的分子基础可能会导致改进的基因转移技术。 ***

9513662 Binns The movement of DNA from Agrobacterium tumefaciens into plant cells depends upon the activities of the virulence (vir) genes of the Ti plasmid. Several of the Vir proteins are required for the production of a single-stranded DNA-binding protein, VirE2. This "T-complex" is thought to be the transferred intermediate. VirB proteins are proposed to form a membrane-localized apparatus responsible for T-complex transfer. Consistent with this hypothesis, VirB proteins are membrane associated and required for virulence. Others have shown, surprisingly, that a T-DNA containing a mutant virE2 (minus) strain could function to deliver its T-DNA to plant cells if, just prior to plant infection, it was mixed with a strain carrying wild type vir genes (VirE2 donor) but lacking the T-DNA. This "extracellular complementation" suggests that VirE2 can move into plant cells independent of T-DNA transfer. Because VirB genes are required for a bacterium to serve as a VirE2 donor, it appears that the VirB complex may also function to transfer proteins. In this project, three aims are designed to further characterize the interaction of VirB proteins amongst themselves and with transported substrates. First, continued genetic analysis of the virB operon is required in order to understand how the various VirB proteins interact to form a membrane bound complex capable of macromolecular transfer. These studies will focus on virB5, 7, 8, 9, and 10. Data suggest that virB7-10 interact in a fashion that is critical for the construction of a transfer complex, and the genetic basis of this interaction will be defined; virB5 is of interest because it has been proposed to encode a protein that interacts with the transported substrate, but has not been extensively characterized. Second, the phenomenon of extracellular complementation indicates that Agrobacterium may be capable of transporting proteins into plant cells independent of the T-DNA. Despite this intriguing observation, and the similarity o f the virB operon to genes encoding the protein secretion apparati of certain bacteria, there has been virtually no genetic or cellular characterization of this process. A quantitative analysis of extracellular complementation at the single plant cell level will be carried out, and the genetic and cellular basis of the proposed VirE2 movement into plant cells will be characterized. Methods to monitor the presence of VirE2 in plant cells will be developed and used to characterize VirE2 transfer after initiation of plant-bacterial interaction, and a genetic analysis of the regions of VirE2 necessary for transport will be carried out. Third, the inhibition of macromolecular transfer by the incQ RSF1010 derivative plasmid pjW323 will be analyzed. These experiments will increase our understanding of the mechanism whereby DNA is specifically transferred from Agrobacterium tumefaciens to plant cells. Agrobacterium-mediated DNA transfer is a significant gene transfer method in agricultural biotechnology. A better understanding of the molecular basis of this process may lead to improved gene transfer technologies. ***