VirB Mediated DNA and Protein Transfer from A.tumefaciens

农杆菌DNA进入植物细胞的运动取决于Ti质粒毒力（vir）基因的活性。一些Vir蛋白是产生单链dna结合蛋白VirE2所必需的。这种“t复合物”被认为是转移的中间体。VirB蛋白被认为形成一个膜定位的装置，负责t复合物的转移。与这一假设一致，VirB蛋白与膜相关，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，将含有突变型病毒2（负）菌株的T-DNA与携带野生型病毒基因（病毒2供体）但缺乏T-DNA的菌株混合，则含有突变型病毒2（负）菌株的T-DNA可以发挥作用，将其T-DNA传递给植物细胞。这种“细胞外互补”表明，VirE2可以独立于T-DNA转移进入植物细胞。由于细菌需要VirB基因作为VirE2供体，因此VirB复合体似乎也具有转移蛋白质的功能。在这个项目中，设计了三个目标来进一步表征VirB蛋白之间以及与转运底物的相互作用。首先，需要继续对virB操纵子进行遗传分析，以便了解各种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体。这些研究将集中在virB5、7、8、9和10上。数据表明，virB7-10以一种对转移复合体的构建至关重要的方式相互作用，这种相互作用的遗传基础将被定义；virB5之所以引起人们的兴趣，是因为有人提出它编码一种与转运底物相互作用的蛋白质，但尚未得到广泛的表征。其次，胞外互补现象表明，农杆菌可能能够独立于T-DNA将蛋白质转运到植物细胞中。尽管有这一有趣的观察结果，并且virB操纵子与编码某些细菌蛋白质分泌装置的基因相似，但实际上没有对这一过程进行遗传或细胞表征。我们将对单个植物细胞水平上的细胞外互补进行定量分析，并对提出的VirE2进入植物细胞的遗传和细胞基础进行表征。研究人员将开发监测植物细胞中VirE2存在的方法，并用于表征植物-细菌相互作用开始后VirE2的转移，并对运输所需的VirE2区域进行遗传分析。第三，分析incQ RSF1010衍生质粒pjW323对大分子转移的抑制作用。这些实验将增加我们对DNA从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。更好地了解这一过程的分子基础可能会导致基因转移技术的改进。＊＊＊

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. ***