VirB Mediated DNA and Protein Transfer from A.tumefaciens

9513662 bnDNA从根癌农杆菌到植物细胞的运动取决于钛质粒的毒力基因的活性。一些Vir蛋白是生产单链DNA结合蛋白VirE2所必需的。这种“T-复合体”被认为是转移的中间体。VIRB蛋白被认为是一个膜定位的装置，负责T-复合体的转移。与这一假设一致，VIRB蛋白是膜相关的，是毒力所必需的。令人惊讶的是，其他研究表明，如果在植物感染之前，含有突变的病毒E2(负)菌株的T-DNA与携带野生型病毒基因的菌株(病毒E2供体)混合，但缺乏T-DNA，它就可以将其T-DNA输送到植物细胞。这种“细胞外互补”表明，病毒E2可以独立于T-DNA转移进入植物细胞。因为VIRB基因是细菌作为病毒E2供体所必需的，所以VIRB复合体似乎也可能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蛋白之间的相互作用以及与运输底物的相互作用。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，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。数据表明，virB7-10以一种对构建转移复合体至关重要的方式相互作用，这种相互作用的遗传基础将被确定；virB5之所以令人感兴趣，是因为它被提议编码一种与运输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将蛋白质运送到植物细胞中。尽管有这一有趣的观察，而且VIRB操纵子与编码某些细菌的蛋白质分泌装置的基因相似，但几乎没有关于这一过程的遗传或细胞特征。将在单个植物细胞水平上进行细胞外互补的定量分析，并将描述拟议的VirE2进入植物细胞的遗传和细胞基础。将开发监测病毒E2在植物细胞中存在的方法，并用于在植物-细菌相互作用开始后表征病毒E2的转移，并将对病毒E2运输所必需的区域进行遗传分析。第三，分析了INQ 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从根癌农杆菌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带来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. ***