VirB Mediated DNA and Protein Transfer from A. tumefaciens

农杆菌是感染受损植物的细菌。细菌DNA被转移到植物的细胞核，在基因工程的过程中，这个过程被用来将所需的基因转移到植物中。重要的是要理解这个过程是如何发生的，因为它代表了跨王国DNA转移的自然发生，以及实际重要性。DNA从根癌农杆菌转移到植物细胞中依赖于Ti质粒毒力基因的活性。一些Vir蛋白是生产单链DNA所必需的，该单链DNA在VirD2的5‘末端共价连接。长期目标是了解VIRB综合体是如何组装和运作的。为此，已经发展了各种遗传和生化方法来研究VIRB蛋白与自身以及与运输底物的相互作用。最近的研究得出了一个令人惊讶的结果，即VIRB蛋白在受体中的表达极大地提高了VIRB介导的RSF1010在农杆菌之间的结合转移的效率。这一结果导致了关于VIRB蛋白和受体活性的几个问题：a)增加受体活性所需的VIRB蛋白的最低补充是什么，b)受体中RSF1010的存在是否影响这种活性，以及c)这种现象的一般性是什么？为了确定VIRB复合体及其活性，必须了解VIRB蛋白与其自身或运输底物之间的相互作用。在这个项目中，将继续利用化学交联和免疫沉淀进行物理/生化分析；将进行酵母双杂交系统分析，特别是研究其他VIRB蛋白质--以及可能其他未知蛋白质--与VirB7-10组相互作用的可能性；并将对VirB7-10基因进行饱和突变，以帮助确定蛋白质相互作用所需的区域，以及总体上毒力所需的区域。

Agrobacteria are bacteria that infect damaged plants. Bacterial DNA is transferred to the plant nucleus, and this process is used to move desired genes into plants in the process of genetic engineering. It is important to understand how this process occurs, as it represents a natural occurence of inter-kingdom DNA transfer, as well as of practical importance. 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 covalently attached to VirD2 at its 5' terminus. The long term objectives are to understand how the VirB complex assembles and functions. Towards this end, a variety of genetic and biochemical methods have been developed to study the interaction of VirB proteins with themselves and with transported substrates. Recent studies yielded the surprising result that the expression of a subset of the VirB proteins in the recipient vastly increased the efficiency of virB-mediated conjugal transfer of RSF1010 between agrobacteria. This result leads to several questions about the VirB proteins and recipient activity: a) what is the minimal complement of VirB proteins necessary for increased recipient activity, b) does the presence of RSF1010 in the recipient affect this activity and c) what is the generality of this phenomenon? The answer to these questions may allow the identification of a "core" VirB complex that could simplify genetic and biochemical studies, and may have important implications to plasmid transfer in general.In order to define the VirB complex and its activity, the interactions between the VirB proteins and themselves or the transported substrates must be understood.. In this project physical/biochemical analysis, utilizing chemical cross-linking and immunoprecipitation will be continued; yeast two hybrid system analysis will be carried out, looking specifically at the possibility that other VirB proteins - and possibly other unidentified proteins - interact with the VirB7-10 group; and saturation mutagenesis on the virB7-10 genes will be carried out to help define regions required for protein interaction and for virulence in general.