Analysis of VirB Proteins Required for Agrobacterium Mediated DNA Transfer

根癌农杆菌介导DNA从细菌的Ti质粒进入植物细胞，在那里转移的DNA(T-DNA)最终整合到宿主基因组中并表达。细菌内导致“转移中间产物”形成的过程已经被很好地研究，并导致了DNA转移过程类似于细菌结合的假设。转移中间体(由蛋白质和DNA组成)如何离开细菌并进入植物细胞的机制几乎是未知的。钛质粒的VIRB基因编码的蛋白质被认为是形成一个膜定位的“转移装置”，转移的中间产物通过这个装置从细菌进入植物细胞。在这个更新项目中，将进行研究，以继续对假定的VIRB编码转移装置进行遗传、分子和生化分析。将实现以下具体目标。首先，将继续对VIRB操纵子的遗传分析，利用最近构建的转座子，这将使构建非极性突变成为可能。应该有可能确定哪些VIRB基因是毒力所必需的，并对单个VIRB基因进行广泛的点突变。这些突变体将与生化和分子策略一起用于研究VIRB相关活性。其次，将表征假定的VIRB编码设备和传送的衬底之间的关系。分子遗传学方法将被用来检验这样的假设，即pJW323产生的中间体如此丰富，以至于它在进入运输孔方面胜过T-复合体。VIRB编码的转移装置对于各种转移分子的特异性将被表征。此外，还将确定其他VIR基因的过度表达是否会影响PJW323向植物的转移或其抑制T-DNA转移的能力。%这项工作集中在有关DNA从农杆菌转移到植物细胞的基本问题上。从历史上看，这一领域始于对农杆菌如何导致烟草植株冠瘤的研究。今天，利用农杆菌作为载体将DNA转移到植物细胞中，并最终从这些细胞中再生出转基因植物，是基础植物科学和应用植物科学的核心技术。这项技术的优点是，农杆菌介导的转化在技术上易于执行，并且在许多植物中以非常高的效率发生。这项技术的一个主要问题是，对于许多农业上重要的植物来说，它要么效率很低，要么根本没有表现出转化的迹象。了解某些宿主的有效转化所涉及的细胞生物学可能有助于深入了解如何修改程序，以便转化目前在农杆菌有用宿主范围之外的宿主。这将对农业研究和新产品开发具有重要意义。

Agrobacterium tumefaciens mediates the movement of DNA from the Ti plasmid of the bacterium into plant cells, where the "transferred DNA" (T-DNA) is ultimately integrated into the host genome and expressed. Processes within the bacterium that result in the formation of "transfer intermediates" have been well studied and have led to the hypothesis that the DNA transfer process is similar to bacterial conjugation. The mechanism whereby the transfer intermediates (composed of protein and DNA) exit the bacterium and enter the plant cells is virtually uncharacterized. The virB genes of the Ti plasmid encode proteins that are hypothesized to form a membrane localized "transfer apparatus" through which the transferred intermediates move from the bacterium into the plant cell. In this renewal project, studies will be undertaken to continue the genetic, molecular, and biochemical analysis of the putative virB-encoded transfer apparatus. The following specific aims will be pursued. First, the genetic analysis of the virB operon will be continued, taking advantage of a recently constructed transposon that will make possible the construction of non-polar mutations. It should be possible to determine which of the virB genes are required for virulence, and to carry out extensive point mutagenesis on individual virB genes. These mutants will be used in conjunction with biochemical and molecular strategies to study VirB related activities. Second, the relationship between the putative virB encoded apparatus and the transported substrates will be characterized. Molecular genetic approaches will be used to test the hypothesis that pJW323 produces an intermediate in such abundance that it outcompetes the T-complex for access to the transport pore. The specificity of the virB encoded transfer apparatus for the various transferred molecules will be characterized. Further, it will be determined whether overexpression of other vir genes affects either the transfer of PJW323 into plants or its ability to inhibit T-DNA transfer. %%% This work focusses on basic issues concerning DNA transfer from Agrobacterium to plant cells. Historically, the field originated with studies of how Agrobacterium caused Crown Gall Tumors in tobacco plants. Today, the utilization of Agrobacterium as a vector to transfer DNA into plant cells, and, ultimately, the regeneration of transgenic plants from such cells, is a central technology in both basic and applied plant science. The advantages of the technique are that Agrobacterium-mediated transformation is technically simple to execute and, in many plants, occurs at remarkably high efficiency. A major problem with the technique is that, for many agriculturally important plants, it is either highly inefficient or hasn't been shown to transform at all. Understanding the cell biology involved in the efficient transformation of certain hosts may provide insight into means by which procedures can be modified so as to transform hosts currently outside the useful host range of Agrobacterium. This would be of major significance to agricultural research and new product development.