Mechanism of DNA Transfer from Agrobacterium to Plants

本研究的目的是阐明T-DNA从细菌到植物的转移机制。根癌农杆菌感染植物并将其肿瘤诱导（Ti-）质粒DNA的区段（T-DNA）稳定地转移到植物核基因组中。 DNA转移需要Ti质粒的毒力（vir）基因。 vir基因产物催化Ti质粒的加工以产生转移中间体，即单链T链DNA。 T-链DNA可能穿过主要由VirB蛋白组成的膜孔。 除了T-DNA，两种蛋白质VirE 2和VirF也被转移到植物中。 为了转运，转运孔必须识别底物。 这种识别可能涉及底物的直接相互作用，也可能由另一种蛋白质介导。 VirD 4被假设为介导识别过程。 这项研究将验证这一假设，并将确定VirD 4在DNA转移中的作用。 免疫电子显微镜将用于研究VirD 4和DNA转移装置是否在细菌细胞中彼此邻近。 VirD 4的功能结构域将通过诱变方法鉴定。 将在virD 4中引入随机突变，并且将分离和表征不能互补或很差地互补virD 4中突变的突变。 将研究突变对virD 4功能的影响。 VirD 4含有核苷酸结合基序序列。 将通过体内和体外方法研究ATP利用在VirD 4功能中的作用（如果有的话）。 通过运输孔识别底物被假设为涉及各种组分之间的相互作用。这些研究将确定这些相互作用，并阐明底物识别的机制。这些研究将采用遗传学和生物化学方法。 致病性和非致病性细菌都使用类似的大分子输出机制。 T-DNA转运系统是IV型转运系统的第一个成员。E.例如，用于通过接合进行质粒转移的大肠杆菌。 了解农杆菌底物识别机制将对理解许多生物过程产生重大影响。

The goal of this research is to elucidate the mechanism of T-DNA transfer from bacteria to plants. Agrobacterium tumefaciens infects plants and stably transfers a segment (T-DNA) of its tumor inducing (Ti-) plasmid DNA into the plant nuclear genome. DNA transfer requires the virulence (vir) genes of the Ti-plasmid. The vir gene products catalyze processing of the Ti-plasmid to generate a transfer intermediate, a single stranded T-strand DNA. The T-strand DNA presumably travels through a membrane pore composedprimarily of the VirB proteins. In addition to the T-DNA two proteins, VirE2 and VirF, are transferred to plants. For translocation the transport pore must recognize the substrates. The recognition may involvedirect interaction of the substrate or it may be mediated by another protein. VirD4 is hypothesized to mediate the recognition process. This research will test this hypothesis and will determine the role of VirD4 in DNA transfer. Immunoelectron microscopy will be used to study whether VirD4 and the DNA transfer apparatus are proximal to each other in the bacterial cell. Functional domains of VirD4 will be identified by mutagenesis methods. Random mutations will be introduced in virD4 and mutations that fail to complement or poorly complement a mutation in virD4 will be isolated and characterized. The effect of a mutation on virD4 function will be investigated. VirD4 contains a nucleotide binding motif sequence. The role of ATP utilization in VirD4 function, if any, will be investigated by in vivo and in vitro methods. Recognition of a substrate by the transport pore is hypothesized to involve interactions among the various components. These studies will identify these interactions and elucidate the mechanism of substrate recognition. Genetic and biochemical methods will be used for these studies. Both pathogenic and non-pathogenic bacteria use a similar mechanism for macromolecule export. The T-DNA transport system is the first member of the type IV transport system. A similar mechanism is used by E. coli for plasmid transfer by conjugation, for example. An understanding of the Agrobacterium substrate recognition mechanism will have a major impact on understanding many biological processes.