Mechanism of DNA Transfer from Agrobacterium to Plants

农杆菌 DNA 转移至植物的机制

• 批准号: 9982804
• 负责人: Anath Das
• 金额: $ 30万
• 依托单位: University of Minnesota-Twin Cities
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9982804&HistoricalAwards=false
• 关键词: Mechanism; DNA; Transfer; Agrobacterium; Plants

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.

本研究的目的是阐明 T-DNA 从细菌转移到植物的机制。根癌农杆菌感染植物并将其肿瘤诱导 (Ti-) 质粒 DNA 的一段 (T-DNA) 稳定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，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 之外，两种蛋白质 VirE2 和 VirF 也被转移到植物中。 为了易位，运输孔必须识别底物。 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。 VirD4 被假设介导识别过程。 这项研究将检验这一假设并确定 VirD4 在 DNA 转移中的作用。 免疫电子显微镜将用于研究 VirD4 和 DNA 转移装置在细菌细胞中是否彼此邻近。 VirD4 的功能域将通过诱变方法进行鉴定。 将在 virD4 中引入随机突变，并且将分离和表征无法补充或不能很好地补充 virD4 中的突变的突变。 将研究突变对 virD4 功能的影响。 VirD4 包含核苷酸结合基序序列。 ATP 利用在 VirD4 功能中的作用（如果有的话）将通过体内和体外方法进行研究。 假设传输孔对底物的识别涉及各种组分之间的相互作用。这些研究将识别这些相互作用并阐明底物识别的机制。这些研究将使用遗传和生化方法。 致病菌和非致病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。例如，大肠杆菌使用类似的机制通过接合进行质粒转移。 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。