FUNCTION & REGULATION OF A. TUMEFACIENS VIRULENCE GENES

这项研究的目的是了解DNA的机制， 从农杆菌转移到植物细胞。 毒力的作用 (vir)基因在DNA转移过程中的作用及转录机制 将研究vir基因的调节。 两种调节蛋白，VirA和VirG，与植物结合 酚乙酰丁香酮控制vir基因表达。 信号分子 （植物酚类物质）由易受影响的寄主植物产生的 调节蛋白质的磷酸化， 激活细菌的致病机制。 突变 将进行研究，以确定信号处理和 VirA和VirG的转录激活子结构域。 这些影响 将研究蛋白质磷酸化的突变。 体外 转录系统将被开发来研究蛋白质的作用。 磷酸化在vir基因表达调控中的作用，并阐明 转录激活机制。 DNA从细菌转移到植物细胞的中间体是一个单一的- 与蛋白质复合的单链DNA（T复合物）。 形成 ssDNA由VirD 2内切核酸酶催化，并且需要存在 VirD 1。 将进行体内研究以确定 VirD 1-VirD 2核酸内切酶作用。 缺失突变研究将 进行这些步骤以限定VirD 2内切核酸酶的功能结构域。 两 VirD 1和VirD 2可能形成共价蛋白质-DNA复合物。 氨基 参与这些络合物形成的酸将是 鉴定 其他VirD蛋白在DNA转移中的作用， 将进行整合研究。 将进行生物化学研究以确定 复合物，并研究其与细菌膜的相互作用。 突变 in vir基因将被用来鉴定参与 这些反应。 将开发一种体外系统来鉴定Vir和 DNA转移到植物细胞所需的其他蛋白质。 使用的检测方法 可以区分DNA转移和DNA整合， 将研究这些反应中的不同Vir蛋白。 本研究的长期目标是了解 农杆菌致病性 vir基因在其中起着核心作用 过程 阐明这些基因的表达机制， 了解编码基因产物的功能对于 更好地了解细菌致病的机制， 行动上

The goal of the proposed research is to understand the mechanism of DNA transfer from Agrobacterium to plant cells. The role of the virulence (vir) genes in the DNA transfer process and the mechanism of transcription regulation of the vir genes will be investigated. Two regulatory proteins, VirA and VirG, in conjunction with a plant phenolic acetosyringone control vir gene expression. The signal molecules (plant phenolics) generated by a susceptible host plant induce modification of the regulatory proteins presumably via phosphorylation leading to the activation of the pathogenic machinery of the bacterium. Mutational studies will be performed to identify the signal processing and transcriptional activator domains of VirA and VirG. The effect of these mutations on protein phosphorylation will be investigated. An in vitro transcription system will be developed to study the role of protein phosphorylation in regulation of vir gene expression and to elucidate the mechanism of transcription activation. An intermediate in DNA transfer from bacteria to plant cells is a single- stranded (ss) DNA complexed with proteins (T-complex). Formation of the ssDNA is catalyzed by the VirD2 endonuclease and requires the presence of VirD1. In vivo studies will be performed to determine the mechanism of VirD1-VirD2 endonuclease action. Deletion mutagenesis studies will be performed to define the function domains of the VirD2 endonuclease. Both VirD1 and VirD2 presumably form covalent protein-DNA complexes. The amino acids that participate in the formation of these complexes will be identified. The role of the other VirD proteins in DNA transfer and integration will be studied. Biochemical studies will be performed to determine the composition of the T-complex and to stud its interaction with bacterial membranes. Mutations in vir genes will be used to identify the gene products that participate in these reactions. An in vitro system will be developed to identify Vir and other proteins required for DNA transfer to plant cells. Using assays that can distinguish between DNA transfer and DNA integration, the role of different Vir proteins in these reactions will be investigated. The long term goal of this study is to understand the mechanism of agrobacterial pathogenicity. The vir genes play a central role in this process. Elucidation of the mechanism of expression of these genes and understanding the functions of the encoded gene products are vital towards a better understanding of mechanisms bacteria employ for their pathogenic action.