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Understanding the mechanism of Agrobacterium tumefaciens transformation.

机译：了解根癌农杆菌转化的机制。

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摘要

The soil bacterium, Agrobacterium tumefaciens, causes crown gall disease on dicotyledonous plants and is the primary vector in genetic engineering of plants for research and commercial agriculture. Agrobacterium exports DNA and at least four protein substrates, VirD2, VirE2, VirE3, and VirF, into the plant cell via a transporter complex called the type IV secretion system. This system, which includes a pilus, is comprised of twelve proteins, named VirB1 to VirB11, and VirD4, and is similar to other secretion systems in human pathogenic bacteria and those used for DNA exchange during bacterial mating, or conjugation. While a general structural model of the transporter complex exists, detailed protein subassemblies and the process of protein and DNA export remains unknown. To better understand Agrobacterium transformation, a genetic screen using the yeast Saccharomyces cerevisiae as the host was developed to identify host proteins involved in transformation. While developing this screen, factors that alter Agrobacterium transformation of yeast were identified. Among these, nitrocellulose filters, recovery after heat-shock transformation, thick plates, and use of galactose as a carbon source, all increase yeast transformation efficiency. To study the first stage of substrate export during Agrobacterium transformation, protein interactions between the exported proteins and the type IV secretion system were identified using the yeast two-hybrid assay. Fluorescent protein fusions to exported proteins indicated that while VirD4 targets VirE2 to the type IV secretion system, VirF is targeted by either VirB4 or an assembled secretion system. These results suggest there are two distinct pathways for protein export in the Agrobacterium type IV secretion system. Additional studies also indicate the secreted proteins are targeted to the type IV secretion system via a conserved C-terminal transport sequence. Lastly, to identify protein subassemblies in the transporter complex, the yeast two-hybrid system was used to assay for interactions between proteins of the secretion system. The results support a new role of VirB1 in pilus assembly. Together, these studies provide insights into transporter complex protein subassemblies, the process of protein export during Agrobacterium transformation, and further contribute to understanding homologous systems in human pathogens and bacterial conjugation.
机译：土壤细菌根癌农杆菌会在双子叶植物上引起冠and病，并且是用于研究和商业农业的植物基因工程中的主要载体。农杆菌通过称为IV型分泌系统的转运复合物将DNA和至少四种蛋白质底物VirD2，VirE2，VirE3和VirF输出到植物细胞中。该系统包括菌毛，由十二种蛋白质组成，分别称为VirB1至VirB11和VirD4，与人类病原细菌中的其他分泌系统以及在细菌交配或结合过程中用于DNA交换的那些分泌系统相似。虽然存在转运蛋白复合物的一般结构模型，但是详细的蛋白质子装配以及蛋白质和DNA输出的过程仍然未知。为了更好地理解农杆菌属转化，开发了使用酵母酿酒酵母作为宿主的遗传筛选，以鉴定参与转化的宿主蛋白。在开发此筛选时，已发现了改变酵母农杆菌转化的因素。其中，硝酸纤维素滤膜，热休克转化后的回收，厚板以及半乳糖作为碳源的使用，均可提高酵母转化效率。为了研究农杆菌转化过程中底物输出的第一阶段，使用酵母双杂交测定法鉴定了输出蛋白质与IV型分泌系统之间的蛋白质相互作用。荧光蛋白与输出蛋白的融合表明，虽然VirD4将VirE2靶向IV型分泌系统，但VirF却被VirB4或组装的分泌系统靶向。这些结果表明在农杆菌IV型分泌系统中有两种不同的蛋白质输出途径。进一步的研究还表明，分泌的蛋白质通过保守的C末端转运序列靶向IV型分泌系统。最后，为了鉴定转运蛋白复合物中的蛋白质组件，使用了酵母双杂交系统来分析分泌系统蛋白质之间的相互作用。结果支持VirB1在菌毛组装中的新作用。总之，这些研究提供了关于转运蛋白复杂子装配体，农杆菌转化过程中蛋白输出过程的见解，并进一步有助于理解人类病原体中的同源系统和细菌结合。

著录项

  • 作者

    Hackworth, Cheryl Anne.;

  • 作者单位

    University of California, Berkeley.;

  • 授予单位 University of California, Berkeley.;
  • 学科 Agriculture Plant Pathology.
  • 学位 Ph.D.
  • 年度 2006
  • 页码 182 p.
  • 总页数 182
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 植物病理学;
  • 关键词

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