Formation and characterization of the Agrobacterium T-complex in plant cells

植物细胞中农杆菌 T 复合物的形成和表征

• 批准号: 0919931
• 负责人: Stanton Gelvin
• 金额: $ 40万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0919931&HistoricalAwards=false
• 关键词: Formation; characterization; Agrobacterium; complex; plant

Intellectual merit: This project will investigate the formation, composition, and sub-cellular site(s) of localization of the Agrobacterium T-complex as it is assembled in and transported through the plant cell. Agrobacterium transfers single-strand DNA (T-strands), covalently linked to VirD2 protein, through a Type IV Secretion System (T4SS) into plant cells. Several other effector virulence proteins, including VirE2, are separately transferred to the plant via the T4SS. VirE2 is a single-strand DNA binding protein that, in vitro, can complex with and coat T-strands. A current favored hypothesis is that in plant cells, VirE2 coats also helps target T-strands to the nucleus, where T-strands become double-stranded and integrate into the plant genome. Targeting T-strands through the cytoplasm and into the nucleus is likely directed by plant proteins such as importin-alpha and VIP1. In vitro and in plant cells, importin-alpha interacts with VirD2, VirE2, and VIP1, and these proteins have been hypothesized to form a mature or super-T-complex with T-strands in planta. However, these studies have all investigated protein-protein interactions in plant cells in the absence of T-strands. In addition, expression of VirE2 has been from a strong plant promoter, resulting in high levels of protein that forms aggregates. Bimolecular Fluorescence Complementation (BiFC) technology can be used to track VirE2 as it exits Agrobacterium and interacts with proteins within the plant cell. BiFC fluorescence technology, along with a modified T-DNA immunoprecipitation assay, will be used to monitor the assembly and intra-cellular trafficking of T-complexes in living plant cells. Under these conditions, the various putative T-complex components are synthesized at natural levels, and in their native organisms, prior to assembly in the plant cell. The results of these studies are important to resolve much conflicting data in the literature regarding the roles of various putative T-complex components in T-DNA trafficking through the plant cell. Broader impacts of the proposed research: Horizontal gene transfer has been recognized as a major component of evolution, and Agrobacterium represents one of the best studied examples of horizontal gene flow. Agrobacterium-mediated genetic transformation is also the major mechanism to generate transgenic plants for basic research and for agricultural biotechnology purposes. Understanding how T-DNA traffics through the plant cell is important for understanding how extra-cellular protein-nucleic acid complexes (including viral genomes) target the nucleus after entering a cell. Because many of the steps in nuclear targeting may be rate-limiting, understanding the process will be important for preventing disease (such as Crown Gall caused by Agrobacterium, or viral diseases), and for improving the transformation of recalcitrant crop species. In addition to training research scientists and graduate students, this project will be used to conduct a vigorous outreach program with Brooklyn College to identify undergraduate students from under-represented minority groups and introduce them, through summer and academic year collaborations, to the conduct of scientific research. Multi-year training of these students will be encouraged to solidify their interest in pursuing a career in science. When they return to their home institution, they will expose additional students to the techniques learned in our laboratory. The under-represented minority students trained during the summer will continue the projects at their home institutions, thus broadening the number of their peers who will come into contact with scientific research. These efforts will encourage undergraduate students to select a career in science.

智力优势：该项目将研究农杆菌t复合物在植物细胞内组装和运输时的形成、组成和定位的亚细胞位点。农杆菌通过IV型分泌系统（T4SS）将与VirD2蛋白共价连接的单链DNA （t链）转移到植物细胞中。其他几种效应毒力蛋白，包括VirE2，分别通过T4SS转移到植物中。VirE2是一种单链DNA结合蛋白，在体外可以与t链复合并包裹在t链上。目前比较受欢迎的一种假设是，在植物细胞中，VirE2外壳也有助于将t链靶向到细胞核，在那里t链变成双链并整合到植物基因组中。通过细胞质靶向t链进入细胞核可能是由植物蛋白如importin- α和VIP1指导的。在体外和植物细胞中，importin- α与VirD2、VirE2和VIP1相互作用，这些蛋白被假设在植物中与t链形成成熟的或超t复合物。然而，这些研究都是在没有t链的情况下研究植物细胞中的蛋白质-蛋白质相互作用。此外，VirE2的表达来自一个强大的植物启动子，导致高水平的蛋白质形成聚集体。双分子荧光互补（BiFC）技术可用于跟踪VirE2从农杆菌中退出并与植物细胞内蛋白质相互作用的过程。BiFC荧光技术，以及改进的T-DNA免疫沉淀测定，将用于监测活植物细胞中t复合物的组装和细胞内运输。在这些条件下，在植物细胞中组装之前，各种假定的t复合物成分在自然水平上和在其原生生物体中合成。这些研究的结果对于解决文献中关于各种假定的t复合物组分在T-DNA通过植物细胞运输中的作用的许多相互矛盾的数据是重要的。所提出研究的更广泛影响：水平基因转移已被认为是进化的主要组成部分，而农杆菌是水平基因流动研究得最好的例子之一。农杆菌介导的遗传转化也是产生用于基础研究和农业生物技术目的的转基因植物的主要机制。了解T-DNA如何在植物细胞中运输对于理解细胞外蛋白-核酸复合物（包括病毒基因组）在进入细胞后如何靶向细胞核非常重要。由于核靶向中的许多步骤可能是限速的，因此了解这一过程对于预防疾病（如由农杆菌或病毒性疾病引起的冠瘿病）和改善抗性作物品种的转化将是重要的。除了培养研究科学家和研究生外，该项目还将与布鲁克林学院开展一项积极的外展计划，从代表性不足的少数群体中识别本科生，并通过夏季和学年合作，将他们介绍给科学研究。鼓励对这些学生进行多年的培训，以巩固他们从事科学事业的兴趣。当他们回到自己的学校后，他们会让更多的学生接触到在我们实验室学到的技术。在夏季接受培训的少数族裔学生将在其本国机构继续这些项目，从而扩大与科学研究接触的同龄人的数量。这些努力将鼓励本科生选择科学领域的职业。