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; 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.

智力优点：该项目将研究农业T-复合物的定位的形成，组成和亚细胞位点，因为它被组装成并通过植物细胞运输。 农杆菌通过IV型分泌系统（T4SS）转移了与VIRD2蛋白共价链接到植物细胞的单链DNA（T链）。 包括Vire2在内的其他几种效应毒力蛋白通过T4SS分别转移到植物中。 Vire2是一种单链DNA结合蛋白，在体外可以与T链和外壳T链复合。当前有利的假设是，在植物细胞中，Vire2涂层还有助于将T链靶向核，在该细胞核中，T链成为双链并整合到植物基因组中。 通过细胞质和细胞核靶向T链可能是由植物蛋白（例如Importin-Alpha和Vip1）引导的。 在体外和植物细胞中，Importin-Alpha与VIRD2，VIRE2和VIP1相互作用，并且已经假设这些蛋白质在植物园中形成与T链形成成熟或超级-T-复合物。 但是，这些研究均研究了在没有T链的情况下植物细胞中蛋白质蛋白质相互作用。 此外，Vire2的表达来自强大的植物启动子，导致形成聚集体的高水平的蛋白质。 双分子荧光互补（BIFC）技术可用于跟踪Vire2，因为它退出农业杆菌并与植物细胞中的蛋白质相互作用。 BIFC荧光技术以及修饰的T-DNA免疫沉淀测定法，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，各种推定的T复合成分在自然水平及其天然生物中合成，在植物细胞中组装之前。 这些研究的结果对于解决文献中有关各种假定​​的T复合成分在通过植物细胞中T-DNA运输中的作用的大量相互矛盾的数据很重要。 拟议研究的更广泛的影响：水平基因转移已被认为是进化的主要组成部分，而农杆菌代表了水平基因流量最佳研究的例子之一。 农杆菌介导的遗传转化也是生成基础研究和农业生物技术目的的转基因植物的主要机制。 了解植物细胞中T-DNA运输对于了解细胞外蛋白质核酸复合物（包括病毒基因组）如何靶向细胞后的细胞核如何至关重要。 由于核靶向的许多步骤可能是限制的，因此了解该过程对于预防疾病（例如由农业杆菌或病毒疾病引起的冠状胆）和改善顽固作物物种的转化至关重要。 除了培训研究科学家和研究生外，该项目还将用于与布鲁克林学院进行激烈的外展计划，以确定来自代表性不足的少数群体的本科生，并通过夏季和学年的合作将其引入科学研究。 将鼓励对这些学生进行多年培训，以巩固他们从事科学职业的兴趣。 当他们返回家庭机构时，他们将使更多的学生接触到我们实验室中学到的技术。 夏季培训的代表性不足的少数族裔学生将继续在其家庭机构继续这些项目，从而扩大将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职业。