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

智力优点：本计画将探讨农杆菌T复合体在植物细胞内组装及运输时的形成、组成及定位的亚细胞位置。 农杆菌通过IV型分泌系统（T4 SS）将与VirD 2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VirE 2，通过T4 SS分别转移到植物中。 VirE 2是一种单链DNA结合蛋白，在体外可以与T链复合并包被T链。目前流行的一种假说是，在植物细胞中，VirE 2外壳也有助于将T链靶向细胞核，在那里T链变成双链并整合到植物基因组中。 靶向T链通过细胞质并进入细胞核可能是由植物蛋白如importin-alpha和VIP 1指导的。 在体外和植物细胞中，importin-alpha与VirD 2，VirE 2和VIP 1相互作用，并且这些蛋白质已被假设在植物中与T链形成成熟或超T复合物。 然而，这些研究都是在缺乏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，VirE 2的表达来自强植物启动子，导致形成聚集体的高水平蛋白质。 双分子荧光互补（BiFC）技术可用于跟踪VirE 2，因为它退出农杆菌和与植物细胞内的蛋白质相互作用。 BiFC荧光技术，沿着一种改良的T-DNA免疫沉淀试验，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复合物组分在天然水平下合成，并且在它们的天然生物体中，然后在植物细胞中组装。 这些研究的结果是重要的，以解决许多相互矛盾的数据，在文献中的各种假定的T-复合体成分的作用，在T-DNA运输通过植物细胞。 拟议研究的更广泛影响：水平基因转移已被认为是进化的一个主要组成部分，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获得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如何通过植物细胞运输对于了解细胞外蛋白质-核酸复合物（包括病毒基因组）如何在进入细胞后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，因此理解该过程对于预防疾病（例如由农杆菌引起的冠瘿病或病毒性疾病）和改善柑橘作物物种的转化将是重要的。 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开展一项积极的外联方案，以确定来自代表性不足的少数群体的本科生，并通过夏季和学年合作向他们介绍科学研究的开展。 将鼓励对这些学生进行多年培训，以巩固他们对从事科学事业的兴趣。 当他们回到自己的家乡机构，他们将接触更多的学生在我们的实验室学到的技术。 在夏季接受培训的人数不足的少数民族学生将继续在其家乡的机构开展项目，从而扩大他们的同龄人接触科学研究的人数。 这些努力将鼓励本科生选择科学职业。

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.