Collaborative Research: Novel Proteins Required for Gene Transfer to Plants

知识价值。物种之间的基因流动是一个自然的过程。无处不在的土壤细菌农杆菌和根状芽孢杆菌自然地将它们的一些DNA转移到植物中，导致树冠瘿和毛状根病的产生。这种转移DNA的能力已经被用于基因工程，现在是将基因传递到植物细胞的首选方法。尽管被广泛使用，关于基因转移如何发生的机制问题仍然存在。在瘤胃芽孢杆菌中，VirE2蛋白通过包裹转移的DNA并促进其进入植物细胞核发挥主要作用。然而，这似乎不是一个普遍的机制，因为一些菌株有效地转移DNA，但缺乏VirE2。相反，这些菌株含有另一种蛋白质，GALLS，它可以替代VirE2，从而为核摄取转移的DNA提供了另一种途径。显然，在进入植物细胞后，GALLS定位于细胞核内，在那里它与附着在转移DNA上的细菌先导蛋白和植物蛋白相互作用（GALLS- interacting protein； GIP）。GALLS被认为是一个“分子牵引器”，将转移的DNA拉入细胞核。为了验证这一想法，将使用多种实验方法来了解GALLS和GIP如何通过不依赖于vire2的途径促进基因转移。更广泛的影响。这些研究将增加对农杆菌介导的基因转移的理解，并确定这一极其重要的基因传递系统的其他未知成分。本项目将促进俄勒冈州立大学一名博士生和普渡大学一名博士后的教育和培训。此外，来自布鲁克林学院的少数族裔本科生将被招募到普渡大学参加暑期研究实习。暑期实习结束后，学生们将在各自所在的机构继续从事相关的实践研究。

Intellectual Merit. Gene flow between species is a natural process. The ubiquitous soil bacteria Agrobacterium tumefaciens and A. rhizogenes naturally transfer some of their DNA to plants, leading to production of crown gall and hairy root diseases. This ability to transfer DNA has been exploited for genetic engineering and is now the preferred method for delivering genes to plant cells. Despite the widespread use, many mechanistic questions remain about how the gene transfer takes place. In A. tumefaciens, the VirE2 protein plays a major role by coating the transferred DNA and facilitating its uptake into the nucleus of the plant cell. However, this does not appear to be a universal mechanism, as some strains of A. rhizogenes transfer DNA efficiently, but lack VirE2. Instead, these strains contain another protein, GALLS, that can substitute for VirE2, thus providing an alternate route for nuclear uptake of transferred DNA. Apparently, upon entry into plant cells, GALLS localizes inside the nucleus where it interacts with a bacterial pilot protein attached to the transferred DNA and with a plant protein (GALLS-Interacting Protein; GIP). GALLS is hypothesized to act as a "molecular tractor" to pull transferred DNA into the nucleus. To test this idea, multiple experimental approaches will be used to understand how GALLS and GIP promote gene transfer via a VirE2-independent pathway. Broader Impacts. These studies will increase understanding of Agrobacterium-mediated gene transfer and identify additional unknown components of this extremely important gene delivery system. This project will promote education and training of a doctoral student at Oregon State University and a postdoctoral associate at Purdue University. In addition, minority undergraduates from Brooklyn College will be recruited to participate in summer research internships at Purdue. After their summer experiences, the students will continue to engage in related hands-on research at their home institution.