Collaborative Research: Novel Proteins Required for Gene Transfer to Plants

智力上的功绩。物种间的基因流动是一个自然的过程。普遍存在的土壤细菌根癌农杆菌和发根农杆菌会将它们的部分DNA自然转移到植物身上，导致冠瘤和毛状根疾病的产生。这种转移DNA的能力已经被用于基因工程，现在是向植物细胞传递基因的首选方法。尽管被广泛使用，但关于基因转移是如何发生的，许多机械性的问题仍然存在。在根癌农杆菌中，病毒E2蛋白通过包裹转移的DNA并促进其进入植物细胞的细胞核而发挥主要作用。然而，这似乎并不是一个通用的机制，因为一些发根曲霉菌株有效地转移DNA，但缺乏病毒E2。相反，这些菌株含有另一种蛋白质，GALS，可以替代病毒E2，从而为转移的DNA的核摄取提供了另一种途径。显然，在进入植物细胞后，虫胆定位于细胞核内，在那里它与附着在转移DNA上的细菌引导蛋白相互作用，并与植物蛋白(虫胆相互作用蛋白；GIP)相互作用。GALS被认为是一个“分子拖拉机”，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GALS和GIP是如何通过不依赖病毒E2的途径促进基因转移的。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了解，并确定这一极其重要的基因传递系统的其他未知成分。该项目将促进俄勒冈州立大学博士生和普渡大学博士后助理的教育和培训。此外，布鲁克林学院的少数民族本科生将被招募参加普渡大学的暑期研究实习。在暑期体验结束后，学生们将继续在他们的家乡机构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.