Pathways for Colonization of Plant Genome by Agrobacterium

农杆菌是一种可以对寄主进行基因改造的迷人微生物，是植物生物学的重要研究课题，也是农业的主要生物技术工具。尽管进行了40年的研究，但农杆菌介导的转化过程的关键方面仍然模糊不清。最耐人寻味但却最不为人所知的方面之一是细菌如何利用其蛋白质效应器，将其与转移的(T-)-DNA一起转移到宿主细胞中，以“定植”宿主基因组；为T-DNA在其他整合位点的入侵做好准备，招募细菌和宿主DNA修复机制，并抑制宿主防御，以确保T-DNA的最佳表达。该项目旨在通过研究农杆菌效应器蛋白与宿主细胞伙伴之间的功能相互作用来提供这些缺失的信息。由此产生的数据的更广泛的重要性在于加强我们对植物-细菌相互作用和农杆菌致病性的基本理解，将所开发的模型转化为更广泛的细菌-宿主相互作用的模型，并为设计更安全、更有效的基因转化程序提供科学基础，从而造福于科学、农业和社会。这项研究将作为本科和社区大学科学教学的平台，并将年轻科学家引入实验植物微生物学和生物学领域。农杆菌一直被认为是一种独特的原核生物，它编码一种蛋白质机制，用于DNA转移和整合到真核基因组中。这一教条受到了最近发现的挑战，即其他不同的细菌物种也编码有功能的DNA转移机制。因此，加强关于农杆菌遗传转化的基础知识，将促进对植物-病原菌相互作用的理解，并促进农杆菌作为农业基因转移工具，但也将为翻译到广泛的真核宿主-原核病原菌相互作用提供信息。这个项目的目标是了解农杆菌如何产生双链DNA断裂(DSB)，这些双链DNA断裂被用作首选整合位点，T-DNA分子和适当的细胞DNA修复因子如何被招募到DSB，以及农杆菌如何克服能够沉默T-DNA表达的宿主防御。目的1研究农杆菌在植物基因组DNA中富含DSB的机制。将检验一种假设，即细菌毒力(VIR)效应蛋白(例如，VirE3)与宿主S1型核酸内切酶相互作用，启动DSB的产生。目的2研究农杆菌将T-DNA靶向DSB并招募宿主DNA聚合酶整合T-DNA并修复DSB的机制。将检验一种假设，即与T-DNA相关的细菌Vir效应器(例如，VirE2)与宿主DSB结合因子(例如，BRCA样蛋白)相互作用以将T-DNA导向DSB，而另一与T-DNA相关的Vir效应器(例如，VirD2)将宿主DNA聚合酶(例如，Pol theta)招募到相同的DSB。目的3将研究农杆菌如何抑制宿主防御性RNA沉默以促进T-DNA的表达。一项假设将得到检验，即农杆菌利用其F-box蛋白效应器之一，通过SCF途径将宿主RNA沉默机制的组件靶向蛋白酶体降解。这一奖项反映了NSF的法定使命，并已通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Agrobacterium, a fascinating microorganism that can genetically modify its plant hosts, represents an important research subject for plant biology and a major biotechnological tool for agriculture. Despite four decades of research, critical aspects of the Agrobacterium-mediated transformation process remain obscure. One of the most intriguing, yet least understood, aspects is how the bacterium utilizes its protein effectors, which it transfers with the Transferred (T-)-DNA, into the host cell, to "colonize" the host genome; prepare it for T-DNA invasion at additional integration sites, recruit bacterial and host DNA repair machinery, and repress host defenses to assure optimal expression of the T-DNA. This project aims to provide these missing pieces of information by investigating functional interactions between Agrobacterium effector proteins and their host cell partners. The broader importance of the resulting data lies in the enhancement of our basic understanding of plant-bacterium interactions and Agrobacterium pathogenicity, translation of the developed models to a broader range of bacterium-host interactions, and in providing a scientific foundation for the design of safer and more efficient genetic transformation procedures that benefit science, agriculture and the society. This research will serve as a platform for teaching undergraduate and community college science and introducing young scientists into the fields of experimental plant microbiology and biology.Agrobacterium has been considered a unique prokaryote that encodes a protein machinery for DNA transfer and integration into eukaryotic genomes. This dogma is challenged by recent discoveries that other diverse bacterial species, also encode functional DNA transfer machineries. Enhancing fundamental knowledge about genetic transformation with Agrobacterium, therefore, will advance understanding of plant-pathogen interactions and improve Agrobacterium as a gene transfer tool for agriculture, but also will inform translation to a broad range of eukaryotic host-prokaryotic pathogen interactions. This project's goals are to understand how Agrobacterium creates double-stranded DNA breaks (DSBs), which are used as preferred integration sites, how the T-DNA molecule and the appropriate cellular DNA repair factors are recruited to DSBs, and how Agrobacterium overcomes host defenses that can silence T-DNA expression. Aim 1 examines the mechanism by which Agrobacterium enriches DSBs in the plant genomic DNA. A hypothesis will be tested that a bacterial virulence (Vir) effector protein (e.g., VirE3) interacts with a host S1-type endonuclease to initiate generation of DSBs. Aim 2 examines the mechanism by which Agrobacterium targets the T-DNA to the DSB and recruits the host DNA polymerase to integrate the T-DNA and repair the DSB. A hypothesis will be tested that a bacterial Vir effector associated with the T-DNA (e.g., VirE2) interacts with a host DSB-bound factor (e.g., a BRCA-like protein) to direct the T-DNA to DSB, and another T-DNA-associated Vir effector (e.g., VirD2) recruits a host DNA polymerase (e.g., Pol theta) to the same DSB. Aim 3 will examine how Agrobacterium suppresses the host defensive RNA silencing to facilitate expression of T-DNA. A hypothesis will be tested that Agrobacterium utilizes one of its F-box protein effectors to target the components of the host RNA silencing machinery to proteasomal degradation via the SCF pathway.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.