CAREER: Stable transformation of STEM identity by integrating cell cycle regulation with persistent infection in Agrobacterium

该项目将确定和详细说明病原植物相关细菌在其整个生命周期中如何生长和发育，它们如何从与宿主植物联系转变为在土壤中自由生活，以及发病机制。特别值得一提的是，该项目使用了“大自然的基因工程师”农杆菌作为研究的模型细菌。该项目的成果将用于提高利用这种生物进行基因工程的效率，并以可预测的、有针对性的方式培育作物。该项目的研究成果也可应用于多种植物物种遗传操作的基础研究问题，为了解多种植物物种的基因组学和进化提供一套新的工具。最后，这个项目的结果可以应用于农杆菌的近亲，其中一些与宿主有有益的关系，另一些对植物、牲畜和人类有潜在的致病性。该项目提供教育和推广活动，强调巩固第一代和经济或种族少数的学生作为科学家的身份。这些活动包括将研究经验纳入本科课程，并支持附近社区学院的学生和教师在一个活跃的研究实验室担任暑期研究科学家。这两组活动都将要求参与者进行假设驱动的实验工作，以支持项目的研究目标。该项目的总体目标是详细说明农杆菌肿瘤细胞周期控制的遗传框架，以及细胞周期控制如何在生态相关环境中被调节，从而促进持续定植。本研究验证的中心假设是，在农杆菌中，PdhS-DivK-CtrA调控途径是环境响应性细胞周期调控的中心点，能够有效地实现宿主定植、生态位构建和发病机制。第一个研究目标将确定PdhS传感器激酶协调调节CtrA活性的分子机制，特别是探索该途径中蛋白质组分的体内和体外相互作用。第二个研究目标将使用转录组学方法对CtrA转录控制下的全套遗传位点进行编目。第三个研究目标是通过植物侵染过程中的竞争分析，量化PdhS-DivK-CtrA通路活性对生态位构建和发病机制的贡献。该项目的基本原理是，它将建立靶向细胞周期调控控制植物定植的有效性。该项目将有助于我们了解在农杆菌中植物相互作用过程中观察到的细胞周期变化的生态学意义以及这些变化是如何影响的。这项工作将扩大我们对农杆菌细胞周期调控的机制理解，以及细胞周期控制如何与其他表型相结合。这项工作将使转化植物病理学的后续工作成为可能，包括开发新的工具和目标，以操纵宿主植物-微生物界面的相互作用。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project will identify and detail relationships between how pathogenic plant-associated bacteria grow and develop throughout their life cycle, how they switch from associating with a host plant and being free-living in the soil, and pathogenesis. In particular, this project uses “nature’s genetic engineer,” Agrobacterium tumefaciens, as the model bacterium for study. Results from this project will be applied to increasing the efficiency of using this organism to genetically engineer and breed crops in a predictable, directed manner. Results from this project may also be applied to basic research questions involving genetic manipulation of a variety of plant species, providing a new set of tools for understanding the genomics and evolution of multiple plant species. Finally, results from this project may be applied to close relatives of Agrobacterium, some of which are involved in beneficial relationships with their hosts and some of which are potentially pathogenic to plants, livestock, and humans. This project provides education and outreach activities that emphasize solidifying the identity of first-generation and economically or racially minoritized students as scientists. These activities include incorporating research experiences into undergraduate coursework and supporting students and faculty from nearby community colleges as summer research scientists in an active research lab. Both of these sets of activities will have participants performing hypothesis-driven experimental work in support of the research objectives of the project.The overall goal of this project is to detail the genetic framework governing cell cycle control in Agrobacterium tumefaciens and how cell cycle control is modulated in ecologically relevant contexts, contributing to persistent colonization. The central hypothesis tested in this work is that in Agrobacterium the PdhS-DivK-CtrA regulatory pathway is a central point of environmentally-responsive cell cycle regulation enabling efficient host colonization, niche construction, and pathogenesis. The first research objective will determine the molecular mechanism by which the PdhS sensor kinases coordinately regulate CtrA activity, specifically exploring the in vivo and in vitro interactions of protein components of this pathway. The second research objective will catalogue the full suite of genetic loci under CtrA transcriptional control using transcriptomics approaches. The third research objective will quantify the contribution of PdhS-DivK-CtrA pathway activity to niche construction and pathogenesis through competition assays during plant infection. The rationale for this project is that it will establish the validity of targeting cell cycle regulation to control plant colonization. This project will contribute to our understanding of the ecological significance of observed cell cycle changes during plant interactions in Agrobacterium and how such changes are effected. This work will expand our mechanistic understanding of cell cycle regulation in Agrobacterium, as well as how cell cycle control integrates with other phenotypes. This work will enable follow-on work in translational phytopathology, including development of new tools and targets for manipulation of interactions at the host plant-microbe interface.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.