The Plant Signal Landscape Recognized by Agrobacterium Tumefaciens

该项目的目标是了解诱导根癌农杆菌毒力基因表达的分子在植物中的分布，从而导致易感植物组织的转化。 控制该过程的生化系统（VirA、VirG和ChvE蛋白）识别来自植物的至少3种不同的诱导信号：酚、糖和低pH。在该项目中，植物组织将暴露于含有由毒力诱导系统控制的“报告”基因的细菌。 当这个基因被表达时它会产生一种特殊的颜色（例如，来自绿色荧光蛋白）与植物组织显著不同，允许研究人员确定在植物中实际上发现信号的位置。通过改变VirA/VirG/ChvE系统的遗传特性，研究人员将能够，第一次，跟踪诱导分子的个别类别的存在，并将该信息与待转化的组织和细胞的能力相关联。 这些实验检验了这样的假设，即在植物中只有少数区域发现所有三种信号的水平足以进行vir诱导，并且这些信号对细菌意味着存在能够被转化的组织。 最后，研究人员将使用遗传学方法来扩大VirA识别为诱导信号的化合物的范围，然后确定是否可以通过上述方法在植物中检测到这些外来化合物。理解植物中诱导信号的分布与这些相同组织被转化的能力之间的关系将对使用农杆菌作为目前对转化具有抗性的植物的遗传修饰的工具。这可能对农业生物技术产生重要影响。此外，具有改变的化学识别特性的VirA变体的定向进化具有提供廉价、易于使用的分析工具的潜力，该分析工具具有指示植物中存在的各种植物次生代谢物的能力，其结果用于营养和毒素研究。 该项目将涉及本科生作为研究工作的积极参与者，研究生和博士后研究人员作为本科课程教学的合作者，并与当地公立学校合作，本科生在课堂上开发和测试新的生命科学模块。

The goal of this project is to understand the distribution within plants of the molecules that induce expression of the virulence genes of Agrobacterium tumefaciens, which leads to transformation of susceptible plant tissues. The biochemical system that controls this process (the VirA, VirG and ChvE proteins) recognizes at least 3 different inducing signals that come from the plant: phenols, sugars and low pH. In this project plant tissues will be exposed to bacteria that contain a "reporter" gene controlled by the virulence inducing system. When this gene is expressed it yields a particular color (e.g. from the green fluorescence protein) significantly different than the plant tissues, allowing the investigators to determine where in the plant the signals are actually found. By altering the genetic properties of the VirA/VirG/ChvE system the investigators will be able, for the first time, to track the presence of individual classes of inducing molecules and relate this information to the capacity of the tissues and cells to be transformed. These experiments test the hypothesis that there are only a few regions in the plant in which all three signals are found at levels sufficient for vir induction and that these signify to the bacteria the presence of tissues capable of being transformed. Finally, the investigators will use genetic methodologies to broaden the range of chemical compounds that VirA recognizes as inducing signals and then determine whether such foreign compounds can be detected in the plant via the methodologies described above.Broader impact: Understanding the relationship between the distribution of inducing signals in the plant and the capacity of those same tissues to be transformed will have implications for the use of Agrobacterium as a tool for genetic modification of plants that are presently resistant to transformation. This could have important consequences on agricultural biotechnology. Furthermore, the directed evolution of variants of VirA with altered chemical recognition properties has the potential to provide inexpensive, easy to use analytical tools with the capacity to indicate the presence in the plant of a variety of plant secondary metabolites with consequences for both nutrition and toxin studies. The project will involve undergraduate students as active participants in the research efforts, graduate students and postdoctoral researchers as collaborators in undergraduate course instruction, and partnerships with local public schools in which undergraduates develop and test new life science modules in classrooms.