The Plant Signal Landscape Recognized by Agrobacterium Tumefaciens

本项目的目的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，从而导致敏感植物组织的转化。控制这一过程的生化系统(VirA、Virg和ChvE蛋白)至少识别来自植物的三种不同的诱导信号：酚、糖和低pH。在这个项目中，植物组织将暴露在细菌中，这些细菌含有由毒力诱导系统控制的“报告”基因。当该基因被表达时，它产生一种与植物组织显著不同的特定颜色(例如，来自绿色荧光蛋白)，使研究人员能够确定信号在植物中的实际位置。通过改变Vira/Virg/ChvE系统的遗传特性，研究人员将首次能够追踪个别类别诱导分子的存在，并将这一信息与待转化组织和细胞的能力联系起来。这些实验验证了一种假设，即在植物中只有几个区域发现所有三种信号的水平足以诱导VIR，并且这些信号对细菌来说意味着能够转化的组织的存在。最后，研究人员将使用遗传方法来扩大Vira识别为诱导信号的化合物的范围，然后通过上述方法确定是否可以在植物中检测到这种外来化合物。广泛的影响：了解诱导信号在植物中的分布和被转化组织的能力之间的关系，将对利用农杆菌作为工具对目前抵抗转化的植物进行基因改造具有意义。这可能会对农业生物技术产生重要影响。此外，化学识别特性改变的VirA变异的定向进化有可能提供廉价、易于使用的分析工具，能够指示植物中各种植物次生代谢物的存在，从而对营养和毒素研究产生影响。该项目将让本科生积极参与研究工作，研究生和博士后研究人员作为本科教学的合作者，并与当地公立学校建立合作伙伴关系，让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.