New Insight into the Assembly and Function of the Agrobacterium Type IV Secretion System by High Resolution Imaging Techniques

该奖项是根据2009年美国复苏和再投资法案(公法111-5)资助的。根癌农杆菌是一种细菌，也是将DNA转移到植物细胞的最广泛使用的载体。然而，DNA和蛋白质到底是如何从细菌细胞运输到植物细胞的仍不清楚。编码农杆菌T-DNA转运蛋白的基因属于进化上保守的转运蛋白，称为IV型分泌系统(T4SS)。T4SS参与细菌交配和蛋白质毒素输出，是细菌与动植物宿主之间致病相互作用所必需的。PI的长期目标是了解T4SS的结构和功能。这项研究项目将使用高分辨率显微镜技术来确定T4SS在细菌中的定位，以及这种定位是否在与植物细胞结合时发生变化。T4SS有两个主要部分：一个跨膜通道和一个细胞外菌毛(称为T菌毛)，它促进细菌与宿主细胞的附着。新的数据显示，形成T4SS膜通道的蛋白质成分，以及由T4SS运输的蛋白质，以螺旋模式定位在细菌细胞的周围。通过将荧光示踪剂与上述蛋白质融合来检测螺旋定位。值得注意的是，这种融合是有功能的，不会干扰农杆菌将DNA转移到植物细胞的能力。细菌细胞周围的多个T4s可能会增加农杆菌与植物寄主细胞接触的效率。研究的目标1将确定a)T4SS组件本地化的时间进程是什么？B)T4SS是否与T-PILI协同本地化？C)T4SS或其底物蛋白的组成部分是否与细菌细胞骨架(已知形成螺旋阵列)共定位？以及d)T4SS的定位在附着到宿主细胞过程中会改变吗？目的2研究T-DNA转移过程中T-菌毛的特性及其与T4SS功能的关系，并确定a)T-菌毛的组成是什么？B)T-菌毛是否含有DNA或T4SS底物蛋白？以及c)T-菌毛在宿主细胞附着过程中发生了什么？毛发是延长的还是收缩的？这项研究将通过帮助我们了解细菌细胞如何附着并与非细菌细胞相互作用来影响社会。更广泛的影响。与本项目中开展的研究活动并行，首席调查员将开发一门为期15周的新生课程，重点是农杆菌生物学及其作为基础和应用研究工具的使用。荧光探针和荧光显微镜将让学生亲眼看到生物。学生将观察到a)携带定位于细胞骨架、膜或其他特定细胞部位的蛋白质的活细菌，b)携带植物基因的农杆菌转化植物细胞，当表达这些基因时，可以在细胞的不同部分检测到它们的存在，或c)农杆菌介导的带有荧光标记的植物病毒的接种，这种病毒显示在整个植物中移动。将讨论如何取得科学发现和长期的社会影响，如转基因生物。本课程将激励新生在生物科学方面继续深造。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Agrobacterium tumefaciens is a bacterium that also serves as the most widely utilized vector for transferring DNA to plant cells. However, exactly how DNA and proteins are transported out of the bacterial cell and into the plant cell remains unclear. Genes encoding the Agrobacterium T-DNA transporter protein belong to growing class of evolutionarily conserved transporters, called type IV secretion systems (T4SS). T4SS are involved in bacterial mating and protein toxin export, and are required for disease-provoking interactions between bacteria and plant and animal hosts. The PI's long-term goal is to understand the structure and function of the T4SS. This research project will use high-resolution microscopy techniques to determine where the T4SS localizes within the bacteria and whether this localization changes upon binding to plant cells. The T4SS has two major parts: a membrane spanning channel and an extracellular pilus (called the T-pilus) that facilitates bacterial attachment to host cells. New data reveal that protein components that form the T4SS membrane channel, as well as proteins that are transported by the T4SS, localize in a helical pattern around the circumference of the bacterial cell. Helical localization is detected by fusion of a fluorescent tracer to the above proteins. Notably such fusions are functional and do not interfere with the ability of Agrobacterium to transfer DNA to plant cells. Multiple T4SS around the bacterial cell likely increases the efficiency whereby Agrobacterium can make contact with plant host cells. Aim 1 of the research will determine a) what is the time course for the localization of the components of the T4SS? b) does the T4SS co-localize with T-pili? c) do components of the T4SS or its substrate proteins co-localize with the bacterial cytoskeleton (which is known to form helical arrays)? and d) does the localization of the T4SS alter during attachment to host cells? Aim 2 will characterize the T-pilus and its relation to T4SS function during T-DNA transfer and determine a) what is the composition of the T-pilus? b) do T-pili contain DNA or T4SS substrate proteins? and c) what happens to the T-pili during host cell attachment? Do pili extend or contract? The research will impact society by helping us understand how bacterial cells attach to and interact with non-bacterial cells. Broader Impacts. In parellel with research activities pursued in this project, the Principal Investigator will develop a 15-week freshman course centered on the biology of Agrobacterium and its use as a tool for basic and applied research. Fluorescent probes and fluorescence microscopy will allow students to see biology first hand. Students will observe a) live bacteria carrying proteins that localize to the cytoskeleton, membranes or other specific cellular sites, b) transformation of plant cells with Agrobacterium carrying plant genes that when expressed can be detected in different parts of the cell or c) Agrobacterium-mediated inoculation of fluorescently tagged plant viruses that display movement throughout the entire plant. How scientific discoveries are made and long-term societal impacts, such as GMOs (genetically modified organisms) will be discussed. This course will stimulate freshmen to pursue further studies in the biological sciences.