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转移到植物细胞中最广泛使用的载体。然而，DNA和蛋白质究竟是如何从细菌细胞中转运到植物细胞中的仍然不清楚。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（T4 SS）。T4 SS参与细菌交配和蛋白质毒素输出，并且是细菌与植物和动物宿主之间引起疾病的相互作用所必需的。PI的长期目标是了解T4 SS的结构和功能。该研究项目将使用高分辨率显微镜技术来确定T4 SS在细菌内的定位位置，以及这种定位在与植物细胞结合后是否会发生变化。T4 SS有两个主要部分：跨膜通道和细胞外菌毛（称为T菌毛），其促进细菌附着到宿主细胞。新的数据表明，形成T4 SS膜通道的蛋白质组分以及由T4 SS运输的蛋白质以螺旋模式定位在细菌细胞的圆周周围。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，这样的融合是功能性的，并且不干扰农杆菌将DNA转移到植物细胞的能力。细菌细胞周围的多个T4 SS可能增加农杆菌与植物宿主细胞接触的效率。研究的目标1将确定a）T4 SS组件本地化的时间过程是什么？B）T4 SS是否与T-菌毛共定位？c）T4 SS或其底物蛋白的组分与细菌细胞骨架（已知其形成螺旋阵列）共定位吗？和d）T4 SS的定位在附着于宿主细胞期间是否改变？目的2将描述T-DNA转移过程中T菌毛及其与T4 SS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T4 SS底物蛋白？以及c）在宿主细胞附着期间T菌毛发生了什么？皮利是伸展还是收缩？这项研究将通过帮助我们了解细菌细胞如何附着在非细菌细胞上并与之相互作用来影响社会。 更广泛的影响。与本项目的研究活动平行，首席研究员将开发一个为期15周的新生课程，以农杆菌生物学及其作为基础和应用研究工具的用途为中心。荧光探针和荧光显微镜将让学生看到生物学的第一手资料。学生将观察a）携带定位于细胞骨架、细胞膜或其他特定细胞位点的蛋白质的活细菌，B）用携带植物基因的农杆菌转化植物细胞，当表达时，可以在细胞的不同部分检测到，或c）农杆菌介导的荧光标记植物病毒的接种，这些病毒在整个植物中显示运动。将讨论科学发现是如何产生的，以及长期的社会影响，如转基因生物（GMO）。本课程将刺激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.