Arabidopsis 2010: Expression Profiling of Plant Disease Resistance Pathways

这个项目的目标是确定关键转录因子(TF)在植物拟南芥防御-反应信号网络中的功能(参见http://ausubellab.Mgh.atherard.edu/nsf2010/查看当前正在研究的转录因子列表)。转录因子是控制植物对病原体的防御反应的关键开关，利用基因芯片(微阵列)进行表达谱分析是高通量分析转录事件最有效的基因组学方法。分析将在未被研究的防御途径中识别候选的TF，包括由通常与植物和动物的病原体相关的所谓的“PAMP”分子诱导的那些。我们将评估Tf突变体对抗病能力的影响，以确定扰乱基因及其功能是否会影响任何防御反应。通过这种方式鉴定的TF将与绿色荧光蛋白相连，以标记和分类表达TF基因的细胞。将在这些细胞中进行转录图谱分析，以确定其表达模式与TF的表达模式相似的基因(即队列转录本)。转铁蛋白还将被修饰，以便它们进入细胞核的运输可以在实验上得到控制，并将识别它们的目标基因。总的来说，这些实验将提供与防御相关的转录事件的空间、时间和层次信息，这些信息目前是缺乏的，但需要建立相互作用的防御路径的转录网络模型。本项目获得的数据将存入综合微阵列数据系统(http://ausubellab.mgh.harvard.edu/imds))，并及时向公众提供。IMDS是本项目创建的可通过网络访问的公共微阵列数据库，以促进微阵列数据集的存储、检索和分析。它存储实验描述、原始微阵列数据和归一化数据集。IMDS中的数据将通过将其输出到NCBI基因表达总览和拟南芥信息资源来在2010年奖项的有效期之后进行维护。这一多名调查员的努力对2010年总体项目目标的意义是多方面的。植物的防御反应涉及基因表达的全球变化(占基因组的10%)。因此，识别转录因子是理解防御机制的关键。此外，对大型基因组的深入分析需要几个实验室的努力，该项目提供的协调避免了重复工作并削减了成本。最后，添加到IMDS中的每个新的图谱实验都将增强科学界研究人员设计实验的能力，这些实验将进一步阐明植物防御反应的关键特征。该项目为本科生、博士生和博士后提供了一个培训环境。参与者不仅将学习经典的遗传和植物病理学方法，还将学习尖端基因组学和生物信息学技术。一个关键的培训部分是该项目的年度研讨会，学生和博士后在半正式的环境中介绍他们的研究。基因组学和生物信息学方面的本科培训也是该项目的一个重要方面。

The goal of this project is to determine the function of key transcription factors (TFs) in the plant Arabidopsis defense-response signaling network (see http://ausubellab. mgh.harvard.edu/nsf2010/ for the current list of transcription factors under study). TFs are key switches that control plant defense responses against pathogens, and expression profiling using Gene Chips (microarrays) is the most powerful genomics approach available for high through-put analysis of transcriptional events. Profiling will identify candidate TFs in understudied defense pathways, including those induced by so-called "PAMP" molecules that are commonly associated with pathogens of plants as well as animals. We will evaluate the TF mutants for their impact on disease resistance to find whether disrupting the genes and their functions will affect any defense response. The TFs identified in this way will be linked to green fluorescent protein to mark and sort the cells expressing the TF genes . Transcriptional profiling will be conducted in these cells to identify genes whose expression patterns mirror those of the TFs (i.e., cohort transcriptomes). The TFs will also be modified so their transport into the nucleus, where they function, can be controlled experimentally and their target genes will be identified. Collectively, these experiments will provide the spatial, temporal, and hierarchical information about defense-related transcriptional events which is currently lacking, but needed to build a model of the transcriptional network of interacting defense pathways. Data obtained in this project will be deposited in IMDS (integrated microarray data system) (http://ausubellab.mgh.harvard.edu/imds) and made available to the public in a timely manner. IMDS is a public web-accessible microarray database created by this project to facilitate storage, retrieval and analysis of microarray data sets. It stores experimental descriptions, raw microarray data and sets of normalized data. The data in IMDS will be maintained beyond the lifetime of the 2010 award by exporting it to the NCBI Gene Expression Omnibus and the Arabidopsis Information Resource. The significance of this multi-investigator effort to the overall 2010 project objectives is several-fold. Defense responses in plants involve global changes in gene expression (10% of the genome). Therefore, identifying TFs holds the key for understanding the mechanism of defense. Also, an in-depth analysis of large gene sets requires the efforts of several laboratories and coordination afforded by this project avoids duplication of efforts and cuts costs. Finally, each new profiling experiment added to IMDS will enhance the ability of researchers in the scientific community to design experiments that will further elucidate key features of the plant defense response. This project provides a training environment for undergraduates, doctoral students, and postdocs. Participants will learn not only classical genetic and phytopathology methods, but also cutting-edge genomics and bioinformatics technologies. A key training component is the project's annual workshop, where students and postdocs present their research in a semi-formal setting. Undergraduate training in genomics and bioinformatics is also a significant aspect of this project.