Discovery of Novel Cis-Elements and Transcription Factors Controlling the Plant Defense Transcriptome

植物病害是造成农作物生产严重损失的原因。为了保护自己免受微生物的攻击，植物已经进化出一种有效的防御系统，该系统在病原体识别时是可诱导的。植物防御的关键步骤是大量基因的协同活性变化。这种转录重编程改变了转录速率，即这些基因的mRNA拷贝的合成。然而，控制这些防御基因的调控过程的细节在很大程度上是未知的。以往的研究主要集中在单个防御基因的调控上。最近建立的微阵列技术允许并行监测大量基因的mRNA水平。使用该技术，鉴定了几组响应于病原体识别而显示高度协调上调的基因。每个组的基因可能由共同的调控机制控制。基因的表达水平通常受转录因子的调控，转录因子与基因启动子上游的短序列（顺式元件）相互作用，本项目的主要目的是利用功能基因组学方法系统地鉴定拟南芥中与防御相关的顺式元件及其同源转录因子。使用微阵列的几组基因表现出高度协调的反应，病原体识别被确定。发现在其启动子中高度保守的序列基序可能构成负责其协调表达的顺式元件。这些保守的基序将进行测试，以确定它们是否与核蛋白因子相互作用，以及它们是否可以激活病原体识别后的报告基因。通过这些实验鉴定的功能性顺式元件将用于通过DNA相互作用筛选方法（例如酵母单杂交系统）鉴定其同源转录因子。这些转录因子在植物免疫应答中的作用将通过突变分析、基因沉默和过表达来研究。拟南芥模型提供了这种系统方法所需的所有工具，例如完全测序的基因组，覆盖整个基因组的微阵列和几乎所有基因中具有插入突变的突变体的大量集合。 所提出的方法是公正的，并可能导致新类型的顺式元件和转录因子的发现。本研究揭示的拟南芥防御基因的调控机制可能适用于其他植物物种，并将有助于设计新的策略来提高作物的抗病性。拟议项目的一个重要组成部分是主要来自少数群体的本科生的大力参与。UC-Riverside是一所“少数民族高等教育机构”，少数民族学生占其总入学学生人数的30%。该项目将与UC-Riverside植物细胞生物学中心正在进行的NSF-REU植物细胞生物学计划相联系。此外，拟议项目将为培养植物分子生物学、植物病理学和功能基因组学方面的博士后学者和研究生提供一个平台。

Plant diseases are the cause for dramatic losses in crop production. To defend themselves against microbial attack, plants have evolved an effective defense system that is inducible upon pathogen recognition. Key steps of plant defenses are coordinated activity changes of large numbers of genes. Such transcriptional re-programming alters the rate of transcription, the synthesis of mRNA copies of these genes. However, details of regulatory processes controlling these defense genes are largely unknown. Previous studies mainly focused on the regulation of individual defense genes. The recently established micro-array technology allows parallel monitoring of mRNA levels of large numbers of genes. Using this technology several groups of genes showing highly coordinated up-regulation in response to pathogen recognition were identified. Genes of each group are likely controlled by common regulatory mechanisms. Typically expression levels of genes are regulated by transcription factors, proteins that interact with short sequences (cis-elements) localized in the respective gene's promoter (DNA sequences upstream of its coding region).The main goal of this project is to systematically identify defense-related cis-elements and their cognate transcription factors by functional genomics in Arabidopsis thaliana. Using microarrays several groups of genes showing highly coordinated responses to pathogen recognition were identified. Sequence motifs found to be strongly conserved in their promoters are likely to constitute cis-elements responsible for their coordinated expression. Such conserved motifs will be tested to determine if they interact with nuclear protein factors and if they can activate reporter genes upon pathogen recognition. Functional cis-elements identified by these experiments will be used to identify their cognate transcription factors by DNA-interactor screening methods, such as the yeast one-hybrid system. The roles of such transcription factors in plant immune responses will be examined by mutational analyses, gene silencing and over-expression. The model Arabidopsis provides all tools required for such a systematic approach, such as a fully sequenced genome, microarrays covering the whole genome and large collections of mutants with insertion mutations in nearly all of its genes. The proposed approach is unbiased and likely to lead to the discovery of novel types of cis-elements and transcription factors. Mechanisms controlling defense genes in Arabidopsis uncovered in this study are likely to apply to other plant species and will facilitate designing new strategies to improve disease resistance in crops. An important component of the proposed project is strong involvement of undergrad students, mainly from minority groups. UC-Riverside is a "Minority Postsecondary Institute" with 30 % minority students in its total enrolled student population. The project will be linked to the ongoing NSF-REU Plant Cell Biology program within the Center for Plant Cell Biology at UC-Riverside. In addition, the proposed project will provide a platform for training of postdoctoral scholars and graduate students in plant molecular biology, plant pathology and functional genomics.