Functional Dissection of the PTO Resistance Gene Using DNA Shuffling

使用 DNA 改组对 PTO 抗性基因进行功能解析

• 批准号: 0133993
• 负责人: Richard Michelmore
• 金额: $ 42.5万
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0133993&HistoricalAwards=false
• 关键词: Functional; Dissection; PTO; Resistance; Gene

Understanding the early events in disease resistance has both fundamental and practical importance. Molecular dissection of disease resistance is currently one of the most intensively studied areas of plant biology and interesting parallels are being established with the animal-pathogen interactions. Although considerable progress has been made over the past ten years, early recognition events that lead to resistance are only just beginning to be understood. In the long term, understanding the molecular basis of specificity in plant-pathogen interactions and the molecular events resulting in resistance will provide new options for developing disease resistant plants. Our long-term goals are to determine the molecular basis of recognition and signaling in plant disease resistance and to engineer new disease resistance specificities. The specific goal of this proposal is to dissect the functional regions of the Pto protein that confer the ability to bind different pathogen products and to initiate physiological pathways (downstream signaling) necessary for defense against the pathogens in diverse plant species.DNA shuffling is a powerful and novel approach for dissecting protein function that makes few a priori assumptions about function yet provides great resolution to dissect individual regions of the protein. DNA shuffling involves the generation of chimeric genes (recombinations from two gene sources) in the test-tube from fragments of naturally occurring versions of a gene. Our proposed studies will further develop DNA shuffling as an experimental tool and provide statistical approaches for interpreting the resulting data. The interaction between the pathogenic bacterium Pseudomonas syringae and Solanaceous plants has become one of the best-characterized plant-pathogen interactions at the molecular level and the Pto gene is ideally suited to DNA shuffling experiments. We have been studying this interaction for several years and have all the necessary biological materials and methodologies in hand to make rapid progress. The first round of DNA shuffling the Pto gene has demonstrated the power and feasibility of the DNA shuffling approach and revealed several previously unrecognized, potentially important domains for binding to the pathogen derived avirulence protein, AvrPto. In this research program we will conduct three parallel lines of investigation that will provide detailed and complementary data on the domains and amino acid sequences of Pto which are required for binding pathogen derived proteins and for downstream signaling: 1) We will conduct a second generation of DNA shuffling as well as specific amino acid substitutions to test the structure - function inferences derived from our first DNA shuffling experiment. 2) We will screen a shuffled library of chimeric genes to identify variants of Pto with new binding specificities. 3) We will shuffle Pto with genes encoding similar proteins from Arabidopsis to identify chimeras that confer the ability to recognize additional avirulence proteins.The proposed experiments are highly multidisciplinary. They are a combination of molecular biology and statistics that draws on plant breeding strategies as well as making and testing structure-function inferences. The postdoc, graduate student, and undergraduates will receive training in both molecular biology and statistics. In addition, they will gain experience in plant-pathogen interactions.

了解抗病性的早期事件具有基础和实际意义。 抗病性的分子解剖是目前植物生物学最深入研究的领域之一，并且正在建立与动物-病原体相互作用的有趣的相似之处。 尽管在过去十年中取得了相当大的进展，但导致耐药性的早期识别事件才刚刚开始被理解。 从长远来看，了解植物-病原体相互作用特异性的分子基础和导致抗性的分子事件将为开发抗病植物提供新的选择。 我们的长期目标是确定植物抗病性识别和信号传导的分子基础，并设计新的抗病特异性。 这项提议的具体目标是解剖Pto蛋白的功能区域，这些功能区域赋予结合不同病原体产物和启动生理途径的能力（下行信号）DNA改组是一种有效而新颖的蛋白质功能分析方法，它对蛋白质功能的先验假设很少，但却能很好地分析蛋白质的功能。蛋白质的区域。 DNA改组涉及在试管中从天然存在的基因片段产生嵌合基因（来自两个基因来源的重组）。 我们提出的研究将进一步发展DNA改组作为一种实验工具，并提供解释所得数据的统计方法。 病原细菌假单胞菌和茄科植物之间的相互作用已成为分子水平上最具特征的植物-病原体相互作用之一，Pto基因非常适合DNA改组实验。 我们已经研究这种相互作用好几年了，并掌握了所有必要的生物材料和方法，可以迅速取得进展。 Pto基因的第一轮DNA改组已经证明了DNA改组方法的能力和可行性，并揭示了几个以前未被认识到的、潜在重要的结构域，用于结合病原体衍生的无毒蛋白AvrPto。 在这项研究计划中，我们将进行三个平行的调查，将提供详细的和互补的数据的结构域和氨基酸序列的Pto所需的结合病原体衍生的蛋白质和下游信号：1）我们将进行第二代DNA改组以及特定的氨基酸取代，以测试从我们的第一次DNA改组实验中得出的结构-功能推断。 2)我们将筛选嵌合基因的改组文库以鉴定具有新的结合特异性的Pto变体。 3)我们将把Pto与编码来自拟南芥的类似蛋白质的基因进行重组，以识别具有识别其他无毒蛋白质能力的嵌合体。拟议的实验是高度多学科的。 它们是分子生物学和统计学的结合，借鉴了植物育种策略，以及制作和测试结构功能推断。 博士后，研究生和本科生将接受分子生物学和统计学的培训。 此外，他们将获得植物病原体相互作用的经验。