Functional Dissection of the PTO Resistance Gene Using DNA Shuffling

了解抗病性的早期事件具有基础和实际意义。抗病的分子解剖是目前植物生物学中最深入研究的领域之一，与动物-病原体相互作用的有趣相似之处正在建立起来。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，了解植物与病原体相互作用特异性的分子基础以及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识别和信号传导的分子基础，并设计新的抗病特异性。本提案的具体目标是剖析Pto蛋白的功能区域，该功能区域赋予了结合不同病原体产物的能力，并启动了防御不同植物物种病原体所需的生理途径（下游信号传导）。DNA洗濯是一种强大而新颖的蛋白质功能解剖方法，它对功能的先验假设很少，但却为解剖蛋白质的单个区域提供了很大的分辨率。DNA洗牌涉及在试管中从自然产生的基因片段中产生嵌合基因（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洗牌作为一种实验工具，并为解释结果数据提供统计方法。丁香假单胞菌与茄科植物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基因非常适合进行DNA洗牌实验。我们已经研究这种相互作用好几年了，手头上有所有必要的生物材料和方法，可以取得快速进展。Pto基因的第一轮DNA洗牌已经证明了DNA洗牌方法的力量和可行性，并揭示了几个以前未被识别的潜在重要结构域与病原体衍生的无毒蛋白AvrPto结合。在这个研究项目中，我们将进行三条平行的调查，以提供Pto结合病原体衍生蛋白和下游信号所需的结构域和氨基酸序列的详细和互补数据：1)我们将进行第二代DNA洗牌以及特定的氨基酸替换，以测试从我们的第一次DNA洗牌实验中得出的结构-功能推断。2)我们将筛选嵌合基因重组文库，以鉴定具有新结合特异性的Pto变体。3)我们将把Pto与来自拟南芥的编码类似蛋白的基因进行洗牌，以鉴定嵌合体，这些嵌合体赋予了识别额外无毒蛋白的能力。提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，利用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方面的培训。此外，他们将获得植物与病原体相互作用的经验。

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.