Functional Dissection of the PTO Resistance Gene Using DNA Shuffling

了解抗病的早期事件具有重要的基础和现实意义。抗病的分子解剖是目前植物生物学研究最深入的领域之一，与动物-病原菌的相互作用建立了有趣的相似之处。尽管在过去十年中取得了相当大的进展，但人们对导致抵抗的早期识别事件的理解才刚刚开始。从长远来看，了解植物-病原菌相互作用中特异性的分子基础以及导致抗性的分子事件将为培育抗病植物提供新的选择。我们的长期目标是确定识别和信号在植物抗病中的分子基础，并设计新的抗病特异性。这个建议的具体目标是分析PTO蛋白的功能区，这些功能区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菌所需的生理途径(下游信号)。DNA改组是一种强大的、新颖的蛋白质功能解剖方法，它对功能做出了很少的先验假设，但提供了很好的分辨率来剖析蛋白质的个别区域。DNA改组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，并为解释结果数据提供统计方法。紫丁香假单胞菌与茄科植物之间的相互作用已成为分子水平上描述植物-病原菌相互作用的最佳途径之一，而PTO基因非常适合于DNA改组实验。几年来，我们一直在研究这种相互作用，并掌握了所有必要的生物材料和方法，以取得快速进展。第一轮DNA改组PTO基因已经证明了DNA改组方法的有效性和可行性，并揭示了几个以前未被识别的潜在重要结构域，它们与病原菌来源的无毒蛋白AvrPto结合。在这项研究计划中，我们将进行三条平行的调查线，提供关于PTO结构域和氨基酸序列的详细和补充数据，这些结构域和氨基酸序列是结合病原体来源的蛋白和下游信号所必需的：1)我们将进行第二代DNA改组以及特定的氨基酸替换，以测试从我们的第一个DNA改组实验得出的结构-功能推断。2)我们将筛选一个嵌合基因的改组文库，以确定具有新的结合特异性的PTO变体。3)我们将利用拟南芥中编码相似蛋白的基因对PTO进行改组，以确定能够识别额外无毒蛋白的嵌合体。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做出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，他们还将获得植物-病原体相互作用方面的经验。

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.