Collaborative Research: Extracellular DNA: in Defense of Plant Cells

新出现的数据显示，在其他防御策略中，动物免疫系统在细胞外陷阱中使用DNA，使微生物病原体失效。此外，作为对策，已经发现病原体分泌降解细胞外DNA（exDNA）的酶并有助于它们的毒力。来自主要研究者的大量初步数据表明，植物也使用exDNA，通过释放exDNA作为吸引和固定病原微生物的陷阱的一部分来保护它们的根部。该项目的主要目标是表征这种exDNA，并确定其与由两种不同的根感染植物病原体分泌的DNA降解酶（exDNases）的相互作用。该项目将测试一种新的植物免疫模型，该模型假设exDNA是成功的病原体必须克服的关键防御成分。该模型认为，含有基质的exDNA通过防御相关蛋白和其他代谢产物捕获并中和病原微生物。如在动物病原体的情况下，有毒植物病原体分泌exDNA酶，其降解位于根和其它植物组织中的exDNA。要解决的问题包括：植物exDNA的性质是什么，它是如何释放的？是什么病原体信号触发了这种植物防御？是什么样的适应性使根攻击植物病原体能够对抗这种防御？作为植物免疫系统的一个新发现的组成部分，从这项研究中获得的见解exDNA在防御中的作用机制应该为开发病原体抗性作物提供新的策略。这个项目将解决这些问题，在三个独立的，但概念上相关的目标：目标1：外DNA在植物防御的表征。实验目标包括分析exDNA的序列和递送，以及这些过程在防御病原体中的作用;目的2：细菌exDNase在植物致病中的作用。与现有的exDNase突变体的细菌性枯萎病病原体青枯菌，研究人员将测量exDNase的毒力的生物学影响，并表征这些酶的调节和特性，包括底物特异性，反应动力学和化学要求的最佳活动。目的3：真菌胞外DNA酶在植物致病中的作用。使用易处理的玉米病原体模型Cochliobolus heterostrophus的exDNase突变体，将表征exDNase基因敲除对毒力的生物学效应，并且研究者将确定发现在毒力中起作用的病原体exDNases的生化特性。研究人员将植物根，根边缘细胞和exDNA的深厚专业知识与具有共同目标的遗传多样性微生物的专业化结合起来：植物根。拟议的研究整合了植物和植物病原体动力学，以建立一个新的防御-反防御过程的多维理解。该项目更广泛的影响包括为大学生，特别是妇女和少数民族，提供结构化的独立研究培训，并开展实践活动，展示exDNA在微生物捕获中的作用。这些将部署在各种社区论坛的K-12学生和他们的家庭。

Emerging data have revealed that among other defense strategies animal immune systems employ DNA in extracellular traps that disable microbial pathogens. Moreover, as a countermeasure pathogens have been found to secrete enzymes that degrade extracellular DNA (exDNA) and contribute to their virulence. Extensive preliminary data from the principal investigator demonstrate that plants also employ exDNA, defending their roots by releasing exDNA as part of a trap that attracts and immobilizes pathogenic microbes. The primary goal of this project is to characterize this exDNA and define its interaction with the DNA degrading enzymes (exDNases) secreted by two distinct root-infecting plant pathogens. This project will test a new model for plant immunity that hypothesizes exDNA to be a key defensive component that successful pathogens must overcome. The model contends that an exDNA containing matrix traps and neutralizes pathogenic microbes via defense-related proteins and other metabolites. As in the case of animal pathogens, virulent plant pathogens secrete exDNases that degrade the exDNA located in roots and other plant tissues. Questions to be addressed include: What is the nature of the plant exDNA and how is it released? What pathogen signals trigger this plant defense? What adaptations enable root-attacking plant pathogens to counter this defense? As a newly discovered component of the plant immune system, the insights gained from this research into the mechanisms underlying the role of exDNA in defense should provide novel strategies for developing pathogen resistant crops. This project will address these questions in three independent but conceptually related objectives: Objective 1: Characterization of exDNA in plant defense. Experimental goals include analysis of the sequence and delivery of exDNA, and the roles of these processes in defense against pathogens; Objective 2: Role of bacterial exDNase in plant pathogenesis. Working with existing exDNase mutants of the bacterial wilt pathogen Ralstonia solanacearum, investigators will measure the biological impact of exDNase on virulence, and characterize the regulation and properties of these enzymes, including substrate specificity, reaction kinetics, and chemical requirements for optimal activities. Objective 3: Role of fungal exDNAse in plant pathogenesis. Using exDNase mutants of Cochliobolus heterostrophus, a tractable model corn pathogen, the biological effects of exDNase gene knockouts on virulence will be characterized, and the investigators will determine the biochemical properties of pathogen exDNases found to play a role in virulence. The investigators unite deep expertise on plant roots, root border cells, and exDNA with specialization in phylogenetically diverse microbes that share a common target: the plant root. The proposed research integrates plant and plant pathogen dynamics to build a multidimensional understanding of a novel defense-counter-defense process. The project's broader impacts include structured independent research training for undergraduates, particularly women and minorities, and development of hands-on activities demonstrating the role exDNA in microbial trapping. These will be deployed in a variety of community forums for K-12 students and their families.