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.