Analysis of reactive oxygen species kinetics in pathogenic fungi of barley and maize using the HyPer sensor
Date
2019
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Publisher
University of Delaware
Abstract
Magnaporthe oryzae and Cochliobolus heterostrophus represent two important fungal pathogens that pose a threat to global food production. The hemi-biotroph M. oryzae has a broad host range with the ability to infect rice, barley, millet, and other grasses. C. heterostrophus on the other hand is a necrotroph with a host range of maize, soybeans, millet, wheat and teosinte. The fungal infection in host plant tissue is a complex process that is in part defined by regulation of Reactive Oxygen Species (ROS). It is well established that plants use ROS as a key component of innate immunity, growth and development. Analogously, fungi need ROS for development and production of necessary infection structures. In both organisms, ROS production must be carefully regulated to prevent deleterious effects. This research is exploring the ROS dynamics in both fungal systems. It is hypothesized that these pathogens will utilize and regulate ROS in a different manner during infection. ☐ To aid in the understanding of this process, I am utilizing a genetically encoded reporter for hydrogen peroxide called HyPer. Three different microscopy approaches for ROS kinetics were conducted. The first was an in-situ analysis of conidia in a perfusion chamber, characterizing the response kinetics of each fungus when treated with oxidative (hydrogen peroxide) or reductive (dithiothreitol) solutions. The second approach was imaging in planta ROS levels through select infection stages. The last approach was the generation of a high throughput imaging workflow to analyze HyPer sensor mutants in future forward genetic screens. With a baseline of how each fungus responds to ROS, we plan to analyze HyPer mutant libraries under development for forward genetic screens, to identify genes related to ROS generation and attenuation. These genes can provide targets for the development of new fungicides or breeding of more resistant plants.