An imaging-based approach to aid in the identification of fungal genes regulating the Reactive Oxygen Species infection response in rice and maize

Timothy Chaya 1,3 Jessica Cooper 2 Jonathan Neifert 2 Danielle Mikolajewski 2 Nicole Donofrio 2 Jeffrey Caplan 1,3
1Department of Biology, University of Delaware, Newark, DE, USA
2Department of Plant and Soil Sciences, University of Delaware, Newark, DE, USA
3Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA

Reactive oxygen species (ROS) have been shown to be vital for plant responses to fungal infection as well as plant developmental signals. The ability of a fungus to withstand ROS that is produced both internally and externally by a plant’s defense response is key to a successful invasion. In collaboration with the Horwitz lab, we aim to explore the mechanisms of ROS creation and responses in the hemi-biotroph Magnaporthe oryzae and the necrotroph Cochilobolus heterostrophus, which cause rice blast and Southern Leaf Blight, respectively. The infection cycle of both is being analyzed with advanced, three-dimensional (3D) confocal microscopy and image analysis to characterize the progression of these pathogens. Our preliminary 3D data was acquired by using the ScaleP technique to clear barley and rice leaves inoculated with M. oryzae. We captured data over a fourth dimension (4D), time, to show pathogenesis progression from germination to full infection into the host tissue. We also will utilize a genetically-encoded ROS sensor, called HyPer, in both M. oryzae and C. heterostrophus coupled with various ROS staining techniques in the host tissue. A time course of the HyPer sensor in M. oryzae (MoHyPer) shows the increase in ROS production through the early infection stages on the leaf surface. These tools will allow us to quantify the ROS response from both perspectives and make a comparative analysis between ROS levels and time of infection in these two distinct pathosystems. From these data we will determine the optimal time in pathogenesis to conduct a forward genetic screen to generate a mutant library of ROS-related mutants. These mutants will then be sequenced to determine the genes that have altered the ROS response. The identified genes will further elucidate the ROS infection response and could be potential targets for novel crop defense strategies or antifungals.