A two-pronged forward genetics approach to identify Magnaporthe oryzae genes that activate reactive oxygen species in rice

Nicole Donofrio 1 Jessica Cooper Pancake 1 Danielle Mikolajewski 1 Jonathon Neifert 1 Timothy Chaya 2 James Sweigard 3 Jeffrey Caplan 4
1Plant and Soil Sciences, University of Delaware, Newark, Delaware, USA
2Biological Sciences, University of Delaware, Newark, Delaware, USA
3DuPont, Stine-Haskell, Newark, Delaware, USA
4Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, USA

Rice Blast disease, caused by the fungus Magnaporthe oryzae, results in devastating loss to crops worldwide, including economically important rice and barley. Research from other labs repeatedly demonstrates this pathogen’s ability to overcome single gene resistance, as well as fungicide applications. Using forward genetics, we seek to further understand and harness host plant basal immunity for better protection against virulent pathogens. Our aim is to identify M. oryzae genes involved in sensing or detoxifying reactive oxygen species (ROS) from the plant. Previous work from our lab characterized the HYR1 gene, which in the deletion mutant triggers a strong, plant-produced ROS burst and lowered susceptibility. Our hypothesis is that a two-pronged forward genetic approach will identify additional fungal genes with this phenotype. We are currently screening ~50,000 random insertion mutant library generated in the early 2000’s (FGSC-m), and a library we are developing (HyPer-m). The HyPer-m library is being developed in a fungal strain that contains a live ROS sensor gene, HyPer-sensor (HS). The HS gene fluoresces in the presence of ROS, thus providing a quantifiable measurement of this basal defense response. Our insertion construct consists of a gene for resistance to the drug Hygromycin, surrounded by two outward facing fungal promoters, to increase chances of maximum gene disruption. We have currently screened about 4,000 and 600 mutants from the FGSC-m and HyPer-m libraries, respectively, and results will be shared.