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Disruption of the Rice Blast genome to identify genes involved in production of Reactive Oxygen Species

Jessica Pancake: University of Delaware

<div>Rice Blast disease, caused by the fungus <em>Magnaporthe oryzae, </em>results in devastating losses to crops worldwide, especially South Asia. To better understand how to take advantage of basal immunity in rice against <em>M. oryzae, </em>we are performing forward genetic screens. We use a barley-infecting strain of <em>M. oryzae</em>, 4091, that had previously been transformed with a live sensor of reactive oxygen species (ROS) called Hyper-sensor (HS) gene into the fungus, which gives 4091 the ability to fluoresce when in the presence of ROS. We are in the process of randomly disrupting the genome of 4091-HS via random integration of a DNA construct carrying resistance to Hygromycin (Hyg), and double, outward-facing promoters. Our goal is to identify genes that disrupt the process of ROS detection and production by the fungus, by altering the quantifiable level of the HyPer sensor. Such genes could potentially play a role in ROS production, directly linked to basal immunity. Changes in ROS levels will be detected using confocal microscopy, and currently, we have over 500 transformed candidates that have gone through a primary screen. Results will be presented on our screening strategy, the initial screen and some preliminary data of our secondary and confocal screens.</div>