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Danielle Mikolajewski (Plant and Soil Sciences) was advised by Dr. Nicole Donofrio (Plant and Soil Sciences) on her project 鈥淒isruption of the Genome of Rice Blast to Identify Genes Involved in Production of Reactive Oxygen Species.鈥�

Rice Blast disease, caused by the fungus Magnaporthe oryzae, causes severe loss to crops worldwide, especially in South Asia. It is a virulent pathogen, which can cause complete devastation when conditions are favorable. To better understand how to take advantage of basal immunity, a mechanism by which plants can protect themselves even against virulent pathogens, we are performing forward genetic screens with rice and M. oryzae. Previous work in our lab used a live sensor gene called Hyper-sensor (HS) to measure detection of reactive oxygen species (ROS) in a well-characterized strain of M. oryzae 4091. ROS is a major contributor to a plant鈥檚 basal immune system. The HS gene gives 4091 the ability to fluoresce when in the presence of ROS, thus providing a quantifiable measurement of ROS. We are in the process of randomly disrupting the genome of 4091-HS by transformation with a DNA insert constructed with a gene for resistance to the drug Hygromycin (Hyg). This construct has two degenerate ends so that it will generate random insertions in the genome, disrupting as many genes as possible. This ability to bind anywhere could disrupt genes that play a role in ROS production in the fungus, or triggering and detection of ROS in the plant, potentially identifying genes directly linked to basal immunity. Through screening the randomly mutagenized library of 4091-HS, we will be able to identify changes in ROS using confocal microscopy. Currently we have over 1100 transformed candidates that have been characterized through a primary and secondary screen whose results will be discussed and 300 mutants waiting further analysis. Results will be presented on our screening strategy and some preliminary data from a similar screen on a previously mutagenized population.