Assays for MAP Kinase Activation in Magnaporthe oryzae and Other Plant Pathogenic Fungi

Mitogen-activated protein (MAP) kinases have the hallmark motif TXY and function in key signal transduction pathways in eukaryotic organisms. Most ascogenous plant pathogenic fungi have three MAPK pathways that regulate different developmental and infection processes. In the rice blast fungus Magnaporthe oryzae, the Pmk1 and Mps1 MAP kinases with the TEY motif are essential for appressorium formation, penetration, and invasive growth. Osm1 is the third MAP kinase that has the TGY motif and functions in osmoregulation. Although orthologs of Pmk1 and Mps1 are important for pathogenesis in all the plant pathogens studied, Osm1 orthologs have species-specific roles in stress responses and pathogenesis. Because of their functions in fungal development and pathogenesis, it is important to determine the expression and activation of MAP kinases under different growth conditions or infection stages. In this chapter, we describe methods for protein extraction and detection of the activation of the three MAP kinases in M. oryzae with the commercially available anti-TpEY or anti-TpGY phosphorylation-specific antibodies. Similar approaches can be used to monitor MAP kinase activation in other plant pathogenic fungi.