Further characterization of MAPK pathways in the rice blast fungus

In the rice blast fungus, Magnaporthe oryzae, three mitogen-activated protein kinase (MAPK) pathways are widely involved in pathogenesis and stress responses. The PMK1 MAPK pathway regulates appressorium formation and invasive growth and the MPS1 MAPK pathway is required for the maintenance of cell wall integrity as well as plant infection. The third MAPK, OSM1, is involved in osmotic stress response. Mst50 is an adaptor protein in the PMK1 pathway and essential for appressorium formation. An affinity approach was used to identify proteins that interacted with Mst50 in vivo. The results indicated that Mst50 regulated the MPS1 MAPK pathway by interacting with Mps1 upstream activators Mck1 and Mkk2. Mst50 is also involved in osmotic regulation by interacting with the osmosensor Hik1 in the OSM1 MAPK pathway. Mst12 is one of the transcription factors functioning downstream from Pmk1 that is required for appressorium penetration and invasive growth. The interaction between Mst12 and Pmk1 was shown by protein co-immunoprecipitation assays. Site-directed mutagenesis of Mst12 revealed that the MAPK-docking region and MAPK- phosphorylation site were essential for the function of MST12. However, the protein kinase A- phosphorylation site of Mst12 was dispensable for plant infection. Mutation either of the two tandem zinc finger domains of Mst12 reduced fungal virulence. Expression of MST12 functionally rescued the invasive growth defects of the yeast ste12 mutant, and electrophoretic mobility shift assay (EMSA) analysis suggested that Mst12 binds to a sequence similar to the PRE sequence (TGAAACA) recognized by yeast Ste12. RNA-seq was used to compare transcriptional profiles between the wild-type strain and the mst12 mutant at the appressorium stage. PRE-like sequence was found in the promoters of 52 genes that were down-regulated in the mst12 mutant. Five genes with two or three putative PREs in their promoters were selected for functional characterization. Gene mutagenesis revealed that MDG6, which encoded a novel zinc finger domain-containing protein, was involved in plant infection. The conclusions drawn from this study provided better understanding of how Mst50 was involved in multiple MAPK pathway regulation, and this study also identified a novel gene that was regulated by Mst12 in M. oryzae