A Pmk1-Interacting Gene Is Involved in Appressorium Differentiation and Plant Infection in Magnaporthe oryzae

In the rice blast fungus Magnaporthe oryzae, the PMK1 mitogen-activated protein (MAP) kinase gene regulates appressorium formation and infectious growth. Its homologs in many other fungi also play critical roles in fungal development and pathogenicity. However, the targets of this important MAP kinase and its interacting genes are not well characterized. In this study, we constructed two yeast two-hybrid libraries of M. oryzae and screened for Pmk1-interacting proteins. Among the nine Pmk1-interacting clones (PICs) identified, two of them, PIC1 and PIC5, were selected for further characterization. Pic1 has one putative nuclear localization signal and one putative MAP kinase phosphorylation site. Pic5 contains one transmembrane domain and two functionally unknown CTNS (cystinosin/ERS1p repeat) motifs. The interaction of Pmk1 with Pic1 or Pic5 was confirmed by coimmunoprecipitation assays. Targeted gene deletion of PIC1 had no apparent effects on vegetative growth and pathogenicity but resulted in a significant reduction in conidiation and abnormal germ tube differentiation on onion epidermal cells. Deletion of PIC5 led to a reduction in conidiation and hyphal growth. Autolysis of aerial hyphae became visible in cultures older than 4 days. The pic5 mutant was defective in germ tube growth and appressorium differentiation. It was reduced in appressorial penetration and virulence on the plant. Both PIC1 and PIC5 are conserved in filamentous ascomycetes, but none of their orthologs have been functionally characterized. Our data indicate that PIC5 is a novel virulence factor involved in appressorium differentiation and pathogenesis in M. oryzae.