ABSTRACTThe cell wall integrity signaling (CWIS) pathway is involved in fungal cell wall biogenesis. This pathway is composed of sensor proteins, protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) pathway, and it controls the transcription of many cell wall-related genes. PKC plays a pivotal role in this pathway; deficiencies in PkcA in the model filamentous fungusAspergillus nidulansand in MgPkc1p in the rice blast fungusMagnaporthe griseaare lethal. This suggests that PKC in filamentous fungi is a potential target for antifungal agents. In the present study, to search for MgPkc1p inhibitors, we carried outin silicoscreening by three-dimensional (3D) structural modeling and performed growth inhibition tests forM. griseaon agar plates. From approximately 800,000 candidate compounds, we selected Z-705 and evaluated its inhibitory activity against chimeric PKC expressed inSaccharomyces cerevisiaecells in which the kinase domain of nativeS. cerevisiaePKC was replaced with those of PKCs of filamentous fungi. Transcriptional analysis ofMLP1, which encodes a downstream factor of PKC inS. cerevisiae, and phosphorylation analysis of the mitogen-activated protein kinase (MAPK) Mpk1p, which is activated downstream of PKC, revealed that Z-705 specifically inhibited PKCs of filamentous fungi. Moreover, the inhibitory activity of Z-705 was similar to that of a well-known PKC inhibitor, staurosporine. Interestingly, Z-705 inhibited melanization induced by cell wall stress inM. grisea. We discuss the relationships between PKC and melanin biosynthesis.IMPORTANCEA candidate inhibitor of filamentous fungal protein kinase C (PKC), Z-705, was identified byin silicoscreening. A screening system to evaluate the effects of fungal PKC inhibitors was constructed inSaccharomyces cerevisiae. Using this system, we found that Z-705 is highly selective for filamentous fungal PKC in comparison withS. cerevisiaePKC. Analysis of theAGS1mRNA level, which is regulated by Mps1p mitogen-activated protein kinase (MAPK) via PKC, in the rice blast fungusMagnaporthe grisearevealed that Z-705 had a PKC inhibitory effect comparable to that of staurosporine. Micafungin induced hyphal melanization inM. grisea, and this melanization, which is required for pathogenicity ofM. grisea, was inhibited by PKC inhibition by both Z-705 and staurosporine. The mRNA levels of4HNR,3HNR, andSCD1, which are essential for melanization inM. grisea, were suppressed by both PKC inhibitors.
Protein kinase
C
is essential for viability of the rice blast fungus
M
agnaporthe oryzae