MAP kinase Slt2 orthologs play similar roles in conidiation, trap formation, and pathogenicity in two nematode-trapping fungi.

Mitogen-activated protein (MAP) kinase Slt2 is a key player in the cell-wall integrity pathway of budding yeast. In this study, we functionally characterized Slt2 orthologs AoSlt2 and MhSlt2 from the nematode-trapping fungi Arthrobotrys oligospora and Monacrosporium haptotylum, respectively. We found that disruption of AoSlt2 and MhSlt2 led to reduced mycelial growth, increased sensitivity to environmental stresses such as sodium dodecyl sulfate, Congo red, and H2O2, and an inability to produce conidia and nematode-trapping structures. Real-time polymerase chain reaction-based analyses showed that the transcription of sporulation-related (AbaA, Sep2, and MedA) and cell wall synthesis-related (Chs, Glu, and Gfpa) genes was down-regulated in the mutants compared with the wild-type strains. Moreover, the mutant strains showed reduced extracellular proteolytic activity and decreased transcription of three homologous serine protease-encoding genes. These results show for the first time that MAP kinase Slt2 orthologs play similar roles in regulating mycelial growth, conidiation, trap formation, stress resistance, and pathogenicity in the divergent nematode-trapping fungal species A. oligospora and M. haptotylum.

Random mutagenesis analysis and identification of a novel C2H2-type transcription factor from the nematode-trapping fungus Arthrobotrys oligospora.

Arthrobotrys oligospora is a typical nematode-trapping fungus. In this study, 37 transformants of A. oligospora were obtained by REMI (restriction enzyme mediated integration) method and phenotypic properties of nine transformants were analyzed. The nine transformants showed differences in growth, conidiation, trap formation, stress tolerance, and/or pathogenicity among each other and with those of the parental wild-type strain (WT). The insertional sites of the hph cassette were identified in transformants X5 and X13. In X5, the cassette was inserted in the non-coding region between AOL_s00076g273 (76g273) and AOL_s00076g274 (76g274) and the transcription of 76g274, but not 76g273, was enhanced in X5. 76g274p had two conserved domains and was predicted as a nucleoprotein, which we confirmed by its nuclear localization in Saccharomyces cerevisiae using the green fluorescent protein-fused 76g274p. The transcription of 76g274 was stimulated or inhibited by several environmental factors. The sporulation yields of 76g274-deficient mutants were decreased by 70%, and transcription of several sporulation-related genes was severely diminished compared to the WT during the conidiation. In summary, a method for screening mutants was established in A. oligospora and using the method, we identified a novel C2H2-type transcription factor that positively regulates the conidiation of A. oligospora.