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

Abstract

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.

Peroxin Pex14/17 Is Required for Trap Formation, and Plays Pleiotropic Roles in Mycelial Development, Stress Response, and Secondary Metabolism in Arthrobotrys oligospora

“…The plasmid pCSN44 containing the selective marker hygromycin resistance gene ( hph ) was stored in the Escherichia coli strain DH5α (TaKaRa, Shiga, Japan), and the knockout vector was created using the plasmid PRS426 ( 48 ). As previously mentioned, S. cerevisiae (FY834) was cultivated in yeast extract peptone dextrose (YPD) medium for the recombinational cloning technique ( 49 ). To investigate mycelial development and associated phenotypic characteristics, the following media were used: TYGA (10 g tryptone, 5 g yeast extract, 10 g glucose, and 5 g molasses per 1 L), TG (10 g tryptone and 10 g glucose per 1 L), and CMY (20 g maizena [corn starch] and 5 g yeast extract per 1 L); additionally, 20 g agar should be added per 1 L of the above media ( 50 ).…”

Involvement of AoMdr1 in the Regulation of the Fluconazole Resistance, Mycelial Fusion, Conidiation, and Trap Formation of Arthrobotrys oligospora

“…A wild-type (WT) strain of A. oligospora (ATCC24927) and mutant strains were maintained in a potato dextrose agar (PDA) medium at 28 • C. The pCSN44 plasmid was used for the amplification of the hygromycin resistance gene (hph) preserved in Escherichia coli strain DH5a (Takara, Dalian, China); the pRS426 plasmid was used for the construction of the knockout vector [27]. The FY834 strain of Saccharomyces cerevisiae was used to construct the homologous recombinant vector for the knockout of the Aomdr1 gene, which was cultured in yeast extract-potato dextrose (YPD) (10 g/L yeast extract, 20 g/L peptone, and 20 g/L dextrose) [28]. The FY834 strain with the correct vector was selected using an SC-Ura medium (2 g/L synthetic drop-out mix minus uracil without a yeast nitrogen base, 26.7 g/L drop-out base with glucose, and 20 g/L agar).…”

Methylglyoxal Has Different Impacts on the Fungistatic Roles of Ammonia and Benzaldehyde, and Lactoylglutathione Lyase Is Necessary for the Resistance of Arthrobotrys oligospora to Soil Fungistasis

“…Its genome, containing a 40.07-Mb assembled sequence, was reported by our lab in 2011 (Yang et al, 2011). Plasmid pCSN44 was stored in Escherichia coli strain DH5а, and used to amplify the hygromycin B resistance gene hph, which is a selection marker for a gene knockout (Jiang et al, 2017;Zhang et al, 2019). Plasmid pRS426 was used as a backbone plasmid for constructing a gene knockout plasmid, and Saccharomyces cerevisiae FY834, a uracil auxotrophic strain, was used as a host strain for recombinational cloning procedures (Xie et al, 2019;Xie et al, 2020).…”