Routine Cultivation and Genetic Analysis of N. crassa

Routine cultivation of N. crassa was performed on Vogel’s minimal medium with 2% sucrose (plus 1.5% agar for solid medium). Asexual spores were obtained by growing strains in glass tubes with slanted medium for one week until full sporulation was evident. Mutant strains were obtained from the Fungal Genetics Stock Center [7 (link)]. The genomic sequence of NCU09974 in the acr-3 mutant was obtained using a routine sequencing methodology [12 (link)]. Radial growth and spot assays were conducted as previously described [10 (link)]. Staurosporine was obtained from LC Laboratories and acriflavine and malachite green from Sigma-Aldrich. For quantitative real-time PCR experiments (qRT-PCR), asexual spores at a concentration of 10⁶ cells/mL were grown at 26 °C in liquid VMM for 7 h; RNA was then isolated using the ZR Fungal/Bacterial RNA MicroPrep kit (Zymo Research), used for cDNA preparation using the SuperScript First-Strand Synthesis System kit (Life Technologies) and the relative expression of abc-3 in different strains using actin (NCU04173) as the reference gene was obtained using the 2^−ΔΔCt calculation method from mixes containing previously described primers [13 (link)] and SYBR Green (Bio-Rad) that were analyzed in a Corbett Research Rotor-Gene 6000 thermocycler. Statistical analysis of qRT-PCR results was conducted on Prism 6 software (Graphpad).