Quantitative Analysis of Fungal Gene Expression

RNA isolation and reverse transcription quantitative polymerase chain reaction (RT‑qPCR) analyses were performed as described by Zhang et al. (2018 (link)) with some modifications. Briefly, total RNA from mycelia was extracted using the FastRNA Pro Red Kit (MPbio, Irvine, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized from total RNA using the TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech, Beijing, China). For RT-qPCR, the transcriptional levels of cbh1 (encoding cellobiohydrolase I), egl1 (encoding endoglucanase I), xyr1 (encoding the main factor XYR1), ace3 (encoding the main factor ACE3), crz1 (calcineurin-responsive zinc finger transcription factor 1, Trire2:36391), sod1 (copper/zinc superoxide dismutase, Trire2:123029), and cat1 (catalase, Trire2:70600) were analyzed using PerfectStart™ Green qPCR SuperMix (TransGen Biotech). The 2^−ΔΔCt method was used for calculations (Livak and Schmittgen 2001 (link)). The sar1 gene was used as an internal reference to normalize the data. The primers used for RT-qPCR are described in Additional file 2: Table S1.