Quantitative Gene Expression Analysis by RT-qPCR

The levels of gene-specific mRNA were assessed using RT-qPCR based on our previous research with some modifications [67 (link)]. Briefly, the total RNA from pre-frozen mycelia was extracted carefully using a FastRNA Pro Red Kit (MPbio, Irvine, CA, USA), according to the manufacturer’s instructions. The extracted RNA (500 ng) was reverse transcribed to cDNA using the PrimeScript RT Reagent Kit with gDNA eraser (TaKaRa Bio, Shiga, Japan), following the manufacturer’s instructions. Quantitative PCR was conducted using the TransStart TipTop Green qPCR SuperMix (TransGen, Shanghai, China) with 200 nM of forward and reverse primers (Additional file 11: Table S8). SYBR green assays along with ABI StepOne thermocycler (Applied Biosystems, Foster City, CA, USA) were used to analyze gene transcriptional levels using the 2^−ΔΔCt method. Gene expression levels were normalized with the reference genes sar1 [64 (link), 68 (link), 69 (link)].