Transcriptional Analysis of SA Signaling Pathway

The samples of mycelia growing on the surface of the substrate under four treatments and the control were collected 3 h after the treatments (referring to the results of Han et al., 2022 (link)) for the determination of transcriptional levels of SA signaling pathway genes by RT-qPCR. All the samples were collected in equal mass (0.5 g) and frozen in liquid nitrogen for RNA extraction. Total RNA was extracted according to the standard method described in the instructions of the Omega E.Z.N.A. plant RNA kit (Omega Bio-Tek, USA). When the A260/A280 ratio of RNA is 2.0 to 2.1 (measured using Thermo Science NanoDrop Lite; USA) and the concentration is diluted to 500 ng/μl, it is used for cDNA synthesis. The cDNA was synthesized by reverse transcription using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit (TransGen, China). Three internal reference genes stably expressed in F. filiformis (RNB, V-ATP, and β-TUB) were used for the standardization of RT-qPCR (Yang et al., 2022 (link)). All the primers for RT-qPCR are shown in Supplementary Table S1. The RT-qPCR was performed according to the standard method described in the instructions of PerfectStart Uni RT and qPCR Kit (TransGen Biotech, Beijing). The relative gene expression levels were determined according to the 2^−ΔΔCt method.