*3.2. RNA Isolation and Real-Time Quantitative PCR*

Total RNA was isolated using RNAiso plus (TaKaRa, Kusatsu, Japan) according to the manufacturer's instructions [37]. The total RNA concentration and purity were detected using a Nano Drop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA; 2.0 < A260/A280 < 2.2). The integrity of RNA was checked by electrophoresis on 1% agarose gel, and the three bands of 28S, 18S and 5S could be clearly observed (Supplementary Figure S1).

Then, 20 μL cDNA was synthesized from 1 μg of total RNA using the HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) kit (Vazyme Biotech, Nanjing, China) according to the manufacturer's instructions. Next, the cDNA was two-fold diluted with double-distilled water and stored at −20 ◦C for quantitative RT-PCR analysis. Specific primers were designed for quantitative RT-PCR analysis of the tested genes, such as *Ggtl,* encoding Leggt (γ-glutamyl transpeptidase); *Csl*, encoding Lecsl (*L. edodes* C-S lyase, *L. edodes* genome Gene ID: LE01Gene02830) and β-actin gene (*Actinl*, encoding *L. edodes* β-actin, *L. edodes* genome Gene ID: LE01Gene01050; Supplementary Table S1) [33].

Quantitative RT-PCR was performed using a CFX Connect real-time PCR system (BIO-RAD). Each reaction consisted of 0.4 μL each of the forward and reverse primers (10 μM), 1 μL of two-fold diluted cDNA, 5 μL of 2 × Taq Master Mix (Vazyme Biotech, Nanjing, China) and 3.2 μL of double-distilled water. The qRT-PCR was performed at 95 ◦C for 3 min, followed by 40 cycles of 95 ◦C for 20 s, 60 ◦C for 30 s, 72 ◦C for 30 s and then maintaining at 72 ◦C for 10 min in a 96-well reaction plate. The specificity and identity of PCR products were verified by melting curve analysis to distinguish specific PCR products from the primer dimmer-caused nonspecific PCR. The existence of a single peak proved each PCR product was specific.

The relative expression was calculated using the 2−ΔΔCT method as previously described [38]. The expression of *Actinl* was used as an internal reference [39]. The expressions during the mycelium stage were taken as control. All PCR experiments were performed in three biological and three technical replications (the maximum difference in Ct was 0.5).
