Cyclocybe aegerita dikaryotic strain YSG deposited in our laboratory was used in the transformation experiments. The mycelia were cultured at 25°C on a yeast extract peptone dextrose (YPD) medium plate (0.2% yeast extract, 0.2% peptone, 2% dextrose, and 1.5% agar) followed by culturing in the sawdust medium (64% hardwood sawdust, 15% wheat bran, 1% gypsum, and 20% cottonseed shells in 50% water) to produce fruiting bodies. The protoplast regeneration medium contained 205 g of sucrose, 2 g of tryptone, 2 g of yeast extract, and 10 g of agar per liter of dH₂O. The DH5α strain of E. coli, to be used for plasmid amplification, was cultured at 37°C in Luria–Bertani (LB) medium containing 100 μg/ml of ampicillin. In order to conduct the growth rate test, equal amounts of mycelia were inoculated in both solid and liquid YPD media for the evaluation of colony diameter and biomass, respectively.

Cyclocybe aegerita YSG strain was cultured in YPD medium at 25°C in the dark for 8 days. The genomic DNA was extracted from the mycelia obtained from this culture of C. aegerita using the Fungal gDNA Isolation Kit (Biomiga, United States). Total RNA was extracted from the fungus using the RNAiso Plus in accordance with the manufacturer’s instruction (TaKaRa, Dalian, China). The complete genomic DNA and cDNA products of the cagch1 gene were amplified in a polymerase chain reaction (PCR) using the primer pair gch1F and gch1R based on the genomic information of C. aegerita monokaryotic strain YSG. The amplified products/fragments were then cloned into the pMD19-T vector (TaKaRa, Dalian, China) and sent for sequencing (Tsingke Biotechnology Co., Ltd.).

The transformation vector pAGH (given by Dr. Zhang, Kunming University of Science and Technology) was used for constructing the RNAi vector using the method described in a previous report (Zhang et al., 2021). The plasmid pAGH comprised the C. aegerita actin promoter, the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter, the CaMV35S terminator, the hygromycin B phosphotransferase gene, and the gpd promoter from Ustilago maydis. A 487-bp antisense fragment, amplified using primer Gch1-1F and Gch1-1R (Supplementary Table 1), of the cagch1 cDNA was also inserted into the EcoRV enzyme site. All fragments were sequentially ligated into the pAGH vector using the ClonExpress MultiS One Step Cloning Kit (Vazyme, Nanjing, China) in accordance with the manufacturer’s instructions.

The transformation was performed by following the PEG-CaCl₂ protocol described in previous reports (Tao et al., 2020) with a few modifications. In brief, the protoplasts were prepared by digesting the C. aegerita mycelia from the liquid culture with a lysing enzyme solution containing 1% cellulase R-10 (Japan), 1% Lywallzyme, and 1% lysing enzymes from Trichoderma harzianum (Sigma-Aldrich) in 0.6 M mannitol buffer as the osmotic stabilizer. The obtained protoplasts were washed in the STC solution [1.2 M sorbitol, 10 mM CaCl2, and 50 mM Tris–HCl (pH 7.0)] and adjusted to a final concentration of 1 × 10⁸ protoplasts ml^–1. Subsequently, a volume of 100 μl of protoplasts was mixed with 1 μg of plasmid DNA digested with the KpnI enzyme and the reaction was allowed to occur for 30 min on ice. Afterward, 1 ml of the PTC solution (40% w/v polyethylene glycol 4,000, 60 mM CaCl2, and 1.2 M sorbitol) was added to the mixture, followed by incubation for 30 min at room temperature. Finally, the cells were added to 10 ml of molten YPD regeneration medium and then poured on plates. After 5 days of incubation at 25°C, 10 mL of molten YPD medium containing 150 μg/ml hygromycin B was poured over the plates for the screening of transformants.

The colonies resistant to hygromycin B were verified using the primer pair adhF and adhR targeted to the flanking region of the inserted fragment. Quantitative PCR (qPCR) was performed in the CFX96 Real-Time PCR detection system (Bio-Rad, Hercules, CA, United States). The PCR reaction was performed in a total volume of 20 μL using the iTaq™ Universal SYBR Green Supermix in accordance with the manufacturer’s instructions (Bio-Rad). The thermal cycling parameters for the PCR reactions were as follows: 20 s at 95°C for initial polymerase activation, followed by 40 cycles of 5 s at 95°C, and 30 s at 60°C. The primers used are listed in Supplementary Table 1. Amplification of each sample was performed in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GPD) was used as the reference gene for the normalization of the qPCR data. The relative expression levels were calculated using the 2^–ΔΔCT method.

Total RNA was isolated from the mature YSG fruiting bodies as well as the transformants T5, T10, and T11. The corresponding transcriptomic data were obtained from six cDNA libraries sequenced on an Illumina sequencing platform at the Beijing Genome Institute (BGI, Shenzhen, China). The differentially expressed genes (DEGs) were classified into GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

On the basis of the genome annotation and sequence analysis, gDNA and cDNA were employed as templates for the amplification of the gene encoding CaGCH1 (the cagch1 gene), and the amplification products were sequenced and analyzed. The gDNA sequence of cagch1 was 1,067 bp in length and contained 3 introns and 4 exons (Accession Number OL303985). The cDNA of cagch1 was 894 bp in length and contained 297 amino acids (Figure 1A).

The protein sequences were aligned using ClustalW (soft package included in Mega 6) (Tamura et al., 2013) and the result revealed that the CaGCH1 protein shared a high level of similarity with the GCH1 proteins of C. aegerita (Accession Number CAA7260263), Cyclocybe pediades (Accession Number KAF9569406), and S. cerevisiae (Accession Number P51601). All four proteins contained seven conserved regions (C1–7) and three Zn²⁺ binding residues (Figure 1B). The phylogenetic analysis of the GCH1 protein sequences was performed with MEGA6 software using neighbor-joining, minimum evolution (Tamura et al., 2013), which indicated that CaGCH1 was the closest to that of the Cyclocybe species (previously used genus name Agrocybe) (Figure 1C).

In order to study the effect of cagch1 on the developmental process, the silence transformants were obtained by constructing a dual RNAi plasmid (Figure 2A). After the screening of transformants on the YPD medium containing hygromycin B, the specific primer pair adhF and adhR targeted to the actin and gpd promoter regions was used for confirming the positive transformants through PCR amplification (Figure 2B). Finally, correct PCR products were detected in three strains T5, T10, and T11 (Figure 2C).

Mycelia were collected from WT, T5, T10, and T11 and subjected to total RNA extraction. The cDNA obtained after the reverse transcription of the extracted RNA was used for quantifying the expression of cagch1. It was observed that the expression level of cagch1 in all three transformants had decreased to a low level – approximately 17.63% in T5, 13.87% in T10, and 13.53% in T11 compared to that in the WT strain (Figure 2D).

After 7 days of culture on YPD plates, the transformants colonies appeared denser with smooth edges than WT colonies (Figure 3A). The colony diameter was measured for growth rate analysis, and it was observed that the WT strains spread faster than the transformants (Figure 3D). The microscopic observation revealed that transformants exhibited relatively greater branching in the hyphal tips growing on the cover glass (Figure 3B).

The equal mycelium block was cultured in the liquid YPD medium under shaking conditions of 180 rpm/min for 7 days (Figure 3C). It was observed that the mycelial biomass of all three transformants was decreased significantly compared to the WT strain, with the mycelium weight of the T5 strain approximately one-half that of the WT strain (Figure 3E).

The WT strain and the three mutant strains were cultured in sawdust bags at 25°C for 40 days to investigate the potential effect of cagch1 on the fruiting bodies of C. aegerita. The development of the transformant strains was observed to be blocked, and there were bottle-shaped stipes (Figure 4A). The longitudinal section of young fruiting bodies revealed that the stipes of the mutant strains comprised flocculent flesh relative to the compact flesh of the WT strain (Figure 4B). At the late stage, the stipe elongation in the mutant strain was significantly inhibited, and the length and the weight of fresh fruiting bodies were approximately one-sixth and one-fifteenth that of the wild type, respectively (Figures 4C–E). Moreover, the morphologies of all strains were stable in the subsequent two culture processes.

Furthermore, the expression level of cagch1 in the mature fruiting bodies was evaluated using the qPCR method. As depicted in Figure 5, the expression level of cagch1 in the three transformants exhibited different degrees of decrease—approximately 21.83% in T5, 23% in T10, and 48% in T11 compared to that in the WT strain (Figure 5).

In order to evaluate the effect of cagch1 knockdown on the expression levels of different genes, the expression difference in the mating bodies of WT was compared to those of each of the three transgenic strains using the transcriptional profile analysis (PRJNA813015).

A total of 11,723 differentially expressed unigenes (DEGs) were identified, including 211 and 218 special genes in the WT fruiting bodies and in the mutant fruiting bodies, respectively (Figure 6A). Furthermore, the 544 DEGs involved in the KEGG metabolic pathway were categorized into five major types comprising 20 categories, among which a few pathways were associated with mycelial growth and development, such as cell wall remodeling in signal transduction pathway and polysaccharide anabolism pathway (Figure 6B). Among these 544 genes, most annotated in the MAPK pathway (Figure 6C) and upregulated in the mutant strains were possibly involved in cell wall remodeling and the mating process (Figure 6D).