Carbon dioxide is commonly used as one of the significant environmental factors to control pileus expansion during mushroom cultivation. However, the pileus expansion mechanism related to CO₂ is still unknown. In this study, the young fruiting bodies of a popular commercial mushroom Flammulina filiformis were cultivated under different CO₂ concentrations. In comparison to the low CO₂ concentration (0.05%), the pileus expansion rates were significantly lower under a high CO₂ concentration (5%). Transcriptome data showed that the up-regulated genes enriched in high CO₂ concentration treatments mainly associated with metabolism processes indicated that the cell metabolism processes were active under high CO₂ conditions. However, the gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with cell division processes contained down-regulated genes at both 12 h and 36 h under a high concentration of CO₂. Transcriptome and qRT-PCR analyses demonstrated that a high CO₂ concentration had an adverse effect on gene expression of the ubiquitin–proteasome system and cell cycle–yeast pathway, which may decrease the cell division ability and exhibit an inhibitory effect on early pileus expansion. Our research reveals the molecular mechanism of inhibition effects on early pileus expansion by elevated CO₂, which could provide a theoretical basis for a CO₂ management strategy in mushroom cultivation.

Flammulina filiformis is a popular mushroom which has been regarded as a potential model fungus for mycelium growth, fruiting body development, and stress response studies. Based on a genome-wide search, four genes encoding heterotrimeric G protein α subunits were identified in F. filiformis. The data of conserved domain analysis showed that these genes contain only one subgroup I of Gα subunit (Gαi), similar to many other fungi. To explore the function of Gαi, FfGa1 over-expression (OE) and RNA interference (RNAi) strains were generated using the Agrobacterium tumefaciens-mediated transformation (ATMT) approach. RNAi transformant strains showed remarkably reduced growth on PDA medium and added sensitivity to cell wall-enforcing agents with maximum growth inhibition, but showed better growth in response to hypertonic stress-causing agents, while OE strains exhibited more resistance to thermal stress and mycoparasite Trichoderma as compared to the wild-type and RNAi strains. Taken together, our results indicated that FfGa1 positively regulates hyphal extension, and is crucial for the maintenance of cell wall integrity and protection against biotic and abiotic (hypertonic and thermal) stress.

β-1, 6-glucan synthase is a key enzyme of β-1, 6-glucan synthesis, which plays a vital role in the cell wall cross-linking of fungi. However, the role of the β-1, 6-glucan synthase gene in the development of the fruiting body and the stress response of macrofungi is largely unknown. In this study, four overexpression transformants of the β-1, 6-glucan synthase gene (FfGS6) were successfully obtained, and gene function was studied in Flammulina filiformis. The overexpression of FfGS6 can increase the width of mycelium cells and improve the tolerance ability under mechanical injury and oxidative stress. Moreover, FfGS6 gene expression fluctuated in up-regulation during the recovery process of mycelium injury but showed a negative correlation with H₂O₂ concentration. Fruiting body phenotype tests showed that mycelia’s recovery ability after scratching improved when the FfGS6 gene was overexpressed. However, primordia formation and the stipe elongation ability were significantly inhibited. Our findings indicate that FfGS6 is involved in regulating mycelial cell morphology, the mycelial stress response, and fruit body development in F. filiformis.

As a potential medicine for the treatment of depression, psilocybin has gradually attracted attention. To elucidate the molecular mechanism regulating psilocybin synthesis in Gymnopilus dilepis, ultra-performance liquid chromatography (UPLC) was used to detect the changes in psilocybin content after S-adenosyl-l-homocysteine (SAH) treatment and the changes of psilocybin content in different parts (stipe and pileus), and RNA-Seq was used to explore the mechanism of psilocybin content changes. In this study, the psilocybin content in G. dilepis mycelia treated with SAH was significantly lower than that in the control group, and the content of psilocybin in the stipe was significantly higher than that in the pileus. Transcriptome analysis revealed that differential expression genes (DEGs) were associated with cysteine and methionine metabolism. In particular, the transcription levels of genes encoding Cystathionine gamma-lyase (CTH) in different treatments and different parts were positively correlated with psilocybin content. In addition, we found that the exogenous addition of CTH activity inhibitor (DL-propargylglycine, PAG) could reduce the content of psilocybin and L-serine, and the content of psilocybin and L-serine returned to normal levels after L-cysteine supplementation, suggesting that psilocybin synthesis may be positively correlated with L-cysteine or CTH, and L-cysteine regulates the synthesis of psilocybin by affecting L-serine and 4-hydroxy-L-tryptophan. In conclusion, this study revealed a new molecular mechanism that affects psilocybin biosynthesis, which can provide a theoretical basis for improving psilocybin synthesis and the possibility for the development of biomedicine.

Construction of the genome of Hericium coralloides, a species of edible mushroom, and identification of the genes involved in terpenoid biosynthesis can determine the biology and genetics of terpenoids. The present study describes the assembly of a high-quality chromosome-scale genome of H. coralloides using Pacbio HiFi sequencing and Hi-C technology. This genome consisted of 13 chromosomes, a total size of 43.6 Mb, contigs of N50 3.6 Mb, GC content at 54%, and BUSCOs integrity of 96.9%. Genes associated with terpenoid biosynthesis were predicted by KEGG enrichment analysis and homologous alignment. The Her011461 and Her008335 genes, encoding proteins in the terpenoid backbone synthesis pathway, were found to encode geranylgeranyl pyrophosphate and farnesyl diphosphate synthases, key enzymes in the biosynthesis of geranylgeranyl diphosphate, a precursor of several diterpenoids. Her011463 was found to be involved in regulating diterpene cyclase. The Her005433, Her006724, Her010605, and Her010608 genes were found to encode sesquiterpene synthesis. Most of these genes were more highly expressed in dikaryotic mycelia than in the primordium and fruiting bodies, indicating that terpenoids may be more abundant in dikaryotic mycelia. To our knowledge, this study is the first to assemble the H. coralloides genome at the chromosome scale and to identify the genes involved in terpenoid biosynthesis.