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.

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.

The key agronomic traits for evaluating the vegetative and reproductive growth capacities of edible mushrooms are the abundance of aerial mycelia and stipe length. These traits directly affect mycelial vitality and fruiting body yield. However, the molecular mechanisms regulating these traits are poorly understood. In this study, Flammulina filiformis mutant with a T-DNA insert exhibited sparse aerial mycelia and longer stipes, which were caused by upregulated expression of a novel self-activating protein FfGH79. The FfGH79 protein, consisting of 238 amino acids and with a relative molecular mass of 27.45 kDa, was localized in the nucleus, but lacked known structural domains. Alignment of homologous proteins from 12 fungi that produce fruiting bodies identified four typical alpha helices in the N-terminal region and one beta turn in the C-terminal region, both of which were conserved tertiary structures. FfGH79 gene overexpression and knockdown strains were constructed to confirm that this gene negatively regulates mycelial branch distance, which results in sparse aerial mycelia. Further, FfGH79 gene positively regulates stipe elongation ability, thereby increasing fruiting body yield. Transcriptomic analysis revealed that most lectin-like and reactive oxygen species catabolism genes were downregulated in the gh79 mutant. These findings provide a new research target for understanding mycelial growth, the mechanisms of fruiting body development, and molecular breeding of edible mushrooms.

Psilocybin has demonstrated efficacy in the treatment of mental health conditions, including anxiety and depression. It is evident that G. dilepis possesses the capacity to produce psilocybin. Therefore, conducting in-depth research into this species will contribute to the understanding of the biosynthetic mechanisms of psilocybin and provide a more comprehensive insight into its biosynthesis. We have assembled the G. dilepis genome at the chromosomal level. G. dilepis is characterized by a chromosome number of 13, a genome size of 54.2 Mb, and a total of 15,211 predicted genes and 16,411,190 transposable elements. The evolutionary analysis suggests that G. dilepis can produce psilocybin due to its close relationship with the Psilocybe genus of mushrooms. Furthermore, transcriptomic analysis revealed that the biosynthesis of psilocybin is associated with several amino acid metabolic pathways, particularly those involving cysteine and methionine metabolism, arginine and proline metabolism, and tryptophan metabolism. These pathways may supply the precursor substances required for psilocybin biosynthesis. This study presents a high-quality genome assembly for G. dilepis and reveals a close relationship between amino acid metabolism and psilocybin biosynthesis, providing a theoretical basis for exploring the genetic relationships in G. dilepis and the genes that regulate the biosynthesis of psilocybin.