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.

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.

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.