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.