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Fungal hydrophobins have many important physiological functions, such as maintaining hydrophobicity and affecting virulence, growth, and development. In Ganoderma lucidum, the molecular regulation mechanisms of hydrophobins in mushroom are unclear. In this study, we investigated a hydrophobin protein 1 (Hyd1) in G. lucidum, which belongs to the fungal Class I hydrophobins. The hyd1 gene was highly expressed during the formation of primordia, and expression was the lowest in fruiting bodies. Through the construction of hyd1 silenced strains, we found that primordia formation was not initiated in these strains. This finding indicated that Hyd1 played an important role in the development of G. lucidum. Second, AreA, a key transcription factor in nitrogen metabolism, negatively regulated the expression of hyd1. In an areA silenced strain, the expression of hyd1 increased by approximately 14-fold compared with that of the wild-type (WT) strain. Electrophoretic mobility shift assays (EMSA) indicated binding of AreA to the promoter of hyd1. Additionally, expression of hyd1 was determined in the presence of different nitrogen sources. Compared with that in the ammonia nitrogen source, the expression of hyd1 in nitrate nitrogen source significantly increased. Finally, we found that hyd1 plays important roles not only in nitrogen regulation but also in the resistance to other abiotic stresses. After silencing of hyd1, the resistance to heat, cell wall, and salt stresses decreased. Our findings reveal the important roles of Hyd1 in the development and resistance to abiotic stresses in G. lucidum and provide insights into the nitrogen regulation mechanism of hydrophobins in higher basidiomycetes.

Fungal AreA is a key nitrogen metabolism transcription factor in nitrogen metabolism repression (NMR). Studies have shown that there are different ways to regulate AreA activity in yeast and filamentous ascomycetes, but in Basidiomycota, how AreA is regulated is unknown. Here, a gene from Ganoderma lucidum with similarity to nmrA of filamentous ascomycetes was identified. The NmrA interacted with the C-terminal of AreA according to yeast two-hybrid assay. In order to determine the effect of NmrA on the AreA, 2 nmrA silenced strains of G. lucidum, with silencing efficiencies of 76% and 78%, were constructed using an RNA interference method. Silencing nmrA resulted in a decreased content of AreA. The content of AreA in nmrAi-3 and nmrAi-48 decreased by approximately 68% and 60%, respectively, compared with that in the WT in the ammonium condition. Under the nitrate culture condition, silencing nmrA resulted in a 40% decrease compared with the WT. Silencing nmrA also reduced the stability of the AreA protein. When the mycelia were treated with cycloheximide for 6 h, the AreA protein was almost undetectable in the nmrA silenced strains, while there was still approximately 80% of the AreA protein in the WT strains. In addition, under the nitrate culture, the content of AreA protein in the nuclei of the WT strains was significantly increased compared with that under the ammonium condition. However, when nmrA was silenced, the content of the AreA protein in the nuclei did not change compared with the WT. Compared with the WT, the expression of the glutamine synthetase gene in nmrAi-3 and nmrAi-48 strains increased by approximately 94% and 88%, respectively, under the ammonium condition, while the expression level of the nitrate reductase gene in nmrAi-3 and nmrAi-48 strains increased by approximately 100% and 93%, respectively, under the nitrate condition. Finally, silencing nmrA inhibited mycelial growth and increased ganoderic acid biosynthesis. Our findings are the first to reveal that a gene from G. lucidum with similarity to the nmrA of filamentous ascomycetes contributes to regulating AreA, which provides new insight into how AreA is regulated in Basidiomycota.

Hydrogen sulfide (H2S) is considered to be a novel gaseous signalling molecule with multiple physiological functions. Recently, the identification of sulfhydrated proteins has become a new hotspot in the analysis of the underlying mechanism of H2S. Our preliminary study has shown that H2S negatively regulates the heat-induced accumulation of ganoderic acids (GAs)，a major secondary metabolite in Ganoderma lucidum. However, a comprehensive understanding of its mechanism is lacking. In this study, sulfhydrated proteins in G. lucidum were quantified by quantitative proteomic mass spectrometry (MS), and the role of H2S in maintaining redox homeostasis under heat stress (HS) was determined. A redox-regulated protein, thioredoxin 1 (Trx1), was selected as a potential target of H2S. Further research revealed that the activity of Trx1 was provoked by sulfhydration at Cys31 and Cys34, contributing to the negative regulation of H2S to ROS accumulation and GAs biosynthesis under HS in G. lucidum. Our results provide a novel target for investigating the molecular mechanism of H2S physiological function. Moreover, new evidence is provided regarding the interaction mechanism between the H2S and ROS signalling pathways.

Shiitake mushroom, Lentinula edodes, is the second largest edible fungus in the world, with a characteristic aroma. 1,2,3,5,6-pentathioheterocycloheptane, commonly known as lenthionine, is the main source of this aroma. Lenthionine has high commercial value, and if we explore the possible induction mechanism of citric acid in lenthionine synthesis, we can provide a reference for the effective application of citric acid as an inducer. In this paper, the single-factor treatment of Lentinula edodes with variable citric acid concentration and treatment duration showed that the best citric acid concentration for L. edodes was 300 μM, and the best treatment duration was 15 days. Additionally, the optimal design conditions were obtained using the response surface method (RSM); the treatment concentration was 406 μM/L, the treatment duration was 15.6 days, and the lenthionine content was 130 μg/g. γ-Glutamyl transpeptidase (LEGGT) and cystine sulfoxide lyase (LECSL) are the key enzymes involved in the biosynthesis of lanthionine. The expression levels of LEGGT and LECSL genes increased significantly under citric acid treatment. Additionally, the lenthionine content of the silenced strains of LEGGT and LECSL was significantly decreased.