Nitrate reductase-dependent nitric oxide plays a key role on MeJA-induced ganoderic acid biosynthesis in Ganoderma lucidum.

Ganoderma lucidum, which contains numerous biologically active compounds, is known worldwide as a medicinal basidiomycete. Because of its application for the prevention and treatment of various diseases, most of artificially cultivated G. lucidum is output to many countries as food, tea, and dietary supplements for further processing. Methyl jasmonate (MeJA) has been reported as a compound that can induce ganoderic acid (GA) biosynthesis, an important secondary metabolite of G. lucidum. Herein, MeJA was found to increase the intracellular level of nitric oxide (NO). In addition, upregulation of GA biosynthesis in the presence of MeJA was abolished when NO was depleted from the culture. This result demonstrated that MeJA-regulated GA biosynthesis might occur via NO signaling. To elucidate the underlying mechanism, we used gene-silenced strains of nitrate reductase (NR) and the inhibitor of NR to illustrate the role of NO in MeJA induction. The results indicated that the increase in GA biosynthesis induced by MeJA was activated by NR-generated NO. Furthermore, the findings indicated that the reduction of NO could induce GA levels in the control group, but NO could also activate GA biosynthesis upon MeJA treatment. Further results indicated that NR silencing reversed the increased enzymatic activity of NOX to generate ROS due to MeJA induction. Importantly, our results highlight the NR-generated NO functions in signaling crosstalk between reactive oxygen species and MeJA. These results provide a good opportunity to determine the potential pathway linking NO to the ROS signaling pathway in fungi treated with MeJA. KEY POINTS: • MeJA increased the intracellular level of nitric oxide (NO) in G. lucidum. • The increase in GA biosynthesis induced by MeJA is activated by NR-generated NO. • NO acts as a signaling molecule between reactive oxygen species (ROS) and MeJA.

Functions of reactive oxygen species in apoptosis and ganoderic acid biosynthesis in Ganoderma lucidum.

Ganoderma lucidum is a medicinal fungus that is widely used in traditional medicine. Fungal PacC is recognized as an important transcription factor that functions during adaptation to environmental pH, fungal development and secondary metabolism. Previous studies have revealed that GlPacC plays important roles in mycelial growth, fruiting body development and ganoderic acid (GA) biosynthesis. In this study, using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, we found that the apoptosis level was increased when PacC was silenced. The transcript and activity levels of caspase-like proteins were significantly increased in the PacC-silenced (PacCi) strains compared with the control strains. Silencing PacC also resulted in an increased reactive oxygen species (ROS) levels (∼2-fold) and decreased activity levels of enzymes involved in the antioxidant system. Further, we found that the intracellular ROS levels contributed to apoptosis and GA biosynthesis. Adding N-acetyl-cysteine and vitamin C decreased intracellular ROS and resulted in the inhibition of apoptosis in the PacCi strains. Additionally, the GA biosynthesis was different between the control strains and the PacCi strains after intracellular ROS was eliminated. Taken together, the findings showed that silencing PacC can result in an intracellular ROS burst, which increases cell apoptosis and GA biosynthesis levels. Our study provides novel insight into the functions of PacC in filamentous fungi.

The Slt2-MAPK pathway is involved in the mechanism by which target of rapamycin regulates cell wall components in Ganoderma lucidum.

The fungal cell wall is very important for cell growth and survival during stress, and the target of rapamycin (TOR) pathway plays a major role in regulating cell growth in response to environmental cues. Ganoderma lucidum is an important edible and medicinal fungus, and the function of TOR in this organism remains unclear. As shown in the present study, the TOR pathway regulates cell wall integrity (CWI) in G. lucidum. Inhibition of TOR signaling by RNA interference (RNAi) or rapamycin treatment reduced the growth of G. lucidum mycelia, increased contents of the cell wall components chitin and ß-1,3-glucan, and increased cell wall thickness. Furthermore, inhibition of TOR signaling enhanced the relative level of phosphorylated Slt2, a member of the MAPK cascade involved in CWI signaling. Moreover, when treated with rapamycin, significantly lower chitin and ß-1,3-glucan contents were observed in Slt2-silenced strains than in WT strains, indicating that TOR regulates the synthesis of these cell wall components through the Slt2-MAPK pathway. These results indicate a potential relationship between TOR signaling and CWI signaling. Additionally, participation of Slt2-MAPK in TOR-mediated regulation of cell wall component production has not previously been reported in a microorganism.

Ganoderma lucidum phosphoglucomutase is required for hyphal growth, polysaccharide production, and cell wall integrity.

Phosphoglucomutase (pgm) is an important enzyme in carbohydrate metabolism that is located at the branching point between glycolysis and the Leloir pathway. pgm catalyzes the reversible conversion reaction between glucose-6-phosphate (Glc-6-P) and glucose-1-phosphate (Glc-1-P). The glpgm gene was cloned in Escherichia coli, and the recombinant pgm protein from Ganoderma lucidum was purified in this study. The activity of native pgm was also detected to demonstrate that this predicted gene was functional in G. lucidum. Interestingly, silencing the glpgm gene in the fungus reduced hyphal growth. Moreover, glpgm silencing was associated with declining extracellular polysaccharide (EPS) production (approximately 20-40% of that in the WT strain) and increasing intracellular polysaccharide (IPS) production (approximately 1.7-fold that in the WT strain). Additionally, in our research, cell wall components were also shown to differ according to the glpgmi strain. Compared with WT, chitin significantly increased by 1.5-fold; however, the content of ß-1,3-glucan was observably reduced to 60-70% that of the WT. Further research showed that the cell wall component changes were associated with the transcription of related genes. These findings provide references for further study on the potential physiological function of pgm in G. lucidum.