Sirtuin GlSIRT1-Mediated Deacetylation of Phosphoglucose Isomerase GlPGI Regulates hyphal growth and branching in Ganoderma lucidum
Description
The commercial value of Ganoderma lucidum is fundamentally constrained by mycelial growth rate, which dictates fermentation cycle duration and metabolite yields in submerged culture, and determines fruiting body and spore production in solid-state cultivation. Here, we demonstrate that the NAD⁺-dependent deacetylase GlSIRT1 is a critical regulator of mycelial growth and branching in G. lucidum. GlSIRT1 knockdown impaired growth rate and promoted hyperbranching, whereas overexpression enhanced growth and suppressed branching. Integrated acetylome and immunoprecipitation–mass spectrometry analyses identified phosphoglucose isomerase (GlPGI) as substrate of GlSIRT1. GlSIRT1 physically interacts with and deacetylates GlPGI at lysine 364 (K364), a highly conserved residue across fungal taxa. Site-directed mutagenesis confirmed that K364 acetylation status directly dictates GlPGI enzymatic activity. The deacetylation-mimetic K364R variant exhibited enhanced catalytic efficiency, whereas the acetylation-mimetic K364Q variant showed markedly reduced activity. Consequently, GlSIRT1-mediated deacetylation of GlPGI promotes glycolytic flux, elevating intracellular pyruvate and ATP levels, while concurrently modulating chitin and β-1,3-glucan contents, thereby coordinating hyphal growth and branching. Collectively, these findings establish that GlSIRT1 deacetylation of GlPGI regulates hyphal growth and branching in filamentous fungi through coordinating energy metabolism and cell wall integrity.
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Hyphal Branching Assay The Glsirt1 and Glpgi knockdown and overexpression strains were inoculated onto solid CYM plates and incubated at 28 °C for 3 days. Under sterile conditions in a laminar flow hood, sterile coverslips were inserted into the agar at the advancing margins of the colonies using forceps, followed by continued incubation at 28 °C. Once the hyphae had sufficiently colonized the coverslips, they were stained with a 2.5 ug/mL Calcofluor White (CFW) solution for 5 minutes. The stained mycelia were subsequently imaged under ultraviolet (UV) excitation using a Nikon Eclipse Ti-S fluorescence microscope. Finally, the internodal distances and branching angles of the hyphae were quantified utilizing Image-Pro Plus 6.0 software. GST pull down The purified GST-PGI or GST protein was incubated with GST-beads (C650031, Sangon Biotech, Shanghai) in binding buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, pH 7.4) overnight at 4°C. Following incubation, the mixture was centrifuged to remove the supernatant. The resulting beads were then incubated with purified His-GlSIRT1 protein for 3 hours at room temperature. Subsequently, the beads were washed thoroughly with washing buffer (0.1 M glycine, pH 2.0). The samples were then boiled and analyzed by Western blot to verify the protein interaction. Immunoprecipitation Mycelia were ground to a fine powder in liquid nitrogen and lysed on ice in IP lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, pH 7.4). The lysate was incubated with the antibody overnight at 4 °C. Protein A/G magnetic beads (Cat. No. PB101–1, Vazyme, Nanjing, China) were pre-washed with washing buffer (1× PBS, 0.5% Tween 20, pH 7.4) and added to the mixture, followed by incubation at room temperature for 3 hours. The magnetic beads were washed three times with washing buffer, eluted with 2× SDS-PAGE buffer (125 mM Tris-HCl [pH 6.8], 2% SDS, 20% glycerol, 100 mM dithiothreitol [DTT], 0.1 mM phenylmethylsulfonyl fluoride [PMSF]), and subjected to Western blot analysis.
Institutions
- Nanjing Agricultural UniversityJiangsu, Nanjing