The APSES transcription factor Swi6B upregulates CATALASE 1 transcription to enhance oxidative stress tolerance of Ganoderma lucidum

Abstract

As sessile organisms, fungi often encounter various stresses throughout their lifespan, resulting in the overproduction of reactive oxygen species, which impedes the normal growth of fungi. Previously, we revealed that the Swi6B transcription factor is involved in the stress tolerance of Ganoderma lucidum. However, the underlying molecular mechanism is unclear. The present study demonstrated that oxidative stress increased the levels of Swi6B. Direct binding of Swi6B to the promoter region of catalase 1 (CAT1) resulted in increased transcription of CAT1 and reduced H₂O₂ levels in SWI6B overexpressing strains. In addition to increased Swi6B protein, the phosphorylation level of Swi6B was increased by H₂O₂ treatment. The overexpression of SLT2, a mitogen-activated protein kinase that interacts with Swi6B, increased the phosphorylation level of Swi6B, and treatment with H₂O₂ further enhanced this increase. As a result, both the binding of Swi6B to the CAT1 gene and tolerance to H₂O₂ were increased in SLT2-overexpressing strains. The present findings revealed that the Slt2-Swi6B-CAT1 pathway responds to oxidative stress and contributes to improving the survival of G. lucidum in adverse environments.IMPORTANCEIn fungi, environmental stress leads to the accumulation of intracellular reactive oxygen species and leads to oxidative stress. Here, we found that the overexpression of the APSES transcription factor Swi6B enhances tolerance to oxidative stress in Ganoderma lucidum. Swi6B binds to the promoter region of CAT1, which increases CAT1 transcription and reduces the H₂O₂ levels. In addition, the phosphorylation of Swi6B by Slt2 promotes the regulation of CAT1 by Swi6B. The Slt2-Swi6B-CAT1 pathway is important for the response of G. lucidum to oxidative stress.

Keywords: CAT1; Ganoderma lucidum; Swi6B; oxidative stress.

Fig 1

Swi6B responses and enhances the tolerance of G. lucidum to oxidative stress. (A and B) The representative pictures (A) and relative growth rates (B) of different genotype strains under oxidative stressor treatment. All strains were cultured on CYM solid medium supplemented with 8  mM H₂O₂ or 8  mM VK₃. The relative growth rate of each strain was calculated as the diameter of hyphae growth under H₂O₂ or VK₃ treatment divided by that under control condition. The data were presented as a percentage. SWI6B-OE, SWI6B overexpression strains; swi6-kd, SWI6 knockdown strains; CK, empty vector strains. (C) Western blotting analysis of the Swi6B protein in the WT strain grown on CYM solid medium supplemented with or without oxidant. The intensity of bands was analyzed with Image J software (v1.8.0). (D) Quantitative real-time PCR analysis of the expression levels of SWI6B in the WT strain cultured with or without oxidant treatment. For panels B and D, the different letters indicate significant differences according to Duncan’s multiple range test (P  <  0.05). For panels C and D, the values of control were set to 1.00 to normalize the values under H₂O₂ (8  mM) or VK₃ (8  mM) treatment.

Fig 2

Overexpression of SWI6B promotes the expression and enzyme activity of CAT1 to reduce H₂O₂ content. (A) Heat map of the expression levels of genes encoding classical antioxidant enzymes with or without H₂O₂ treatment. The expression levels of genes in all the genotypes were analyzed by RT-qPCR. (B) Western blotting analysis of the CAT1 protein in the SWI6B-OE, swi6-kd, WT, and CK strains. All strains grown on CYM solid medium were added with or without H₂O₂. The intensity of bands was analyzed with Image J software (v1.8.0), and the values of WT treated without H₂O₂ were set to 1.00. (C and D) Fold change of relative CAT activity (C) or the H₂O₂ content (D) in different genotype strains cultured under H₂O₂ treatment. The relative enzyme activity or H₂O₂ content of each strain was calculated as the CAT activity or H₂O₂ content in the presence of H₂O₂ divided by the control, respectively. (E and F) The representative pictures (E) and hyphae radius (F) of SWI6B-OE and WT strains cultured on H₂O₂ (8 mM) containing CYM solid medium supplemented with or without 3-amino-1,2,4-triazole (3-AT; 2 mM). (G and H) Fold change of relative CAT activity (G) or the H₂O₂ content (H) in SWI6B-OE and WT strains. All strains were cultured as described (E). The relative enzyme activity or H₂O₂ content of each strain was calculated as the CAT activity or H₂O₂ content in the presence of H₂O₂ + 3-AT divided by that under SWI6B-OE and WT, respectively. For panels C, D, F, and H, the different letters indicate significant differences according to Duncan’s multiple range test (P < 0.05).

Fig 3

H₂O₂ treatment improves the binding of Swi6B with the promoter of CAT1 to promote CAT1 accumulation. (A) Schematic diagram of the Swi6B binding elements (CAT1^WT) and mutation elements (CAT1^mutant) present in the CAT1 promoter region. (B and C) Y1H (B) and EMSA assays exhibited the interaction between Swi6B and the CAT1 ^WT but not the CAT1^mutant fragments. (D) ChIP-qPCR analysis showed the binding of Swi6B to the promoter of CAT1 in the SWI6B-OE and WT strains treated with or without H₂O₂. The different letters indicate significant differences according to Duncan’s multiple range test (P  <  0.05). (E) Western blotting analysis of the H₂O₂-induced CAT1 protein in the SWI6B-OE and WT strains. All strains grown on CYM solid medium supplemented with or without H₂O₂. The intensity of bands was analyzed with Image J software (v1.8.0), and the values of WT under control condition were set to 1.00.

Fig 4

Knockdown of CAT1 results in enhanced H₂O₂ accumulation and reduced tolerance to H₂O₂ treatment. (A and B) Analysis of the transcription level of the CAT1 gene by qRT-PCR (A) or the abundance of CAT1 protein by western blotting (B) in the strains as indicated. (C) Relative CAT activity in WT, CK, and cat1-kds strains. The activity values were normalized, and the control was set to 100%. (D) Relative H₂O₂ content determination of WT, CK, and cat1-kds strains. The H₂O₂ content values were normalized, and the control was set to 100%. (E and F) Representative 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining pictures (E) and the relative fluorescence intensity (F) of ROS in the WT, CK, and cat1-kds strains. The fluorescence was monitored using a confocal laser scanning microscope with consistent exposure time, and fluorescence signal intensity was determined using ZEN 2011 SP2 software. Values were normalized, and the WT was set to 100%. (G and H) The representative pictures (G) and relative growth rates (H) of different genotype strains under (8 mM) H₂O₂ treatment. All strains grown on CYM solid medium supplemented with or without H₂O₂. The relative growth rate of each strain was calculated as the diameter of hyphae growth under H₂O₂ treatment divided by that under control condition. For panels A, C, D, F, and H, the different letters indicate significant differences according to Duncan’s multiple range test (P < 0.05).

Fig 5

Overexpression of SLT2 enhances the binding of Swi6B to the CAT1 by improving the phosphorylation of Swi6B. (A and B) The representative pictures (A) and hyphae radius (B) of SLT2-OE and WT strains cultured under different conditions as indicated. (C) ChIP-qPCR detection of the enrichment degree of Swi6B on the CAT1 promoter in the SLT2-OE and WT strains grown on CYM solid medium added with or without H₂O₂. Immunoprecipitation was performed with anti-Swi6B antibody, and the enriched DNA fragments were used as a template for qPCR. Values were shown as the mean ± SD (n = 3) of the cycle threshold. (D) Western blotting analysis of the phosphorylation levels of Swi6B in the SLT2-OEs and WT strains grown on CYM solid medium added with or without H₂O₂. All strains grown on CYM solid medium were added without H₂O₂ (8 mM) were set as control. The intensity of bands was analyzed with Image J software (v1.8.0), and the values of WT treated without H₂O₂ were set to 1.00. For panels B and C, the different letters indicate significant differences according to Duncan’s multiple range test (P < 0.05).