Effects of 5-Azacytidine on Growth and Hypocrellin Production of Shiraia bambusicola

Abstract

Hypocrellins, fungal perylenequinones of Shiraia bambusicola are developed as important photodynamic therapy agents against cancers and viruses. Due to the limitation of the wild resources, the mycelium culture is a promising alternative for hypocrellin production. As DNA methylation has profound effects on fungal growth, development and secondary metabolism, we used both McrBC cleavage and HPLC analysis to reveal the status of DNA methylation of S. bambusicola mycelium. We found that DNA methylation is absent in mycelia, but DNA methylation inhibitor 5-azacytidine (5-AC) still induced the fluffy phenotype and decreased hypocrellin contents significantly. Simultaneously, a total of 4,046 differentially expressed genes were induced by 5-AC, including up-regulated 2,392 unigenes (59.12%) and down-regulated 1,654 unigenes (40.88%). Gene ontology analysis showed 5-AC treatment changed expression of genes involved in membrane composition and oxidation-reduction process. The fluffy phenotype in 5-AC-treated S. bambusicola was closely related to strong promotion of developmental regulator WetA and the repression of the sexual developmental actor VeA and LaeA. It was a surprise finding that 5-AC reduced reactive oxygen species (ROS) production significantly in the mycelia via the inhibition of NADPH oxidase gene (NOX) expression and NOX activity. With the treatment of vitamin C and H₂O₂, we found that the reduced ROS generation was involved in the down-regulated expression of key genes for hypocrellin biosynthesis and the decreased hypocrellin production. To our knowledge, this is the first attempt to examine DNA methylation level in S. bambusicola. Our results suggested that the mediation of ROS generation could not be ignored in the study using 5-AC as a specific DNA methylation inhibitor.

Keywords: 5-azacytidine; DNA methylation; Shiraia bambusicola; fluffy; hypocrellin; reactive oxygen species; transcriptome analysis.

FIGURE 1

Effects of 5-AC on mycelial growth and hypocrellin contents of Shiraia bambusicola S8. (A) Morphologic characteristics and red pigments secretion. The mycelia of S. bambusicola were kept on PDA with or without 5-AC (0.8 mM) for 10 days. (a,b) Macroscopic colony appearance; (c,d) the secretion of pigments on PDA medium; (e,f) hyphal morphology on dissecting microscopy (40×); (g,h) the pycnidium formation and conidium generation. Arrow (red) indicates the pycnidium. Arrow (blue) indicates the conidium. (B) Effect of 5-AC (0.8 mM) on the generation of conidia of S. bambusicola on day 8. (C) Effects of 5-AC at different concentrations (0–1.0 mM) on mycelia biomass, hypocrellin contents. (D) Time profiles of mycelia biomass and hypocrellin contents in S. bambusicola submerged cultures with the addition of 5-AC at 0.8 mM. 5-AC was added on day 3 of the culture. Arrow (black) indicates the time point of 5-AC addition. Values are mean ± SD from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01.

FIGURE 2

DNA methylation in mycelium of S. bambusicola S8 after 5-AC treatment. (A) McrBC digestion of genomic DNA of S8 strain. (+), McrBC digestion system; (–), a system contains digestion buffer but not McrBC enzyme. Genomic DNA from mycelia treated with 5-AC (lane 1+, –) or without 5-AC (lane 2+, –). 5-AC at 0.8 mM was added on day 3 and mycelium cultures continued to day 8. DMSO-treated group was used as negative control (lane 3+, –). A plasmid with a digestion site of McrBC was used as positive control (lane 4+, –). (B) Detection of 5-mdC in the genomic DNA of S8 strain. (a) Chromatogram of five standard deoxynucleotides. dA, dC, dG, 5-mdC and dT represent 2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine, 5′-methyl-2′-deoxycytidine and 2′-deoxythymidine, respectively. (b) The chromatogram of hydrolysed DNA isolated from S. bambusicola S8.

FIGURE 3

Gene ontology (GO) function categories of the DEGs in S. bambusicola under 5-AC treatment at 0.8 mM. Unigenes were assigned to three categories: (A) biological processes, (B) cellular components, and (C) molecular functions.

FIGURE 4

Effects of 5-AC on the expression of genes associated with fluffy mycelia phenotype of S. bambusicola S8. (A) Quantitative real-time PCR (qPCR) analysis of these genes expression in mycelia treated by 5-AC at 0.8 mM for 4 days. (B) Time profiles of expression of VeA gene in submerged cultures of S. bambusicola with or without addition of 5-AC. 5-AC was added on day 3 of the fermentation and cultured at 28°C. Values are mean ± SD from three independent experiments. ^∗∗p < 0.01 versus control group.

FIGURE 5

Effects of 5-AC on the expression of hypocrellin biosynthetic genes of S. bambusicola S8. 5-AC was added on day 3 of the culture. After 1 day of treatment the mycelia were harvested. Values are mean ± SD from three independent experiments.

FIGURE 6

Effects of 5-AC on ROS generation of S. bambusicola S8. (A) Bright-field image (left) and fluorescence microscopy (485 nm excitation and 528 nm emission) of DCFH-DA-stained mycelium (right) in cultures with or without 5-AC. 5-AC at 0.8 mM was added on day 3 of the culture. After 2 days of treatment the mycelia were harvested and observed. The O₂^- (B) and H₂O₂ (C) contents of mycelia were detected under 5-AC treatment. Arrow indicates the time point of 5-AC addition. Values are mean ± SD from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01 versus control.

FIGURE 7

(A) Effects of 5-AC on the activity of NADPH oxidase (NOX) of S. bambusicola S8. 5-AC at 0.8 mM was added on day 3 of the culture. Arrow indicates the time point of 5-AC addition. (B) Effects of 5-AC on the expression of different NOX-related genes. (C) Time profiles of expression of NOX-A gene (CL4189Contig1) in submerged cultures of S. bambusicola with or without addition of 5-AC. 5-AC at 0.8 mM was added on day 3. After 1 day of treatment the mycelia were harvested and measured. Values are mean ± SD from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01 versus control.

FIGURE 8

The effect of decreased ROS on the mycelial growth, hypocrellin production, and related gene expression after 5-AC treatment. Effects of H₂O₂ (A) and Vc (B) on mycelial biomass and hypocrellin contents. H₂O₂ and Vc were added on day 3 of the culture. After 5 days of treatment the mycelia were harvested. Effects of H₂O₂ and Vc on hypocrellin contents (C) and gene expression (D) after 5-AC treatment. H₂O₂ (0.01 mM) and Vc (0.01 mM) were added 1 h prior to 5-AC treatment at 0.8 mM on day 3. Hypocrellin contents and the gene expression levels were measured on day 8 and day 4, respectively. PKS: polyketide synthase (CL954Contig1), Mono: monooxygenase (CL1046Contig1). Values are mean ± SD from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01 versus control, ^#p < 0.05, ^##p < 0.01 versus 5-AC alone group.