Harnessing Epigenetic Modifiers Reveals MAPK-Mediated Regulation Mechanisms in Hadal Fungi of Alternaria alternata Under High Hydrostatic Pressure

Abstract

High hydrostatic pressure (HHP) significantly modulates microbial metabolism, while chemical epigenetic modifiers are known to reactivate silent biosynthetic gene clusters and induce novel natural products. However, the mechanisms by which these epigenetic modifiers regulate fungal responses under differential pressure conditions, and how such regulation affects natural product biosynthesis, remain completely unexplored. Here, we investigated the hadal fungus Alternaria alternata CIEL23 isolated from 7332 m sediments in the Mariana Trench under epigenetic modifier treatment with contrasting pressures (0.1 MPa vs. 40 MPa). Our results revealed that epigenetic perturbations and high pressure significantly altered fungal phenotypes, gene expression, and secondary metabolite composition. Transcriptome-level analysis of epigenetic regulatory mechanisms under epigenetic modifiers in both pressure conditions (0.1 MPa and 40 MPa) demonstrated that the addition of epigenetic modifiers regulated MAPK pathway-related gene expression in response to the environment stimuli. Under dual stress conditions, the IG, CWI, and HOG branches of the MAPK pathway showed significantly altered activity patterns. These changes were associated with differential the regulation of genes related to hyphal growth, cell wall remodeling, cell cycle progression, and osmolyte synthesis, suggesting the coordinated modulation of multiple cellular processes. These findings provide the mechanistic link between epigenetic modification induced HHP-response changes and regulation in hadal fungi. Our study not only advances understanding of hadal fungal response to dual stressors but also unlocks new possibilities for harnessing their stress-driven metabolic versatility for biotechnological applications.

Keywords: Alternaria alternata; epigenetic modification; high hydrostatic pressure; regulation mechanism; transcriptomic analysis.

Figure 1

Phenotypes and the PKS gene expression of A.alternata CIEL 23. (a) the colony phenotypes of A. alternata CIEL 23, which was cultured with four concentrations of 5-AzaC (0, 50 μM, 500 μM, and 1000 μM) on PDA at 28 °C under the different culture pressures. The mycelia and spore phenotypes were taken under a ×40 microscope with a scale of 10 μm. The arrow points to the spore elongated beaks. (b) the PKS gene expression of A.alternata CIEL 23 cultured at different concentrations of epigenetic modifiers under different culture pressures. The X-axis was the different concentrations of 5-AzaC, and the Y-axis was the relative expression. Compared with black controls, results were considered to be significant at the level of p (NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Secondary metabolites of A. alternata CIEL 23 at different concentrations of 5-AzaC under different conditions. (a) the UPLC-MS/MS spectra of secondary metabolites of A. alternata CIEL 23 under atmospheric pressure condition. (b) the UPLC-MS/MS spectra of the secondary metabolites of A. alternata CIEL 23 under HHP condition. The X-axis was the retention time (min), and the Y-axis was the Relative response (%). The total ion chromatogram (TIC) of the products produced by the strain in media containing different concentrations of 5-AzaC (the number in the upper right corner of the picture indicates the concentration of 5-AzaC) was indicated by different colored lines (black-0, red-50 μM, green-500 μM, and blue-1000 μM). The different signal peaks were marked in the red box. (c) the antibacterial activities of secondary metabolite of A. alternata CIEL 23 under atmospheric pressure condition. (d) the antibacterial activities of secondary metabolite of A. alternata CIEL 23 under HHP condition. The number on the left of the picture indicates the concentration of 5-AzaC. The additive concentration group inducing maximal response was boxed in red.

Figure 3

Graph of differentially expressed genes analyzed in group B\A. (a) the Venn diagram of gene co-expression. The graphs showed the number of genes uniquely expressed in each group, and the overlapping areas showed the number of genes co-expressed in two groups. (b) the volcano map of differential genes. The X-axis was the log2Fold Change value, and the Y-axis was the −log10 padj or −log10 p-value. The blue dashed line indicated the threshold line for the differential gene screening criteria. (c) the scatter plot of GO pathway enrichment. (d) the scatter plot of KEGG pathway enrichment. In (c,d), the X-axis was the Gene Ratio, and the Y-axis was the name of the pathway. The size of the padj was indicated by the color of the dots. The smaller the padj was, the closer the color was to red. The number of differential genes contained under each pathway was indicated by the size of the dots.

Figure 4

Graph of differentially expressed genes analyzed in group D\C. (a) the Venn diagram of gene co-expression. The graphs showed the number of genes uniquely expressed in each group, and the overlapping areas showed the number of genes co-expressed in two groups. (b) the volcano map of differential genes. The X-axis was the log2Fold Change value, and the Y-axis was the −log10 padj or −log10 p-value. The blue dashed line indicated the threshold line for the differential gene screening criteria. (c) the scatter plot of GO pathway enrichment. (d) the scatter plot of KEGG pathway enrichment. In (c,d), the X-axis was the Gene Ratio, and the Y-axis was the name of the pathway. The size of the padj was indicated by the color of the dots. The smaller the padj was, the closer the color was to red. The number of differential genes contained under each pathway was indicated by the size of the dots.

Figure 5

Diagram of the MAPK signaling pathway. In the figure, the upregulation was marked in red, and the downregulation was marked in green. The solid lines represent direct effects, and the dotted lines represent indirect effects. The arrows represent promotion, and the vertical lines represent inhibition.

Figure 6

Diagram of reactive oxidative stress in mycelia and conidia. (a) the ROS of mycelia. (b) the ROS of conadia. The X-axis was the pressure of the cultured, and the Y-axis was the absorbance value detected by the fluorescent microplate reader. Compared with black controls, results were considered to be significant at the level of p (*** p < 0.001).

Figure 7

Diagram of the pathway of chitin metabolism and starch and sugar metabolism. (a) the chitin metabolism in D\C group. (b) the starch and sugar metabolism in D\C group. The upregulation was marked in red, the downregulation was marked in green, and both were marked in yellow. The arrow indicates the direction of synthesis.