Involvement of PaSNF1 in Fungal Development, Sterigmatocystin Biosynthesis, and Lignocellulosic Degradation in the Filamentous Fungus Podospora anserina

Abstract

The sucrose non-fermenting 1/AMP-activated protein kinase (SNF1/AMPK) is a central regulator of carbon metabolism and energy production in the eukaryotes. In this study, the functions of the Podospora anserina SNF1 (PaSNF1) ortholog were investigated. The ΔPaSNF1 mutant displays a delayed development of mycelium and fruiting bodies and fails to form ascospores. The expression of the PaSNF1 gene in the strain providing female organs in a cross is sufficient to ensure fertility, indicating a maternal effect. Results of environmental stress showed that ΔPaSNF1 was hypersensitive to stress, such as osmotic pressure and heat shock, and resistant to fluconazole. Interestingly, the knockout of PaSNF1 significantly promoted sterigmatocystin (ST) synthesis but suppressed cellulase [filter paperase (FPA), endoglucanase (EG), and β-glucosidase (BG)] activity. Further, transcriptome analysis indicated that PaSNF1 made positive regulatory effects on the expression of genes encoding cellulolytic enzymes. These results suggested that PaSNF1 may function in balancing the operation of primary and secondary metabolism. This study suggested that SNF1 was a key regulator concerting vegetative growth, sexual development, and stress tolerance. Our study provided the first genetic evidence that SNF1 was involved in the ST biosynthesis and that it may also be a major actor of lignocellulose degradation in P. anserina.

Keywords: Podospora anserina; lignocellulose degradation; secondary metabolism; sexual development; stress tolerance; sucrose non-fermenting 1.

FIGURE 1

Phylogenetic analysis of PaSNF1 and SNF1 orthologs identified in other organisms. The phylogenetic tree was reconstructed using a neighbor-joining method. Numbers at nodes indicate bootstrap values with 1,000 replications. GenBank accession number of a given SNF1-coding gene was parenthesized following the name of each fungal species. Distance scale: scale that represents the number of differences between sequences (e.g., 0.05 means 5% differences between two sequences). SNF1, sucrose non-fermenting 1; PaSNF1, Podospora anserina SNF1.

FIGURE 2

Growth and development of WT and mutants on M2 medium. (A) Colony size of WT and ΔPaSNF1 after 3 days of incubation. (B) Fertility on M₂ medium developed after 5 days. Perithecia are visible as small black dots. All the strains produced the usual ring of mature perithecia in the center of the Petri dish. (C) Microscopic observation of perithecia of WT and ΔPaSNF1. (D) Microscopic observation of ascospores inside perithecia. (E) Microscopic observation of ascospores erupting from fruiting bodies. (F,G) Colony diameter and fruiting bodies number, respectively; fruiting body number was expressed with respect to that of WT considered as 100%. Error bars are standard deviations of triplicate samples. Differences in the data were assessed by the T-test. WT, wild type; PaSNF1, Podospora anserina SNF1.

FIGURE 3

Spreading experiments. Podospora anserina is a heterothallic species with two mating types called mat+ and mat-. Each mating type differentiates male and female organs. Male organs (spermatia) are single cells that can be spread by water all over the plate. They are recognized by female organs of opposite mating type, which fuse with the male cells and subsequently differentiate in mature fruiting bodies. Crosses were made by inoculating the strains 1.5 cm apart. After 3 days of growth, 1.5 ml of water was added and spread all over the plate. The pictures were taken 4 days after fertilization.

FIGURE 4

Tolerance analysis to environmental stresses. (A) Colony sizes of WT, ΔPaSNF1, and CPPaSNF1 were incubated separately on M2 medium, supplemented with one of the osmotic stress reagents (0.5 M of NaCl, 0.5 M of KCl, 0.5 M of sorbitol, and 0.75 M of glycerol), oxidative reagents (0.01% H₂O₂ and 50 μM of menadione), and 2.5 μg/ml of fluconazole; and fresh mycelium was incubated at 45°C in water bath for 2 h. M2 medium without the supplements was served as the control. (B) Relative inhibition rate. All cultures were incubated at 27°C for 3 days, and colony size was measured. Three plates were set for each treatment, and the experiment was repeated once. Error bars are standard deviations of triplicate samples. Differences in the data were assessed by the T-test. WT, wild type; PaSNF1, Podospora anserina SNF1.

FIGURE 5

Identification and analysis of ST. (A) HPLC analysis of SM production with WT and ΔPaSNF1 mutant. The strains were grown on M₂ media for 7 days. (B) The chemical structure of ST by NMR. (C) Relative amounts of ST in ΔPaSNF1 strain compared with WT. Error bars are standard deviations of triplicate samples. Differences in the data were assessed by the T-test. ST, sterigmatocystin; HPLC, high-performance liquid chromatography; SM, secondary metabolite; WT, wild type; PaSNF1, Podospora anserina SNF1; NMR, nuclear magnetic resonance.

FIGURE 6

Knockdown of PaSNF1 led to defects in cellulose-utilizing ability. (A) Enzyme assays in WT and ΔPaSNF1 mutant. The mycelia of 2-day-old cultures grown on M2 medium of the indicated strains were harvested and smashed in proper extraction buffer. Activity was expressed with respect to that of WT considered as 100%. Error bars are standard deviations of triplicate samples. Differences in the data were assessed by the T-test. (B) Fertility of WT and ΔPaSNF1 on medium with MCC and straw medium. Fertility was measured by counting the number of mature perithecia on plates containing MCC and straw medium as sole carbon source. Pictures of mycelia of WT and ΔPaSNF1 mutant were taken after 7 days of growth. Fruiting bodies (perithecia) are visible as small black dots. PaSNF1, Podospora anserina SNF1; WT, wild type; MCC, microcrystalline cellulose.

FIGURE 7

Comparative transcriptomics analysis between ΔPaSNF1 and WT grown in Avicel medium. (A) Differentially expressed genes in the mutant ΔPaSNF1 relative to WT. The expression profile was calculated for conditions including Avicel. Genes that showed differential expression identified by DESeq2 package are shown. Up-regulated and down-regulated genes are shown in red and green, respectively. (B) Relative mRNA abundance of all CAZy genes in ΔPaSNF1 mutant versus WT. The significantly up-regulated genes are shown in red, whereas down-regulated genes are shown in blue (based on adjusted p-value). (C) Total expression of genes encoding major cellulases from RNA-Seq data in WT and ΔPaSNF1 strains. (D) Total expression of genes encoding LPMO from RNA-Seq data in WT and ΔPaSNF1 strains. (E) Regulation of putative transporter genes by PaSNF1. Values in heatmaps were calculated by log2 (Gene_FPKM in ΔPaSNF1/Gene_FPKM in WT). FPKM mapped. (F) Expression level validation of DEGs using RT-qPCR. Comparisons of log2 ratios of 10 up-regulated and 10 down-regulated genes from the RNA-Seq data and RT-qPCR experiments with paired primers (Supplementary Table S1). PaSNF1, Podospora anserina SNF1; WT, wild type; CAZy, Carbohydrate-Active Enzymes; DEG, differentially expressed genes.