The Duality of the MAPK Signaling Pathway in the Control of Metabolic Processes and Cellulase Production in Trichoderma reesei

Abstract

In this study, through global transcriptional analysis by RNA-Sequencing, we identified the main changes in gene expression that occurred in two functional mutants of the MAPK genes tmk1 and tmk2 in Trichoderma reesei during sugarcane bagasse degradation. We found that the proteins encoded by these genes regulated independent processes, sometimes in a cross-talk manner, to modulate gene expression in T. reesei. In the Δtmk2 strain, growth in sugarcane bagasse modulated the expression of genes involved in carbohydrate metabolism, cell growth and development, and G-protein-coupled receptor-mediated cell signaling. On the other hand, deletion of tmk1 led to decreased expression of the major genes for cellulases and xylanases. Furthermore, TMK1 found to be involved in the regulation of the expression of major facilitator superfamily transporters. Our results revealed that the MAPK signaling pathway in T. reesei regulates many important processes that allow the fungus to recognize, transport, and metabolize different carbon sources during plant cell wall degradation.

Figure 1

Expression profile of MAPK genes in the QM6a parental strain grown in sugarcane bagasse and glucose. (A) Relative expression of tmk1, tmk2, and tmk3 upon growth in glucose. Expression levels were calibrated according to the comparative 2^−ΔCt method, using the constitutively expressed gene β-actin as an endogenous control (ANOVA followed by Tukey’s pairwise comparison P < 0.05). *Significantly different from 24 h (P < 0.05). (B) Relative expression of tmk1, tmk2, and tmk3 in the presence of sugarcane bagasse. Expression levels were calibrated according to the comparative 2^−ΔΔCt method, using the constitutively expressed gene β-actin as an endogenous control and glycerol samples the reference group (ANOVA followed by Tukey’s pairwise comparison P < 0.0001). *Significantly different from 48 h, 72 h, and 96 h (P < 0.0001). These results are based on three replicates of three independent experiments and are expressed as mean ± standard deviation.

Figure 2

Gene Regulatory Network (GRN) of differentially expressed genes in the mutant strains grown in glucose, sugarcane, and glycerol. (A) GRN of 402 differentially expressed genes in the Δtmk1 compared to the QM6a (Δtmk1/QM6a) in the presence of glucose, glycerol, and sugarcane bagasse. Genes are represented as nodes (circles), and interactions are represented as edges (red lines: upregulated interactions, green lines: downregulated interactions), connecting the nodes: 382 interactions, (B) GRN of 2575 differentially expressed genes in the Δtmk2 compared to the QM6a (Δtmk2/QM6a) in the presence of glucose, glycerol, and sugarcane bagasse. Genes are represented as nodes (shown as circles), and interactions are represented as edges (red lines: upregulated interactions, green lines: downregulated interactions), connecting the nodes: 1980 interactions.

Figure 3

Expression pattern of genes commonly expressed between the Δtmk1 and Δtmk2 representing the number of differentially expressed genes in the presence of glucose and sugarcane bagasse. (A) Comparative Venn diagram of commonly expressed genes between the Δtmk1 and Δtmk2 strains in the presence of glucose and sugarcane bagasse. Venn diagram clustering was designed using Venny 2.1 tools. (B) Gene Ontology (GO) enrichment analysis of commonly expressed genes between the Δtmk1 and Δtmk2 strains in the presence of glucose and sugarcane bagasse. The enriched GO terms according to molecular, cellular component, and biological process in T. reesei. Significantly enriched categories (P ≤ 0.05) are shown. The threshold for calling differentially expressed genes was P ≤ 0.05.

Figure 4

Cellulase gene expression profile of T. reesei QM6a, Δtmk1, and Δtmk2 strains during growth in glucose (gluc), glycerol (glyc), and sugarcane bagasse (SCB). (A) Heatmap of differentially expressed CAZy genes in the Δtmk1 and QM6a strains showing all the conditions of this study. (B) Heatmap of differentially expressed CAZys in Δtmk1 and QM6a strains grown in sugarcane bagasse. (C) Heatmap of differentially expressed CAZys of Δtmk1 and QM6a strains grown in glucose. (D) Heatmap of differentially CAZy genes in the Δtmk2 and QM6a strains showing all the conditions of this study. (E) Heatmap of differentially expressed CAZys in Δtmk2 and QM6a strains grown in sugarcane bagasse. (F) Heatmap of differentially expressed CAZys from Δtmk2 and QM6a strains grown in glucose. (G) Heatmap of differentially expressed CAZys from Δtmk2 and QM6a strains grown in glycerol. The hierarchical clustering was performed using the R pheatmap package. Complete linkage method and Euclidean distance of row centered and scaled TPM values were used for hierarchical clustering of the differentially expressed genes in all analyzed conditions.

Figure 5

Holocellulolytic activities of QM6a, Δtmk2, and Δtmk1 grown in sugarcane bagasse. (A) Endoglucanase activity (CMCase), (B) β-glucosidase, (C) β-xylosidase and (D) Xylanase activities from culture supernatant of T. reesei, QM6a parental strain, Δtmk2, and Δtmk1 grown in the presence of sugarcane bagasse for the indicated times. ***Significantly different from the QM6a parental strain (P < 0.001) and *Significantly different from the QM6a parental strain (P < 0.05).

Figure 6

Global view of the MAPK signaling pathway in Δtmk2 and Δtmk1 mutant strains grown in glucose, glycerol, and sugarcane bagasse. (A) Functional reconstruction of the main MAPK signaling pathways involved in pheromone response, cell wall stress, high osmolarity, and starvation in T. reesei. This analysis was performed using the KEGG MAPK signaling pathway in Yeast (https://www.genome.jp/kegg/pathway/sce/sce04011.html) as reference. (B) Heatmap showing expression of the differentially expressed genes belonging to the MAPK signaling pathway in the T. reesei mutant strains. The hierarchical clustering was performed using the R pheatmap package. Complete linkage method and Euclidean distance of row centered and scaled TPM values were used for hierarchical clustering of the differentially expressed genes in all analyzed conditions. The gene IDs represent the genes differentially expressed in the mutant strains grown in glucose, glycerol, and sugarcane bagasse.

Figure 7

Heat map of predicted MAPK phosphorylation sites of MFS transporters in the Δtmk1 and Δtmk2 strains grown in sugarcane bagasse. ST: serine and threonine residues are phosphorylated; Y: phosphorylation occurs in tyrosine residues and ST + Y: phosphorylation sites in all three residues, serine, threonine, and tyrosine.