Trichoderma reesei is a saprophytic fungus that produces large amounts of cellulases and is widely used for biotechnological applications. Cerato-platanins (CPs) are a family of proteins universally distributed among Dikarya fungi and have been implicated in various functions related to fungal physiology and interaction with the environment. In T. reesei, three CPs are encoded in the genome: Trire2_111449, Trire2_123955, and Trire2_82662. However, their function is not fully elucidated. In this study, we deleted the Trire2_123955 gene (named here as epl2) in the wild-type QM6aΔtmus53Δpyr4 (WT) strain and examined the behavior of the Δepl2 strain compared with WT grown for 72 h in 1% cellulose using RNA sequencing. Of the 9143 genes in the T. reesei genome, 760 were differentially expressed, including 260 only in WT, 214 only in Δepl2, and 286 in both. Genes involved in oxidative stress, oxidoreductase activity, antioxidant activity, and transport were upregulated in the Δepl2 mutant. Genes encoding cell wall synthesis were upregulated in the mutant strain during the late growth stage. The Δepl2 mutant accumulated chitin and glucan at higher levels than the parental strain and was more resistant to cell wall stressors. These results suggest a compensatory effect in cell wall remodeling due to the absence of EPL2 in T. reesei. This study is expected to contribute to a better understanding of the role of the EPL2 protein in T. reesei and improve its application in biotechnological fields.

Recent research has revealed the calcium signaling significance in the production of cellulases in Trichoderma reesei. While vacuoles serve as the primary calcium storage within cells, the function of vacuolar calcium transporter proteins in this process remains unclear. In this study, we conducted a functional characterization of four vacuolar calcium transport proteins in T. reesei. This was accomplished by the construction of the four mutant strains ∆trpmc1, ∆tryvc1, ∆tryvc3, and ∆tryvc4. These mutants displayed enhanced growth when subjected to arabinose, xylitol, and xylose. Furthermore, the mutants ∆trpmc1, ∆tryvc1, and ∆tryvc4 showed a reduction in growth under conditions of 100 mM MnCl₂, implying their role in manganese resistance. Our enzymatic activity assays revealed a lack of the expected augmentation in cellulolytic activity that is typically seen in the parental strain following the introduction of calcium. This was mirrored in the expression patterns of the cellulase genes. The vacuolar calcium transport genes were also found to play a role in the expression of genes involved with the biosynthesis of secondary metabolites. In summary, our research highlights the crucial role of the vacuolar calcium transporters and, therefore, of the calcium signaling in orchestrating cellulase and hemicellulase expression, sugar utilization, and stress resistance in T. reesei.