The Cerato-Platanin EPL2 from Trichoderma reesei Is Not Directly Involved in Cellulase Formation but in Cell Wall Remodeling

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.


Introduction
Trichoderma reesei is a saprophytic fungus present in different habitats [1]. Due to its capacity to produce cellulolytic enzymes, it has excellent biotechnological applications [2]. In the presence of inducing carbon sources, such as cellulose, T. reesei produces high levels of cellulases that degrade the substrate. The sugars generated are transported across the cytoplasmatic membrane and metabolized [3]. Despite being a widely studied microorganism, how T. reesei detects and recognizes the external environment, such as the available carbon sources, metal ions, and pH, is not fully elucidated [3].
Proteins of the cerato-platanin (CP) family are small fungal-exclusive proteins found in both Ascomycota and Basidiomycota [4]. They are related to the early development of fungi and fungus-host interactions [5]. These proteins are found abundantly in secretomes during hyphal growth, mycelial mass formation, sporulation, and spore maturation [6]. They are involved in cell wall remodeling and enlargement during hyphal growth and chlamydospore formation [7]. CPs are characterized by four cysteines forming two disulfide bridges and can be identified by the cerato-platanin domain (IPR010829). Structural

Cladogram Construction
The amino acid sequences of proteins characterized or cited as CPs were extracted from the NCBI database [18]. Similarly, amino acid sequences of Trichoderma spp. proteins predicted as CPs were obtained from the Joint Genome Institute (JGI) [19]. The sequences were aligned using MAFFT [20], and the cladogram was inferred using FastTree 2 [21]. The complete list of proteins, including their identifications, organisms, and references, is available in Table S1.

T. reesei epl2 Gene Deletion
To delete the epl2 gene, the epl2 open reading frame (ORF) was replaced with the selection marker pyr4 encoding the orotidine-5 -phosphate decarboxylase enzyme. The complete deletion cassette was assembled using S. cerevisiae SC9721 (genotype: MATa his3∆200 ura3-52 leu2∆1 lys2∆202 trp1∆63) by homologous recombination [22]. To transform S. cerevisiae, the 5 UTR and 3 UTR regions of the epl2 gene and the pyr4 gene were amplified from T. reesei DNA (Table S2) and, together with the linearized pRS426 vector, were used to transform the yeast. The resulting deletion cassette was amplified by PCR and used to transform T. reesei QM6a∆tmus53∆pyr4 (WT).

Transformation of T. reesei
The transformation of the T. reesei WT was performed as described by Gruber and coworkers [23]. A total of 20 µg of linear DNA from the deletion cassette was used in the assay. After transformation, the plates were incubated at 30 • C for 3 to 4 days until spores were visible. The candidates were submitted to three rounds of selection on MA medium with and without 0.1% Triton X-100 (GE Healthcare, Uppsala, Sweden). Cassette integration in the genome locus was verified by PCR using a specific set of primers for pyr4 and epl2 (Table S2). The expression profile of the epl2 gene was also analyzed by RT-qPCR (Table S3).

RNA Extraction and Transcript Analysis Using Quantitative PCR (RT-qPCR)
The mycelia from the WT T. reesei strain and the ∆epl2 strain grown on cellulose for 24, 48, and 72 h (after being pre-grown in glycerol for 24 h) were filtered through Miracloth (Merck, Darmstadt, Hesse, Germany), frozen in liquid nitrogen, and macerated. According to the manufacturer's instructions, the total RNA was isolated using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA). The samples were quantified, and their integrity was verified using a 1% agarose gel.
To analyze gene expression by RT-qPCR, 1 µg of RNA was treated with DNAse I (ThermoFisher Scientific, Waltham, MA, USA) to remove genomic DNA. After DNA removal, cDNA synthesis was performed using a Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. cDNA was diluted in water (1:50) and used for RT-qPCR analysis in Bio-Rad CFX96TM equipment using SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) for the detection signal according to the manufacturer's instructions. The actin gene was used for endogenous control to normalize the total amount of cDNA in each reaction. The genes analyzed are represented in Table S3.

RNA Sequencing
To perform the sequencing, RNA from the WT and ∆epl2 strains grown in glycerol 1% for 24 h and cellulose 1% for 72 h (MA medium) was isolated in biological triplicate. For growth in cellulose, the strains were pre-cultured in 1% glycerol for 24 h and then transferred to a new flask containing fresh medium with 1% cellulose. The RNAs obtained from the WT and ∆epl2 strains of T. reesei were diluted in water and quantified, and 20 µg was treated with the Qiagen RNAeasy Kit (QIAGEN, Hilden, North Rhine-Westphalia, Germany) according to the manufacturer's recommendations. Sequencing was performed by Novogene Corporation Inc. in Sacramento, California, USA, using Illumina technology.

Data Analysis
FastQC v0.11.5 [24] was used to assess the quality of the data obtained by sequencing. The removal of adapters was performed with Cutadapt v3.4 [25] using the following configuration: removal of reads with N > 10%, Qscore ≤ 5, and minimum size < 150 bp. Sequences were mapped based on the Trichoderma reesei v2.0 reference genome provided by the Joint Genome Institute (JGI) Genome Portal (http://genome.jgipsf.org/Trire2/Trire2 .home.html, accessed on 20 June 2021) using STAR v2.7.9 [26]. HTSeq-count v0.13.5 [27] was used to count the mapped reads, and expression values were calculated using the DESeq package version 1.31.21 [28]. The genes present in this analysis were annotated using InterProScan version 5.51-85 [29]. Differential expression was represented in log2 fold change, and genes were considered upregulated when the log2 fold change was ≥1 and downregulated when the log2 fold change was ≤−1 using an adjusted p-value < 0.05 as a threshold). In addition, to identify the genes differentially expressed in ∆epl2 in relation to WT in cellulose, we subtracted the log 2 fold change values. A difference of at least 1 was set as the cut-off. A Venn diagram was created using the Euler package in R [30]. In accordance with Wright and coworkers, functional categorization was performed with Gene Ontology (GO) terms [31].

Test of Sensitivity to Cell Wall Stressors
To assess how the fungal strains respond to cell wall stressors, 10 µL of a solution containing 10 6 conidia/mL from the WT and ∆epl2 strains of T. reesei were inoculated on the center of minimal media (MM) plates (1 g/L MgSO 4 ·7H 2 O (Synth, Diadema, SP, Brazil), 10 g/L KH 2 PO 4 (Synth, Brazil), 6 g/L (NH 4 ) 2 SO 4 (Synth, Diadema, SP, Brazil), 3 g/L Na 3 C 6 H 5 O 7 (Synth, Diadema, SP, Brazil), 20 mL/L trace elements, and 20 g/L bacteriological agar (Kasvi, Madrid, Spain)) with 2% glucose (Synth, Diadema, SP, Brazil) in the absence or presence of different concentrations of Calcofluor White (CFW) (Sigma-Aldrich, St. Louis, MO, USA) (20 and 40 µg/mL) or Congo Red (CR) (Sigma-Aldrich, St. Louis, MO, USA) (100 and 200 µg/mL). After incubation for 3 days, the mycelia diameter under the indicated conditions was recorded. Three replicates of each experiment were conducted. Statistical tests were performed using one-way analysis of variance (ANOVA) followed by Bonferroni's test (available in Prism software v8.0) for comparing the growth of the parental and mutant strains. CFW and CR stains were used for chitin and β-1,3-glucan; the former interacts preferentially with chitin, and both interfere with cell wall assembly and integrity [34]. The use of these stressors allows the study of fungi strains with a role in cell wall maintenance.

Microscopy Analysis
The methodology used for the cell wall thickness analysis was in accordance with Ribeiro et al. [33], with some modifications. The WT and the mutant strain ∆epl2 were cultivated in microculture dishes using the modified Riddell technique [35]. Briefly, conidia from WT and ∆epl2 were inoculated into small blocks (~1 cm 2 ) of MEA medium between sterile coverslips for 48 h at 30 • C. After growing the cultures, the coverslips were removed from the culture dish, stained with 0.001% Calcofluor White (Sigma-Aldrich, St. Louis, Microorganisms 2023, 11,1965 5 of 16 MO, USA) for 5 min, and washed three times with phosphate-buffered saline (PBS) 1×. Coverslips were inspected on a Leica SP5 confocal microscope using a 63× objective oil immersion lens. For the Calcofluor White (CFW) stain, the excitation wavelength was 405 nm, and the emission wavelength was 433 nm. The differential interference contrast (DIC) and confocal images were captured with PMTs and HyD detectors and processed using the software LAS AF 2.7.3.9723. After the images were acquired, they were submitted for wall thickness analysis using the ImageJ software (https://imagej.nih.gov/ij/index. html, accessed on 31 March 2023). Statistical tests were performed using one-way analysis of variance (ANOVA) followed by Bonferroni's test (available in Prism software v8.0) to compare the cell wall thickness of the parental (n = 42) and mutant (n = 39) strains.

Deletion of the epl2 Gene in T. reesei
To obtain a better understanding of the function of the cerato-platanin protein from T. reesei, the ORF of the epl2 gene was replaced with the auxotrophic marker gene pyr4 in the strain QM6a∆tmus53∆pyr4, referred to here as the WT strain, resulting in the ∆epl2 strain.
Confirmation of the absence of the epl2 gene (753 pb) ( Figure 2A) and the pyr4 gene insertion (3.246 pb) was performed by PCR using specific primers (Table S2). The correct integration of the deletion cassette in the ∆epl2 strain was also verified by PCR using primers that bind at 200 bp before the 5 UTR region and 200 bp after the 3 UTR region. The results showed the presence of amplicons with the expected size ( Figure 2B). Strains 8,9,11,12,and 14 show bands corresponding to the expected fragment sizes ( Figure 2B). In addition, no epl2 mRNA was detected by RT-qPCR ( Figure 2C). These results confirmed the complete deletion of the epl2 gene from the fungal genome.

Global Analysis of the Genes Regulated by Δepl2 in the Presence of Cellulose
The WT and Δepl2 strains of T. reesei were initially grown in biological triplicate in a medium containing cellulose 1% (w/v) as the sole carbon source. The global transcriptome analysis was performed using RNA-Seq. The preparation of the cDNA libraries generated many reads. They were processed and mapped to the Trichoderma reesei v2.0 genome (https://mycocosm.jgi.doe.gov/Trire2/Trire2.home.html, accessed on 31 March 2023). Overall, 98% of the reads were mapped to the reference genome (Table S4), and a principal

Global Analysis of the Genes Regulated by ∆epl2 in the Presence of Cellulose
The WT and ∆epl2 strains of T. reesei were initially grown in biological triplicate in a medium containing cellulose 1% (w/v) as the sole carbon source. The global transcriptome analysis was performed using RNA-Seq. The preparation of the cDNA libraries generated many reads. They were processed and mapped to the Trichoderma reesei v2.0 genome (https://mycocosm.jgi.doe.gov/Trire2/Trire2.home.html, accessed on 31 March 2023). Overall, 98% of the reads were mapped to the reference genome (Table S4), and a principal component analysis demonstrated the reliability of the RNA-Seq samples ( Figure S1).
The global expression of the ∆epl2 and WT strains revealed that of the 9143 T. reesei genes, 2338 were differentially expressed in the WT, and 2074 were expressed in ∆epl2 (cellulose in relation to glycerol) ( Figure S2). After the screening, 760 genes were considered differentially expressed between these strains, with 260 unique to WT (166 upregulated and 97 downregulated), 214 unique to ∆epl2 (177 upregulated and 37 downregulated), and 286 in common for both strains (252 upregulated and 33 downregulated) ( Figure 3).

Top Genes Regulated in the Δepl2mutant in the Cellulose Condition
The upregulated and downregulated genes in the WT and Δepl2 were categorized using GO terms. To verify the biological processes ( Figure S3A), molecular functions (Figure S3B), and cellular components ( Figure S3C) involved in cellulose culture, a hierarchical tree cluster analysis was performed. The most enriched biological processes for both strains in cellulose in relation to glycerol are the glucosamine-containing metabolic processes, amino sugar metabolic processes, oxidative stress responses, and lipid-catabolic processes. Among the upregulated processes for Δepl2 are organic acid transport, oxidative stress responses, oxidoreductase activity, antioxidant activity, and transportation.
The ten genes most upregulated and downregulated in the Δepl2 strain compared to WT after cellulose culture are listed in Table 1. The gene with the highest expression en-

Top Genes Regulated in the ∆epl2mutant in the Cellulose Condition
The upregulated and downregulated genes in the WT and ∆epl2 were categorized using GO terms. To verify the biological processes ( Figure S3A), molecular functions ( Figure S3B), and cellular components ( Figure S3C) involved in cellulose culture, a hierarchical tree cluster analysis was performed. The most enriched biological processes for both strains in cellulose in relation to glycerol are the glucosamine-containing metabolic processes, amino sugar metabolic processes, oxidative stress responses, and lipid-catabolic processes. Among the upregulated processes for ∆epl2 are organic acid transport, oxidative stress responses, oxidoreductase activity, antioxidant activity, and transportation. The ten genes most upregulated and downregulated in the ∆epl2 strain compared to WT after cellulose culture are listed in Table 1. The gene with the highest expression encodes a protein of the glucose-methanol-choline oxidoreductase family, known to have a FAD-binding domain and a substrate-binding domain, which can be various sugars, alcohols, cholesterol, and choline [43]. The most downregulated gene encodes a long-chain fatty acid Acyl-CoA ligase protein, which is involved in lipid metabolism. This enzyme forms Acyl-CoA, allowing fatty acids to move into the β-oxidation pathway [44]. It should be noted that several genes that encode proteins with unknown functions were found to be both up-and downregulated. Furthermore, a gene that encodes for a candidate protein for glucan degradation was upregulated in the ∆epl2 mutant, indicating the participation of EPL2 in cell wall remodeling (Table 1).
We also decided to analyze the expression of cellulase genes using RT-qPCR at different times of cultivation in the presence of cellulose. As can be seen in Figure 4, the genes that encode for β-glucosidase (cel3a) ( Figure 4A), xyloglucanase (cel74a) (Figure 4B), and exoglucanase (cel6a) ( Figure 4D) are downregulated in the mutant ∆epl2 in relation to the WT. On the other hand, the cel7a gene that encodes for exoglucanase is upregulated in the mutant ∆epl2 in relation to the WT ( Figure 4C). However, the up-or downregulation of cellulase genes in the ∆epl2 mutant is not reflected in the cellulolytic enzyme activity compared to the WT ( Figure S4). The results suggest that EPL2 does not act directly in regulating cellulase formation. It seems that the epl2 gene is fundamental for the cell wall remodeling process [46,47]. . The values presented are the means of three groups of experiments, with the corresponding deviations. For statistical analysis, a two-way ANOVA test was performed (95% confidence interval). Asterisks indicate groups that showed significant differences when compared with the WT strain. ** = p ≤ 0.01. and *** = p ≤ 0.001.

EPL2 Is Involved in Cell Wall Remodeling
To investigate the influence of EPL2 in cell wall remodeling, we first evaluated the growth and sensitivity of the WT and Δepl2 strains in the presence of cell wall stressors such as Calcofluor White (CFW) and Congo Red (CR) ( Figure 5). As shown in Figure 5, the Δepl2 strain was more tolerant to CFW and CR stressors even at concentrations as high as 40 ug/mL and 200 ug/mL, respectively ( Figure 5B). For statistical analysis, a two-way ANOVA test was performed (95% confidence interval). Asterisks indicate groups that showed significant differences when compared with the WT strain. ** = p ≤ 0.01. and *** = p ≤ 0.001.

EPL2 Is Involved in Cell Wall Remodeling
To investigate the influence of EPL2 in cell wall remodeling, we first evaluated the growth and sensitivity of the WT and ∆epl2 strains in the presence of cell wall stressors such as Calcofluor White (CFW) and Congo Red (CR) ( Figure 5). As shown in Figure 5, the ∆epl2 strain was more tolerant to CFW and CR stressors even at concentrations as high as 40 µg/mL and 200 µg/mL, respectively ( Figure 5B).  , and the results are expressed as radial growth of treatment/radial growth of control (without any stressor). For statistical analysis, a one-way analysis of variance (ANOVA) followed by Bonferroni's test was performed (95% confidence interval). Asterisks indicate groups that showed significant differences when compared with the WT. * = p ≤ 0.05, *** = p ≤ 0.01, and **** = p ≤ 0.001.
Tolerance to the cell wall damage caused by CFW and CR indicates that the ∆epl2 strain can alter the fungal cell wall's composition and organization. Therefore, we investigated whether the deletion of epl2 affects cell wall organization in T. reesei. Confocal microscopy analysis using CFW showed that epl2 deletion causes an increase in cell wall thickness, with cell wall thicknesses of 0.35 ± 0.05 µm for the mutant and 0.27 ± 0.025 µm for the WT strain (p < 0.01) ( Figure 6A).
Microorganisms 2023, 11, x FOR PEER REVIEW 13 of 18 cultivation in the WT strain. In contrast, the Δepl2 strain showed higher expression levels of these two genes at the end of cultivation. Furthermore, the gene-encoding glucan endo-1,3-α-glucosidase (Trire2_71532) showed increased expression levels during the cultivation of the Δepl2 strain, unlike the WT strain. In contrast, both T. reesei strains exhibited low expression levels of the Trire2_108672 and Trire2_73643 genes encoding glucanase and β-1,6-N-acetylglucosaminyltransferase, respectively ( Figure 6B). The fungal cell wall comprises polysaccharides, including chitin, β-1,3-glucan, and β-1,6-glucan. Polysaccharides from the cell wall were extracted and hydrolyzed, and HPLC analyzed the composition of fungal cell walls from the WT and Δepl2 strains. The HPLC results showed that the deletion of epl2 alters the composition of the cell wall, causing an increase in chitin oligomers (chit4, chit3, and chit2), N-acetyl-glucosamine (Nag), and glucose ( Figure 6C). These results showed that EPL2 has an essential role in cell wall integrity maintenance in T. reesei, affecting the expression of genes involved in cell wall remodeling and participating in cell wall composition and organization. For statistical analysis, a one-way analysis of variance (ANOVA) followed by Bonferroni's test was performed (95% confidence interval). Asterisks indicate groups that showed significant differences when compared with the WT. Trire2_ is abbreviated as Tr_ in the figure. Tetraacetylchitotetraose (Chit4), triacetylchitotriose (Chit3), diacetyl-β-D-chitobiose (Chit2), N-Acetil-β-D-glucosamina (nag), and glucose (gluc). ** = p ≤ 0.01.

Discussion
CPs are found exclusively in fungi and are involved in growth, development, recognition, adhesion, cell wall morphogenesis, and parasitism [42,[48][49][50]. Despite several studies of CPs in fungi, these proteins' biological function remains unclear in T. reesei.
In this work, we constructed a cladogram that showed four clades for Trichoderma spp. In the EPL1 (SM1) clade, all the proteins were shown to be involved in plant resistance. We found that EPL1 from Trichoderma atroviride, which forms protein layers, increases the polarity of aqueous solutions and surfaces, binds to chitin, and induces plant growth and resistance to phytopathogens [12,42]; Epl1-Tas from Trichoderma asperellum For statistical analysis, a one-way analysis of variance (ANOVA) followed by Bonferroni's test was performed (95% confidence interval). Asterisks indicate groups that showed significant differences when compared with the WT. Trire2_ is abbreviated as Tr_ in the figure. Tetraacetylchitotetraose (Chit4), triacetylchitotriose (Chit3), diacetyl-β-D-chitobiose (Chit2), N-Acetil-β-D-glucosamina (nag), and glucose (gluc). ** = p ≤ 0.01. Furthermore, we performed RT-qPCR using genes involved in fungal cell wall remodeling (Table S3) and sugar quantification by HPLC. For gene expression, we selected genes involved in cell wall remodeling (N-acetyl-glucosaminidases, chitinases, and glucanases) differentially expressed in the RNA-Seq data and assessed their profile in WT and ∆epl2 strains grown in cellulose. Our results revealed a significant increase in the expression of all genes in the ∆epl2 strain at the final cultivation time (72 h) compared to the WT strain ( Figure 6B). In contrast, the WT strain showed the highest gene expression levels at 48 h of cultivation. This result suggests a compensation response to the absence of EPL2 protein.
Notably, the Trire2_59791 and Trire2_54242 genes, which encode chitinase and glucanase proteins, displayed higher expression levels during the early hours of cultivation in the WT strain. In contrast, the ∆epl2 strain showed higher expression levels of these two genes at the end of cultivation. Furthermore, the gene-encoding glucan endo-1,3-α-glucosidase (Trire2_71532) showed increased expression levels during the cultivation of the ∆epl2 strain, unlike the WT strain. In contrast, both T. reesei strains exhibited low expression levels of the Trire2_108672 and Trire2_73643 genes encoding glucanase and β-1,6-N-acetylglucosaminyltransferase, respectively ( Figure 6B). The fungal cell wall comprises polysaccharides, including chitin, β-1,3-glucan, and β-1,6-glucan. Polysaccharides from the cell wall were extracted and hydrolyzed, and HPLC analyzed the composition of fungal cell walls from the WT and ∆epl2 strains. The HPLC results showed that the deletion of epl2 alters the composition of the cell wall, causing an increase in chitin oligomers (chit4, chit3, and chit2), N-acetyl-glucosamine (Nag), and glucose ( Figure 6C). These results showed that EPL2 has an essential role in cell wall integrity maintenance in T. reesei, affecting the expression of genes involved in cell wall remodeling and participating in cell wall composition and organization.

Discussion
CPs are found exclusively in fungi and are involved in growth, development, recognition, adhesion, cell wall morphogenesis, and parasitism [42,[48][49][50]. Despite several studies of CPs in fungi, these proteins' biological function remains unclear in T. reesei.
In this work, we constructed a cladogram that showed four clades for Trichoderma spp. In the EPL1 (SM1) clade, all the proteins were shown to be involved in plant resistance. We found that EPL1 from Trichoderma atroviride, which forms protein layers, increases the polarity of aqueous solutions and surfaces, binds to chitin, and induces plant growth and resistance to phytopathogens [12,42]; Epl1-Tas from Trichoderma asperellum elicits woody plant resistance to pathogens [39]; Epl-1 from Trichoderma harzianum is involved in mycoparasitism, self-recognition, and the expression of defense-related genes in plants [14,39]; and SM1 from Trichoderma virens induces the production of reactive oxygen species and elicits the expression of defense-related genes in plants [13,37].
In the EPL2 clade, we also identified proteins involved in plant resistance. EPL-2 from T. atroviride is slightly expressed during cultivation with glucose and strongly expressed during cultivation on colloidal chitin or fungal conidiation. However, its knock-out strain shows no effect on diverse growth and development tests, including biomass formation on shake flasks, agar plate growth, hydrophobicity, osmotic stress, and cell wall stress [42]. There is also SM2 from T. virens, which is not expressed on shake flask cultivation with glucose but is expressed during conidiation on potato dextrose agar plates. Similar to EPL2 from T. atroviride, its knock-out strain shows no phenotypic difference from the parental strain; however, both proteins have been shown to be involved in plant protection [38]. In addition, this clade includes the protein analyzed in this study: EPL2 from T. reesei.
In the EPL3 clade, we identified EPL3 from T. atroviride, which was described by Frischmann et al. [42]. However, its expression was not detected in most of the analyzed situations, including during cultivation with colloidal chitin or stages of spore formation. In this clade, we also observed SM3 from T. virens, the transcript of which was not detected during shake flask cultivation with glucose, nor was it detected on potato dextrose agar plates [38].
In the EPL4 clade, we identified proteins exclusively from T. atroviride, T. virens, and T. asperellum, consistent with the study conducted by Gao and colleagues [15]. Furthermore, we observed that the proteins from plant pathogens were grouped in different clades. This observation aligns with previous findings that these proteins play a role in virulence [6,[9][10][11], a characteristic not present in Trichoderma spp. (Figure 1).
To better understand the function of epl2 in T. reesei, we deleted this gene in the QM6a∆tmus53∆pyr4 strain, referred to here as the WT. To gain novel insights into the role of this gene in T. reesei physiology, we performed a global expression analysis comparing the T. reesei ∆epl2 mutant strain with its WT strain. The functional categorization of genes differentially expressed in the ∆epl2 strain demonstrated processes related to transport, oxidoreductase activity, polysaccharide binding, and carbohydrate binding. Considering that T. reesei was subjected to a hydrolytic enzyme-inducing condition (i.e., growth in cellulose), these processes can be related to recognizing the carbon source present in the medium, suggesting that the absence of the EPL2 protein could cause an increase in activities associated with the extracellular medium. Nevertheless, no role has so far been reported for the EPL2 protein in T. reesei.
Studies demonstrated that CPs could bind to and weaken lignocellulosic materials, acting similarly to plant expansins and facilitating access to cellulolytic enzymes [50,51]. This characteristic has been reported in the CP of Ceratocystis populicola [51], Moniliophthora perniciosa [52], Fusarium graminearum [53], and Trichoderma harzianum [54]. In T. reesei, CPs were found in the secretome during fermentation processes in sugarcane bagasse [55]. To assess the influence of epl2 deletion on cellulose recognition and degradation, a CAZymes analysis was performed. Nine genes were shown to be upregulated in the ∆epl2 mutant, indicating that EPL2 may be involved in cellulase gene expression. In addition, we conducted a temporal analysis of the expression of the main cellulase genes in both the WT and ∆epl2 strains when grown on 1% cellulose at 24, 48, and 72 h. Although we observed differences in gene expression between the two strains, our enzymatic activity assays and protein dosage showed no significant difference between them. This suggests that the role of EPL2 may primarily occur at the transcriptional level.
Interestingly, our results indicated that EPL2 plays a role in cell wall remodeling, as the ∆epl2 strain showed tolerance to cell wall stressors, increased cell wall thickness, upregulation of genes involved in cell wall remodeling, and the composition of polysaccharides from the cell wall. It seems that the absence of the epl2 gene generates a compensatory effect in the fungus, which starts to express some enzymes responsible for synthesizing cell wall components in greater quantity. Thus, we observed higher chitin, glucan, and Nag content. This is in accordance with T. reesei's remarkable capacity of adapting to changing environmental conditions [56]. One way that T. reesei achieves this is by remodeling its cell wall, which involves the deposition, degradation, and modification of different cell wall components, including chitin, glucans, and proteins [57].
Although CPs are abundant in fungal secretomes, they have not been studied systematically in the genus Trichoderma. In some Trichoderma species, the genes for CPs are present at higher levels during the early processes of fungal development. In T. atroviride, epl2 expression occurred during hyphae growth and mycelium development [42]. Similarly, the sm1 gene of T. virens was expressed in germinating spores. Nevertheless, studies performed with strains deleted for epl2 did not show differences in growth, conidia germination, and hyphae development [13,37]. In T. reesei, deleting the epl2 gene did not cause significant development changes in a cellulose medium.
In summary, our results suggest the involvement of EPL2 in cell wall remodeling and the growth of T. reesei and demonstrate that it is indirectly related to increasing cellulase production. More studies are necessary to explain the real role of CPs in cellulase production/recognition. The three CPs in T. reesei probably work together in different stages of development and hydrolase production.

Conclusions
Our analysis of the T. reesei transcriptome identified several genes that were affected by the epl2 deletion, including cellulolytic genes and genes related to cell wall remodeling. However, most genes were indirectly influenced by epl2. Our results also suggest that the EPL2 protein could be involved in cellulose recognition processes and cell wall remodeling. Further study of the two other cerato-platanins from T. reesei may provide additional insights into the function of these proteins in this fungus and their potential applications in biotechnology.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/microorganisms11081965/s1; Figure S1: Principal component analysis (PCA) plot of each condition analyzed. Figure S2: Differentially expressed genes in wild type (WT) and ∆epl2 strains. Figure S3: Gene Ontology enrichment analysis of WT and ∆epl2 strains. Figure S4: Enzymatic activity of WT and ∆epl2 strains grown on cellulose. No significant changes were observed. Table S1: Proteins used to infer the cladogram. Table S2: Primer sequences used in this study. Table  S3: Primers used in RT-qPCR experiments. Table S4: Summary of reads obtained by RNA sequencing.