Expression of Saccharomyces cerevisiae RER2 Gene Encoding Cis-Prenyltransferase in Trichoderma atroviride Increases the Activity of Secretory Hydrolases and Enhances Antimicrobial Features

Some Trichoderma spp. exhibit natural abilities to reduce fungal diseases of plants through their mycoparasitic and antagonistic properties. In this study, we created new Trichoderma atroviride strains with elevated antifungal activity. This effect was achieved by improving the activity of cis-prenyltransferase, the main enzyme in dolichol synthesis, by expressing the RER2 gene from Saccharomyces cerevisiae. Since dolichyl phosphate is the carrier of carbohydrate residues during protein glycosylation, activation of its synthesis enhanced the activities of dolichyl-dependent enzymes, DPM synthase and N-acetylglucosamine transferase, as well as stimulated glycosylation of secretory proteins. Cellulases secreted by the transformants revealed significantly higher levels or activities compared to the control strain. Consequently, the resulting Trichoderma strains were more effective against the plant pathogens Pythium ultimum.


Introduction
Some species of the filamentous fungus Trichoderma exhibit biocontrol activity against fungal plant pathogens. Preparations containing Trichoderma spp. are presently used commercially for biological control against plant pathogens such as Pythium spp., Phytophthora spp., Botrytis spp., Rhizoctonia and Fusarium spp. The antagonistic properties of Trichoderma are based on multiple mechanisms [1]. Biocontrol can result from a direct interaction between the pathogen and the biocontrol agent, which involves synthesis of hydrolases and metabolites acting synergistically. Trichoderma attached to the pathogenic fungus produces cell wall-degrading enzymes. They allow assimilation of the released carbohydrates and penetration into the hypha of the pathogen. A comparative transcriptomic analysis of three Trichoderma species, T. reesei, T. atroviride and T. virens, had shown different transcriptomic responses upon recognition before the physical contact with the alien hyphae [2]. The types of genes expressed distinguished T. atroviride and T. virens from T. reesei. T. reesei mainly expressed genes for nutrient acquisition, which could be explained by an attempt to compete for nutrients with the other fungus. T. virens and T. atroviride differ in their antifungal strategy, the former poisoning the pathogenic fungus with gliotoxin and the latter using antibiosis and hydrolytic enzymes. In addition, T. atroviride produces 6-pentyl-α-pyrone, a volatile compound with antifungal activity that is not formed by T. virens [2].
Since T. atroviride carries out a hydrolytic attack on the pathogen cell wall as a strategy of mycoparasitism, we attempted to improve its antifungal characteristics by enhancing its hydrolytic properties.
The hydrolytic enzymes secreted by Trichoderma are heavily glycosylated with both Nand O-linked carbohydrates [3], accounting for as much as 12-24% of their molecular mass [4]. The O-linked glycans are attached in a linker connecting the catalytic and substrate binding domains and are required for their proper relative positioning. Nglycosylation is connected with protein folding, and it is crucial for recognition of misfolded glycoproteins [5,6]. Any dysfunction of glycosylation results in the accumulation of affected proteins in the endoplasmic reticulum and down-regulation of their genes [7,8]. These data suggest that an enhanced glycosylation of secretory proteins in T. atroviride could improve their secretion. Glycoproteins would be folded correctly, more rapidly and in a higher proportion. Furthermore, such effective glycosylation could also improve the activity of the secretory enzymes by stabilizing them outside the cell as well [4,6,[9][10][11].
In this study, we present the effects of expression of the yeast RER2 gene coding for cis-prenyltransferase (dehydrodolichyl diphosphate synthase) ( Figure 1) in the biocontrol T. atroviride strain P1.
be explained by an attempt to compete for nutrients with the other fungus. T. virens and T. atroviride differ in their antifungal strategy, the former poisoning the pathogenic fungus with gliotoxin and the latter using antibiosis and hydrolytic enzymes. In addition, T. atroviride produces 6-pentyl-α-pyrone, a volatile compound with antifungal activity that is not formed by T. virens [2].
Since T. atroviride carries out a hydrolytic attack on the pathogen cell wall as a strategy of mycoparasitism, we attempted to improve its antifungal characteristics by enhancing its hydrolytic properties.
The hydrolytic enzymes secreted by Trichoderma are heavily glycosylated with both N-and O-linked carbohydrates [3], accounting for as much as 12-24% of their molecular mass [4]. The O-linked glycans are attached in a linker connecting the catalytic and substrate binding domains and are required for their proper relative positioning. N-glycosylation is connected with protein folding, and it is crucial for recognition of misfolded glycoproteins [5,6]. Any dysfunction of glycosylation results in the accumulation of affected proteins in the endoplasmic reticulum and down-regulation of their genes [7,8]. These data suggest that an enhanced glycosylation of secretory proteins in T. atroviride could improve their secretion. Glycoproteins would be folded correctly, more rapidly and in a higher proportion. Furthermore, such effective glycosylation could also improve the activity of the secretory enzymes by stabilizing them outside the cell as well [4,6,[9][10][11].
Our previous results showed that, indeed, glycosylation processes and protein secretion in T. reesei were stimulated by overproduction of GDP-mannose and dolichyl phosphate mannose (DPM) and O-and N-glycosylation substrates [12][13][14].
In this study, we present the effects of expression of the yeast RER2 gene coding for cis-prenyltransferase (dehydrodolichyl diphosphate synthase) ( Figure 1) in the biocontrol T. atroviride strain P1.  HMGR-3 hydroxy-3-methylglutaryl-CoA reductase (regulatory enzyme of the mevalonate pathway); FPP synthase-farnesyl diphosphate synthase; SS-squalene synthase; cis-PT (Rer2)-cisprenyltransferase; Nus1-cis-PT complex subunit; DPM synthase-dolichyl phosphate mannose synthase. The names of the pathways are marked in green, and the names of the enzymes analyzed in this study in red.
Cis-prenyltransferase, a key enzyme in dolichol synthesis, is the first enzyme of the polyprenyl branch of the mevalonate pathway. This enzyme catalyzes the elongation of polyprenyl chain by sequential addition of isopentenyl diphosphate (IPP) to farnesyl diphosphate (FPP) [15,16]. The phosphorylated form of dolichols is indispensable as a carbohydrate carrier in protein glycosylation.
Transformants expressing RER2S.c gene had an elevated activity of cis-prenyltransferase and also of the dolichol-dependent DPM synthase as well as N-acetylglucosamine transferase. Moreover, they showed significantly higher levels of Oand N-glycosylation of secretory proteins. The activities of hydrolytic enzymes secreted to the cultivation medium were increased, and their maximum activity correlated with maximum glycosylation. Probably as a consequence of their higher hydrolytic activity, the new strains also had substantially improved antimicrobial ability.

Strains and Cultivation Media
T. atroviride strain P1 ("Trichoderma harzianum" ATCC 74058) was used as the recipient strain for transformation.

Analysis of Fungal Growth
Fungal dry weight was determined by filtering culture samples through G1 sintered glass funnels, washing the biomass with water and drying to a constant weight at 110 • C.
The transformants were then cultivated in liquid MM medium for preparation of DNA. Molecular biology methods

Nucleic Acids Isolation
Chromosomal DNA was isolated from T. atroviride using the Promega Wizard Genomic DNA Purification kit. Total RNA was isolated using the single-step method described by Chomczynski and Sacchi [20]. Other molecular biology procedures were performed according to standard protocols [21].

RER2 Copies Integrated into T. atroviride Genome
Quantitative real-time PCR (qRT-PCR) analysis was performed to estimate the copy number of the transgenes in the Trichoderma genome using the relative standard curve method [22][23][24]. Serial 1:10 dilutions of standard samples containing from 300,000 to 300 copies of the RER2 gene were prepared by mixing the wild-type Trichoderma genomic DNA with pGEM-RER2 plasmid. Each reaction contained an adequate amount of DNA calculated using the online tool (DNA Copy Number and Dilution Calculator, Thermo Fisher Scientific, Warsaw, Poland), 0.5 µM of each primer (Table S1) and 7.25 µL 3color 2xHS-qPCR Master Mix Sybr (A&A Biotechnology, Gdynia, Poland) in a total volume of 15 µL. All reactions were run on a LightCycler ® 96 (Roche Diagnostics GmbH, Mannheim, Germany) using the following program: 360 s 95 • C, 40 times (15 s 95 • C; 60 s 60 • C), followed by a melting curve generated from 65 • C to 90 • C. Four technical replicates were used for both the standard curve and the test samples. The single product was amplified by all primer pars tested. Raw data were processed using LightCycler ® 96 software Version 1.1.0.1320 (Roche Diagnostics GmbH, Mannheim, Germany), and the transgene copy number was calculated automatically.

Quantitative Reverse Transcription PCR (RT-qPCR)
One-step RT-qPCR assay was performed using a LightCycler ® EvoScript RNA SYBR ® Green I Master Kit (Roche Diagnostics GmbH, Mannheim, Germany) and was carried out in a 20 µL reaction, which consisted of 5 µL mRNA (25 pg), 0.4 µM each of forward and reverse primers (Table S1) and 4 µL of Master Mix, 5× concentrated containing enzymes for reverse transcription and PCR, RT-qPCR reaction buffer; dATP, dCTP, dGTP and dUTP; Mg(OAc) 2 ; SYBR ® Green I dye and proprietary additives. For the negative controls, mRNA was substituted with molecular grade water. The assay was performed using a LightCycler96 ® (Roche Diagnostics GmbH, Mannheim, Germany) with an initial incubation at 60 • C for 15 min for RT followed by preincubation at 95 • C for 10 min. Forty-five cycles of amplification were performed using a thermal cycling profile of 95 • C for 10 s, 58 • C for 30 s. Subsequently, a melting curve was recorded by holding at 95 • C for 10 s, cooling to 65 • C for 60 s and then heating at 0.1 • C/s up to 97 • C. The amplification and melting curve data were collected and analyzed using the LightCycler96 ® software 1.0. Biochemical methods

Cell Membrane Preparation
Mycelium was harvested by filtration, washed with water and suspended in 50 mM Tris-HCl, pH 7.4, containing 15 mM MgCl 2 and 9 mM 2-mercaptoethanol. Cells were homogenized in a beadbeater with glass beads (0.5 mm), and the homogenate was centrifuged at 5000× g for 10 min to remove cell debris and unbroken cells. The supernatant was centrifuged at 100,000× g for 1 h. The membrane pellet was homogenized in 50 mM Tris-HCl, pH 7.4, containing 3.5 mM MgCl 2 and 6 mM β-mercaptoethanol and used as the source of enzyme. The whole procedure was performed at 4 • C [18].

Cis-Prenyltransferase (EC 2.5.1.20) Activity
The enzyme activity was assayed in the membrane fraction by incubation (final volume 250 µL) of 500 µg of membrane proteins with 4 µg FPP, 50 mM sodium phosphate buffer pH 7.4, 0.5 mM MgCl 2 , 20 mM 2-mercaptoethanol, 10 mM KF and 3 × 10 5 cpm [ 14 C]IPP (sp. act.: 52 Ci/mol) (ARC, St. Louis, MO, USA). After 90 min incubation at 30 • C, the reaction was terminated by addition of 4 mL of chloroform-methanol (3:2 v/v). The protein pellet was removed by centrifugation, and the supernatant was washed three times with 1/5 volume of 10 mM EDTA in 0.9% NaCl. The organic phase was concentrated under a stream of nitrogen and subjected to thin-layer chromatography on HPTLC RP-18 plates developed in 50 mM H 3 PO 4 in acetone. The zone containing the radiolabeled polyprenols was scraped off, and the radioactivity was measured in a scintillation counter [18,25].   [28]. The reaction was stopped by the addition of 4 mL of chloroform-methanol (3:2 v/v). Formation of radioactive dolichyl diphosphate Nacetylglucosamine and dolichyl diphosphate chitobiose was measured in the organic fraction in a scintillation counter.
2.11. FPP Synthase (EC2.5.1.10) Activity The FPP synthase activity was measured in cell-free extract obtained from transformants and the control T. atroviride P1 strain. After 168 h of cultivation, mycelia of Trichoderma were harvested by filtration, washed with water and suspended in 50 mM phosphate buffer, pH 7.5, containing 1 mM MgCl 2 and 5 mM iodoacetamide. Cells were homogenized in a beadbeater with glass beads (0.5 mm), and the homogenate was centrifuged at 5000× g for 10 min to remove cell debris and unbroken cells. The resulting supernatant was centrifuged again at 100,000× g for 1 h to remove the membrane pellet, and the obtained supernatant was used as the source of FPP synthase. The whole procedure was performed at 4 • C.
The FPP synthase activity was measured in 100 µL of reaction mixture containing 50 mM phosphate buffer, pH 7.5, 1 mM MgCl 2 , 5 mM iodoacetamide, 60 µM isopentenyl diphosphate (IPP), 1 × 10 5 cpm [ 14 C] IPP (sp. act.: 52 mCi/mM) (ARC, St. Louis, MO, USA), 120 µM dimethylallyldiphosphate (DMAPP) and 150 µg protein [29,30]. After a 5-min incubation at 37 • C, samples were ice-chilled, and 0.5 mL of water was added, followed by 1 mL of hexane and 0.2 mL of 1 M HCl to dephosphorylate the products. The samples were shaken for 30 min at 37 • C. The mixture was ice-chilled and mixed vigorously. The upper phase was washed three times with water and subjected to TLC on silica gel 60 plates in benzene-ethyl acetate, 7:1. Radioactive spots were localized by autoradiography, identified by co-chromatography with unlabeled standards and then scraped off, and the radioactivity was measured in a scintillation counter [29,30].

Squalene Synthase (EC 2.5.1.21) Activity
Squalene synthase activity was analyzed in the membrane fractions obtained from Trichoderma transformants and the control strain by incubation of 200 µg of membrane fraction with 100 mM potassium phosphate buffer pH 7.4, 5 mM MgCl 2 , 5 mM CHAPS, 10 mM DTT, 2 mM NADPH and 10 µM [ 3 H]FPP in a total volume of 100 µL. Incubation was performed at 37 • C for 20 min, and then the reaction was stopped with 10 µL of 1 M EDTA pH 9.2, and 10 µL of unlabeled 0.5% squalene was added as a carrier. The reaction mixture was applied onto a Silica Gel 60 (Merck) thin-layer chromatography plate and developed in 5% (v/v) toluene in hexane. The radioactive zone containing squalene (R f = 0.5) was scraped off and measured in a scintillation counter [31,32].

Protein Concentration Assay
Protein concentration was estimated according to Lowry et al. [33].

Identification and Quantification of Saccharides Bound to Secreted Proteins
The saccharides bound to proteins isolated from T. atroviride culture filtrates were assayed by the phenol-sulfuric acid procedure [34]. Secreted proteins were precipitated with two volumes of 96% ethanol, washed twice with 70% ethanol and dissolved in distilled water [35]. O-linked carbohydrates were removed from the protein pellet using mild alkaline hydrolysis according to Duk et al. [36] and analyzed in the supernatant, and then the concentration of N-linked carbohydrates was measured in the pellet. The calibration curve was prepared with D-mannose.
Oand Nlinked carbohydrates obtained as above were hydrolyzed with 2 M TFA (trifluoroacetic acid) at 100 • C for 16 h. Monosaccharides were determined by highperformance anion-exchange chromatography using the Dionex ICS-3000 Ion Chromatography System with a Carbo Pac PA10 analytical column. Neutral sugars were eluted with 18 mM NaOH at 0.25 mL/min [37].

Cellulase Activity
The activity of cellulases was measured in the cultivation medium by incubation of 0.5 mL of carboxymethylcellulose (10 g/L) in 50 mM sodium citrate buffer (pH 5.0) at 50 • C for 2 h with 0.2 mL of culture filtrate. The reaction was stopped by boiling for 5 min. The amount of reducing sugars formed was determined by the method of Bernfeld [38] and estimated using a standard curve prepared with glucose. One unit of activity was defined as the amount of enzyme that liberated 1 µg of glucose from the substrate per 2 h.

Extraction and Purification of Polyisoprenoids
Trichoderma biomass was harvested by filtration, suspended in 60% KOH with 0.25% pyrogallol and hydrolyzed at 100 • C for 1 h. Lipophylic products were extracted with di-ethyl ether and evaporated to dryness, and then dissolved in hexane and applied onto a silica gel column equilibrated with hexane. The column was washed with 3% diethyl ether in hexane, and the polyisoprenoid fraction was eluted with 10% and 20% diethyl ether in hexane; the two eluates were pooled, evaporated to dryness, dissolved in isopropanol and subjected to HPLC/UV analysis as described earlier [39], with modifications. Briefly, a linear gradient of methanol: water (9:1, v/v) in methanol:isopropanol:hexane (2:1:1, v/v/v) was used for elution. Fractions containing polyisoprenoids (polyprenols and dolichols) were collected (based on comparison of their retention times with those of yeast dolichols used as external standards) and analyzed. For quantitative analysis, Dol13 was added to the biomass as an internal standard before hydrolysis. The polyprenol and dolichol standards were from the Collection of Polyprenols (IBB PAS).

Squalene Determination
Sterols were extracted according to the method proposed by Bernat et al. [40] with some modifications. First, 30 mg of lyophilized biomass was transferred into Eppendorf tubes containing glass beads, 0.66 mL of methanol and 0.33 mL of chloroform. The homogenization process using a ball mill (FastPrep-24, MP-Biomedicals) was carried out for 2 min. Next, the sample was centrifuged (2 min, 6000× g). The mixture was transferred to another Eppendorf tube. In order to facilitate the separation of two layers, 0.2 mL of H 2 O was added. The lower layer was collected and evaporated. Then, samples were diluted in 1 mL of methanol:chloroform (4:1, v/v). Sterols were measured using an Agilent 1200 HPLC system (Santa Clara, CA, USA) and a 3200 Q-TRAP mass spectrometer (Sciex, Framingham, MA, USA) equipped with the atmospheric pressure chemical ionization (APCI) source operating in the positive ionization mode. Then, 10 µL of the lipid extract was injected onto a Kinetex C18 column (50 mm × 2.1 mm, particle size: 5 µm; Phenomenex, Torrance, CA, USA) heated to 40 • C with a flow rate of 0.8 mL min −1 . Water (A) and methanol (B) were applied as a mobile phase, both consisting of 5 mM ammonium formate. The solvent gradient was initiated at 40% B, increased to 100% B during 1 min and maintained at 100% B for 3 min before returning to the initial solvent composition over 2 min. The following instrumental settings were applied: curtain gas 25.0, ion spray voltage to 5500 nebulizer gas 50, auxiliary gas 50 and ion source temperature of 550 • C, entrance potential to 10

Plate Confrontation Assay
Mycelial disks (5 mm) of T. atroviride RER2 transformants and P. ultimum were placed at opposite sides (7 cm apart) of agar plates with MM supplemented with 1% glucose. The plates were incubated at 28 • C, and photographs were taken after 3 days of incubation. Three replicates were prepared for each experiment and for each transformant.

Growth Inhibition of Fungal Pathogen by Agents Secreted by Trichoderma to Cultivation Medium
To determine its antagonistic activity, Trichoderma was grown on solid minimal medium with lactose covered with cellophane. Following the removal of the cellophane with Trichoderma, the effect of postculture medium containing secreted hydrolytic enzymes and metabolites on growth of P. ultimum was analyzed. A 5 mm mycelial disk of P. ultimum was placed in the center of the Trichoderma-pretreated plate. Plates were incubated at 28 • C for eight days, and the area of growth was measured. As a control, P. ultimum was cultivated on non-pretreated plates.

Statistical Analysis
For statistical analysis, we used the Student's t test to compare the mean and standard deviation obtained for the mutants and the control strain. Standard significance level p-value of 0.05 was used.

Expression of S. cerevisiae RER2 Gene in T. atroviride
The biocontrol T. atroviride strain P1 was transformed with the RER2 gene from S. cerevisiae, and stable transformants were isolated, as detailed in Section 2 The presence of the yeast gene in the genome of the transformants was analyzed by PCR using RER2U (5 CGGGATTCATGGAAACGGATAGTG GTATA 3 ) and RER2L (5 CGGATATCT-TAATTCAACTTTTTTTCTTTC AAATC 3 ) primers [18] and genomic DNA isolated from the transformants ( Figure S1). As a positive and negative control, the PCR reactions were performed on the templates of DNA from S. cerevisiae and untransformed T. atroviride, respectively. Two transformants, RER30/11 and RER32/11, were obtained and subjected to further analysis. A copy number of RER2 gene integrated into the Trichoderma genome was estimated by quantitative real-time PCR (qRT-PCR) analysis using the relative standard curve method [22][23][24]. It was established that transformants differ in the number of copies of the RER2 gene integrated into their genome. Two copies were found in the RER30/11 strain, while only one copy was found in the RER32/11 transformant.
To quantify RER2 expression in the transformed strains, mRNA was isolated, and reverse transcription qPCR analysis was performed. A significant expression of the RER2 gene was observed in both transformants ( Figure 2). The expression was tightly dependent on the number of RER2 copies. atroviride. Trichoderma strains (30-RER30/11; 32-RER32/11) and control strain P1 were grown for 144 h in PDB medium, RNA was extracted and cDNA synthesized. qPCR reactions were performed using a LightCycler 96 instrument. The amplification and melting curve data were collected and analyzed using the LightCycler96 ® software 1.0. Data were obtained from three independent experiments, each determined in triplicate. Expression of the native rer2 gene in the transformed strains was normalized to rer2 expression in the control strain P1.
Furthermore, expression of the native rer2 gene from Trichoderma was increased in the transformants compared to the control strain P1.
Thus, the PCR analyses showed that the RER2 gene was integrated into the genome of the T. atroviride transformants and expressed in both transformants. Moreover, expression of the native rer2 gene was slightly elevated, and expression of nus1, encoding subunit of the cis-prenyltransferase complex, was decreased in these strains compared to the control.

Biochemical Characterization of T. atroviride RER2 Transformants
To reach higher activity of cis-prenyltransferase, RER2 gene from S. cerevisiae was used for expression in T. atroviride since activity of cis-prenyltransferase from yeast is several times higher compared to the homolog from Trichoderma [18,25,41].
Expression of the RER2 gene increased the activity of cis-prenyltransferase in the membrane fraction obtained from the RER2 transformants compared to the control strain P1. The higher increase, by 93%, was observed for the RER30/11 strain, while RER32/11 revealed a 30% increase (Figure 3). The higher activity of cis-prenyltransferase in the RER30/11 transformant is consistent with the higher expression of the RER2 gene in this strain (Figure 2). Furthermore, expression of the native rer2 gene from Trichoderma was increased in the transformants compared to the control strain P1.
Thus, the PCR analyses showed that the RER2 gene was integrated into the genome of the T. atroviride transformants and expressed in both transformants. Moreover, expression of the native rer2 gene was slightly elevated, and expression of nus1, encoding subunit of the cis-prenyltransferase complex, was decreased in these strains compared to the control.

Biochemical Characterization of T. atroviride RER2 Transformants
To reach higher activity of cis-prenyltransferase, RER2 gene from S. cerevisiae was used for expression in T. atroviride since activity of cis-prenyltransferase from yeast is several times higher compared to the homolog from Trichoderma [18,25,41].
Expression of the RER2 gene increased the activity of cis-prenyltransferase in the membrane fraction obtained from the RER2 transformants compared to the control strain P1. The higher increase, by 93%, was observed for the RER30/11 strain, while RER32/11 revealed a 30% increase (Figure 3). The higher activity of cis-prenyltransferase in the RER30/11 transformant is consistent with the higher expression of the RER2 gene in this strain (Figure 2).

Figure 3.
Activities of cis-prenyltransferase and squalene synthase in membrane fraction or FPP synthase in cell-free extracts from RER2 transformed T. atroviride. Trichoderma strains (30-RER30/11; 32-RER32/11) and control strain P1 were processed and analyzed for enzymatic activity, as described in Section 2. Data are presented as mean ± standard deviation from six independent experiments, each determined in triplicate. Differences are statistically significant (p < 0.05; t test).
cis-prenyltransferase uses farnesyl diphosphate (FPP) synthesized by FPP synthase as a substrate. The increased activity of cis-prenyltransferase in the transformants consumes more substrate, and this in turn could stimulate FPP synthase for more intensive production. Analysis of the FPP synthase activity confirmed this assumption: it was increased by 23 (RER32/11) and 29% (RER30/11) in the transformants compared to the control strain (Figure 3). FPP is also consumed by squalene synthase. Our study revealed that activity of squalene synthase was increased by 74 and 29% for RER30/11 and RER32/11, respectively, compared to the control strain ( Figure 3).
The higher activities of cis-prenyltransferase and squalene synthase are reflected in a higher content of dolichols and squalene in the mycelia of the RER2 transformants (Figure 4). Concentration of dolichols was 30% and 21% elevated in the RER30/11 and RER32/11 transformants, respectively, compared to the control strain. Concentration of squalene was also increased. A higher increase, by 52%, was observed for the RER30/11 strain, while RER32/11 revealed only a 5% increase, and the latter difference was not statistically significant. Increase in concentration of the both products, dolichols and squalene, is consistent with higher activity of the corresponding enzymes, cis-prenyltransferase and squalene synthase, respectively (Figures 3 and 4). . Activities of cis-prenyltransferase and squalene synthase in membrane fraction or FPP synthase in cell-free extracts from RER2 transformed T. atroviride. Trichoderma strains (30-RER30/11; 32-RER32/11) and control strain P1 were processed and analyzed for enzymatic activity, as described in Section 2. Data are presented as mean ± standard deviation from six independent experiments, each determined in triplicate. Differences are statistically significant (p < 0.05; t test).
Cis-prenyltransferase uses farnesyl diphosphate (FPP) synthesized by FPP synthase as a substrate. The increased activity of cis-prenyltransferase in the transformants consumes more substrate, and this in turn could stimulate FPP synthase for more intensive production. Analysis of the FPP synthase activity confirmed this assumption: it was increased by 23 (RER32/11) and 29% (RER30/11) in the transformants compared to the control strain ( Figure 3). FPP is also consumed by squalene synthase. Our study revealed that activity of squalene synthase was increased by 74 and 29% for RER30/11 and RER32/11, respectively, compared to the control strain ( Figure 3).
The higher activities of cis-prenyltransferase and squalene synthase are reflected in a higher content of dolichols and squalene in the mycelia of the RER2 transformants ( Figure 4). Concentration of dolichols was 30% and 21% elevated in the RER30/11 and RER32/11 transformants, respectively, compared to the control strain. Concentration of squalene was also increased. A higher increase, by 52%, was observed for the RER30/11 strain, while RER32/11 revealed only a 5% increase, and the latter difference was not statistically significant. Increase in concentration of the both products, dolichols and squalene, is consistent with higher activity of the corresponding enzymes, cis-prenyltransferase and squalene synthase, respectively (Figures 3 and 4). Trichoderma strains (30-RER30/11; 32-RER32/11) and control strain P1 were processed and analyzed for the concentration of products synthesized by cis-prenyltransferase and squalene synthase, dolichols and squalene, respectively. Data are presented as mean ± standard deviation from three independent experiments, each determined in triplicate. Differences are statistically significant (p < 0.05; t test).
Since cis-prenyltransferase produces dolichols and phosphorylated dolichols (Dol-P) serve as acceptors of carbohydrate residues for glycosylation, we examined the activity of dolichyl phosphate mannose (DPM) synthase producing the active form of mannose, DPM, for O-and N-glycosylation of proteins. Both transformants revealed higher activity of DPM synthase compared to the control ( Figure 5). A higher increase, by 37%, was found in the membrane fraction of RER32/11, while in RER30/11, the activity was elevated only by 17%. We also analyzed the activity of N-acetylglucosamine transferase, the first enzyme in the synthesis of Dol-PP-oligosaccharide, used for N-glycosylation. Its activity was elevated by ca. 40% in both transformants compared to the control ( Figure 5). Figure 5. Activities of DPM synthase and N-acetylglucosamine transferase in membrane fraction from RER2 transformed T. atroviride. Trichoderma strains (30-RER30/11; 32-RER32/11) and control strain P1 were processed and analyzed for enzymatic activity as described in Section 2. Data are presented as mean ± standard deviation from six (DPM synthase) or four (N-acetylglucosamine transferase) independent experiments, each determined in triplicate. Differences are statistically significant (p < 0.05; t test).  Since cis-prenyltransferase produces dolichols and phosphorylated dolichols (Dol-P) serve as acceptors of carbohydrate residues for glycosylation, we examined the activity of dolichyl phosphate mannose (DPM) synthase producing the active form of mannose, DPM, for Oand N-glycosylation of proteins. Both transformants revealed higher activity of DPM synthase compared to the control ( Figure 5). A higher increase, by 37%, was found in the membrane fraction of RER32/11, while in RER30/11, the activity was elevated only by 17%. We also analyzed the activity of N-acetylglucosamine transferase, the first enzyme in the synthesis of Dol-PP-oligosaccharide, used for N-glycosylation. Its activity was elevated by ca. 40% in both transformants compared to the control ( Figure 5).  Since cis-prenyltransferase produces dolichols and phosphorylated dolichols (Dol-P) serve as acceptors of carbohydrate residues for glycosylation, we examined the activity of dolichyl phosphate mannose (DPM) synthase producing the active form of mannose, DPM, for O-and N-glycosylation of proteins. Both transformants revealed higher activity of DPM synthase compared to the control ( Figure 5). A higher increase, by 37%, was found in the membrane fraction of RER32/11, while in RER30/11, the activity was elevated only by 17%. We also analyzed the activity of N-acetylglucosamine transferase, the first enzyme in the synthesis of Dol-PP-oligosaccharide, used for N-glycosylation. Its activity was elevated by ca. 40% in both transformants compared to the control ( Figure 5).   Figure 5. Activities of DPM synthase and N-acetylglucosamine transferase in membrane fraction from RER2 transformed T. atroviride. Trichoderma strains (30-RER30/11; 32-RER32/11) and control strain P1 were processed and analyzed for enzymatic activity as described in Section 2. Data are presented as mean ± standard deviation from six (DPM synthase) or four (N-acetylglucosamine transferase) independent experiments, each determined in triplicate. Differences are statistically significant (p < 0.05; t test).

N-acetylglucosamine transferase
A higher production of DPM and DolPP-N-acetylglucosamine seemed likely to enhance the glycosylation of secretory proteins. In turn, it is hypothesized that the enhanced glycosylation of cellulases could influence their enzymatic activity.
To analyze the glycosylation of secretory proteins, Trichoderma was cultivated in minimal medium with lactose as a carbon source. Proteins were precipitated from culture medium after 72 or 96 h of cultivation, and the amount of Oand N-linked carbohydrates was determined.
A nearly 70% increase in the content of O-linked carbohydrates was found for both transformants compared to the control, while the amount of N-linked sugars was elevated more than fourfold ( Figure 6). A qualitative analysis of the Oand N-linked polysaccharides by high-performance anion-exchange chromatography showed that mannose was the dominant sugar in both types of polycarbohydrates. A higher amount of glucose and mannose in the N-linked polycarbohydrates was found for RER30/11 strain, and it was, respectively, 7.4-fold and 3.7-fold that of the control. The O-linked carbohydrates of both transformed strains contained nearly the same amount of galactose as the control, while the amount of mannose was 1.75-fold and 2.1-fold higher for the RER32/11 and RER30/11 transformants, respectively ( Figure 6). A higher production of DPM and DolPP-N-acetylglucosamine seemed likely to enhance the glycosylation of secretory proteins. In turn, it is hypothesized that the enhanced glycosylation of cellulases could influence their enzymatic activity.
To analyze the glycosylation of secretory proteins, Trichoderma was cultivated in minimal medium with lactose as a carbon source. Proteins were precipitated from culture medium after 72 or 96 h of cultivation, and the amount of O-and N-linked carbohydrates was determined.
A nearly 70% increase in the content of O-linked carbohydrates was found for both transformants compared to the control, while the amount of N-linked sugars was elevated more than fourfold (Figure 6). A qualitative analysis of the O-and N-linked polysaccharides by high-performance anion-exchange chromatography showed that mannose was the dominant sugar in both types of polycarbohydrates. A higher amount of glucose and mannose in the N-linked polycarbohydrates was found for RER30/11 strain, and it was, respectively, 7.4-fold and 3.7-fold that of the control. The O-linked carbohydrates of both transformed strains contained nearly the same amount of galactose as the control, while the amount of mannose was 1.75-fold and 2.1-fold higher for the RER32/11 and RER30/11 transformants, respectively ( Figure 6). O-and N-linked carbohydrates were released selectively from proteins secreted by transformants (30-RER30/11; 32-RER32/11) and control strain P1 and determined by high-performance anion-exchange chromatography. Data are presented as mean ± standard deviation from three independent experiments.

Cellulolytic Activity of RER2-Expressing T. atroviride
The aim of this study was to elevate the secreted hydrolytic activity of T. atroviride. Activity of cellulases was measured in the medium during cultivation. Higher activity was found in the cultivation medium of RER30/11 (Figure 7). It was two-fold higher compared to the control strain. The activity registered in the medium of RER32/11 was much lower, but still it was 1.2-fold higher than that in the control.

Cellulolytic Activity of RER2-Expressing T. atroviride
The aim of this study was to elevate the secreted hydrolytic activity of T. atroviride. Activity of cellulases was measured in the medium during cultivation. Higher activity was found in the cultivation medium of RER30/11 (Figure 7). It was two-fold higher compared to the control strain. The activity registered in the medium of RER32/11 was much lower, but still it was 1.2-fold higher than that in the control. Figure 7. Activities of cellulases in cultivation medium from RER2-transformed T. atroviride. Concentration of reducing sugars released from carboxymethylcellulose by cellulases secreted into cultivation medium by the transformants (30-RER30/11; 32-RER32/11) and the parental strain P1 were measured. Data are presented as mean ± standard deviation from six independent experiments. Differences are statistically significant (p < 0.05; t test).
The activity of cellulases reached the maximum after 96 h of cultivation. The higher enzymatic activity of cellulases, secreted by the transformants was taken as an indicator for a pronounced inhibition of the growth of pathogenic Oomycete, particularly of Pythium ultimum containing a high proportion of cellulose in its cell wall. To prove this hypothesis, Trichoderma was cultivated on agar plates covered with cellophane, which was permeable to low-molecular-weight metabolites and hydrolases. After removing the cellophane together with the mycelia, the plant pathogen P. ultimum was cultivated on the pretreated plates. The growth of P. ultimum was inhibited by 70% on plates previously overgrown by the control strain, while on the plates pretreated by the transformants, the inhibition reached 89% (Figure 8). atroviride. Trichoderma strains (30-RER30/11; 32-RER32/11) and control P1strain were cultivated for three days on MM plates covered with cellophane and then removed with the cellophane. P. ultimum was inoculated on the pretreated plates, and its rate of growth was determined as colony diameter after two days. As a control (C), P. ultimum was cultivated on non-pretreated plates. Data are presented as mean ± standard deviation from six independent experiments. Differences are statistically significant (p < 0.05; t test). . Activities of cellulases in cultivation medium from RER2-transformed T. atroviride. Concentration of reducing sugars released from carboxymethylcellulose by cellulases secreted into cultivation medium by the transformants (30-RER30/11; 32-RER32/11) and the parental strain P1 were measured. Data are presented as mean ± standard deviation from six independent experiments. Differences are statistically significant (p < 0.05; t test).
The activity of cellulases reached the maximum after 96 h of cultivation. The higher enzymatic activity of cellulases, secreted by the transformants was taken as an indicator for a pronounced inhibition of the growth of pathogenic Oomycete, particularly of Pythium ultimum containing a high proportion of cellulose in its cell wall. To prove this hypothesis, Trichoderma was cultivated on agar plates covered with cellophane, which was permeable to low-molecular-weight metabolites and hydrolases. After removing the cellophane together with the mycelia, the plant pathogen P. ultimum was cultivated on the pretreated plates. The growth of P. ultimum was inhibited by 70% on plates previously overgrown by the control strain, while on the plates pretreated by the transformants, the inhibition reached 89% (Figure 8).
J. Fungi 2023, 9, x FOR PEER REVIEW 12 of 16 Figure 7. Activities of cellulases in cultivation medium from RER2-transformed T. atroviride. Concentration of reducing sugars released from carboxymethylcellulose by cellulases secreted into cultivation medium by the transformants (30-RER30/11; 32-RER32/11) and the parental strain P1 were measured. Data are presented as mean ± standard deviation from six independent experiments. Differences are statistically significant (p < 0.05; t test).
The activity of cellulases reached the maximum after 96 h of cultivation. The higher enzymatic activity of cellulases, secreted by the transformants was taken as an indicator for a pronounced inhibition of the growth of pathogenic Oomycete, particularly of Pythium ultimum containing a high proportion of cellulose in its cell wall. To prove this hypothesis, Trichoderma was cultivated on agar plates covered with cellophane, which was permeable to low-molecular-weight metabolites and hydrolases. After removing the cellophane together with the mycelia, the plant pathogen P. ultimum was cultivated on the pretreated plates. The growth of P. ultimum was inhibited by 70% on plates previously overgrown by the control strain, while on the plates pretreated by the transformants, the inhibition reached 89% (Figure 8). atroviride. Trichoderma strains (30-RER30/11; 32-RER32/11) and control P1strain were cultivated for three days on MM plates covered with cellophane and then removed with the cellophane. P. ultimum was inoculated on the pretreated plates, and its rate of growth was determined as colony diameter after two days. As a control (C), P. ultimum was cultivated on non-pretreated plates. Data are presented as mean ± standard deviation from six independent experiments. Differences are statistically significant (p < 0.05; t test). atroviride. Trichoderma strains (30-RER30/11; 32-RER32/11) and control P1strain were cultivated for three days on MM plates covered with cellophane and then removed with the cellophane. P. ultimum was inoculated on the pretreated plates, and its rate of growth was determined as colony diameter after two days. As a control (C), P. ultimum was cultivated on non-pretreated plates. Data are presented as mean ± standard deviation from six independent experiments. Differences are statistically significant (p < 0.05; t test).
In addition, we made a confrontation assay between the new Trichoderma strains and P. ultimum (Figure 9). Trichoderma and Pythium were cultivated on a single plate from separate inocula. We observed that Pythium was overgrown more rapidly by both transformants than by the control P1 strain and that the RER30/11 transformant was the most efficient. In addition, we made a confrontation assay between the new Trichoderma strains and P. ultimum (Figure 9). Trichoderma and Pythium were cultivated on a single plate from separate inocula. We observed that Pythium was overgrown more rapidly by both transformants than by the control P1 strain and that the RER30/11 transformant was the most efficient. Figure 9. Plate confrontation assay of T. atroviride against P. ultimum. Mycelial disks of RER30/11 (30), RER32/11 (32) transformants and the control strain (P1) and P. ultimum were placed at opposite sides of agar plates and incubated at 28 °C. Pictures were taken three days after inoculation. Arrows mark the overgrown zone between the two fungi. The overgrown zones were measured, and results are presented as mean ± standard deviation from three separate plates. Differences are statistically significant (p < 0.05; t test).

Discussion
The aim of this study was to construct novel strains of T. atroviride with elevated hydrolytic capabilities. It is well known that during a direct attack, Trichoderma degrades the cell wall of a fungal pathogen with a variety of secreted extracellular enzymes [42]. Based on this knowledge, we decided to increase the hydrolytic activity of the new strains. Our previous studies have shown an interdependence between the level of glycosylation of secretory proteins and their hydrolytic activity. Furthermore, we observed that improved processing of secretory proteins in the endoplasmic reticulum elevated their secretion [12,13,31]. We improved glycosylation by increasing synthesis of dolichols by cis-prenyltransferase.
The basal activity of cis-prenyltransferase in T. atroviride was much lower than that in S. cerevisiae [18,25,41]. Therefore, the use of RER2 gene from S. cerevisiae for expression in Trichoderma was very promising for achieving high activity of cis-prenyltransferase in the transformed strains. In addition, Rer2 protein from yeast had to form a functional complex with Nus1p from Trichoderma, giving increased activity of cis-prenyltransferase in the RER2 transformants of T. reesei [18]. It was shown that Nus1protein together with Rer2p were required for cis-prenyltransferase activity [43].
The glycosylation of secretory proteins has been postulated to protect them against proteolysis, and, moreover, it has been shown to directly affect the conformation and stability of the secreted enzymes [4,6,[9][10][11]. In addition, it has been reported that O-glycosylation is necessary for secretion of hydrolases by Trichoderma [44]. CBHI (cellobiohydrolase I), the main protein secreted by Trichoderma, consists of a catalytic domain and a cellulose binding module joined by a highly O-glycosylated linker peptide. The catalytic domain has four N-linked motifs, three of which are glycosylated. The type and extent of glycan on the catalytic domain are influenced by both the strain and culture condition [45]. In addition, the amount of carbohydrates bound to secretory proteins varies during cultivation [18]. T. atroviride P. ultimum Figure 9. Plate confrontation assay of T. atroviride against P. ultimum. Mycelial disks of RER30/11 (30), RER32/11 (32) transformants and the control strain (P1) and P. ultimum were placed at opposite sides of agar plates and incubated at 28 • C. Pictures were taken three days after inoculation. Arrows mark the overgrown zone between the two fungi. The overgrown zones were measured, and results are presented as mean ± standard deviation from three separate plates. Differences are statistically significant (p < 0.05; t test).

Discussion
The aim of this study was to construct novel strains of T. atroviride with elevated hydrolytic capabilities. It is well known that during a direct attack, Trichoderma degrades the cell wall of a fungal pathogen with a variety of secreted extracellular enzymes [42]. Based on this knowledge, we decided to increase the hydrolytic activity of the new strains. Our previous studies have shown an interdependence between the level of glycosylation of secretory proteins and their hydrolytic activity. Furthermore, we observed that improved processing of secretory proteins in the endoplasmic reticulum elevated their secretion [12,13,31]. We improved glycosylation by increasing synthesis of dolichols by cis-prenyltransferase.
The basal activity of cis-prenyltransferase in T. atroviride was much lower than that in S. cerevisiae [18,25,41]. Therefore, the use of RER2 gene from S. cerevisiae for expression in Trichoderma was very promising for achieving high activity of cis-prenyltransferase in the transformed strains. In addition, Rer2 protein from yeast had to form a functional complex with Nus1p from Trichoderma, giving increased activity of cis-prenyltransferase in the RER2 transformants of T. reesei [18]. It was shown that Nus1protein together with Rer2p were required for cis-prenyltransferase activity [43].
The glycosylation of secretory proteins has been postulated to protect them against proteolysis, and, moreover, it has been shown to directly affect the conformation and stability of the secreted enzymes [4,6,[9][10][11]. In addition, it has been reported that O-glycosylation is necessary for secretion of hydrolases by Trichoderma [44]. CBHI (cellobiohydrolase I), the main protein secreted by Trichoderma, consists of a catalytic domain and a cellulose binding module joined by a highly O-glycosylated linker peptide. The catalytic domain has four N-linked motifs, three of which are glycosylated. The type and extent of glycan on the catalytic domain are influenced by both the strain and culture condition [45]. In addition, the amount of carbohydrates bound to secretory proteins varies during cultivation [18].
In the present report, the highest glycosylation of secretory proteins appeared after 96 h of cultivation of the RER2-transformed strains and correlated with their highest hydrolytic activity.
The hydrolases secreted by Trichoderma to the medium strongly inhibited the growth of P. ultimum, an Oomycetes whose cell wall contains mainly 1,3-β glucans, 1,6-β-glucans and 1,4-β-glucans (cellulose). Chitin, the major constituent of the "typical" fungal cell wall, has been detected in small amounts in few Oomycetes [46]. Since the cellulolytic activity secreted by the Trichoderma transformants was significantly elevated compared to the nontransformed strain, it resulted in substantially higher antimicrobial activity of these strains against P. ultimum.
In summary, these results show that posttranslational modification of secretory proteins is a promising target for improving the effectiveness of the direct attack of Trichoderma on fungal pathogens. This study and our previous results confirm that simple genetic manipulations to enhance the activity of glycosylation-related enzymes, such as DPM synthase, guanylyltransferase, farnesyl pyrophosphate synthase and cis-prenyltransferase, produce pleiotropic effects that ultimately result in a higher activity and/or secretion of hydrolytic enzymes [12][13][14]31,47]. These results are in contrast to more direct attempts to enhance the hydrolytic activity of Trichoderma by overexpression of genes coding for a single lytic enzyme [48,49]. The biocontrol effects of such manipulations were rarely significant, probably because polymers such as cellulose, glucan and chitin require several hydrolytic enzymes for degradation. By improving protein glycosylation, we obtained a more general effect not limited to a single enzyme.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jof9010038/s1. Table S1: Primers used in this studies; Figure S1: The presence of the yeast RER2 gene in the genome of the transformants analyzed by PCR.