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

Abstract

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.

Keywords: T. atroviride; antimicrobial activity; cis-prenyltransferase; glycosylation.

Figure 1

Metabolic pathways analyzed in this study. Abbreviations presented in the figure: HMGR—3 hydroxy-3-methylglutaryl–CoA reductase (regulatory enzyme of the mevalonate pathway); FPP synthase—farnesyl diphosphate synthase; SS—squalene synthase; cis-PT (Rer2)—cis-prenyltransferase; 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.

Figure 2

Transcript levels of RER2 genes from S. cerevisiae (RER2S.c) and T. atroviride (rer2T.a) and nus1 subunit of cis-prenyltransferase (nus1T.a) determined by RT-qPCR in the RER2 transformed T. 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.

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).

Figure 4

Concentration of dolichols and squalene in the mycelia from RER2 transformed T. atroviride. 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).

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).

Figure 6

Content of O- and N-linked carbohydrates in proteins secreted by RER2 transformed T. atroviride after 96 h of cultivation. 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. ★—differences statistically insignificant (p < 0.05; t test).

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).

Figure 8

Growth inhibition of P. ultimum cultivated on plates pretreated with RER2 transformed T. 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).

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).