Integration of additional copies of Trichoderma reesei gene encoding protein Omannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition.
Wioletta Górka-Nieć 1 , Anna Kania 1 , Urszula Perlińska-Lenart 1 , Gabriela
Smoleńska-Sym 2 , Grażyna Palamarczyk 1 , Joanna S. Kruszewska 1*
1.
Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Pawińskiego 5a, 02-106 Warsaw, Poland
2.
Department of Biochemistry, Institute of Haematology and Blood
Transfusion, Chocimska 5, 00-957 Warsaw, Poland
Address for correspondence: Dr. Joanna S. Kruszewska, Institute of
Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-
106 Warsaw, Poland. Phone (+ 48 22) 592 12 09 Fax: (+48 22) 658 46 36
E-mail jsk@ibb.waw.pl
Key words: Trichoderma reesei ; silencing of pmt genes; protein glycosylation; cell wall composition
1

SUMMARY
In fungi, transfer of the first mannosyl residue to proteins during their Oglycosylation is catalyzed by protein O-mannosyltransferases. Integration of additional copies of the pmt1 gene into Trichoderma reesei genome unexpectedly resulted in the silencing of pmt1 expression. Strains carrying the additional copies of pmt1 gene exhibited lower total activity of protein Omannosyltransferases, lower O- and N-glycosylation of secreted proteins and showed defects in their cell wall composition. Moreover, the strains grew slowly on solid medium and were hypersensitive to an antifungal reagent, Calcofluor white. These results indicate that protein O-manosyltransferases are required for proper cell wall formation, and their decreased activity influences not only O- but also N-glycosylation.
2

INTRODUCTION
Trichoderma species play an important role in the biotechnological industry where their protein synthesis and secretory capability are widely exploited for protein production. Some species of Trichoderma secrete up to 40g of protein per liter of culture (Durand et al. 1988). Our previous study indicated that protein production and secretion could be up-regulated simultaneously with the activity of the O-glycosylation pathway (Kruszewska et al. 1999, Perlińska-
Lenart et al. 2005, 2006b). Overexpression of the Saccharomyces cerevisiae
DPM1 gene coding for dolichyl phosphate mannose synthase (DPM synthase) in
T.reesei
resulted in up to seven-fold higher secretion of proteins. Moreover, all those proteins were glycosylated at the wild type level (Kruszewska et al. 1999).
Similarly, expression of the DPM1 gene in Aspergillus nidulans stimulated protein production, but the proteins were arrested between the cell wall and cell membrane (Perlińska-Lenart et al. 2005). This result pointed at a fundamental role of cell wall permeability for protein secretion in Aspergillus . Removal of the cell wall from Trichoderma strains carrying yeast DPM1 gene resulted in a four-fold increase of protein secretion compared with mycelia, which suggested that the cell wall was a barrier for secretion in those strains. At the same time, secretion from protoplasts and mycelia of the control strain was nearly identical, showing that not only the cell wall was a barrier for secretion but also efficient processing of newly synthesized glycoproteins (Perlińska-Lenart et al. 2006b).
3

On the other hand, stimulation of the O- and N-glycosylation pathways by increased production of GDP-mannose or dolichyl phosphate (Dol-P) did not alter cell wall composition and did not stimulate protein production or secretion but resulted in overglycosylation of secreted proteins (Zakrzewska et al. 2003b,
Perlińska-Lenart et al. 2006a).
Furthermore, protein production and secretion could be stimulated by limited glycosylation, as was observed in a Hansenula polymorpha pmt1 mutant. In this strain, N-glycosylated proteins such as heterologously expressed human urinary type plasminogen activator were produced and secreted in higher amounts although they were unglycosylated (Agaphonov et al. 2005). It is important that the stimulation of protein production and secretion concerned only Nglycoproteins whereas production and secretion of the O-glycosylated chitinase was significantly decreased.
All these results pointed to the importance of the O-glycosylation process for protein production and secretion and for the cell wall synthesis.
In this study we concentrated on the enzyme catalyzing the direct transfer of mannosyl residue from dolichyl phosphate mannose (DPM) to the serine/threonine OH group of the protein. These enzymes, called protein Omannosyltransferases, are very strongly represented in yeast by seven proteins encoded by PMT genes (Girrbach et al. 2000).
4

In S.cerevisiae
, the decrease of O-mannosyltransferase activity caused by deletion of some PMT genes resulted in limited protein glycosylation and changes in the cell wall structure (Strahl-Bolsinger et al. 1999).
In A.nidulans
, three Pmt proteins have been identified and it was shown that lack of individual Pmt proteins resulted in cell wall damage, swollen hyphae, reduced or non-conidiation, hyperbranching, increased number of nuclei and presence of non-O-glycosylated proteins (Goto, 2007). The growth repression and swollen hyphae phenotype of the Aspergillus pmtA disruptant was suppressed under high osmolarity conditions.
Up to now, only one pmt gene encoding protein O-mannosyltransferase in
Trichoderma has been cloned (Zakrzewska et al. 2003a), however, a search of the Trichoderma genome data base revealed two more sequences similar to pmt sequences from other filamentous fungi (our unpublished data).
Analysis of the predicted amino acids sequence of the Trichoderma PMTI protein showed the highest 51% identity with S.cerevisiae
Pmt4p. On the other hand, expression of the Trichoderma PMTI protein in S.cerevisiae pmt∆ mutants revealed its functional similarity to the yeast Pmt2 protein. The Trichoderma
PMTI protein was able to form an active O-mannosyltransferase complex with yeast Pmt1p and partially rescued the defective glycosylation pattern of chitinase (Zakrzewska et al. 2003a) and completely restored glycosylation of heterologously expressed cellobiohydrolase II from Trichoderma in the pmt2

S.cerevisiae mutant (Górka-Nieć et al. 2007).
5

In this paper we investigated the influence of additional copies of the pmt1 gene integrated into the genome of T.reesei
on the glycosylation, production and secretion of proteins and on the cell wall composition. Transformation of
T.reesei
with the pmt1 gene resulted in gene silencing which led to a decrease of the overall activity of protein O-mannosyltransferases. Silencing of the pmt genes limited both O- and N-glycosylation of secreted proteins and decreased the amount of

-(1,3) glucan in the cell wall, while the content of both, alkali soluble and alkali insoluble

-(1,6) glucans increased.
6

MATERIALS AND METHODS
Strains and growth conditions
T.reesei QM 9414 (ATCC 26921) was used as a recipient strain for transformation. Escherichia coli strain JM 109 was used for plasmid propagation (Yanish-Perron et al., 1985). T.reesei
was cultivated at 30°C on a rotary shaker (250 r.p.m.) in 2 l shake flasks containing 1 l of minimal medium
(MM): 1 g MgSO
4 x 7H
2
O, 6 g (NH
4
)
2
SO
4
, 10 g KH
2
PO
4
, 3 g sodium citrate x
2H
2
O, and trace elements (25 mg FeSO
4
x 7H
2
O, 2.7 mg MnCl
2
x 4H
2
O, 6.2 mg
ZnSO
4
x 7H
2
O, 14 mg CaCl
2
x 2H
2
O) per liter and 1% (w/v) lactose as a carbon source. The flasks were inoculated with 42 x 10 6 conidia/l medium.
For inhibition of DNA methylation 25

M 5-azacytidine was added to the cultivation medium (Zhou et al. 2001).
Integration of additional copies of the pmt1 gene to the T.reesei genome.
The Trichoderma pmt1 gene was introduced into T. reesei under the Aspergillus nidulans gpdA gene promoter (glyceraldehyde-3 phosphate dehydrogenase) and trpC (indole-3-glycerol phosphate synthase) terminator using the pAN52-1NotI plasmid (NCBI accession number Z32697). The complete coding sequence of the T.reesei pmt1 gene (2435 bp cDNA) was amplified by PCR, using The
Expand High Fidelity PCR System (Boehringer Mannheim). The primers pmt1-
U (5’ CGG GAT CCT CCT CGC TCC AAA CCA CCG CAT AG 3’) and pmt1-L (5’ TCC CCC GGG GAC CGC CCC CTT CTG GCT GTC ATC 3’)
7

were used for gene amplification. The pAN521N plasmid was cleaved between the promoter and the terminator using NcoI, the sticky ends were blunted with
S1 nuclease (Promega) and the PCR product was ligated with the plasmid. The proper orientation and sequence of the pmt1 open reading frame was checked by sequencing.
The resultant plasmid was used in transformation of T.reesei
QM9414 by the protoplast transformation method (Debets et al., 1990;
Yamashiro et al., 1992). The plasmid p3SR2 with the amdS marker gene encoding acetaminidase from A. nidulans was used as a partner in cotransformation (Penttila et al. 1987). Cotransformants were selected on MM medium containing 20 mM acetamide, and stable transformants were isolated by three rounds from selective to nonselective medium.
The transformants were then cultivated in liquid MM medium for preparation of
DNA and RNA.
Molecular biology methods
Chromosomal DNA was isolated from T. reesei using the Promega Wizard
Genomic DNA Purification kit. Total RNA was isolated using the single-step method described by Chomczynski and Sacchi (1987). Other molecular biology procedures were performed according to standard protocols (Sambrook et al.,
1989).
Integration of additional copies of pmt1 gene was shown by Southern blotting analysis of DNA from transformants and the control strain cut with EcoRI
8

restriction enzyme (not cutting inside the pmt1 gene) and hybridized with the 2.5 kb DNA containing coding sequence of T.reesei pmt1 gene.
DNA methylation was confirmed by Southern blotting analysis done on DNA from pmt1 transformants and the control strain cut with HpaII and MspI restriction enzymes and hybridized as described above.
Both Hpa II and Msp I recognize the same tetranucleotide sequence (5’-CCGG-
3’), but display differential sensitivity to DNA methylation. Hpa
II is inactive when any of the two cytosines is fully methylated, but cuts the hemi-methylated
5’-CCGG-3’ at a lower rate compared to the unmethylated sequence; whereas,
Msp
I cuts 5’-C5mCGG-3’, but not 5’-5mCCGG-3’(Fermentas protocol).
The radioactive probe was prepared using

[ 32 P] dATP and the Fermentas
HexaLabel Plus DNA labeling system according to the standard Fermentas protocol.
Semiquantitative RTPCR method used to estimate pmt1 expression in the
strains carrying additional copies of pmt1 gene.
Total RNA (2
 g) obtained from the pmt1 overexpressing strains and the control was reverse transcribed using Advantage ™ RT-for-PCR Kit (Clontech) according to manufactures instruction.
PCR was carried out in a 50
 l reaction volume with 1 U of Taq DNA polymerase (Invitrogen) and 30 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, and polymerization at 72°C for 2 min. The primers used were as follows: pmt-FU (5’ GATATC AAG CGC CGC TAT GGA 3’),
9

pmt-FL (5’ GAT TTC CTG CTG CCT GTA GGC 3’), act1-U (5’CAT GTT
CGA GAC ATT CAA CGC 3’) and act1-L (5’ AGC TCGTAG GAC TTC TCC
AAG 3’). The expression of act1 in each strain was used as a control. The relative level of mRNA isolated from strains carrying additional copies of the pmt1 gene was normalized to the level of mRNA isolated from the parental
T.reesei
QM9414 strain. To determine the effects of overexpression, the level of
RNA isolated from the parental strain was defined as 100%.
Controls without reverse transcriptase were performed for the assessment of
DNA contamination in the RNA samples.
Biochemical methods
Membrane fraction preparation
After 168h of cultivation (early stationary phase) 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

-mercaptoethanol. Cells were homogenized in a beadbeater with glass beads (0.5 mm) and the homogenate was centrifuged at 5,000 x g for 10 min to remove cell debris and unbroken cells. The supernatant was centrifuged at 100,000 x 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

-mercaptoethanol, and used as the source of enzymes. The whole procedure was performed at 4 o C (Pless and Palamarczyk, 1987).
10

Activity of protein O-mannosyltransferases
Protein O-mannosyltransferases activity was assayed in the pelleted membrane fraction by 1 h incubation at 30 o C with GDP[ 14 C]-mannose (sp.act. 288 Ci/mol,
Amersham) and 5 ng of dolichyl phosphate (Dol-P), according to Kruszewska et al. (1989).
Total membrane proteins (about 300
 g) were used as the sugar acceptor. Since only the transfer of the first mannosyl residue from dolichyl phosphate mannose (DPM) to the hydroxyl group of serine or threonine catalyzed by Pmt proteins was to be measured, the reaction mixture was supplemented with 10 mM MgCl
2
. Under these conditions elongation of the Olinked sugar chain does not occur (Sharma et al.1974). The protein Omannosyltransferase activity was expressed in pmoles of [ 14 C]-mannose incorporated into 1 mg membrane protein during 1 h.
N-acetylglucosamine transferase activity
N-acetylglucosamine transferase activity was measured in the membrane fraction by 30 min incubation at 30 o C of 200
 g of membrane protein in a total volume of 50
 l containing 1 x 10 5 cpm UDP[ 14 C]N-acetylglucosamine (sp.act.
249 Ci/mol, Amersham) and 5 ng of Dol-P in 40 mM Tris/HCl buffer pH 7.4 with 10 mM MgCl
2
and 0.1% Nonidet P-40 (Palamarczyk and Hemming, 1975).
The reaction was stopped by addition of 4 ml of chloroform – methanol (3:2 v/v). Formation of radioactive dolichyl diphosphate N-acetylglucosamine and
11

dolichyl diphosphate chitobiose was measured in the organic fraction by a scintillation counter.
Concentration of saccharides bound to secreted proteins
Secreted proteins were precipitated with two volumes of 96% ethanol, washed twice with 70% ethanol and dissolved in distilled water (Ma et al. 1996). Olinked sugars were cleaved by mild alkaline hydrolysis and then remaining proteins containing N-linked sugars were precipitated with two volumes of ethanol and centrifuged.
Total hydrolysis of carbohydrates was done with 2M TFA (trifluoroacetic acid ) at 100 o C with preliminary metanolysis with 1.5M HCL in anhydrous methanol at 85 o C for 16 hours (Zdebska and Kościelak, 1999). Neutral sugars and hexosamines were determined in the hydrolysates by high performance anionexchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a Dionex Series 4500i system. Neutral sugars and hexosamines were eluted with 18mM NaOH at 1ml min -1 .
Protein concentration assay
Protein concentrations were estimated according to Lowry et al. (1951).
Quantification of fungal dry weight
Fungal dry weight was quantified by filtering culture samples through G1 sintered glass funnels, washing the biomass with a threefold volume of tap water, and drying to constant weight at 110oC.
Colony growth rate
12

Colony growth rates were measured as described previously (Gradd et al. 2001,
Oka et al. 2004). Conidia were point-inoculated into the center of agar MM plates with or without Calcofluor white (300
 g ml -1 ) (Oka et al. 2004) and incubated at 30°C. Colony diameter was measured at 48, 72, 96, 120 and 144 h.
Measurements of the growth for all strains were done six times. Data are presented as a percentage of inhibition of growth caused by Calcofluor white compared to the growth on control plates.
Cell wall preparation
T.reesei
strains were cultivated for 168 h and the mycelia were harvested by centrifugation, homogenized in a beadbeater with 0.5 mm glass beads in 50 mM
Tris/HCl pH 7.5 with 1 mM DTT and centrifuged at 1500 x g for 10 min.
The resulting pellet containing cell walls was washed with ice-cold 1 M NaCl until disappearance of absorbance at 260-280 nm (Nemcovic and Farkas, 2001).
Determination of cell wall polysaccharides
The amount of glucans in the cell wall was determined as described previously
(Oka et al. 2004), with a slight modification. For quantification of alkali-soluble

-(1, 6) glucan, 200 mg of cell walls was suspended in 3 % NaOH, heated at
75

C for 1 h and centrifuged. The supernatant was dialyzed overnight at 4 o C against distilled water, lyophilized and the amount of alkali-soluble

-(1, 6) glucan was estimated by the method described by Dubois et al. (1956). The remaining pellet was washed twice with 0.1 M Tris/HCl pH 7.4 and once with
13

10 mM Tris/HCl pH 7.4 and digested overnight with zymolyase 20T (ICN
Biomedicals Inc.) (5 mg/ml in 10 mM Tris/HCl pH 7.4). Then the samples were centrifuged (13 000 rpm, 15 min) and the supernatant was used to estimate the amount of alkali-insoluble

-(1, 3) glucan by the same method (Dubois et al.
1956). The remaining pellets were incubated for 16 h with 70 % sulfuric acid at
4 o C, then diluted ten-fold with water and heated at 100 o C for 8 h. After neutralization with 2 M NaOH the samples were used to estimate the amount of alkali-insoluble

-(1, 6) glucan (Dubois et al. 1956).
For chitin measurements alkaline hydrolysis of cell walls was performed in 6%
KOH for 90 min at 80 o C in order to release cell wall proteins. After neutralization with acetic acid, the cell walls were washed with phosphatebuffered saline and chitinase buffer, pH 6.0, containing 18 mM citric acid and
60 mM dibasic sodium phosphate. Subsequently, the cell walls were treated with chitinase C (InterSpex Products) for 3 h at 37 o C. The level of chitin was measured with Ehrlich’s reagent as described (Reissig et al. 1955).
Microscopic analysis of mycelia
T. reesei transformants and control strains were harvested by centrifugation, washed twice with sterile water, resuspended in warm 1.5 % agarose (A-5030
Type IX from Sigma), allowed to cool and observed under a Nicon Eclipse E
6800 fluorescence microscope. The hyphae diameter and distances between septa were calculated from about one hundred pictures of each strain using
Lucia G program.
14

RESULTS
Integration of additional copies of the pmt1 gene to the T.reesei genome
T.reesei
QM9414 was transformed with pmt1 cDNA encoding protein Omannosyltransferase under the constitutive promoter of the gpdA gene (encoding glyceraldehyde-3-phosphate dehydrogenase) from A. nidulans (Hirano et al.,
2000). More than one hundred transformants growing on a selective medium containing acetamide were collected and isolated by three transfers from selective to nonselective medium, and examined by Southern analysis for integration of additional pmt1 gene copies into the T.reesei
genome (Fig.1).
Thirty- two transformants exhibiting an additional band were subjected to further analysis by Northern blotting. Total RNA isolated from these transformants was examined for overproduction of pmt1 mRNA and no such transformants were found. A positive signal of pmt1 mRNA was found only for
RNA obtained from the parental QM 9414 strain. To determine the level of mRNA in the pmt1 - transformed strains more accurately semiquantitative Real-
Time PCR was performed. When thirty cycles of the reaction were performed, the PCR product for pmt1 mRNA was detected only for the control strain; to detect the product for the transformants we had to prolong the reaction up to 40 cycles. In these conditions the amount of pmt1 mRNA in the transformants calculated from the assay was reduced by 6 up to 45% of the control level
(Fig.2A).
15

For further analysis we randomly chose three strains from the 32 obtained after
Southern and semiquantitative RT-PCR analysis.
To elevate the level of mRNA of genes coding for enzymes of the Oglycosylation pathway we routinely overexpressed the genes and in a number of cases observed the desired increase of the level of the respective mRNA
(Kruszewska et al. 1999, Zakrzewska et al. 2003b). The phenomenon of gene silencing obtained during gene overexpression was observed earlier in plants and was found to be correlated with DNA methylation (Napoli et al. 1990). The same mechanism of silencing was observed in Ascobolus immersus as a result of replicated DNA sequences (Goyon and Faugeron, 1989). To answer the question if the observed silencing of the pmt1 gene expression was caused by DNA methylation we cultivated pmt1 -transformed strains with 5-azacytidine, a drug reducing cytosine methylation, purified RNA and performed semiquantitative
RT-PCR analysis of the pmt1 mRNA level (Fig.2B). The inhibition of DNA methylation resulted in an increased level of pmt1 mRNA up to 95 % of the control level obtained without 5-azacitidine. Inhibition of DNA methylation also increased the pmt1 mRNA level in the control strain. The presence of DNA methylation was also confirmed by Southern blot analysis (Fig.3). Digestion of the DNA from transformants and the control strain with two restriction enzymes
MspI and HpaII characterized by different sensitivity to DNA methylation revealed significant differences in the pattern of the digestion for the pmt1
16

transformed strains compared to the control. These results suggest that in the transformants the coding sequence of pmt1 is methylated.
Effect of pmt1 gene silencing on the total activity of protein O- mannosyltransferases
Protein O-mannosyltransferase I (PMTI protein) encoded by the pmt1 gene catalyzes transfer of the mannosyl residue from dolichyl phosphate mannose
(DPM) to a serine or threonine OH group in the protein. To determined the influence of pmt1 silencing on the total activity of direct O-glycosylation we measured the combined activity of all PMT proteins (Fig.4). The experiment revealed a significant decrease of the activity of protein O-mannosyltransferases in all the strains compared to the control. The most pronounced decrease to 35% of the control activity was noticed for the VGJK17/07 strain, whereas, the activity in the VGJK5/07 and VGJK43/07 strains was similar and decreased to about 69% of the control. All the differences are statistically significant.
Effect of a lower activity of protein O-mannosyltransferases on the growth of
Trichoderma, protein secretion and glycosylation of secreted proteins
Trichoderma strains were cultivated in the lactose-base MM medium and every
24h portions of cultures were collected, mycelia harvested, dried and growth of the transformants was measured as the increase of dry mass of mycelia during time of cultivation. The differences in growth between transformants and the control strain, although observed, were not statistically significant. To make the
17

growth assay more precise the strains were cultivated on plates and colony diameter was measured as described in the Methods. The differences in growth determined in that manner showed that all the transformed strains were growing more slowly than the control strain and the differences were statistically significant (Table 1). We also measured the amount of proteins liberated to the cultivation medium during 216 h of cultivation and found that the amount of proteins secreted by all the transformed strains was similar to that secreted by the control QM9414 strain. Next, secreted proteins were examined for their glycosylation and it was noticed that silencing of the pmt genes’ expression caused a decrease of both O- and N-glycosylation (Table 2). The amount of Olinked carbohydrates bound to the secreted proteins decreased to 25 to 37% of the control, and at the same time, N-glycosylation of the proteins decreased to
38 to 58% of the control. Since N-glycosylation of the secreted proteins was altered we measured the activity of N-acetylglucosamine transferase, the first enzyme engaged in this process and found that this activity remained unchanged in the membrane fraction of the transformants compared to the control strain
(data not shown).
Effect of pmt1 gene silencing on the composition of cell wall of the transformants
It was observed in Aspergillus that limited activity of protein Omannosyltransferases in the pmtA disrupted strain altered the composition of the
18

cell wall of the mutant and caused changes in the sensitivity to antifungal drugs such as Calcofluor white (Oka et al. 2004; 2005).
Trichoderma strains with silenced pmt expression and the control QM9414 strain were cultivated for 168 h, the cell wall was isolated and the content of glucans and chitin was measured (Table 3). Silencing of the pmt genes caused a decrease in the content of

-(1,3) glucan from 20- to 35-fold compared to the control, while the content of both

-(1,6) glucans (alkali-soluble and insoluble) increased from 1.8- to 2.6-fold. Even though the amount of

-(1,6) glucans was elevated compared to the control strain the total content of glucans in the transformants was lower than in the control. Chitin content was nearly the same in the cell wall of the transformants compared to the control strain and the small differences presented in Table 3 were not statistically significant.
Hypersensitivity of pmt-transformed strains to Calcofluor white
Calcofluor white is known to be adsorbed on cell wall chitin and to exhibit antifungal properties as a consequence (Roncero and Duran, 1985). Hyphal growth of pmt1 transformants was significantly inhibited by Calcofluor white
(Table 1). The most pronounced, 36% inhibition of growth was observed for the
VGJK17/07 strain. In contrast to the transformants, growth of the control strain was not sensitive to Calcofluor white at the concentration used.
Microscopic analysis of mycelia
Our T.reesei
mutants showed significant changes in the composition of their cell wall. Since a weak cell wall could influence the structure of mycelia
19

we examined these strains under a phase-contrast microscope and measured the diameter of hyphae and the distance between septa using the Lucia G program.
One-hundred pictures of every transformant and control were analyzed, and we found that the hyphae of the strains bearing additional copies of the pmt1 gene had a diameter about double that of the control (Table 4, Fig.5). At the same time, the distance between septa in the mycelia of the mutants and the control strain were the same.
DISCUSSION
Our earlier studies showed that elevation of DPM synthase, guanyltransferase or cis -prenyltransferase activity in Trichoderma or Aspergillus caused significant changes in the activity of other enzymes engaged in the O-glycosylation pathway and altered protein secretion or glycosylation of secreted proteins
(Zakrzewska et al. 2003b, Perlińska-Lenart et al. 2005, 2006a, 2006b). This study concentrates on protein O-mannosyltransferases catalyzing the direct transfer of mannosyl residues to proteins. We intended to increase the total activity of these enzymes by integration of additional copies of the pmt1 gene into the genome of Trichoderma .
Unexpectedly, transformation of Trichoderma with the pmt1 gene resulted not in its overexpression and elevated activity of protein O-mannosyltransferases but in the silencing of the gene expression and a decrease of the activity of these enzymes.
20

In Schizophyllum commune introduction of additional copies of the SC3 hydrophobin gene resulted in gene silencing acting on both the introduced and endogenous SC3 copies and connected with DNA methylation (Schuurs et al.
1997).
De novo methylation was also detected in duplicated DNA sequences obtained by integration of additional copies of the met2 gene into the Ascobolus immersus genome, or the trp1 gene into the genome of Coprinus cinereus (Goyon and
Faugeron, 1989, Freedman and Pukkila, 1993).
In our case we also dealt with gene silencing caused by integration of additional copies of a gene and the mechanism of this silencing was related to DNA methylation, which was shown by cultivation of the VGJK strains with 5azacytidine, a drug reducing cytosine methylation, which resulted in an increased level of pmt1 mRNA. These results were confirmed by Southern blot analysis of DNA cut with enzymes differing in their sensitivity to DNA methylation.
Our results showed that integration of additional copies of the pmt1 gene led to a significant decrease in the total activity of protein O-mannosyltransferases. In spite of the lower activity of protein O-mannosyltransferases in the strains bearing the additional copies of the pmt1 gene, the alteration of the cell wall structure was not profound enough to disable the growth of the VGJK strains without an osmotic stabilizer, in contrast to the Trichoderma strain carrying
21

disruption of the pmt1 gene which could not grow without sorbitol (Górka-Nieć et al. 2008).
The decrease of the activity of protein O-mannosyltransferases slightly affected growth of the transformants and made the strains sensitive to the antifungal agent Calcofluor white. Moreover, microscopic study showed that mycelia of the transformants were about twice wider compared to the control strain.
It is known that a decreased activity of Pmt proteins alters the cell wall composition in fungi (Oka et al. 2004, 2005, Willer et al. 2005).
By analyzing the cell wall composition of strains with the expression of pmt genes silenced we found a simultaneous decrease of the total content of glucan and chitin. This means that the loss of glucan was not compensated by intensive synthesis of chitin because the cell wall compensatory mechanism was apparently not induced in our mutants. A different situation was observed in A. nidulans bearing disruption of the pmtA gene, where production of chitin was elevated and compensated the decreased amount of glucans (Oka et al. 2004,
2005).
In this study we also examined the influence of a decreased activity of protein
O-mannosyltransferases on the secretion of proteins and their glycosylation. We found that all our mutants secreted similar amounts of proteins compared to the control. A different effect was observed for chitinase secreted by the pmt mutant of Hansenula where an under- O-mannosylated enzyme was secreted in a decreased amount (Agaphonov et al. 2005). On the other hand, glucoamylase I
22

from A.awamori
expressed in the S.cerevisiae pmt1 mutant, although Oglycosylated to a lesser extent, was secreted in the same amount as in a wild type strain (Goto et al. 1999).
In T. reesei , the majority of secretory proteins are not only O-glycosylated but also have N- linked glycans (Palamarczyk, et al., 1998). The decreased activity of protein O-mannosyltransferases resulted in lower O-glycosylation of the secreted proteins and a simultaneous decrease of their N-glycosylation.
Changes in both types of glycosylation were observed for a Hansenula polymorpha pmt mutant, where O-mannosylation of chitinase was partially inhibited together with total inhibition of N-glycosylation of invertase or heterologously expressed human urinary type plasminogen activator
(Agaphonov, et al., 2005). The nature of the influence of a limited protein Omannosyltransferase activity on the N-glycosylation process is not clear, although many authors have reported a close interdependence of the two glycosylation processes (Ecker et al., 2003, Harty et al. 2001, Nakatsukasa et al.
2004). It was shown for an S.cerevisiae
pmt4

mutant that a lack of O-linked sugar bound to the serine or threonine in the vicinity of the N-glycosylation site enabled its N-glycosylation, while in the wild type strain, where the Oglycosylation site was occupied, the N-glycosylation site remained non- glycosylated (Ecker et al., 2003).
On the other hand, glucoamylase I secreted by an A.awamori pmtA mutant was
N-glycosylated to the same extent as the one secreted by the control strain,
23

although O-glycosylation of the protein was strongly decreased (Oka et al.
2005). To make the matter even more complex, one ought to mention that inhibition of N-glycosylation by tunicamycin has been reported to result in over-
O-mannosylation of many endogenous proteins (Harty et al. 2001). Those authors confirmed the competition of N- and O-linked glycosylation and showed that it was a general phenomenon in the ER that affected a large number of proteins. On the other hand, we showed that although N-glycosylation of the proteins secreted by VGJK mutants was decreased the activity of Nacetylglucosamine transferase, the first enzyme of the N-glycosylation pathway was not inhibited. That result suggests that some proteins that failed to be Oglycosylated could become N-glycosylated instead. A substitutive Nglycosylation may be a signal for glycosylation-induced glycoprotein degradation (Nakatsukasa et al. 2004).
In the present paper we showed the importance of at least partial activity of all the protein O-mannosyltransferases for the survival of Trichoderma cells. All the results underlined an essential role of the PMT proteins for the glycosylation of extracellular proteins and for the cell wall formation in Trichoderma .
24

ACKNOWLEDGMENTS
We wish to thank Dr. Marja Paloheimo from Roal Oy, Finland for the p3SR2 plasmid and Robert Ziętek from the Institute of Hematology and Blood
Transfusion for technical assistance. This work was partially supported by the
State Committee for Scientific Research (KBN), Warsaw, Poland, grant No.
6P04B00621, 1307/P01/2006/31 and Operational Programme - Innovative
Economy, Warsaw, Poland, grant No. UDA-POIG.01.03.01-14-038/09 to J.S.
Kruszewska.
25

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FIGURE LEGENDS
Figure 1
Southern blot analysis of EcoRI digested DNA of the control strain (QM9414) and the pmt1 transformants (VGJK5/07, VGJK17/07, VGJK43/07) hybridized
DNA probe containing coding sequence of T.reesei pmt1 gene.
Figure 2
Semiquantitative RTPCR analysis of pmt1 expression.
A. PCR product representing pmt1 mRNA obtained after 40 cycles of reaction.
B. Influence of 5-azacytidine on the transcription of the pmt1 gene. The columns show the signal intensity in arbitrary units after normalization for the actin band.
Black columns – Trichoderma growing without 5-azacitidine; gray columns-
Trichoderma growing with 5-azacitidine. Below are shown PCR products representing pmt1 mRNA and act mRNA obtained after 40 cycles of reaction.
Figure 3
Southern blot analysis of DNA methylation.
Autoradiography of MspI- and HpaII-digested total DNA from transformants,
(5)- VGJK5/07, (17) - VGJK17/07, (43) - VGJK43/07, and the control strain
(C).
Figure 4
Total activity of protein O-mannosyltransferases in the membrane fraction from
T.reesei
strains carrying additional copies of the pmt1 gene (VGJK strains) in comparison to the control strain QM9414.
33

Strains were cultivated for 168 h in the lactose-base MM medium.
The data are presented as mean

standard deviation from three separate cultures.
Figure 5
Hyphae morphology of the pmt1 transformed strains VGJK5/07, VGJK17/07 and VGJK43/07 in comparison to the control QM9414 strain.
Magnification 100 x. The presented picture is one from about 100 analysed for each strain.
34