Cloning of genes expressed early during cellulase induction in Hypocrea jecorina by a rapid subtraction hybridization approach

Fungal Genetics and Biology 41 (2004) 877–887 www.elsevier.com/locate/yfgbi

Cloning of genes expressed early during cellulase induction in Hypocrea jecorina by a rapid subtraction hybridization approach Monika Schmoll,* Susanne Zeilinger, Robert L. Mach, and Christian P. Kubicek Division of Gene Technology and Applied Biochemistry, Institute for Chemical Engineering, Vienna University of Technology, Getreidemarkt 9/1665, A-1060 Wien, Austria Received 15 April 2004; accepted 8 June 2004 Available online 17 July 2004

Abstract The cellulase system of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) is encoded by several cellobiohydrolase, endoglucanase and b-glucosidase genes, which are co-ordinately expressed upon induction by cellulose or the disaccharide sophorose. To identify genes, which are specifically expressed under these inducing conditions and possibly related to the induction process, we applied rapid subtraction hybridization (RaSH) to sophorose induced mRNAs from the wild-type strain H. jecorina QM9414 and a mutant strain H. jecorina QM9978, which is defective in the induction of cellulase gene expression. From a total of 224 clones, 22 gene fragments representing 20 different genes were analyzed. These included one gene encoding a PAS-domain protein with similarity to the Neurospora clock modulator VIVID; one gene similar to Podospora anserina ami1 involved in nuclear migration and the genes encoding translation elongation factor 1a, the transcriptional activator Hap5, and myo-inositol-1-phosphate synthase; in addition, several genes were detected, whose function is unknown. Some of them did not even have potential homologues in the Neurospora or Fusarium genome databases. The differential regulation of expression of those 20 genes by sophorose in wild-type and mutant was verified by Northern blotting. Their consistent response to additional inducing conditions (cellulose) confirms their interconnection with cellulase formation. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Hypocrea jecorina; Trichoderma reesei; Cellulose signal transduction; Subtraction hybridization

1. Introduction The cellulolytic enzyme system of the ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) has received considerable attention because of its applications in industry (Buchert et al., 1998; Galante et al., 1998a,b; Ulmasov et al., 1997), and because of the use of the cellulase promoters to drive heterologous protein production by this fungus (de Faria et al., 2002; Keranen and Penttila, 1995; Schmoll and Kubicek, 2003; Uusitalo et al., 1991). A detailed analysis of two cellulase promoters (cbh1 and cbh2) is now available, and the involvement of at least three transcriptional activators (Ace1 (Saloheimo et al., 2000), Ace2 (Aro et al., 2001) *

Corresponding author. Fax: +43-1-58801-17299. E-mail address: [email protected] (M. Schmoll). URL: http://www.vt.tuwien.ac.at. 1087-1845/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2004.06.002

and Hap2/3/5 (Zeilinger et al., 2001)) and of one repressor (Cre1 (Ilmen et al., 1996a,b) has been demonstrated. However, the mechanism how expression of these genes is turned on by the presence of cellulose is still unclear: in vivo footprinting of the cbh2 promoter and analysis of its chromatin architecture revealed that its transacting factors are permanently bound to the DNA (Zeilinger et al., 1998, 2003). The cellulose-specific signal must therefore be delivered by a protein not directly binding to the promoter. Identification of genes involved in the signalling of cellulose to cellulase gene expression is a difficult task in fungi like Trichoderma, as gene cloning by mutant complementation is difficult because of lack of extrachromosomal plasmid replication and absence of a convenient selection system for gain of cellulase formation. Alternative strategies such as REMI mutagenesis also failed in Trichoderma (Zeilinger, personal

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communication). Another approach to detect genes involved in this process is to use differential screening to identify and characterize genes that are uniquely transcribed in cellulase producing cultures. Yet another approach would be based on the assumption that the major components of signalling processes are expressed in higher abundance in the presence of the respective signal, and that they could thus be cloned by screening for differentially expressed transcripts using methods such as suppression subtractive hybridization (Diatchenko et al., 1998), cDNA microarrays or RaSH. We chose rapid subtraction hybridization (RaSH; Jiang et al., 2000) because of its simplicity and the possibility to apply variable stringency, which is an essential advantage over SSH-PCR. Ji et al. (2002) emphasized that the latter will enrich only at least 5-fold differentially expressed genes, which—because of considerable background amplification—in practice need to be present in at least 125-fold abundance under the investigated condition. Because of the variable stringency, RaSH can detect much smaller changes in gene expression, which was considered as more useful for the purpose of our study. Here, we have applied RaSH to cDNAs prepared from mycelia of the wild-type strain QM9414 and the cellulase-negative mutant strain QM9978 (Torigoi et al., 1996; Zeilinger et al., 2000) after replacement to cellulase inducing conditions and finally identified 20 genes with a clear response to cellulase inducing conditions as proven by Northern analysis. The results confirm that RaSH detects genes which show only 2-fold differences in transcript abundance upon induction and also such which show different transcript sizes reflecting alternative splicing events.

2. Materials and methods 2.1. Microbial strains and culture conditions Trichoderma reesei (H. jecorina) wild-type strain QM9414 (ATCC 26921) and the cellulase-negative mutant QM9978 (obtained from Dr. K.O’ Donnell, U.S. Department of Agriculture, Peoria, IL) were used throughout this study. They were kept on malt–agar and subcultured monthly. H. jecorina was grown in liquid culture in 1-L Erlenmeyer flasks on a rotary shaker (250 rpm) at 28 °C in 200 ml of medium as described by (Mandels and Andreotti, 1978) using 108 conidia/L (final concentration) as inoculum. Carbon sources used are specified in legends to the respective figure, and were used as 1% (w/v) in batch cultures. To induce cellulase formation by sophorose, the replacement technique described by (Sternberg and Mandels, 1979) was used: mycelia, grown for 24 h on 1% (w/v) glycerol as the carbon source under otherwise

similar conditions as described above, were cautiously filtered through Miracloth (Calbiochem, EMD Biosciences, La Jolla, CA), washed with tap water to prevent osmotic shock and to remove the repressing carbon source glycerol (Zeilinger et al., 2003), and resuspended in minimal medium (Mandels and Andreotti, 1978) lacking the carbon source. Sophorose (1.5 mM, final concentration) was then added, and the incubation continued for 1, 3, and 5 h (for cDNA libraries), or 2, 4, and 5 h (for Northern blots). To guarantee equal treatment of the two cultures (wild-type and mutant, respectively), they were done in parallel. Escherichia coli JM109 (Yanisch-Perron et al., 1985) was used for the propagation of vector molecules and DNA manipulations. 2.2. Preparation of PCR-based cDNA libraries The experiment was performed essentially as described by (Jiang et al., 2000). mRNA was isolated from total RNA using the PolyATtract mRNA Isolation System (Promega, Madison, USA). For preparation of cDNA the Universal RiboClone cDNA Synthesis System (Promega, Madison, USA) was used. Tester and driver cDNA were digested with EcoRII (a 4.5 cutter with the recognition sequence CCWGG; New England Biolabs, Beverly, USA). Purification of cDNA was performed by the aid of the QIAquick PCR Purification Kit (Qiagen, GesmbH, Hilden, Germany). 2.3. Subtraction hybridization and generation of subtracted libraries Hundred nanogram of tester cDNA (isolated and combined from H. jecorina QM9414, induced by sophorose for 1, 3, or 5 h, respectively) were added to 3 lg of driver cDNA (isolated from H. jecorina QM9978, similarly induced by sophorose for 1, 3, or 5 h) and treated as described. Various amounts of the purified hybridization mixture were ligated with calf intestinal phosphatase-treated pBluescript SK (Stratagene, La Jolla, CA, USA) plasmids digested with XhoI and transformed into Escherichia coli JM109. Bacterial colonies were picked randomly, grown in 100 ll LBamp medium for 2 h and 1 ll of the culture was used for insert screening PCR. The PCR-amplified inserts were analysed by agarose gel electrophoresis. 2.4. Dot blot and Reverse Northern hybridization For the dot blot, PCR products were denatured with an equal volume of denaturing solution (3 M NaCl, 1 M NaOH) and 1.5 ll of each (224 in total) spotted manually onto a Hybond-N membrane (Amersham–Pharmacia Biotech, Uppsala, Sweden). Reverse Northern blotting was performed using 2.4 lg of PCR amplified

M. Schmoll et al. / Fungal Genetics and Biology 41 (2004) 877–887

and subsequently [a-32 P] radiolabelled cDNA from H. jecorina QM9414 (sophorose-induced) and QM9978 (sophorose-induced), respectively, as probes. Hybridization was done at 64 °C for 20 h in 6
SSC, 0.5% SDS, 5
Denhardts solution and 125 lg/ml denatured herring sperm DNA. The candidates for a more detailed analysis were chosen by visual inspection first, and then this decision was cross-checked by quantitative measurements using the Bio-Rad Geldoc Imaging system and Bio-Rad Quantity One software, both for three different expositions of the dot blot. 2.5. Nucleic acid isolation and hybridization Fungal mycelia were harvested by filtration, washed with tap water, frozen, and ground in liquid nitrogen. For extraction of DNA, mycelial powder was suspended in buffer A (1.2 M NaCl, 5 mM EDTA, 0.1 M Tris–HCl, pH 8.0), incubated for 20 min at 65 °C, cooled down on ice, mixed with 1 vol. phenol:chloroform:isoamylalcohol 49:49:2 (v/v/v) and centrifuged (21000g, 15 min). Following an extraction with 1 vol. of chloroform:isoamylalcohol 24:1 (v/v), the DNA was precipitated with 1 vol. of isopropanol, and washed with 70% (v/v) ethanol. Total RNA was isolated by the guanidinium thiocyanate method (Chomczynski and Sacchi, 1987). Following electrophoretic separation on a 1.2% agarose-gel containing 2.2 M formaldehyde in 1
Mops buffer (0.04 M Mops, 0.001 M EDTA, pH 7.0), RNA was blotted onto nylon membranes (Hybond-N; Amersham–Pharmacia Biotech, Uppsala, Sweden) and hybridized in 50% formamide, 10% dextransulfate, 0,5% SDS, 5
Denhardts solution and 125 lg/ml denatured herring sperm DNA at 42 °C for 20 h. XhoI-fragments of the isolated clones were [a-32 P] radiolabelled using random primers and the Klenow fragment (Fermentas, Vilnius, Lithuania). Washing was performed with 2
SSC +0.1% SDS at 42 °C (2
10 min). Normalization of gene expression was performed according to the following formula: {(cfa gene expression, time point)/(cfa gene expression in wild-type, 5 h sophorose)}/{(control 18SrRNA, time point)/(18S rRNA wild-type, 5 h sophorose)}. The quantitative measurements were performed using the Bio-Rad Geldoc Imaging system which allows for detection of 256 shades of gray, and Bio-Rad Quantity One software. 2.6. Sequence analysis and identification To enable a detailed analysis of the results, the number of fragments to be investigated was limited to approximately 20, thus only the most promising ones showing clear differential expression in the Reverse Northern blot were selected. Then plasmids containing differentially expressed cDNA fragments as suggested by

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the dot blot were sequenced using the T7 promoter within pBluescript as primer binding site. The respective sequences were used for BLASTX searches of the swissprot and nr database on the NCBI server (http:// www.ncbi.nlm.nih.gov/BLAST/) and of the Neurospora crassa, Fusarium graminearum, and Aspergillus fumigatus genome databases (http://www-genome.wi.mit.edu/ resources.html). Because of the small size of the EST fragments (resulting in probability coefficients of sometimes >e)5), we also recorded the degree of similarity of encoded aa-sequences for identified nearest neighbours in related organisms. To find T. reesei EST-sequences corresponding to the gene fragments found in the experiment, a BLASTN search of the EST-database on the NCBI server, that contains both the EST-databases described by (Chambergo et al., 2002) and (Foreman et al., 2003), was performed. We also searched yeast, and E. coli databases but did not find significant hits.

3. Results 3.1. Isolation of expressed sequence tags which are differentially expressed in a H. jecorina wild-type strain and a cellulase non-inducible mutant on sophorose We used the differences in the ability of H. jecorina QM 9414 and the cellulase-non inducible mutant QM 9978 to express cellulase genes in the presence of the cellulase inducer sophorose to screen for mRNAs which are more abundant in the wild-type strain under these conditions. To prevent missing transcripts with only transient accumulation upon incubation with sophorose, mRNAs were isolated from mycelia after 1, 3, and 5 h of incubation, and combined. As we applied a relatively low stringency, we expected to be able to detect not only genes that are not at all present in the cellulase negative mutant strain QM9978, but also some that actually are, but with different abundance, expressed during growth on inducing carbon sources. Consequently, 224 putatively positive ESTs were isolated and tested by Reverse Northern blotting (Fig. 1) (Huang et al., 1999a; Jiang et al., 2000; Kang et al., 1998). The lengths of the respective cDNA sequences were 100–300 bp on an average. Based on the signal intensity on the Reverse Northern Blot, 22 EST fragments clearly being differentially expressed between wild-type and cellulase negative mutant and representing 20 different genes were consecutively chosen for further investigation (Fig. 1). These EST-fragments were termed cfa (cellulase f ormation associated; for accession numbers see Table 1). Since the Reverse Northern blot can only be considered a preliminary analysis due to certain limitations such as manual spotting or the possibility that the plasmid could contain more than one

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For those EST fragments which showed essentially the same expression patterns in Northern analysis (Fig. 2), full-length chromosomal clones were isolated and sequenced to assess whether these ESTs would be fragments of one and the same mRNA. However, with the exception of cfa4B, cfa25C, and cfa90D, which indeed were parts of the same mRNA (therefore only one Northern blot is given in Fig. 2), this was not the case with the other clones. Interestingly, none of the cellulase genes, particularly the abundantly expressed gene cbh1, was detected in this study. A similar phenomenon was already observed by Jiang et al. (2000) during first development of RaSH. The reason for it, however, is still unclear—further remarks are given in Section 4. The absence of restriction sites yielding fragments of appropriate lengths required for RaSH in some genes may possibly explain this finding, but other reasons cannot be ruled out. 3.2. Identification of ESTs

Fig. 1. Reverse Northern blot analysis of 224 differentially expressed sequence tags identified by RaSH. PCR-amplified products from bacterial clones of the subtracted library were spotted manually onto nylon membranes. As probes 2.4 lg of PCR-amplified and [a-32 P] labeled cDNAs from the wild-type strain QM9414 and the cellulase noninducible mutant strain QM9978 were used.

insert, we investigated the actual expression patterns of all 20 genes by Northern blotting. To unequivocally prove the involvement of a certain gene in the process of cellulase signalling, we isolated RNA from cultures replaced to sophorose, and moreover from growth on cellulose as well as under repressing conditions (glucose, glycerol; Zeilinger et al., 2003) (Fig. 2, Table 2). The expression of the major cellulase gene cbh1 was used as a control for the actual differences in cellulase expression in wild-type and mutant. Expression of different cellulase genes has been shown to be co-ordinately regulated (Ilmen et al., 1997; Schmoll and Kubicek, 2003), and thus a single cellulase gene was considered to be sufficient for this purpose (Fig. 3). Northern blotting with probes derived from the selected putatively differentially expressed sequences revealed, that also some clones were identified whose expression pattern was more or less the same in the wild-type and the mutant strain, but they nevertheless showed regulation during the induction process by sophorose, which was consistently observed under all inducing conditions (for example cfa14D).

A H. jecorina genome sequence was not available at the time this study was carried out. Therefore, safe identification of the clones obtained in this work was done by the following strategy: first, we performed a BLASTN search against the NCBI EST-database that includes the T. reesei EST-databases described by (Chambergo et al., 2002) and (Foreman et al., 2003). Criterion for identification was absolute identity between the nt-sequences. In cases where this failed, the sequences were then subjected to BLAST searches of the genome databases of N. crassa (http://www-genome.wi.mit.edu/ annotation/fungi/neurospora/) and F. graminearum (http://www-genome.wi.mit.edu/resources.html). As the degree of identity between proteins from these two fungi (particularly with Fusarium, which also belongs to the family Hypocreales) to H. jecorina is high, we only took aa-similarities >50% of the encoded protein fragment as a criterion of putative identity. We also routinely used the A. fumigatus genome database for BLAST analyses, but in positive cases the similarities were lower than with Neurospora or Fusarium, or (if no matches with these two were obtained) searches in A. fumigatus were also negative. In cases where the best match of an EST was with an ORF of unknown function, a full-length version of this ORF was subjected to GenBank BLASTP search. BLAST hits
Table 1 Characteristics and putative functions of clones isolated with RaSH Clone ID

Frequency

Length of fragment (bp)

GenBank Accession No. of fragments

Transcript size (bp)

5

104

CF653646

1400

cfa4B

1

164

CF653647

1300

cfa8C cfa11C

1 4

166 206

CF653648 CF653649

1100 600

cfa14D cfa23C cfa25C

1 1 2

191 107 122

CF653650 CF653652 CF653664

2000 1400 1300

cfa36C

1

252

CF653653

1400

cfa37C

1

77

CF653654

1900

cfa39C

2

265

CF653655

2000

cfa48C

1

56

CF653656

1300

Translation elongation factor 1a (tef1) VIVID PAS protein (vvd) Unknown Unknown

Expect

Aminoacid similarity with nearest neighbour (within fragment) (%)

Accession No. of identified gene or nearest neighbour in F. graminearum or N. crassa database (WICGR) Trichoderma Fusarium

Neurospora

Genbank Accession No. H. jecorina EST sequences covering fragment

Z23012

3.00E)11

Unknown 1.00E)08 Unknown VIVID PAS 2.30E)02 protein (vvd) ATP synthase beta chain 3.20E)01 Related to nuclear migration protein ami1 myo-inositol1-phosphate synthase Outer mitochondrial membrane porin

Protein synthesis

75

AF338412

CB903326 CB905696 CB902519 CB902005 CB902479 CB909036 CB895542 82

Putative function

Contig 1.112

Light response protein Unknown Unknown

Unknown BM076786

69

AF338412 BM076422

92

BM076422 CAC28706

BM076397

BM076457

Light response protein ATP synthesis Nuclear migration

CB903696 BM076397 CB896228 BM076457 CB896986 CB905628 CB905005 CB895300 CB896789

M. Schmoll et al. / Fungal Genetics and Biology 41 (2004) 877–887

cfa2D

Identified gene of T. reesei or nearest neighbour

Inositol metabolism Membrane protein

881

882

Table 1 (continued) Clone ID

Frequency

Length of fragment (bp)

Transcript size (bp)

Identified gene of T. reesei or nearest neighbour

cfa48D cfa58C

1 1

208 159

CF653657 CF653659

2700 2000

Unknown Unknown

cfa65D

1

245

CF653660

1200

CCAATbinding protein subunit HAP5 (hap5) gene

cfa67C cfa71C cfa77C cfa80C

1 1 2 1

218 106 126 127

CF653661 CF653662 CF653663 CF653665

1300 1500 600 1500

cfa85D cfa90D

1 1

252 290

CF653666 CF653667

2700 1300

Unknown Unknown

Unknown VIVID PAS protein (vvd) (identified by sequencing cDNA)

Expect

Aminoacid similarity with nearest neighbour (within fragment) (%)

Accession No. of identified gene or nearest neighbour in F. graminearum or N. crassa database (WICGR) Trichoderma Fusarium

Neurospora

Genbank Accession No. H. jecorina EST sequences covering fragment

Putative function

CB906728 CB904258 AF120159

Required for DNA-binding of the HAP complex

7.00E)12 5.00E)08

51 76

Contig 1.383 Contig 1.196

7.00E)06

75

Contig 1.318

2.00E)24

94

Contig 1.34 AF338412

Unknown Unknown Unknown Related to Cytochrome P450 monooxygenases (mycotoxin biosynthesis) Unknown Light response protein

M. Schmoll et al. / Fungal Genetics and Biology 41 (2004) 877–887

GenBank Accession No. of fragments

M. Schmoll et al. / Fungal Genetics and Biology 41 (2004) 877–887

883

Fig. 2. Analysis of transcript abundance of the cfa (cellulase formation associated) clones isolated during this study by Northern blotting. Twenty milligrams of total RNA were applied per slot. WT indicates strain QM9414; MU indicates mutant strain QM9978. The cultures were grown for the respective time containing the carbon source (1%, w/v) as indicated. For sophorose-induction, mycelia were precultured for 24 h on 1% (w/v) glycerol, and replaced to minimal medium containing 1.5 mM sophorose (Sternberg and Mandels, 1979). Hybridizations were performed with the sequence tags as obtained by the RaSH experiment except for cfa4B, cfa25C, and cfa90D, where a PCR fragment spanning the whole genomic region containing the PAS domain was used. The actin-encoding gene act1, and a fragment of the 18S rRNA were used as hybridization controls, and the ethidium bromide stained agarose gel is given as a loading control.

Only two genes already described from H. jecorina were thereby identified (tef1 (Nakari et al., 1993) and hap5 (Zeilinger et al., 2001)) (Table 1). Putative orthologues in Neurospora or Fusarium could be detected for the majority of the clones obtained, but several of them encoded putative proteins without any match in the databases. Many of the EST fragments were isolated several times (Table 1), indicating that they are predominant expression products under the conditions used. Notable examples are cfa2D, cfa31C, cfa42D, cfa67D, cfa83D, and cfa63D which are identical to Translation Elongation Factor 1a of H. jecorina; cfa39C and cfa84C, which share similarity with myo-inositol-1-phosphate synthase; and, cfa4B, cfa25C, and cfa90D, representing three different regions within the mRNA of the same gene. Isolation of a chromosomal full-length copy of this gene approved this, and showed that it encodes a polypeptide con-

taining a PAS domain with considerable similarity to VIVID of N. crassa (Heintzen et al., 2001; Schmoll, 2003). Only one fragment representing a certain gene is given in Table 1 for multiple isolated ESTs (for example. cfa2D, cfa25C, and cfa39C). Among the less frequently accumulated clones, cfa37C encodes a protein fragment with high similarity to a short aa-sequence of the Podospora anserina ami1 protein, a regulator of nuclear migration (Graia et al., 2000); cfa65D is identical to H. jecorina hap5, a component of the Hap2/3/5 transcriptional regulator complex (Zeilinger et al., 2001); cfa80C encodes a P450dependent monooxygenase; and cfa36C codes for a gene with similarity to the b-chain of the mitochondrial ATPase. Orthologues of unknown function were obtained for the clones cfa67CA and cfa71C. No significant similarity to genes in available databases was found

3.77 0.68 0.99 0.01 1.10 1.68 1.77 1.65 4.69 1.90 1.59 0.43 1.94 1.07 1.41 6.02 1.18 0.96 5.12 1.03 0.93 0.05 1.22 2.15 1.46 3.26 6.85 2.63 1.17 0.48 2.35 1.74 1.66 1.54 1.37 0.90 3.31 0.68 0.94 0.31 1.92 2.29 1.16 3.59 3.64 1.80 1.06 0.68 2.29 1.23 2.47 2.62 1.57 1.16

4.28 1.33 1.17 1.00 4.30 2.38 1.75 1.33 5.00 2.15 1.71 0.70 3.24 2.04 2.48 8.50 1.52 1.38

4.07 1.07 1.17 0.53 3.85 2.19 1.63 1.59 3.73 2.36 1.52 0.62 2.67 1.96 2.10 5.83 1.52 1.13

1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

0.83 0.63 0.72 0.16 1.05 1.13 1.02 0.50 1.26 0.89 0.90 0.34 0.75 0.70 0.98 2.11 0.91 0.72

3.63 5.00 1.16 0.01 1.44 1.36 0.97 4.05 3.85 1.23 1.43 0.41 1.29 0.87 1.14 1.13 0.95 0.66

0.88 0.89 0.90 0.56 0.79 1.19 1.19 0.57 2.44 0.82 0.85 0.38 1.19 0.63 0.85 4.61 0.97 0.71

4.30 0.51 0.86 0.01 0.79 1.68 1.53 4.08 4.74 2.20 1.02 0.39 1.75 2.31 1.46 1.40 1.16 0.83 7.31 1.53 1.33 0.21 2.47 2.88 1.30 6.61 5.00 2.35 2.78 0.89 5.75 3.24 4.07 4.17 2.08 1.39 cfa2D cfa4B cfa8C cfa11C cfa14D cfa23C cfa36C cfa37C cfa39C cfa48C cfa48D cfa58C cfa65D cfa67C cfa71C cfa77C cfa80C cfa85D

WT glycerol 24 h MU glucose 20 h WT MU WT MU WT MU WT cellulose MU cellulose WT glucose sophorose 2 h sophorose 2 h sophorose 4 h sophorose 4 h sophorose 5 h sophorose 5 h 48 h 48 h 20 h Clone

Table 2 Quantification of transcript abundances of the selected ESTs under cellulase inducing and non-inducing conditions, normalized to wild-type 5 h after replacement to sophorose

3.61 0.45 0.85 0.03 1.00 1.61 1.23 3.37 2.81 1.90 1.44 0.36 1.77 0.67 1.28 3.79 1.02 0.77

M. Schmoll et al. / Fungal Genetics and Biology 41 (2004) 877–887 MU glycerol 24 h

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Fig. 3. Northern analysis of cbh1 transcript accumulation. Twenty milligrams of total RNA were applied per slot. WT, strain QM9414; MU, mutant strain QM9978. The cultures were grown for the respective time containing the carbon source (1%, w/v) as indicated. For sophorose-induction, mycelia were precultured for 24 h on 1% (w/v) glycerol, and replaced to minimal medium containing 1.5 mM sophorose (Sternberg and Mandels, 1979).

for cfa8C, cfa11C, cfa14D, cfa23C, cfa48D, cfa58C, cfa77C, and cfa85D. 3.3. Verification of differential expression of the isolated genes The clones described above were isolated because they accumulated to different levels in the Reverse Northern blot (Fig. 1). To verify their suggested pattern of expression, the abundance of their transcripts was analysed by Northern blotting of sophorose-induced, cellulose induced, and carbon catabolite repressed (glycerol, glucose; Zeilinger et al., 2003) mRNA from the wild-type strain H. jecorina QM9414 and the cellulase negative mutant QM9978, using the original EST fragments, as obtained from the RaSH experiments, as probes. As can be seen in Fig. 2, not all of the clones indeed confirmed the differences in expression pattern between the wild-type strain and the mutant as detected on the dot blots; however, all of them clearly showed significant up-regulation during cellulase induction even if (as in one case) no differences in expression in mutant and wild-type were detected (Fig. 2, Table 2). While most of the clones were up-regulated by the presence of sophorose and cellulose, the expression patterns of cfa11C and cfa77C showed an intriguingly different pattern: in the wild-type strain, both were strongly up-regulated after replacement to sophorose whereas no transcript was found on cellulose. In contrast, their transcript did not accumulate in the mutant strain upon induction by sophorose, but was abundant in the presence of cellulose. In addition, the transcripts of several clones (cfa4B, cfa25C, cfa90D, cfa48C, and cfa58C) displayed a different size when the wild-type and the mutant strain, or cellulose/sophorose and glucose cultures or different

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time points after sophorose induction were compared. Whether this is due to alternative splicing, or different lengths of the 50 and 30 untranslated region remains to be studied. For two genes (cfa19C with similarity to translation elongation factor 1b and cfa52C, a putative aspartyl protease) differential expression and cellulase-induction specific transcription could not unequivocally be confirmed and therefore they will not be discussed further.

4. Discussion In the present study, we aimed to isolate genes which are expressed early during contact of H. jecorina with cellulose, and which do not show up in a H. jecorina mutant which is impaired in cellulase induction. The rationale for this approach was based on the assumption that sophorose either is or mimics the inducer which is formed by initial degradation of cellulose. The Northern analysis in this paper however shows that this assumption has to be treated with caution as several clones isolated in this study showed slight to severe differences in the effect of sophorose and cellulose on their expression. Nevertheless, all of the clones isolated exhibited a cellulose-specific transcription pattern, and thus the goal of the approach was reached. The complete lack of identification of any of the cellulase genes was puzzling. However, in addition to the technical explanation given above, screening methods used in differential expression analyses often reveal only a subset of genes (Huang et al., 1999b; Jiang et al., 2000) and only a combination of several methods (for example SSH, cDNA microarrays, or RaSH) and variations in stringency with RaSH and also in the restriction sites used can be expected to cover the whole expression spectrum. Thus, we consider the absence of cellulase genes not as an indication that the conditions used were not appropriately chosen, especially because all 20 genes selected from the RaSH experiment were shown to respond to the cellulase inducing conditions. Nevertheless, it can be assumed that a more extensive screening (and using variations as discussed above) of these two strains would yield additional genes being differentially expressed under these conditions. Two genes, which were detected by this approach had already been cloned from H. jecorina before: hap5, a member of the CCAAT-binding complex that binds to the cellulose activating element within the cbh2 promoter of T. reesei (Zeilinger et al., 1998, 2000); and tef1, which encodes translation elongation factor 1a (Nakari et al., 1993). Both genes were up-regulated only 2 h after induction by sophorose, but their transcripts returned to the basal expression level thereafter. As this up-regulation did not occur in the cellulase-negative mutant strain, it is likely a specific event during cellulase in-

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duction. It is well-known that the nutrient status of the cell, such as availability of amino acids or glucose can modulate the rate of translation initiation (Proud, 2002), and this may also be the case when a battery of secretory proteins such as cellulases is induced. In addition, the mammalian homologue ELF 1a is an activator of phosphatidyl inositol 4 kinase (Chang et al., 2002), and ER-stress induced apoptosis (Talapatra et al., 2002), and it may thus be speculated whether tef1 could be involved in cellulose signalling in H. jecorina. A number of clones exhibited reasonable similarity with genes identified from other fungi, so that their identity seems to be clear. One, for which several ESTs were obtained, encodes an orthologue of myo-inositol 1phosphate synthase (Ino1; EC 5.5.1.4), which catalyzes the conversion of glucose 6-phosphate to myo-inositol1-phosphate. Although its transcript accumulated to highest and similar abundance in the wild-type and the mutant strain during growth on glucose, its expression on cellulose, and other carbon sources was weaker in the cellulase negative mutant. Ino1 is essential for de novo biosynthesis of myo-inositol in all organisms studied to date (Majumder et al., 1997), and therefore also for inositol phosphates involved in cellular signal transduction. An involvement of a phosphoinositol-signalling pathway in cellulose signalling would also coincide with the phosphatidyl inositol 4 kinase-regulating activity of Tef1, as discussed above. Whether or not this putative myo-inositol-1-phosphate synthase plays a role in cellulase induction remains to be elucidated, however. Three other clones, one of them isolated twice, (cfa4B, cfa25C, and cfa90D) turned out to be fragments of the transcript of the same gene, which encodes a member of the PAS superfamily, a class of sensory proteins named after Drosophila period (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded (SIM) (Pellequer et al., 1998; Ponting and Aravind, 1997; Taylor and Zhulin, 1999; Zhulin et al., 1997). PAS-proteins are reported to play a role in signal transduction processes by mediating protein–protein interactions and sensory functions (Taylor and Zhulin, 1999). The polypeptide encoded by a chromosomal gene fragment containing the ORFs of all three ESTs showed high similarity to VIVID of N. crassa (Heintzen et al., 2001), which defines a clock associated feedback loop and influences gating of the light response. Light response in H. jecorina has not been studied so far, but sporulation in other Trichoderma spp. (e.g., T. atroviride) requires blue light (Berrocal-Tito et al., 1999, 2000). Whether, cfa4B/ cfa25C/cfa90D therefore encodes a functional homologue of VIVID is unclear. However, the isolation of several ESTs of this gene under sophorose induction, its strong up-regulation on cellulose, and the inductionspecific differences in transcript sizes between the wildtype and the cellulase negative mutant strain of

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H. jecorina strongly support the assumption that this gene is involved in cellulase induction. The function for some other genes cloned is less clear: cfa37C encoded a short protein fragment with high similarity to ami1 of P. anserina, an orthologue of the Aspergillus nidulans apsA gene, which are both involved in nuclear migration. Two other clones (cfa36C and cfa48C) encoded proteins involved in mitochondrial activity. This is interesting in view of the work by (Abrahao-Neto et al., 1995) on the necessity of mitochondrial function for cellulase gene expression. The mechanism which relates mitochondrial function to cellulase expression has not been clarified yet. No significant matches were found for several clones (cfa8C, cfa11C, cfa14D, cfa23C, cfa48D, cfa58C, cfa67CA, cfa71C, cfa77C, and cfa85D) in any of the existing databases. These data indicate the presence of a number of genes which are specific for H. jecorina, and thus most probably involved in cellulose signalling. Among them, the expression patterns of cfa11C and cfa77C are most intriguing, as they respond differently to the presence of cellulose and sophorose, being both strongly up-regulated after replacement to sophorose in the wild-type strain but not in the mutant, whereas no transcript is detected on cellulose in the wild-type, but it is clearly present in the mutant. A differential effect of cellulose and sophorose on cellulase-induction has also been shown in an ace2 (a transcriptional regulator of cellulase expression; (Aro et al., 2001))—deletion strain of H. jecorina, in which cellulose—but not sophorose— induction is altered. While both cellulose and sophorose induce cellulase gene transcription, their signal transduction obviously involves at least in part different cellular routes.

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