Isolation, Characterization, and Expression Patterns of a DMC1 Homolog from the Basidiomycete Pleurotus ostreatus

Fungal Genetics and Biology 33, 59 – 66 (2001) doi:10.1006/fgbi.2001.1265, available online at http://www.idealibrary.com on

Isolation, Characterization, and Expression Patterns of a DMC1 Homolog from the Basidiomycete Pleurotus ostreatus

T. S. P. Mikosch, A. S. M. Sonnenberg, and L. J. L. D. Van Griensven Department of Genetics and Breeding, Mushroom Experimental Station, P.O. Box 6042, 5960 AA Horst, The Netherlands

Accepted for publication March 6, 2001; published online April 25, 2001

Mikosch, T. S. P., Sonnenberg, A. S. M., and Van Griensven, L. J. L. D. 2001. Isolation, characterization, and expression patterns of a DMC1 homolog from the basidiomycete Pleurotus ostreatus. Fungal Genetics and Biology 33, 59 – 66. Here we describe the isolation of a Pleurotus ostreatus gene PoDMC1. The predicted amino acid sequence of the oyster mushroom gene is 62% identical to the yeast DMC1 and 60% identical to human DMC1. The highest degree of amino acid identity (88%), however, was shown with Coprinus CoLIM15, a DMC1 homolog recently found in Coprinus cinereus. The exact matching of sizes and positions of most introns in both basidiomycete genes underlines the close relationship between these DMC1 orthologs. The RecA homolog DMC1 from yeast and its orthologs from other species have been reported to be meiosis specific and essential for sporulation. Here we show that PoDMC1 is exclusively expressed in the lamellae/basidiospore fraction of fruit bodies and not in somatic cells of fruiting bodies or in vegetative mycelium. Furthermore, the gene is not expressed in the lamellae/basidiospore fraction of a nonsporulating mutant of P. ostreatus. Since one of the major problems in cultivating the oyster mushroom is the abundant sporulation that causes allergic reactions in man, PoDMC1 could be

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an important target gene in constructing sporeless Pleurotus strains. © 2001 Academic Press The central role of RecA from Escherichia coli in pairing and strand exchange during homologous recombination is well established (for reviews see Roca and Cox, 1990; Kowalczykowski, 1991; West, 1992; Clark and Sandler, 1994; Camerini-Otero and Hsieh, 1995). Eukaryotic homologs of the E. coli RecA were first discovered in Saccharomyces cerevisiae (Basile et al., 1992; Shinohara et al., 1992; Bishop et al., 1992) and later in a variety of eukaryotes. Phylogenetic analyses of eukaryotic RecA homologs reveal a gene duplication during evolution which gave rise to two phylogenetically distinct subclasses of RecA-like genes, the RAD51 group and the DMC1 group (Stassen et al., 1997). In S. cerevisiae RAD51 is important for double-strand break repair and recombination (Game, 1993). Mutants in this gene are sensitive to DNA-damaging agents (methyl methanesulfonate and ionizing radiation) and are defective in meiosis. The DMC1 gene of S. cerevisiae is necessary for meiosis, but appears to be not involved in mitotic DNA repair (Bishop et al., 1992). RAD51 and DMC1 mutants have very similar phenotypes in yeast, and both genes are essential for completion of the meiotic cell cycle and consequently the production of

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viable spores (Shinohara et al., 1992; Bishop et al., 1992). Unlike RAD51 mutants, which undergo meiosis and spore formation to some extent (Shinohara et al., 1992), cells lacking DMC1 arrest in meiotic prophase (Bishop et al., 1992). RAD51 protein forms nucleoprotein filaments, on both single- and double-stranded DNA, that resemble the filaments made by RecA (Ogawa et al., 1993; Sung and Robberson, 1995) and can catalyze homologous DNA pairing and strand exchange in an ATP-dependent manner (Sung, 1994). During meiosis in yeast, RAD51 and DMC1 proteins colocalize on synaptonemal complexes prior to chromosome synapsis in a regulated order in which RAD51 is apparently required for the formation of DMC1 complexes (Bishop, 1994). In S. cerevisiae, transcription of RAD51 is induced by irridation with UV light (Aboussekhra et al., 1992) or X rays (Basile et al., 1992), by treatment with methyl methanesulfonate, and during meiosis (Shinohara et al., 1992). DMC1 is induced during meiosis (Bishop et al., 1992), but not by treatment with DNA-damaging agents (Bishop, 1994). RAD51 and DMC1 homologs have been isolated from organisms as diverse as fungi, animals, and plants (for a listing see Stassen et al., 1997). In vitro and in vivo studies have shown that across different systems these proteins share common properties and resemble characteristic features of their S. cerevisiae homologs. It has therefore been concluded that RAD51 is expressed during mitotic and meiotic DNA metabolism, whereas DMC1 is specifically expressed during meiosis (Doutriaux et al., 1998). We are currently studying the processes of meiosis and spore formation in the basidiomycete Pleurotus ostreatus (oyster mushroom). Worldwide production of this edible mushroom ranges third behind Agaricus bisporus (white button mushroom) and Lentinus edodes (shiitake) (Chang, 1996). One of the major drawbacks of oyster mushroom cultivation is the large number of spores produced by P. ostreatus fruit bodies. Within 24 h, oyster mushroom caps produce up to 6 ⫻ 10 8 spores per gram of fruit body (Sonnenberg et al., 1996). Mushroom workers can develop an extrinsic allergic alveolitis (EAA) after inhalation of these spores. The characteristic symptoms of EAA are fever, cough, dyspnoea, chills, muscle pain, and headaches starting within a few hours after inhalation of the spores (Cox et al., 1988). Continuous exposure to the antigen can cause permanent lung damage due to fibrosis of lung tissue (Van den Bogard et al., 1993). Therefore mushroom workers have to use appropriate face masks to prevent inhalation of spores and the development of lung fibrosis.

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Mikosch, Sonnenberg, and Van Griensven

Developing a sporeless Pleurotus strain would therefore be an option to eliminate the health risks associated with oyster mushroom cultivation. Attempts to make improved variants of the oyster mushroom using either a spontaneous mutant with a sporeless phenotype (Eger, 1970; LealLara, 1978) or poorly sporulating mutants generated by chemical and physical mutagenesis (Imbernon and Labarere, 1989) for breeding were not successful mainly because of poor production yields and abnormal fruit body morphology. We therefore aim to isolate genes expressed during meiosis. These sequences could be used as markers in breeding or as targets for knock-out experiments to generate nonsporulating mushroom strains. In this article we describe the isolation, characterization, and expression patterns of a DMC1 ortholog from the basidiomycete P. ostreatus. The deduced amino acid sequence of P. ostreatus DMC1 (PoDMC1) shows greatest sequence similarities with DMC1 of Coprinus cinereus, yeast, and humans. Northern analysis shows that expression of PoDMC1 is found only in lamellar tissue and is not induced after DNA damage. Furthermore, PoDMC1 is not expressed in lamellar tissue of a nonsporulating mutant strain.

MATERIAL AND METHODS Strains and Cultivation P. ostreatus strain Somycel 3015 and the sporeless mutant ATCC58937 were grown on MMP medium (1% malt extract, 0.5% mycological peptone, 10 mM KMOPS (3[N-morpholino] propansulfonic acid), pH 7.0, 1.5% agar) at 24°C. For DNA and RNA isolation, mycelium was grown on MMP plates covered with cellophane disks. Laboratory-scale fruiting experiments were performed according to Baars et al. (2000). For MMS treatment, mycelium was grown overnight in liquid medium; samples were taken before MMS addition and at 1, 2, 4, 6, and 16 h following treatment with 0.02% MMS (Arcos Organics, The Netherlands).

Bacterial Strains and Growth Conditions E. coli strain DH5␣ was used for cloning, amplification, and maintenance of plasmids. E. coli DH5␣ was grown in LB medium (10 g/L casein, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L agar). E. coli strain LE 392 was used as host

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DMC1 Homolog from Pleurotus ostreatus

strain for bacteriophage ␭-EMBL3. For bacteriophage ␭-EMBL3 infection, E. coli LE392 was grown in LM medium (LB medium supplemented with 0.2% maltose and 10 mM MgSO 4). E. coli strain XL1-Blue MRF ⫺ was used as host strain for bacteriophage ␭-Uni-ZAP XR. E. coli XL1-Blue MRF ⫺ was grown on LB medium supplemented with 12.5 ␮g/ml tetracycline for general purposes and for infection in LM medium.

Cloning and DNA Manipulations All standard DNA manipulations and cloning were performed according to Sambrook et al. (1989) or following manufacturer’s instructions.

PCR with Degenerated Oligonucleotide Primers PCR was carried out in 25 ␮l containing 0.3 ␮g of P. ostreatus (Somycel 3015) genomic DNA, 5 ␮M primers A and B, 0.2 mM dNTP, and 0.3 units Super TAQ (HC) (HT Biotechnology LTD) in buffer prepared as recommended by HT Biotechnology LTD. The first PCR consisted of an initial denaturation step for 5 min at 94°C, followed by a touch-down PCR with the following parameters: denaturation for 1 min at 94°C, annealing starting at 65°C with a decrease of 2°C after every second cycle, and amplification for 2 min at 72°C. After the touch-down PCR reached an annealing temperature of 43°C, a PCR for 10 cycles under the following conditions was performed: 1 min at 94°C, 2 min at 43°C, and 2 min at 72°C. The final cycle was followed by an additional 5 min at 72°C. For a second amplification, 5 ␮l of the first PCR was used as template for a PCR under the same conditions as described above. Primer A is the oligonucleotide 5⬘-GGNGARTTYMGNWSNGGNAAR-3⬘ and primer B is 5⬘-YTCNCCTCKNCCTSWRWARTC-3⬘, where N is A, C, G, or T, K is G or T, M is A or C, R is A or G, S is C or G, W is A or T, and Y is C or T. Primer A is the sense strand and primer B is the antisense strand primer. PCR products were cloned in the vector pGEM-T (Promega) under conditions recommended by Promega.

DNA Sequences All sequencing reactions were perfomed by BaseClear, Leiden, The Netherlands. The GenBank Accession No. for PoDMC1 is AJ311528.

Northern Blot Analysis Total RNA was isolated from tissue or mycelium after being frozen in liquid nitrogen and the sample was ground with a mortar and pestle. About 100 mg of the powdered samples was transferred to a tube containing 3 ml extraction buffer (100 mM Tris/HCl, pH 9.0, 10 mM EDTA, 1% (w/v) SDS). Subsequently the samples were extracted once with PCI (phenol/chloroform/isoamylalcohol, 25:24:1) and twice with CI (chloroform/isoamylalcohol, 24:1). For phase separation the tubes were centrifuged at 15,000g, 4°C for 10 min. The RNA was precipitated with LiCl at a final concentration of 2 M LiCl at 4°C overnight. RNA was sedimented by centrifugation and washed twice with 80% (v/v) ethanol. The isolated RNA was dissolved in H 2O and stored in portions of 30 ␮g at ⫺20°C. For gel electrophoreses the RNA was denaturated with glyoxal/ dimethyl sulfoxide (DMSO) (1 M glyoxal, 45% (v/v) DMSO, 30 mM Bis–Tris, 10 mM 1,4-piperazinebis(ethanesulfonic acid) (Pipes), 1 mM EDTA, 0.1% (w/v) bromophenol blue, pH 6.5) for 1 h at 55°C and separated in a 1% BTPE–agarose gel (30 mM Bis–Tris, 10 mM Pipes, 1 mM EDTA, pH 6.5). The RNA was blotted onto Hybond N nylon membranes by capillary transfer with 20⫻ SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0). Blots were cross-linked for 5 min with UV light and used for hybridizations after being baked for 2 h at 80°C. UVshadowing to visualize rRNA on the Nylon membranes was performed according to Mikosch (1999). Northern hybridization was performed according to the standard protocol of the Boehringer Mannheim DIG (digoxygenine) system for filter hybridizations with DIG-labeled RNA probes. Single-strand probes were generated from cDNA containing plasmids by use of T7 and T3 RNApolymerases and the Boehringer Mannheim DIG RNA labeling mix. To generate runoff transcripts the plasmids were digested on one side of the cDNA inserts prior to in vitro transcription according to the manufacturer’s protocol. The coding strands of cDNA’s were used as negative controls.

5⬘-RACE To identify the 5⬘ end of transcripts the SMART RACE cDNA amplification Kit (Clontech) was used; 1 ␮g of total RNA was used as starting material and a primer annealing about 130 bp downstream of the longest cDNA sequence was used for 5⬘ amplification according to the manufac-

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turer’s protocol. The primer had the sequence AAGAGTCGGAAGTCC.

RESULTS AND DISCUSSION Isolation and Characterization of a DMC1 Homolog from P. ostreatus To isolate a DMC1 homolog from P. ostreatus we designed two degenerate primers for codons of amino acid sequences conserved among members of the DMC1 gene family of different organisms (see Materials and Methods). These primers were used to amplify a fragment of a DMC1 homolog from chromosomal DNA of P. ostreatus. Based on the position of the primers the amplification product should have a length of approximatly 600 bp. Sequence analysis of PCR products of the expected length identified a 620-bp fragment that codes for a protein homologous to known DMC1 proteins. Database searches using the sequence analysis program BlastX (Altschul et al., 1997) revealed that this sequence could encode a polypeptide that is about 40% identical to DMC1 proteins from S. cerevisiae, C. cinereus, and other DMC1 homologs. The PCR product was used as probe to screen a P. ostreatus EMBL3 genomic library to isolate the genomic sequence. A ␭ZAP cDNA library made from lamellae as starting material was screened to isolate the corresponding cDNA sequence. From the genomic library we isolated two overlapping clones spanning 3.3 kb, and the single cDNA clone isolated contained a 1.3-kb insert. To identify the transcription start of the Pleurotus DMC1, 5⬘-RACEs were perfomed with different primers based on the cDNA sequence. Screening a large number of products, we succeeded in extending the transcript 58 bp beyond the start codon. As shown in Fig. 1, the cDNA sequence contains a 1044-bp open reading frame (ORF) encoding the Pleurotus ortholog of the Saccharomyces DMC1. The deduced protein sequence has 347 amino acid residues. Interestingly, the recently published sequence of the C. cinereus ortholog CoLIM 15 has excactly the same length (Nara et al., 1999). Homology studies of the deduced protein sequence using BlastP (Altschul et al., 1997) revealed that the Pleurotus sequence shows a high degree of amino acid identity to the Coprinus CoLIM15 (88% identity), the yeast DMC1 (63% identity), and the human DMC1 (60% identity) proteins. Furthermore, the characteristic nucle-

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Mikosch, Sonnenberg, and Van Griensven

otide binding domains of the RecA family were present within the predicted protein sequence (Fig. 1). Therefore the sequence was designated PoDMC1 (P. ostreatus DMC1). Comparison of the Pleurotus genomic and cDNA sequences revealed that the ORF is interrupted by 18 introns (Fig. 1) ranging in size from 43 to 68 bp, a size consistent with those found in other fungi (Gurr et al., 1987). The only other fungal DMC1 ortholog with that many introns is the Coprinus CoLIM15 with 11 introns (Nara et al., 1999). A comparison of the exon/intron distribution revealed that the introns of CoLIM15 do exactly match with the position and size of 11 introns in PoDMC1 (Fig. 2), underlining the close phylogenetic relation of these two basidiomycete DMC1 orthologs. The three regulatory sequences URS1, UAS H, and T 4C located upstream of DMC1 and other early meiotic genes in S. cerevesiae (Bowdish and Mitchell, 1993; Mitchell, 1994) were not found within the promotor region of PoDMC1. We, therefore, aligned the promotor sequences of PoDMC1 and CoLIM15 using the ClustalW program (Thompson et al., 1994) to identify similar sequence stretches within both promoters. This revealed sequences that resemble parts of the regulatory sequences URS1, UAS H, and T 4C found in the yeast promoter database (SCPD, http://cgsigma.cshl.org/jian/) within the promoter sequence of PoDMC1 and CoLIM15 (Fig. 3). Although the sequences do not match exactly, they might be useful starting points for future promoter studies concerning the two basidiomycete DMC1 genes.

Northern Analysis In yeast and Coprinus the RAD51 gene is induced after DNA damage and during meiosis (Shinohara et al., 1992; Stassen et al., 1997). The DMC1 gene, however, is specifically induced in both fungi during meiosis (Bishop et al., 1992; Nara et al., 1999). To determine whether the PoDMC1 gene is also specifically induced during meiosis, total RNA was isolated from vegetative mycelium, fruit body stipes, and lamellae/ basidia of P. ostreatus. The PoDMC1 transcript was detected only in lamellae/basidia and was completely absent in vegetative mycelium and fruit body stipes (Fig. 4). The yeast DMC1 gene is essential for completion of meiosis. DMC1 mutant yeast strains arrest in meiotic prophase and do not produce spores (Bishop et al., 1992). Since a sporeless P. ostreatus strain (ATCC58937) had been described by Eger (1970), we were interested in the

DMC1 Homolog from Pleurotus ostreatus

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FIG. 1. Sequence of PoDMC1. Capital letters indicate coding sequences. Introns and noncoding sequences are represented by lowercase letters. The single-letter designations for the amino acids encoded by the ORF are placed under the third base of a codon. Core promoter element (tatatat) and polyadenylation signal (aatataa) are boldface italic. The 5⬘ end of the mRNA and the beginning of the poly(A) tail are indicated in boldface and ATP binding motifs are underlined.

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Mikosch, Sonnenberg, and Van Griensven

FIG. 2.

Comparison of the genomic organization of CoLIM15 and PoDMC1. Thin lines represent introns and boxes exons.

expression of PoDMC1 in this mutant strain. So far no genetic analysis of this strain has been published and the mutation responsible for the sporeless phenotype has not been characterized. As shown in Fig. 4, the expression of PoDMC1 is almost absent in lamellae/basidia from the mutant strain ATCC58937. Nara et al. (1999) have shown for Coprinus that expression of CoLIM15 is induced shortly after karyogamy, is most abundant at zygotene, and diminishes in later stages of meiosis. We are aware of the fact that a missing expression of PoDMC1 in the lamellae/ basidia of ATCC58937 is no evidence for a PoDMC1 mutation in this strain, but this result suggests that a defect before meiosis (i.e., karyogamy) or early in meiosis might be responsible for the mutant phenotype of ATCC58937, because it seems improbable that a defect in later stages of meiosis should affect a gene that is most likely expressed early in meiosis. In yeast many genes expressed during meiosis are coregulated at the transcript level by transcriptional cascades (Chu et al., 1998; Klein et al., 1994) and it

FIG. 3. Alignment of regulatory sequences present in yeast DMC1 and other early meiotic genes with the PoDMC1 and CoLIM15 promoter sequences.

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has been shown that genes expressed early in yeast meiosis have similar regulatory elements, i.e., URS1, UAS, and T 4C (Bowdish and Mitchell, 1993). If similar regulatory systems are present in Pleurotus, a missing expression of an activating protein of (early) meiotic genes could also affect PoDMC1 expression in the mutant strain. For example, does a missing expression of the inducer proteins IME1 and IME2 in yeast affect expression of meiotic genes (Bowdish and Mitchell, 1994).

FIG. 4. (A) Northern hybridization with the PoDMC1 cDNA as probe; sp⫹, sporulating strain; sp⫺, sporeless strain. Lanes 1 ⫹ 2 RNA from lamellae/basidia; lanes 3 ⫹ 4 RNA from fruit body stipes; lanes 5 ⫹ 6 RNA from vegetative mycelium; each lane 30 ␮g of total RNA. (B) Loading control. Same blot as in A. Detection of the rRNA bands on the nylon membrane by UV-shadowing (Mikosch, 1999).

DMC1 Homolog from Pleurotus ostreatus

To investigate whether PoDMC1 expression is induced by DNA damage, we incubated mycelium from P. ostreatus and the mutant strain up to 16 h in the presence of 0.02% MMS. As can be expected for DMC1 orthologs, Northern hybridization showed that PoDMC1 expression is not induced following MMS treatment in either strain (data not shown). At present, attempts are being made to construct PoDMC1 knock-outs. They will be used to reveal the exact role of this gene in Pleurotus during meiosis and sporulation. Furthermore, the introduction of PoDMC1 in the mutant strain ATCC58937 will clarify whether a PoDMC1 defect is involved in the sporeless phenotype.

ACKNOWLEDGMENTS This work was funded by the Dutch Ministry of Agriculture, Nature Management and Fisheries (LNV). The authors thank Mrs. Brigitte Lavrijssen, Karen den Hollander, and Jose´ in t’Zandt-Linders for technical assistance.

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