The basidiomycetous mushroom Lentinula edodes white collar-2 homolog PHRB, a partner of putative blue-light photoreceptor PHRA, binds to a specific site in the promoter region of the L. edodes tyrosinase gene.

We isolated a cDNA homolog of Neurospora crassa wc-2 from the basidiomycetous mushroom Lentinula edodes and termed it phrB cDNA. The deduced PHRB (313 amino acid residues) contained a PAS domain and a zinc-finger motif. Random binding-site selection analysis of the PHRB produced in Escherichia coli revealed that it bound to a 7-bp sequence with the consensus sequence 5'GATA/TTG/T/AC3'. Electrophoretic mobility-shift assay showed that it also bound to the consensus sequence 5'GATATTC3' in the promoter region of the L. edodes tyrosinase gene (Le.tyr). In vitro GST-pulldown immunoblot analysis disclosed that PHRB interacts with a putative blue-light photoreceptor of L. edodes (PHRA), the homolog of N. crassa WC-1, through the PAS B- and/or PAS C domain of PHRA. The expression of phrB and Le.tyr genes in pre-primordial mycelia of L. edodes is induced by light exposure, suggesting that PHRB can regulate the expression of the Le.tyr gene in a light-dependent manner.

Stimulative effects of light and a temperature downshift on transcriptional expressions of developmentally regulated genes in the initial stages of fruiting-body formation of the basidiomycetous mushroom Lentinula edodes.

Quantitative reverse transcription PCR experiments were done to analyze the effects of light and a temperature downshift on transcriptional expressions of a variety of developmentally regulated genes in the initial stages of fruiting-body formation of Lentinula edodes wild-type strain FMC2. It was revealed that both proper light conditions and a temperature downshift are required for high levels of transcriptions of the genes and the formation of fruiting bodies. In L. edodes MIL-LEW-M13-1 strain, which has been reported previously to be capable of forming fruiting bodies without a low-temperature treatment, the expressions of the developmentally regulated genes were enhanced by light exposure.

Basidiomycete Lentinula edodes CDC5 and a novel interacting protein CIPB bind to a newly isolated target gene in an unusual manner.

We isolated a target gene for the Lentinula edodes putative transcription factor Le.CDC5 that contains a c-Myb-type DNA-binding domain. The gene, termed ctg1, encodes a novel protein (159 amino acid residues) with a leucine zipper-like sequence and contains a 7-bp Le.CDC5-binding sequence, 5'GCAATCT3', in its transcribed region downstream of the start codon. Chromatin immunoprecipitation analysis strongly suggested that intracellular Le.CDC5 binds to this 7-bp sequence on L. edodes chromatin. Binding was most efficient on chromatin from the stipes of mature fruiting bodies. Two Le.CDC5-interaction partners were identified in L. edodes and named CIPA and CIPB. The CIPB protein (127 amino acid residues) binds to a 6-bp sequence with the consensus sequence 5'CAACAC/T/G3'. The ctg1 gene contains nine 6-bp consensus (or consensus-like) sequences, six are in the 5'-upstream region and three in the transcribed region downstream of the start codon. At least two each of the upstream and downstream sequences appear to bind CIPB in vitro. We suggest that Le.CDC5 and CIPB can cooperatively regulate the expression of ctg1.

Sequence analysis and expression of a blue-light photoreceptor gene, Le.phrA from the basidiomycetous mushroom Lentinula edodes.

We cloned and sequenced a photoreceptor gene (Le.phrA) from the basidiomycete Lentinula edodes. The product of Le.phrA, Le.PHRA (924 aa residues) contained a serine-rich region, an LOV domain and two PAS domains. It was clearly smaller than other fungal LOV domain-containing blue-light photoreceptors such as Coprinopsis cinerea Dst1 (1,175 aa), Neurospora crassa WC-1 (1,167 aa), and Cryptococcus neoformans WC-1 (1,141 aa). The Le.phrA gene was found to be transcribed at all stages of the fruiting-body formation of L. edodes, but it was most abundantly transcribed in the immature fruiting body. Fully-matured fruiting body also contained relatively large amounts of Le.phrA transcript. Although the transcript level was lower, preprimordial aggregated mycelial cells grown under a light environment contained larger amounts of the transcript than those grown under continuous darkness, suggesting a light-enhanced expression of the Le.phrA gene. Hymenophore-depleted pileus contained a markedly higher level of the transcript than the stipe.

Long terminal repeat of the mushroom retrotransposon functions as a dual terminator to transcription.

The 474-bp long terminal repeat (LTR) of the basidiomycete Lentinula edodes retrotransposon was found to contain a different type of transcription termination-poly (A)-addition signal on each strand: TATGT- - -TATG- - -TCT, first reported in yeast genes, and an AATAA (or ACATAA)- - -GT(Py)-rich sequence, frequently found in mammalian genes.

The fruiting-specific Le.flp1 gene, encoding a novel fungal fasciclin-like protein, of the basidiomycetous mushroom Lentinula edodes.

To understand the molecular mechanisms of fruiting body formation of basidiomycetous mushrooms, we have isolated over a 100 of developmentally regulated genes that were specifically transcribed during fruiting body development in Lentinula edodes (Shiitake-mushroom) by a subtractive hybridization, cDNA-RDA (cDNA representational difference analysis). One of these genes, named Le.flp1, was isolated from the primordial cDNA library of L. edodes, and the expression product of Le.flp1 and putative fungal homologues contained a characteristic region, homologous to the Fas domain of fasciclin family proteins, which are capable of promoting cell adhesion through Fas domain-mediated homophilic interactions in various organisms. RT-PCR analyses suggested that Le.flp1 was specifically expressed in primordia and mature fruiting bodies. In situ hybridization indicated that Le.flp1 transcripts were distributed distinctly in the following tissues: the inside of gills of fruiting bodies, especially at the boundary between the subhymenium and trama, where there is active proliferation of basidium cells for producing basidiospores; peripheral regions of the primordium, pileus and stipe; and both inner tissue and outer regions of the stipe. Our results suggest the hypothesis that Le.flp1 plays a role in cellular differentiation and development in ubiquitous tissues during fruiting body formation in L. edodes, possibly through cell adhesion.

Developmental regulator Le.CDC5 of the mushroom Lentinula edodes: analyses of its amount in each of the stages of fruiting-body formation and its distribution in parts of the fruiting bodies.

Immunoblot analysis of Le.CDC5 (842 amino acid residues), the expressed product of the cDNA of Le.cdc5 gene that has been previously reported to be most actively transcribed in primordia and small immature fruiting bodies of the basidiomycete Lentinula edodes, showed that the primordia, immature fruiting bodies and mature fruiting bodies contain similar amounts of Le.CDC5 protein. This indicates that the Le.CDC5 protein molecules synthesized in the beginning and early stage of fruiting-body formation remains in mycelial tissues even after small immature fruiting bodies developed and matured. Immunohistochemical analysis showed that Le.CDC5 is present everywhere in the mycelial tissues of immature fruiting body, but prehymenophore, the border between pileus and stipe, and the bottom of stipe seem likely to contain larger amounts of Le.CDC5. Within the hymenophore of mature fruiting body, the hymenium (in/on which a large number of basidia and basidiospores are formed) contains the Le.CDC5 most exclusively.

Expression of the autofluorescent protein, DsRed2, in the recombinants of the ectomycorrhizal basidiomycete, Suillus grevillei, generated by Agrobacterium-mediated transformation.

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5' gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.

Intron-dependent accumulation of mRNA in Coriolus hirsutus of lignin peroxidase gene the product of which is involved in conversion/degradation of polychlorinated aromatic hydrocarbons.

The homobasidiomycete Coriolus hirsutus coding sequences of a lignin peroxidase (LiP) gene (lip, containing six (I-VI) introns), a lip cDNA (lipc), and three lipc derivatives containing one (I), three (I-III), or five (I-V) introns were inserted into chromosome-integrating expression vector. These recombinant plasmids were introduced into C. hirustus monokaryotic strain. The transformant carrying the promoter-lipc-terminator cassette did not contain enough mRNA molecules to be detectable by Northern-blot analysis. On the other hand, all the transformants carrying cassettes of genomic lip and intron(s)-containing lipc sequences contained sufficient amounts of mRNAs to be easily detected by Northern-blot analysis. LiP activities in the culture supernatants of these transformants were found to be about five times as high as those of transformants carrying the lipc cassette (or no cassette). The culture supernatants of the transformants with high LiP activity showed remarkably high conversion activity toward pentachlorophenol (PCP) and degradation activity toward 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). These results indicate that at least one intron (intron I) is required for accumulation of lip mRNA and its subsequent translational expression in C. hirsutus.

Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basidiomycetous promoters.

Coprinopsis cinerea laccase gene lcc1 was expressed in this basidiomycete under naturally non-inductive conditions using various homologous and heterologous promoters. Laccase expression was achieved in solid and liquid media with promoter sequences from the C. cinerea tub1 gene, the Agaricus bisporus gpdII gene, the Lentinus edodes priA gene and the Schizophyllum commune Sc3 gene. As measured by enzyme activity in liquid cultures, a 277-bp gpdII promoter fragment, followed by a 423-bp priA fragment, was most efficient. A shorter priA sequence of 372 bp was inactive. tub1 promoter fragments were reasonably active, whereas the S. commune Sc3 promoter sequence was less active, in comparison. Irrespective of the promoter used, addition of copper to the medium increased enzymatic activities for highly active transformants by 10- to 50-fold and for less active transformants for 2- to 7-fold. The highest enzymatic activities (3 U/ml) were reached with the gpdII promoter in the presence of 0.1 mM CuSO(4).

Analysis of recQ gene transcript in fruiting bodies of basidiomycetous mushroom Lentinula edodes.

Quantitative reverse transcription PCR analysis and total RNA staining demonstrated that Lentinula edodes recQ gene (Le.recQ) transcript is present in all the parts of the fruiting body, but in hymenophore at the highest density. Results of in situ RNA-RNA hybridization showed that the Le.recQ transcript level within the hymenophore is higher in the hymenium, subhymenium, and the outer region of the trama. Trama cells themselves contain a lower level of the transcript.

Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus.

Rat cytochrome P450, CYP1A1, has been reported to play an important role in the metabolism of mono-trichlorodibenzo-p-dioxins (M-TriCDDs). To breed lignin (and M-TetraCDDs)-degrading basidiomycete Coriolus hirsutus strains producing rat CYP1A1, an expression cassette [C. hirsutus gpd promoter-C. hirsutus gpd 5' portion (224-bp of 1st exon-8th base of 4th exon)-rat cyp1a1 cDNA-Lentinula edodes priA terminator] was constructed and inserted into pUCR1 carrying the C. hirsutus arg1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1 + arg1) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078 (Arg(-), Leu(-)), obtaining three good Arg(+) transformants. These transformants [ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)] were estimated to carry nine, six, and seven copies of the expression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants produce similar amounts of rat CYP1A1 enzyme. ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), ChTF5-6(CYP1A1) and recipient OJ1078 were cultivated in a liquid medium containing 2,7/2,8(at a ratio of 1:1)-dichlorodibenzo-p-dioxins (2,7/2,8-DCDDs) and the amount of intra- and extracellular 2,7/2,8-DCDDs remaining was measured. The results showed that all three transformants efficiently transform 2,7/2,8-DCDDs through the action of the recombinant rat CYP1A1 enzyme.

Specific distribution in homobasidiomycete hymenophores of the transcripts of Ras protein and G-protein alpha-subunit genes.

Results of in situ RNA-RNA hybridization showed a complementary distribution of the transcripts of Ras protein gene (ras) and trimeric G-protein alpha-subunit gene (ga1) in the hymenophores of white-rot basidiomycete Lentinula edodes and ectomycorrhizal basidiomycete Lyophyllum shimeji. The ras gene is present mostly in outer region of trama (the region branching out into the subhymenium) and in trams cells, while the ga1 gene is present mostly in hymenium (on which a large number of basidiospores are formed) and in subhymenium (on the top of which hymenium is formed). This suggests that the ras and ga1 genes play distinct physiological roles in the hymenophores of both basidiomycete fungi.

A recQ gene homolog from the basidiomycetous mushroom Lentinula edodes: sequence analysis and expression.

We cloned and sequenced a recQ gene homolog from the basidiomycetous mushroom Lentinula edodes (Le.). This gene, named Le.recQ, was found to have a coding capacity of 945 amino acids (aa) and be interrupted by 11 small introns. The deduced Le.RECQ protein was clearly smaller than other fungal RecQ proteins such as Neurospora crassa QDE3 (1955 aa), Schizosaccharomyces pombe Rqh1 (1328 aa), and Saccharomyces cerevisiae SGS1 (1447 aa). It exhibited the highest homology to the Arabidopsis thaliana RecQl4A protein (1182 aa) in its size and aa sequence. Northern-blot analysis showed that the Le.recQ gene is transcribed at similar levels during mycelial development in L. edodes fruiting-body formation on a sawdust-corn bran medium. The L. edodes dikaryotic mycelial cells, which had been vegetatively grown in SMY liquid medium, were found to contain a clearly larger amount of Le.recQ transcript than the L. edodes two compatible monokaryotic mycelial cells. Expression of Le.recQ cDNA in S. cerevisiae might partially complement defects associated with the loss of its homolog S. cerevisiae SGS1 gene.

A cDNA homologue of Schizosaccharomyces pombe cdc5(+) from the mushroom Lentinula edodes: characterization of the cDNA and its expressed product.

A cDNA homologue of Schizosaccharomyces pombe cdc5(+) was isolated from the basidiomycete mushroom Lentinula edodes and it was named Le.cdc5 cDNA. The deduced Le.CDC5 (842 amino acid residues) possessed N-terminal amino acid sequence highly homologous to those of S. pombe cdc5(+) gene product (Sp.cdc5p) and Sp.cdc5p-related proteins (SPCDC5RPs). The N-terminal 185 amino acid peptide of Le.CDC5 (Le.CDC5(1-185) peptide) produced in Escherichia coli was subjected to random binding-site selection analysis, revealing that Le.CDC5(1-185) peptide binds to a 7-bp sequence with the consensus sequence of 5'GCAATGT3' (complementary; 5'ACATTGC3'). Genomic binding-site (GBS) cloning by using Le.CDC5(1-185) peptide resulted in an isolation of the DNA fragment that contained three sets of 7-bp consensus-like sequence and TATA box. The Le.CDC5 protein contained two putative phosphorylation sites of cAMP-dependent protein kinase (A kinase) in its C-terminus. There exists a possible leucine zipper between the two phosphorylation sites. The Le.CDC5 fragment containing the two phosphorylation sites was actually phosphorylated by commercially available A kinase. Yeast two-hybrid analysis suggested the homodimerization of Le.CDC5 protein probably through the leucine zipper. Northern blot analysis showed that Le.cdc5 gene is most actively transcribed in primordia and small immature fruiting bodies of L. edodes, implying that Le.cdc5 may play a role in the beginning and early stage of fruiting-body formation.

Overproduction of recombinant laccase using a homologous expression system in Coriolus versicolor.

One of the major extracellular enzymes of the white-rot fungus Coriolus versicolor is laccase, which is involved in the degradation of lignin. We constructed a homologous system for the expression of a gene for laccase III (cvl3) in C. versicolor, using a chimeric laccase gene driven by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase (gpd) from this fungus. We transformed C. versicolor successfully by introducing both a gene for hygromycin B phosphotransferase (hph) and the chimeric laccase gene. In three independent experiments, we recovered 47 hygromycin-resistant transformants at a transformation frequency of 13 transformants microg(-1) of plasmid DNA. We confirmed the introduction of the chimeric laccase gene into the mycelia of transformants by a polymerase chain reaction in nine randomly selected transformants. Overproduction of extracellular laccase by the transformants was revealed by a colorimetric assay for laccase activity. We examined the transformant (T2) that had the highest laccase activity and found that its activity was significantly higher than that of the wild type, particularly in the presence of copper (II). Our transformation system should contribute to the efficient production of the extracellular proteins of C. versicolor for the accelerated degradation of lignin and aromatic pollutants.

Target genes of the developmental regulator PRIB of the mushroom Lentinula edodes.

Using the method of genomic binding-site cloning, we identified three target genes of the developmental regulator, the product of priB gene (PRIB) in Lentinula edodes: the previously cloned priB and uck1 (UMP-CMP kinase gene) and a new gene, which we named mfbC. Identification of the former two genes was expected, because the promoter regions of priB and the gene encoding UMP-CMP kinase (uck1) have been shown to contain four or two consensus-like sequences of PRIB binding respectively. The mfbC gene contained two consensus-like sequences of PRIB binding in its promoter region and the PRIB protein bound them. The deduced 330 amino acid sequence of the product of mfbC gene (MFBC) was highly homologous to the 325 amino acid sequence of S. cerevisiae YJR070C/Lia1, the protein interacting with a putative translation initiation factor. Only the mature fruiting body of L. edodes was shown to contain the transcript of the mfbC gene almost exclusively, suggesting that mfbC may play a role in the final stage of fruiting-body formation.

Catalytic Reaction of Basidiomycete Lentinula edodes Cytochrome P450, Le.CYP1 Enzyme Produced in Yeast.

The basidiomycete Lentinula edodes (Le.) cytochrome P450, Le.CYP1 was functionally expressed in Saccharomyces cerevisiae. The microsomal fraction containing Le.CYP1 was prepared from the recombinant yeast and the Le.CYP1 was analyzed. The 7-ethoxycoumarin and benzo(a)pyrene were found to be the substrates of Le.CYP1 enzyme. Le.CYP1 converted 7-ethoxycoumarin to 7-hydroxycoumarin.

A promoter activity in Saccharomyces cerevisiae of the 3'-noncoding region of the basidiomycetous mushroom gene.

The 3'-noncoding region of the priB gene derived from the basidiomycete Lentinus edodes was found to contain one GC box-like sequence, two CAAT boxes, two TATA box-like sequences and two pyrimidine-rich stretches (CT-motifs). It also contained a 16-bp sequence similar to the consensus sequence for PRIB protein binding and a short (61 bp) intron. Single-strand-specific S1 nuclease analysis of plasmid pBR322 DNA containing the 3'-noncoding region propagated in Escherichia coli revealed that this region forms an unusual, extended open structure within/around the downstream pyrimidine/purine (CT/AG)-biased sequence. To examine the promoter activity of the priB 3'-noncoding region in Saccharomyces cerevisiae, in which an autonomously replicating plasmid vector is available, we constructed two plasmids, YEp3'NCR-lacZ and YEp3'UTR-lacZ. The former contains the priB 3'-noncoding region and the reporter E. coli beta-galactosidase gene while the latter contains the priB 3'-noncoding region lacking the intron and the reporter gene. The yeast transformants obtained through introductions of these plasmids showed beta-galactosidase activity and the activity conferred by YEp3'NCR-lacZ was about 50% of that conferred by YEp3'UTR-lacZ. The primer extension analysis showed that transcription of the reporter gene on both plasmids starts at three alternative sites all of which are located within the downstream CT-motif, suggesting a role for the unusual structure of the CT/AG-biased sequence in the initiation of transcription.

Cloning and expression of cytochrome P450 genes, belonging to a new P450 family, of the basidiomycete Lentinula edodes.

Three cytochrome P450 genes, named Le. cyp1, Le. cyp2, and Le. cyp3, were isolated from the basidiomycete Lentinula edodes. Le. cyp1 and Le. cyp2 contained coding regions of 1500 bp and 1497 bp, respectively, but Le. cyp3 was found to be a defective gene. The deduced amino acid sequences of Le. CYP1 (CYP510A1, 500 amino acids) and Le. CYP2 (CYP510A3, 499 amino acids) were highly similar to each other (87% identical) and they had 32-33% identities to that of Coprinus cinereus P450 (CYP502) and 27-28% identities to those of two Aspergillus P450s (CYP64 family). Quantitative RT-PCR analysis of the transcripts of Le. cyp1 and Le. cyp2 genes in the course of fruiting-body development of L. edodes showed that the primordium seems to contain larger amounts of these transcripts. The transcript levels of both of these genes in the stipe of the premature fruiting body were higher than those in the whole pileus and gill tissue.