Toshikazu Irie

Carboxin resistance transformation of the homobasidiomycete fungus Pleurotus ostreatus.

Abstract A novel selection marker gene for transformation of the white-rot basidiomycete Pleurotus ostreatus was developed by introducing a point mutation in a gene which encodes the iron-sulfur protein (Ip) subunit of succinate dehydrogenase. The mutant gene, CbxR, encodes a modified Ip subunit with an amino-acid substitution (His239 to Leu) and confers resistance to the systemic fungicide, carboxin. The DNA sequence was integrated ectopically in the chromosome of the transformants. This is the first report of a homologous marker gene which is available for the molecular breeding of an edible mushroom.

Serial Analysis of Gene Expression (SAGE) of Magnaporthe grisea: genes involved in appressorium formation

Treatment with cyclic AMP (cAMP) induces appressorium formation in the phytopathogenic fungus Magnaporthe grisea, the causative agent of rice blast disease. In a search for the M. grisea genes responsible for appressorium formation and host invasion, SAGE (Serial Analysis of Gene Expression) was carried out using mRNA isolated from fungal conidia germinating in the presence and absence of cAMP. From cAMP-treated conidia 5087 tags including 2889 unique tags were isolated, whereas untreated conidia yielded 2342 unique tags out of total of 3938. cAMP treatment resulted in up- and down-regulation of genes corresponding to 57 and 53 unique tags, respectively. Upon consultation of EST/cDNA databases, 22 tags with higher representation in cAMP-treated conidia were annotated with putative gene names. Furthermore, 28 tags corresponding to cAMP-induced genes could be annotated with the help of the recently published genome sequence of M. grisea. cAMP-induced genes identified by SAGE included many genes that have not been described so far, as well as a number of genes known to be involved in pathogenicity, e.g. MPG1, MAS1 and MAC1. RT-PCR of 13 randomly selected genes confirmed the SAGE results, verifying the fidelity of the SAGE data.

Stable transformation of Pleurotus ostreatus to hygromycin B resistance using Lentinus edodes GPD expression signals.

Abstract. It was reported that Pleurotus ostreatus was transformed unstably using recombinant plasmids containing a hygromycin B phosphotransferase gene (hph) under the control of Aspergillusnidulans expression signals, and that the plasmids were maintained extrachromosomally in the transformants. Here we report a stable and integrative transformation of the fungus to hygromycin B resistance, using a recombinant hph fused with Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase expression signals. Restriction-enzyme-mediated integration (REMI) was also tried and increased the transformation efficiency about ten-fold.

Isolation and characterization of a fruiting body-specific exo-β-1,3-glucanase-encoding gene, exg1, from Lentinula edodes

An exo-β-1,3-glucanase-encoding gene was isolated from Lentinula edodes to investigate the relationship between the cell wall lytic enzyme and mushroom morphogenesis. The deduced amino acid sequence of the protein corresponding to the exg1 gene displayed 67% identity to AbEXG1 of Agaricus bisporus and approximately 40% identity to yeast exo-β-1,3-glucanases. Two conserved glutamic acids within the catalytic active site in yeast exo-β-1,3-glucanases were conserved in exg1 of L. edodes. The exg1 gene was expressed in fruiting bodies, but not in vegetative mycelia. Expression was higher in the stipe than in the pileus of young fruiting bodies. The gene was additionally expressed in the gills of mature fruiting bodies. We purified a glucanase from the stipes of young fruiting bodies that had an N-terminus identical to that of the putative exg1 product. These results collectively indicate that exg1 is involved in L. edodes fruiting body development, including stipe elongation.

Homologous expression of recombinant manganese peroxidase genes in ligninolytic fungus Pleurotus ostreatus

Abstract. Pleurotus ostreatus is a white-rot fungus known as an efficient degrader of lignin and also various organo-pollutants. Using a DNA-mediated transformation system, a molecular breeding approach to isolate overproducers of a manganese peroxidase isozyme, MnP3, was carried out. Recombinant mnp3 constructs under the control of P. ostreatus sdi1 expression signals were introduced into the wild-type P. ostreatus strain by co-transformation with a carboxin-resistant vector plasmid, pTM1. One of the recombinants obtained by a mating between two monokaryotic transformants, TMG9-C1, showed a several times higher level of MnP activity than the wild-type control in the early stage of liquid culture. Predominant transcription of the recombinant mnp3 in the strain was demonstrated by RT-PCR experiments. This is the first report of a genetically modified P. ostreatus strain with an expression system for recombinant genes.

Isolation of cDNA and genomic fragments encoding the major manganese peroxidase isozyme from the white rot basidiomycete Pleurotus ostreatus

We have isolated the cDNA and genomic sequences encoding the major isozyme of manganese peroxidase, MnP3, from the white rot basidiomycetePleurotus ostreatus strain IS1. The genemnp3 is interrupted by 10 introns and encodes a mature protein of 357 amino acid residues with a 26-amino-acid signal peptide. The amino acid residues known to be involved in peroxidase function and those that form the Mn-binding site in thePanerochaete chrysosporium MnP isozyme are conserved in MnP3. Comparison of the deduced primary structure of MnP3 with those of other peroxidases from various white rot fungi suggested that MnPs fromP. ostreatus andTrametes versicolor belong to a subgroup that is more similar to the lignin peroxidases than MnPs fromP. chrysosporium orCeriporiopsis subvermispora.

Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase

Laccase (Lcc) is a lignin-degrading enzyme produced by white-rot fungi and has been the subject of much interest in the field of bioremediation due to its ability to oxidize phenolic compounds. In this report, we describe the isolation and characterization of lcc1, a novel gene of Lentinula edodes that encodes Lcc1, and demonstrate that recombinant Lcc1 is expressed in an active, secreted form in tobacco BY-2 cells in culture. The open reading frame of lcc1 was 1,557 base pairs in length and encoded a putative protein of 518 amino acids. We introduced a chimeric form of lcc1 (CaMV35Sp:clcc1) into tobacco BY-2 cells and obtained several stable clcc1 transformants that expressed active Lcc1. Lcc1 activity in BY-2 culture media was higher than in cellular extracts, which indicated that recombinant Lcc1 was produced in a secreted form. Recombinant Lcc1 had a smaller apparent molecular weight and exhibited a different pattern of posttranslational modification than Lcc1 purified from L. edodes. The substrate specificity of purified recombinant Lcc1 was similar to L. edodes Lcc1, and both enzymes were able to decolorize the same set of dyes. These results suggest that heterologous expression of fungal Lcc1 in BY-2 cells will be a valuable tool for the production of sufficient quantities of active laccase for bioremediation.

Construction of a homologous selectable marker gene for Lentinula edodes transformation

We cloned a gene for the iron sulfur protein (Ip) subunit from an edible mushroom, Lentinula edodes, and introduced a point mutation that confers carboxin resistance into it. The mutant gene successfully transformed L. edodes with high efficiency (9 transformants/2.5 μg vector DNA). Restriction enzyme-mediated integration (REMI) increased the transformation efficiency by about two-fold.

Changes in the Gene Expression of the White Rot Fungus Phanerochaete chrysosporium Due to the Addition of Atropine

We constructed a LongSAGE (Long Serial Analysis of Gene Expression) library from a 3-d culture of Phanerochaete chrysosporium supplemented with atropine, which inhibits the production of lignin-degrading enzymes. The library (the atropine library) contains 13,108 LongSAGE tags and 6,783 unique tags. The gene expression profile represented by the tags was compared with those of two previously constructed libraries, one of which was constructed using 2-d cultures in which the fungus had not yet produced ligninolytic enzymes (the 2-d library) and the other was constructed using 3-d cultures in which the fungus had just started to produce the enzymes (the 3-d library). We found a total of 595 genes that were at least twice more highly or at least twice less highly expressed in the 3-d library than in the 2-d library or the atropine library, and the fluctuations were statistically significant. The relationships among these 595 genes were considered using cluster analysis. Of the 595 genes, 164 showed expression patterns similar to those of four ligninolytic enzyme genes, which were more expressed on day 3 than under any other conditions. Many of these 164 genes comprised genes possibly involved in lignin degradation, lipid metabolism, xenobiotic degradation, stress response, or signal transduction pathways.

Cloning and characterization of the gene encoding the iron-sulfur protein of succinate dehydrogenase from Pleurotus ostreatus.

Genomic and cDNA fragments encoding the iron-sulfur protein (Ip) subunit of dehydrogenase (EC 1.3.99.1) have been cloned from the edible basidiomycetous fungus, Pleurotus ostreatus. The gene is interrupted by five introns and is predicted to encode a polypeptide of 268 amino acid residues. Sequence comparison with the Ip subunit from other species identified three conserved cysteine-rich clusters. One of these contains a critical histidine residue implicated in carboxin sensitivity in the heterobasidiomycete Ustilago maydis.