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.

An exo-beta-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-beta-1,3-glucanases. Two conserved glutamic acids within the catalytic active site in yeast exo-beta-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.

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.

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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 over-producers 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.

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