Molecular genetic approaches have advantages in investigating various physiological aspects of mushrooms. We have been engaged in developing genetic transformation system mainly in wood rot mushrooms. In this presentation, our recent trials to accelerate mushroom science beyond the post-genomic era will be presented. They include development of research tools both in forward and reverse genetics; a transfection system and its utilization in gene expression signal analysis in the selective white rot fungus Ceriporiopsis subvermispora, multiple gene targeting system in Pleurotus ostreatus and Coprinopsis cinerea, which was used to do knock-out or knock-in of reporter constructs on the chromosome of these fungi, and a “powered” forward genetics combined with genome information was used to identify new gene responsible for lignin-degradation in P. ostreatus. They may contribute to elucidate uncovered molecular mechanisms in unique processes in the mushrooms and to make them as a new industrial tool in the next era. Furthermore, our recent results in on-going genome editing experiments will be also reported.

Ceriporiopsis subvermispora is a unique white rot fungus degrading plant cell wall lignin without severe loss of cellulose. Recombinant plasmids containing homologous gene expression signals fused to the coding sequence of Escherichia coli hph which encodes for hygromycin phosphotransferase were introduced in protoplasts of wild type C. subvermispora using PEG/CaCl2 protocol. A number of hygromycin B resistant strains were isolated on a screening plate containing 100㎍/ml of hygromycin B: whereas, no colonies were observed when protoplasts were treated with no DNA as a negative control. It was demonstrated that most of the isolates lost their drug resistance during successive cultivations in the presence of the same concentration of hygromycin B, but some of the isolates showed stable drug resistance after five times repeated screening. They did not lose the drug resistance even after the cultivation in the absence of hygromycin B and incorporation of the hph sequence was confirmed by specific amplification of the target sequence in PCR experiments and Southern hybridization analysis. The stable transformation system will make it possible to do molecular genetic analysis, as well as breeding of genetically modified strains, in C. subvermispora. Moreover, it was demonstrated that recombinant constructs with truncated promoter showed reduced number of the drug resistant isolates on the first screening plate, in response with the length of the remaining promoter sequence. These findings indicated that unstable drug resistance observed in these isolates should originate from transient expression of the introducing marker genes, and that a promoter assay system has been developed for the first time in basidiomycete. This system is practically not affected by the positional effect of the integrated recombinant gene in the host chromosome.