In the bipolar basidiomycete Pholiota nameko, a pair of homeodomain protein genes located at the A mating-type locus regulates mating compatibility. In the present study, we used a DNA-mediated transformation system in P. nameko to investigate the homeodomain proteins that control the clamp formation. When a single homeodomain protein gene (A3- hox1 or A3-hox2) from the A3 monokaryon strain was introduced into the A4 monokaryon strain, the transformants produced many pseudo-clamps but very few clamps. When two homeodomain protein genes (A3-hox1 and A3-hox2) were transformed either separately or together into the A4 monokaryon, the ratio of clamps to the clamp-like cells in the transformants was significantly increased to approximately 50%. We, therefore, concluded that the gene dosage of homeodomain protein genes is important for clamp formation. When the sip promoter was connected to the coding region of A3-hox1 and A3-hox2 and the fused fragments were introduced into NGW19-6 (A4), the transformants achieved more than 85% clamp formation and exhibited two nuclei per cell, similar to the dikaryon (NGW12 -163 × NGW19-6). The results of real-time RT-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. nameko. So, we concluded that nearly 100% clamp formation requires high expression levels of homeodomain protein genes and that altered expression of the A mating-type genes alone is sufficient to drive true clamp formation.

Basidiomycetes can degrade lignocellulosic biomass, and some basidiomycetes produce alcohol dehydrogenase, so it is feasible to produce alcohol from basidiomycetes. Agaricus blazei, Flammulina velutipes and Tricholoma matsutake have been used for mushroom fermentation to produce alcohol. To investigate whether Pholilta nameko can be used for mushroom fermentation, and find out the relationship between mannitol-1-phosphate dehydrogenase and alcohol dehydrogenase, we cloned mannitol-1-phosphate dehydrogenase gene from P. nameko, which is a zinc-containing long- chain alcohol dehydrogenase. Mpd, the gene encoding mannitol-1-phosphate dehydrogenase (MPD), has been sequenced and characterized from basiodiomycete P. nameko. The length of the coding region is 1360bp. The gene encodes a putative protein of 359 amino acids; predicted protein molecular weight is 38.6 kDa and an isoelectric point is PI = 7.34. The locations of exons and introns in the gene were deduced on the basis of interruptions in the amino acid sequence that were homologous to those in the MPD of Laccaria bicolor, the coding region was split into 6 exons and 5 introns. The protein deduced from the gene MPD showed more than 46% sequence identity to 20 fungal MPDs or alcohol dehydrogenases documented in the Gene bank protein database, based on BLASTP analysis, and was phylogenetically close to the MPDs of L. bicolor and Coprinopsis cinerea. This protein shared the same conserved domain with the alcohol dehydrogenase.