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.

ABSTRACT
 Introduction
 Materials and methods
  Fungal strains
  Mycelium preparation, DNA and RNA extraction
  Amplification of A3-hox1 and A3-hox2 genes
  Co-transformation method
  DAPI and Fluorescent Brightener 28 staining andmicroscopic observation
  Construction of two plasmid vectors for overexpressionof the A3-hox1 and A3-hox2 genes
  Southern hybridization
  Real-time PCR assay
 Results and discussions
  A single introduced hox gene is insufficient to inducetrue clamps in high frequency
  Two separated, introduced hox gene increases thefrequency of clamps
  Two introduced combined hox gene also increasethe frequency of fused hook cell
  Greater expression of the hox genes drive the realclamp formation
  Different growth condition was observed in differentkinds of transformants
  Different expression amount of four hox gene indifferent kinds of transformants
 REFERENCES

• Ruirong Yi(The United Graduate School of Agricultural Sciences, Tottori University, 4-101 Koyama-cho Minami, Tottori-shi, Tottori 680-8553, Japan)
• Hiroyuki Mukaiyama( Faculty of Agriculture, Tottori University, 4-101 Koyama-cho Minami, Tottori-shi, Tottori 680-8553, Japan) | Hiroyuki Mukaiyama
• Takashi Tachikawa( Faculty of Agriculture, Tottori University, 4-101 Koyama-cho Minami, Tottori-shi, Tottori 680-8553, Japan) | Takashi Tachikawa
• Norihiro Shimomura( Faculty of Agriculture, Tottori University, 4-101 Koyama-cho Minami, Tottori-shi, Tottori 680-8553, Japan) | Norihiro Shimomura
• Tadanori Aimi( Faculty of Agriculture, Tottori University, 4-101 Koyama-cho Minami, Tottori-shi, Tottori 680-8553, Japan) | Tadanori Aimi