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In the bipolar basidiomycete Pholiota microspora, 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. microspora 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 transformed into the A4 monokaryon strain, the transformants produced many pseudoclamps 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 clamplike cells in the transformants was significantly increased to ca. 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 reverse transcription-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. microspora. Thus, 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.In basidiomycetous mushrooms, mating compatibility is controlled by one or two sets of multiple allelomorphic genes known as bipolar or tetrapolar mating systems, respectively (37). In tetrapolar mushrooms, such as Coprinopsis cinerea (5, 13), Laccaria bicolor (8,11,19), and Schizophyllum commune (10), the mating-type loci A and B, which are located on different chromosomes, regulate mating and clamp formation (9,15,27,28). The A locus comprises multigenes encoding homeodomain proteins, and the B locus comprises multigenes encoding pheromones and pheromone receptor proteins (6,13,21,25,26,30,31,32,35,36). On the basis of the homeodomain sequence, the mating-type proteins of the A locus are divided into two subgroups: HD1 and HD2 (20,21). When an HD1 protein from one mate heterodimerizes with an HD2 protein from the other mate to form a functional regulatory protein, sexual compatibility is intracellularly recognized, and the A developmental pathway is initiated (3,17,23).Few studies have examined the composition and function of mating-type loci in bipolar basidiomycetes. In a landmark study, Bakkeren and Kronstad (2) discovered that in bipolar fungus, Ustilago hordei, the A-and B-mating-type loci were fused into one nonrecombining mating-type region with two alleles. However, subsequent studies revealed that although both A-and B-mating-type homologs are found in bipolar mushrooms, they are present on different chromosomes, and only the A-mating-type homologs are related to mating compatibility (1, 16).Although Ph...

We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the effi ciency of carboxin resistance transformation was about 88.8 transformants/μg pMBsip2 DNA using 5 × 10 6 protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the effi ciency of hygromycin B resistance transformation was about 122.4 transformants/μg pMBhph1 DNA using 5 × 10 6 protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more.

We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the effi ciency of carboxin resistance transformation was about 88.8 transformants/μg pMBsip2 DNA using 5 × 10 6 protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the effi ciency of hygromycin B resistance transformation was about 122.4 transformants/μg pMBhph1 DNA using 5 × 10 6 protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more.