Analysis of pcc 1 , a Gene That Leads to A-Regulated Sexual Morphogenesis in Coprinus cinereus

A homokaryotic strain (5337) in our culture stock of Coprinus cinereus produced fertile fruit bodies after prolonged culture. Microscopic examination revealed that hyphae dedifferentiated from the tissues of one of the fruit bodies, as well as all basidiospore derivatives from the fruit body, exhibited pseudoclamps, whereas vegetative hyphae of 5337, from which the fruit body developed, had no clamp connections. Genetic analysis showed that the formation of pseudoclamps results from a recessive mutation in a gene designated pcc1 (pseudoclamp connection formation), which is distinct from the A and B mating type genes. Cloning and sequencing of the pcc1 gene and cDNA identified an ORF of 1683 bp interrupted by one intron. Database searches revealed that pcc1 encodes an SRY-type HMG protein. The HMG box shared 44, 41, and 29% sequence identities (.80 amino acids) to those of FPR1 of Podospora anserina, MAT-Mc of Schizosaccharomyces pombe, and prf1 of Ustilago maydis, respectively. Northern analysis revealed that the level of pcc1 expression is higher in the dikaryon, in homokaryons in which the A and B mating type developmental sequences are individually activated, than in the homokaryon in which these sequences are not active. Sequencing of the pcc1-1 mutant allele revealed that the mutant carries a nonsense mutation at serine 211, a residue located between the HMG box and the C terminus. Based on these results, possible roles of the pcc1 gene in the sexual development of homobasidiomycetes are discussed. T homobasidiomycete Coprinus cinereus is a tethas been shown in both C. cinereus and U. maydis that a compatible mating is recognized by the ability of allelic rapolar species in which mating type is determined by genes at two unlinked loci known as A and B (Kimura forms of HD1 and HD2 proteins to heterodimerize (Banham et al. 1995; Kämper et al. 1995). Heterodimeri1952). Genes at these loci confer multiple mating specificities and determine compatibility of mating cells by zation between two classes of homeodomain proteins in C. cinereus has been shown to bring together potential regulating initial steps in sexual development. The asexDNA-binding and activation domains (Asante-Owusu ual homokaryons are compatible when genes at both et al. 1996) of a putative dikaryon-specific transcription the A and B loci are different, and the operation of factor. These findings strongly suggest that there are both the A and B regulated steps in sexual development target genes specific for the A protein heterodimer. As gives rise to fertile dikaryotic hyphae that have characteryet, no target gene has been identified. istic structures known as clamp connections (Raper In this paper, we describe the analysis of a mutation 1966). When only the A genes are different, only the A in a gene distinct from the A-mating type locus of sequence operates, resulting in hyphae with unfused C. cinereus that leads to constitutive operation of A-regupseudoclamp connections (Raper 1966). Recent moleclated sexual morphogenesis, i.e., pseudoclamp connecular analyses have shown that the genes that determine tion formation, in the absence of the A protein heterothe A-mating specificity in C. cinereus encode two dissimidimer. lar classes of proteins, designated HD1 and HD2, on the basis of conserved but distinct homeodomain motifs (Kües et al. 1992; Tymon et al. 1992). Similar homeodoMATERIALS AND METHODS main sequences have also been found in the deduced polypeptides of the A genes of another homobasidiomyStrains and culture conditions: Strains of C. cinereus (Schaeff. cete, Schizophyllum commune (Stankis et al. 1992; Specht ex Fr.) S. F. Gray sensu Korn. listed in Table 1 were used. CY-1 et al. 1992), and the b mating type genes of the heteroagar plates in 9-cm petri dishes (Kamada et al. 1982) were used for homokaryotic fruiting of 5337. The minimal medium basidiomycetes Ustilago maydis (Gillissen et al. 1992) was that of Shahriari and Casselton (1974) modified by and Ustilago hordei (Bakkeren and Kronstad 1993). It Binninger et al. (1987). Malt extract–yeast extract–glucose medium (Rao and Niederpruem 1969) solidified with 2% (w/v) agar in petri dishes 9 cm in diameter was used for routine mycelial cultures. Slants of the same medium in test Corresponding author: Takashi Kamada, Department of Biology, Factubes were used for dikaryotic fruiting, the same medium ulty of Science, Okayama University, Okayama 700, Japan. E-mail: kamada@cc.okayama-u.ac. jp without agar in Erlenmeyer flasks for mycelial cultures for Genetics 149: 1753–1761 (August 1998) 1754 Y. Murata et al.