Ursula Kües

Life History and Developmental Processes in the Basidiomycete Coprinus cinereus

SUMMARY Coprinus cinereus has two main types of mycelia, the asexual monokaryon and the sexual dikaryon, formed by fusion of compatible monokaryons. Syngamy (plasmogamy) and karyogamy are spatially and temporally separated, which is typical for basidiomycetous fungi. This property of the dikaryon enables an easy exchange of nuclear partners in further dikaryotic-monokaryotic and dikaryotic-dikaryotic mycelial fusions. Fruiting bodies normally develop on the dikaryon, and the cytological process of fruiting-body development has been described in its principles. Within the specialized basidia, present within the gills of the fruiting bodies, karyogamy occurs in a synchronized manner. It is directly followed by meiosis and by the production of the meiotic basidiospores. The synchrony of karyogamy and meiosis has made the fungus a classical object to study meiotic cytology and recombination. Several genes involved in these processes have been identified. Both monokaryons and dikaryons can form multicellular resting bodies (sclerotia) and different types of mitotic spores, the small uninucleate aerial oidia, and, within submerged mycelium, the large thick-walled chlamydospores. The decision about whether a structure will be formed is made on the basis of environmental signals (light, temperature, humidity, and nutrients). Of the intrinsic factors that control development, the products of the two mating type loci are most important. Mutant complementation and PCR approaches identified further genes which possibly link the two mating-type pathways with each other and with nutritional regulation, for example with the cAMP signaling pathway. Among genes specifically expressed within the fruiting body are those for two galectins, β-galactoside binding lectins that probably act in hyphal aggregation. These genes serve as molecular markers to study development in wild-type and mutant strains. The isolation of genes for potential non-DNA methyltransferases, needed for tissue formation within the fruiting body, promises the discovery of new signaling pathways, possibly involving secondary fungal metabolites.

Restriction enzyme-mediated DNA integration in Coprinus cinereus

Restriction enzyme-mediated DNA integration (REMI) has recently received attention as a new technique for the generation of mutants by transformation in fungi. Here we analyse this method in the basidiomycete Coprinus cinereus using the homologous pab1 gene as a selectable marker and the restriction enzymes BamHI, EcoRI and PstI. Addition of restriction enzymes to transformation mixtures results in an earlier appearance of transformants and influences transformation rates in an enzyme- and concentration-dependent manner. Low concentrations of restriction enzyme result in increased numbers of transformants compared to no addition of enzymes. Transformation rates decrease with higher enzyme concentrations. If protoplasts are made from cells stored in the cold, the transformation rates drop drastically even in the presence of low amounts of enzyme. In several transformants, plasmid integration directly correlated with the action of restriction enzyme at random chromosomal restriction sites. In some cases, restriction enzymes appear to reduce the number of integration events per transformant. Simultaneously, mutation rates can be enhanced due to the presence of restriction enzymes. Although restriction enzymes clearly promote plasmid integration into the host genome they also have cytotoxic and possibly mutagenic effects that result from processes other than plasmid integration. In consequence, for any given enzyme used in REMI mutagenesis, the enzyme concentration that gives the highest number of transformants must be defined experimentally. Such optimal transformation conditions should give the highest probability of obtaining mutations caused by a single restriction enzyme-mediated integration of the selection marker.

Cellular and molecular mechanisms of sexual incompatibility in plants and fungi.

Plants and fungi show an astonishing diversity of mechanisms to promote outbreeding, the most widespread of which is sexual incompatibility. Sexual incompatibility involves molecular recognition between mating partners. In fungi and algae, highly polymorphic mating-type loci mediate mating through complementary interactions between molecules encoded or regulated by different mating-type haplotypes, whereas in flowering plants polymorphic self-incompatibility loci regulate mate recognition through oppositional interactions between molecules encoded by the same self-incompatibility haplotypes. This subtle mechanistic difference is a consequence of the different life cycles of fungi, algae, and flowering plants. Recent molecular and biochemical studies have provided fascinating insights into the mechanisms of mate recognition and are beginning to shed light on evolution and population genetics of these extraordinarily polymorphic genetic systems of incompatibility.

Two Classes of Homeodomain Proteins Specify the Multiple A Mating Types of the Mushroom Coprínus cínereus

The A mating type locus of the mushroom Coprinus cinereus regulates essential steps in sexual development. The locus is complex and contains several functionally redundant, multiallelic genes that encode putative transcription factors. Here, we compare four genes from an A locus designated A42. Overall, the DNA sequences are very different (approximately 50% homology), but two classes of genes can be distinguished on the basis of a conserved homeodomain motif in their predicted proteins (HD1 and HD2). Development is postulated to be triggered by an HD1 and an HD2 gene from different A loci. Thus, proteins encoded by genes of the same locus must be distinguished from those encoded by another locus. Individual proteins of both classes recognize each other using the region N-terminal to the homeodomain. These N-terminal specificity regions (COP1 and COP2) are predicted to be helical and are potential dimerization interfaces. The amino acid composition of the C-terminal regions of HD1 proteins suggests a role in activation, and gene truncations indicate that this region is essential for function in vivo. A corresponding C-terminal region in HD2 proteins can be dispensed with in vivo. We will discuss these predicted structural features of the C. cinereus A proteins, their proposed interactions following a compatible cell fusion, and their similarities to the a1 and alpha 2 mating type proteins of the yeast Saccharomyces cerevisiae.

A FUNGAL MATING TYPE PROTEIN THAT REGULATES SEXUAL AND ASEXUAL DEVELOPMENT CONTAINS A POU-RELATED DOMAIN

The A mating type factor of the fungus Coprinus cinereus regulates essential steps in sexual development. Here we describe features of one of the four specificity genes of the A42 factor. By transformation we show that the gene regulates not only sexual development but also asexual sporulation. DNA sequence analysis shows that the gene beta 1–1, encodes a protein with a DNA binding motif and is thus likely to be a transcription factor. The DNA binding domain is an unusual homeodomain with D replacing the normally invariant N in the recognition helix and apparent absence of helix II. The homeodomain is linked to a helical region related to the POUs domain, which is part of a bipartite DNA binding domain of certain animal transcription factors. Like POU factors, the beta 1–1 protein has regions rich in serine, threonine and proline which are possible transactivation domains. Putative dimerization domains and sites for post‐translational modification are described.

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12 h light/12 h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain.

The laccase gene family in Coprinopsis cinerea (Coprinus cinereus)

Abstract In this study, we isolated and sequenced eight non-allelic laccase genes from Coprinopsis cinerea (Coprinus cinereus) homokaryon AmutBmut. These eight genes represent the largest laccase gene family identified so far in a single haploid fungal genome. We analyzed the phylogenetic relationships between these genes by intron positions, amino acid sequence conservation and similarities in promoter sequences. All deduced protein products have the laccase signature sequences L1–L4, the typical conserved cysteine and the ten histidine residues which are ligands in the two laccase copper-binding centers, T1 and T2/T3. Proteins Lcc2 and Lcc3 of Coprinopsis cinerea are most similar to the acidic, membrane-associated laccase CLAC2 from Coprinellus congregatus implicated in neutralization of acidic medium. All other laccases from the saprophyte Coprinopsis cinerea, including the well described enzyme Lcc1, form a cluster separate from these three enzymes and from various laccases of wood-rotting and plant-pathogenic basidiomycetes.

A chimeric homeodomain protein causes self-compatibility and constitutive sexual development in the mushroom Coprinus cinereus.

The A mating type genes of the mushroom Coprinus cinereus encode two classes of putative transcription factor with distinctive homeodomain motifs (HD1 and HD2). A successful mating brings together different allelic forms of these genes and this triggers part of a developmental sequence required for sexual reproduction. In this report we provide evidence that this developmental programme is promoted by a physical interaction between the two classes of homeodomain protein. Rare dominant mutations conferring self‐compatibility map to the A locus and result in constitutive operation of the A‐regulated developmental pathway. Our molecular analysis of one of these mutations shows that it has generated a chimeric gene by inframe fusion of an HD2 and an HD1 gene. Fusion has overcome the normal incompatibility between two proteins coded by genes of the same A locus and generated a protein that is sufficient to promote development in the absence of any other active A mating type genes. The fusion protein retains most of the HD2 sequence, but only the C‐terminal part of the HD1 protein. It has only the HD2 homeodomain motif as a potential DNA binding domain fused to an essential C‐terminal region of the HD1 protein, which in a normal HD1‐HD2 protein complex may be the major activation domain.

Fungal mating type genes — regulators of sexual development

The mating type genes of the fungi determine sexual compatibility between different haploid individuals. In both ascomycetes and basidiomycetes the genes are seen to influence directly or indirectly the expression of developmentally regulated genes. Mating type genes from several species have now been cloned and sequenced and alternative forms within the same species are generally found to be unrelated or very dissimilar in sequence. In most cases the genes encode proteins with characteristic DNA binding motifs (transcription factors). In the yeast, Saccharomyces cerevisiae, sequence-specific DNA binding of purified proteins has been demonstrated and involves different protein associations in different cell types. Evidence for similar protein-protein interactions between mating type encoded transcription factors exists for basidiomycetes but has yet to be demonstrated in other ascomycete fungi. It is interesting to find that the genes for mating pheromones that are subject to regulation by mating type proteins in ascomycete fungi are encoded directly by mating type genes in basidiomycetous yeasts.

Molecular mechanisms of self-incompatibility in flowering plants and fungi — different means to the same end

Hermaphrodite flowering plants and fungi face the same sexual dilemma - how to avoid self-fertilization. Both have evolved ingenious recognition systems that reduce or eliminate the possibility of selfing. These self-incompatibility (SI) systems offer unique opportunities to study recognition and signalling in non-animal cells and also represent model systems for studying the evolution of breeding systems at a molecular level. In this review, the authors discuss recent molecular data that predict an astonishing diversity in the cellular mechanisms of SI operating in flowering plants and fungi.