The eln3 gene involved in fruiting body morphogenesis of Coprinus cinereus encodes a putative membrane protein with a general glycosyltransferase domain.

We identified and characterized elongationless3 (eln3-1), a restriction enzyme-mediated integration (REMI) mutation affecting fruiting body morphogenesis in Coprinus cinereus. The mutant produces an aberrant fruiting body in which the stipe hardly elongates during fruiting body maturation. In the wild type, cylindrical stipe cells, elongation growth of which is responsible for stipe elongation, make side-by-side contact with one another and run parallel to the stipe axis, whereas in the mutant, the organization of the stipe tissue is disturbed and much space is produced between stipe cells. This disorganization of the stipe tissue, together with reduced elongation of the stipe cells, causes the mutant stipe short and bulgy. After a plasmid rescue, the eln3 gene was identified as a DNA fragment that complements the eln3-1 mutation. The eln3 ORF is predicted to encode a protein of 927 amino acids with a general glycosyltransferase domain and to be located in the plasma membrane. Transcription of the eln3 gene is specifically activated in rapidly elongating stipes. Possible involvement of the putative Eln3 enzyme in cell-to-cell connection is discussed.

Life history and developmental processes in the basidiomycete Coprinus cinereus.

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, beta-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.

The rad9 gene of Coprinus cinereus encodes a proline-rich protein required for meiotic chromosome condensation and synapsis.

The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stage of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus.

Chromosome dynamics in rad12 mutants of Coprinus cinereus.

We have characterized the phenotypes of three rad12 mutants of the basidiomycete Coprinus cinereus, which were isolated on the basis of sensitivity to ionizing radiation. Electron microscopic studies of meiotic nuclear spreads showed that all three rad12 mutants are defective in chromosomal synapsis. For rad12-1 and rad12-4, very limited assembly of the synaptonemal complex occurs. The phenotype of rad12-15 is less severe and longer stretches of synapsed chromosomes are formed. However, for all three alleles mutant nuclei arrest in a diffuse state with little synaptonemal complex structure. Observations made of spreads of acridine orange-stained meiotic nuclei correlated with the electron microscopic data. In rad12 strains, chromosomes condense but do not pair, and they later arrest in a decondensed state; very few rad12 cells enter metaphase I. Homozygous dikaryons of rad12 mutants produce fruiting bodies with significantly fewer basidiospores than are found in wild-type dikaryons. The viability of these spores is greatly reduced: all spores produced by rad12-1 and rad12-4 mushrooms fail to germinate, while only 16% of rad12-15 spores are viable. Recombination within the tract of the ribosomal RNA gene repeats was not significantly different in the mutants when compared with a wild-type congenic control. Quantitative measurements of oidial survival indicate that all three rad12 alleles are sensitive to gamma radiation but insensitive to UV radiation relative to wild-type strains.

Meiosis in Coprinus. VIII. A time-course study of the fusion and division of the spindle pole body during meiosis.

The time-course study of meiosis in the fungus Coprinus cinereus (C. lagopus) by electron microscopy reveals that two monoglobular spindle pole bodies (SPB's) of prekaryogamy nuclei come together during karyogamy and are fused. The fusion SPB of postkaryogamy nucleus persists through zygotene and pachytene as evidenced by the presence of axial components and synaptonemal complexes. At early diplotene, the SPB divides. The divided SPB takes on a diglobular form, which grows in size to form two daughter SPB's. These separate and move to opposite poles at metaphase I.

A highly infectious 'mycoplasma' that inhibits meiosis in the fungus Coprinus.

We have discovered a cytoplasmically inherited infectious agent that inhibits meiosis in a species of Coprinus, a basidiomycetous fungus. From infectivity, filtration, centrifugation and ultrastructural studies we believe the agent to be a mycoplasma. The agent is highly infectious to several strains of the host species and is capable of spreading rapidly through infected hosts. No pathological effect has been seen on any aspect of growth or differentiation of the fungus except for the inability of infected strains or undergo meiosis. The failure of meiosis results in mushrooms that do not produce the normal black spores and are therefore pale in colour. The paleness represents a simple assay for the presence and activity of the infectious agent. Infected hosts do not display any ultrastructural abnormalities in the vegetative stages, only in the cells in which meiosis should occur. In the meiotic cells, at the time when normal cells are undergoing synapsis and synaptinemal complexes are forming, the vacuoles of the infected cells become occupied with vesicular, membrane-bound bodies resembling in shape and form mycoplasmas. Extracts from infected clones may be filtered through 0.2-mum filters and retain full infectivity. The infectious material may be pelleted from such extracts at only 10 000g. Migration experiments, as well as the filtration studies, rule out involvement directly of nuclei. The high rate of infection and spread of the mycoplasma through the host, combined with the anatomical simplicity of the host, make this an ideal system in which to study the basis of infection. The singularity of the pathological effect make this host-parasite association useful in studying both the underlying mechanisms of mycoplasma pathogenicity and to investigate the regulation of meiosis. This is only the second report of mycoplasmas in fungi.

Growth and ultrastructural studies on the mitochondrial mutant of Coprinus lagopus.

The cytoplasmic acu-10 mutant of Corprinus lagopus has a respiratory deficiency due to an altered cytochrome component and is slower growing than wild type. When growth of wild type and acu-10 monokaryons and dikaryons were compared on solid medium and in liquid culture the mutation was found to restrict growth of the dikaryon more severely than that of the monokaryon. Ultrastructural studies revealed that faster growth of the acu-10 monokaryon occurred at the expense of maintaining the cytoplasmic cell contents and with little increase in the numbers of mitochondria. Cells of the acu-10 dikaryon were comparatively unvacuolated and contained greatly increased numbers of mitochondria. Mitochondria in cells of the mutant had a typical orthodox conformation with clear matrix and well defined cristae. In contrast, mitochondria in wild type cells had a more compact and elongated shape with dense matrix and less obvious cristae. The observed difference in mitochondrial ultrastructure is interpreted as one of conformation rather than structure and is attributed to impaired ability of mutant mitochondria to carry out oxidative phosphorylation. In an old cell of the mutant the mitochondria showed signs of recovering the wild type conformation.