Blue light exposure and nutrient conditions influence the expression of genes involved in simultaneous hyphal knot formation in Coprinopsis cinerea.

Light and nutrients are crucial environmental factors influencing fungal sexual reproduction. Blue light induces simultaneous hyphal knot formation in Coprinopsis cinerea mycelia grown on low-glucose media but not in mycelia grown on high-glucose media. Many hyphal knots are visible in the arc near the edge of the colony one day after 15 min of blue light stimulation. These findings collectively suggest that blue light accelerates hyphal knot induction in nutrient-limited conditions. Transcriptome analysis revealed that gene expression after light exposure is divided into at least two major stages. In the first stage, genes coding for fasciclin (fas1), cyclopropane-fatty-acyl-phospholipid synthases (cfs1 and cfs2), and putative lipid exporter (nod1) are highly expressed after 1 h of light exposure in the mycelial region where the hyphal knot will be developed. These genes are upregulated by blue light and not influenced by glucose condition and mating. These results suggest that although some of the genes are critical for induction of the hyphal knots, they are not sufficient for hyphal knot development. In the second gene expression stage, genes encoding galectins (cgl1-3), farnesyl cysteine-carboxyl methyltransferases, mating pheromone-containing protein, nucleus protein (ich1), and laccase (lcc1) are specifically upregulated at 10-16 h after blue light exposure when the mycelia are cultivated on low-glucose media. These genes might be involved in the architecture of hyphal knots or signal transduction for further fruiting body development. These results contribute to the understanding of the effect of environmental factors on sexual reproduction in basidiomycetous fungi.

The dst1 gene involved in mushroom photomorphogenesis of Coprinus cinereus encodes a putative photoreceptor for blue light.

The homobasidiomycete Coprinus cinereus exhibits remarkable photomorphogenesis during fruiting-body development. Under proper light conditions, fruiting-body primordia proceed to the maturation phase in which basidia in the pileus undergo meiosis, producing sexual spores, followed by stipe elongation and pileus expansion for efficient dispersal of the spores. In the continuous darkness, however, the primordia do not proceed to the maturation phase but are etiolated: the pileus and stipe tissues at the upper part of the primordium remain rudimentary and the basal part of the primordium elongates, producing "dark stipe." In this study we genetically analyzed five strains that produce dark stipes even if light conditions promoting the maturation are given and then characterized one of them, Uar801 (dst1-1). The dst1 gene was cloned as a DNA fragment that rescues the dst1-1 mutation. Dst1 is predicted to be a protein of 1175 amino acids that contains two PAS domains, a coiled-coil structure, and a putative, glutamine-rich, transcriptional activation domain (AD). One of the PAS domains exhibits significant similarity to the LOV domains of known blue-light receptors, suggesting that Dst1 is a blue-light receptor of C. cinereus. The dst1-1 mutation is predicted to truncate the putative AD in the C-terminal region.

The Coprinus cinereus adherin Rad9 functions in Mre11-dependent DNA repair, meiotic sister-chromatid cohesion, and meiotic homolog pairing.

Mitotic sister-chromatid cohesion (SCC) is known to depend in part on conserved proteins called adherins, which although necessary for SCC are not themselves localized between sister chromatids. We have examined mitotic DNA-repair and meiotic chromosome behavior in the Coprinus cinereus adherin mutant rad9-1. Genetic pathway analysis established that Rad9 functions in an Mre11-dependent pathway of DNA repair. Using fluorescence in situ hybridization, we found that the rad9-1 mutant is defective in the establishment of meiotic homolog pairing at both interstitial and subtelomeric sites but in the maintenance of pairing at only interstitial loci. To determine the role of Rad9 in meiotic SCC, we hybridized nuclear spreads simultaneously with a homolog-specific probe and a probe that recognizes both members of a homologous pair. We found that Rad9 is required for wild-type levels of meiotic SCC, and that nuclei showing loss of cohesion were twice as likely also to fail at homolog pairing. To ask whether the contribution of Rad9 to homolog pairing is solely in the establishment of SCC, we examined a rad9-1;msh5-22 double mutant, in which premeiotic DNA replication is inhibited. The msh5-22 mutation partially suppressed the deleterious effects of the rad9-1 mutation on homolog pairing; however, pairing in the double mutant still was significantly lower than in the msh5-22 single mutant control. Because the role of Rad9 in homolog pairing is not obviated by the absence of a sister chromatid, we conclude that adherins have one or more early meiotic functions distinct from the establishment of cohesion.

Multiple roles of Spo11 in meiotic chromosome behavior.

Spo11, a type II topoisomerase, is likely to be required universally for initiation of meiotic recombination. However, a dichotomy exists between budding yeast and the animals Caenorhabditis elegans and Drosophila melanogaster with respect to additional roles of Spo11 in meiosis. In Saccharomyces cerevisiae, Spo11 is required for homolog pairing, as well as axial element (AE) and synaptonemal complex (SC) formation. All of these functions are Spo11 independent in C.elegans and D.melanogaster. We examined Spo11 function in a multicellular fungus, Coprinus cinereus. The C.cinereus spo11-1 mutant shows high levels of homolog pairing and occasionally forms full-length AEs, but no SC. In C.cinereus, Spo11 is also required for maintenance of meiotic chromosome condensation and proper spindle formation. Meiotic progression in spo11-1 is aberrant; late in meiosis basidia undergo programmed cell death (PCD). To our knowledge, this is the first example of meiotic PCD outside the animal kingdom. Ionizing radiation can partially rescue spo11-1 for both AE and SC formation and viable spore production, suggesting that the double-strand break function of Spo11 is conserved and is required for these functions.

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.

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.

Pathway analysis of radiation-sensitive meiotic mutants of Coprinus cinereus.

We have isolated 37 radiation-sensitive mutants of the basidiomycete Coprinus cinereus. Each mutation is recessive, and the collection defines at least ten complementation groups for survival of gamma irradiation. Four complementation groups define the genes rad3, rad9, rad11 and rad12, which are required both for survival of gamma irradiation and for meiosis. Mutants in each of these four groups fail to complete meiosis and produce mushrooms with greatly reduced numbers of viable spores. Propidium iodide staining of meiotic nuclei showed a characteristic terminal appearance for each mutant: few cells of any of the meiotic mutants progress beyond prophase I, and both condensation and fragmentation or dispersal of meiotic chromatin are frequently observed. Scanning electron micrographs showed that the meiotic mutants make varying numbers (0-6) of basidiospore initials and that few of these initials develop into mature spores. When initials are present they are always symmetrically arrayed on the basidium, regardless of initial number. In quantitative measurements of gamma ray sensitivity, double mutants of every tested combination of rad3, rad9, rad11 and rad12 consistently showed the same gamma ray sensitivity as the more sensitive single mutant parent of the cross. Therefore, these four genes are in the same pathway for the repair of gamma radiation damage, and this pathway also represents one or more functions essential for meiosis.

Signal transduction in Coprinus congregatus: evidence for the involvement of G proteins in blue light photomorphogenesis.

This paper reports the presence of several G proteins and light-sensitive GTP-binding proteins in the fungus Coprinus congregatus, a filamentous eukaryote. (Mono)ADP-ribosylation experiments with crude membranes in the presence of the (poly)ADP-ribosyltransferase inhibitor, 3-amino-benzamide, resulted in the detection of a cholera toxin substrate of 52 kDa and two pertussis toxin substrates, 33 and 39 kDa. Two-dimensional polyacrylamide gel analysis of GTP-binding proteins exposed in vivo to [35S]-labeled guanosine 5'-[gamma-thio]-triphosphate in the presence or absence of light demonstrated light enhanced analog binding. These results support the concept of the involvement of G proteins in phototransduction in C. congregatus.

Genetic characterization of the new morphological and UV-sensitive mutants in Coprinus cinereus. I. A UV-sensitive mutation rad 1 associated with elevated frequencies of mitotic and meiotic recombination.

Studies on the effect of an UV-sensitive mutation, rad 1, in meiotic and mitotic recombination in Coprinus indicated that, in homozygous condition, rad 1 increased the spontaneous meiotic recombination by 50% and UV-induced mitotic intergenic recombination by about 5-fold. The homozygous rad 1 diploid was shown to be much more sensitive to the recombinogenic effects of polyfunctional than than of mono- or non-functional alkylating agents.

Mutational synergism between p-fluorophenylalanine and UV in Coprinus lagopus.

Previous studies have shown that the amino acid analogue p-fluorophenylalanine (PFP) is mutagenic to Coprinus lagopus due to its incorporation into proteins [32]. Spontaneous mutations, PFP and UV mutagenesis and PFP/UV synergism have been studied in a UV resistant strain and in two complementing UV sensitive mutant strains. By comparison to the UV resistant strain, one UV sensitive strain shows normal spontaneous mutations, 1.4% PFP-induced mutations and 50-fold UV mutagenesis. The second UV sensitive strain has 19-fold spontaneous mutation frequency, 8% PFP induced mutations and slightly elevated UV mutagenesis. In all 3 strains the PFP/UV synergism is comparable (4--5 times the arithmetic expected). The results indicate that PFP mutagenesis is due to the incorporation of PFP into enzymes normally functioning in the organism but which also participate in UV repair mechanisms. A model is proposed for UV repair which is based on a PFP sensitive excision repair system of at least two enzymes, and alternative "error proof" pathway which is not suscetible to PFP and an "error prone" pathway which is responsible for UV mutagenesis and is susceptible to PFP as shown by the PFP/UV synergism. Because PFP is given before UV treatment, this implies a UV inducible cofactor and a PFP sensitive enzyme which only functions after UV activation.