The agaricomycete (homobasidiomycete) Coprinopsis cinerea has been used as a model to study the molecular mechanism for photomorphogenesis. Molecular genetic analyses of mutants defective in fruiting ...body (mushroom) photomorphogenesis of C. cinerea identified two genes, dst1 and dst2. dst1 encodes a homolog of WC-1, a fungal blue-light photoreceptor first identified in Neurospora crassa, while dst2 encodes a novel protein with a putative flavin adenine dinucleotide (FAD)-binding-4 domain. In addition, reverse genetic analysis revealed that disruption of a C. cinerea gene encoding a WC-2 homolog, the partner of WC-1, causes the same blind phenotype. Searches on the genome data show that both WC-1 and WC-2 homologs are present in some agaricomycetes other than C. cinerea. Furthermore, in an agaricomycete, Lentinula edodes, it has been shown in vitro that the WC-1 and WC-2 homologs interact with each other. These findings suggest that the presumptive mechanism for blue-light sensing in agaricomycetes is fundamentally similar to that in Neurospora crassa, in which the WC-1/WC-2 complex plays a central role. Since the WC-1/WC-2 complex operates as a photoreceptor and a transcription factor, future studies will include identification of the targets of the WC-1/WC-2 complex that regulate photomorphogenesis in agaricomycetes. Another future challenge will be elucidation of the role of the newly identified photomorphogenetic protein, Dst2, in the blue-light-sensing mechanism.
Agaricomycetes exhibit a remarkable morphological differentiation from vegetative mycelia to huge fruiting bodies. To investigate the molecular mechanism underlying the fruiting body development, we ...have isolated and characterized many
Coprinopsis cinerea
mutant strains defective in fruiting initiation to date. Dikaryon formation in agaricomycetes, which is followed by fruiting development, is governed by the mating type loci,
A
and
B
. Recently, mutations in the
Cc.snf5
gene, which encodes a putative component of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF), were shown to cause defects in
A
-regulated clamp cell morphogenesis, as well as in fruiting initiation. Here, we demonstrate that
Cc.arp9
, which encodes a putative actin-related protein associated with two chromatin remodeling complexes, SWI/SNF and remodels the structure of chromatin (RSC), is also essential for fruiting initiation. In contrast to
Cc.snf5
mutants,
Cc.arp9
mutants were not defective in clamp cell formation. The effects of mutations in
Cc.arp9
and
Cc.snf5
on oidia production and the transcriptional expression levels of
clp1
and
pcc1
, which are under the control of the
A
gene, were also examined. These indicated that Cc.Snf5 is involved in
A
-regulated pathways, whereas Cc.Arp9 is not apparently
. Cc.arp9
/
Cc.snf5
double-gene disruptants were generated and their phenotypes were analyzed, which suggested a complicated developmental regulation mechanism mediated by chromatin remodeling.
► We identified a Coprinopsis cinerea mutant defective in fruiting. ► The gene responsible for the mutation encodes a Snf5 protein named Cc.Snf5. ► Cc.Snf5 contains a GATA Zn-finger domain. ► Cc.Snf5 ...is a component of the SWI/SNF chromatin remodeling complex. ► Different targeted disruptions of Cc.Snf5 affect sexual development differently.
We characterized a Coprinopsis cinerea mutant strain, Spe20, defective in fruiting initiation, which was isolated after restriction enzyme-mediated integration (REMI) mutagenesis of a homokaryotic fruiting strain, 326. A plasmid rescue followed by complementation experiments, RACE, and cDNA analyses revealed that the gene, a mutation of which is responsible for the phenotype, is predicted to encode a protein that exhibits a high similarity to yeast Snf5p, a key component of the chromatin remodeling complex SWI/SNF, and named Cc.snf5. Cc.Snf5 is, however, different from Snf5p in that the former has, in addition to an Snf5 domain comprising N-terminal repeat1 (rp1) and C-terminal repeat2 (rp2) subdomains in a middle region, a GATA Zn-finger domain in a C-terminal region. In strain Spe20, plasmid pPHT1 used for REMI is inserted in the ORF encoding rp2. This raised the possibility that in strain Spe20, the disrupted Cc.Snf5 is functionally active albeit incompletely because it retains rp1. Thus, we disrupted the whole SNF5 domain and its downstream peptide and found that the disruption results in inhibition of not only fruiting initiation but also dikaryon development, a prerequisite for fruiting. We also found that specific disruption of the Zn-finger domain results in inhibition of fruiting initiation. These results indicate that Cc.Snf5 plays an essential role in sexual development of C. cinerea.
The formation and proliferation of the dikaryon in the agaricomycete
Coprinopsis cinerea is controlled by the mating type genes,
A and
B. The
B genes, which encode pheromones and pheromone receptors, ...control nuclear migration for dikaryosis as well as the fusion of the clamp cell with the subterminal cell while the
A genes, which encode two classes of homeodomain proteins, control conjugate nuclear division associated with clamp connection development. We characterized the mutant, B28, which was newly isolated as a strain that fails to form a primary hyphal knot, the first visible sign toward fruiting, from a homokaryotic fruiting strain after REMI mutagenesis. Detailed phenotypic analysis revealed that strain B28 exhibits, in addition to the fruiting defect, a defect in
A-regulated clamp cell morphogenesis as well as a defect in
B-regulated nuclear migration for dikaryosis. The mutant clamp cells are unique in that they continue growing like branches without fusing with the subterminal cells, in contrast to the unfused pseudoclamp which are normally formed in
A-on
B-off strains, providing evidence for the existence of an as yet unidentified mechanism for the growth suppression of the clamp cell. Molecular analysis revealed that the gene responsible for the phenotypes, designated
Cc.ubc2, encodes a protein similar to Ubc2, an adaptor protein for filamentous growth, pheromone response and virulence in the smut fungus
Ustilago maydis. In addition, western blot analysis demonstrated that the
Cc.ubc2-1 mutation blocks phosphorylation of a presumptive MAP kinase.
We characterized two developmental mutants of Coprinopsis cinerea, Apa56 and Sac29, newly isolated from a homokaryotic fruiting strain, 326 (Amut Bmut pab1-1), after restriction enzyme-mediated ...integration (REMI) mutagenesis. Both Apa56 and Sac29 exhibited slower mycelial growth than the parental wild-type strain and failed to initiate fruiting when grown on standard malt extract-yeast extract-glucose medium under 12 h light/12 h dark cycle. Both mutants exhibited unusual differentiation in aerial hyphae: differentiated hyphae lacked clamp connections and exhibited irregular shapes. The differentiated hyphae were similar to the component cells of hyphal knots, but did not form hyphal knots: they spread as dense mycelial mats. When the carbon source (glucose) in the medium was substituted with sucrose or galactose, both strains formed as many hyphal knots as the parental wild type. The hyphal knots formed, however, did not develop into fruiting-body initials, but developed into sclerotia. Molecular genetic analysis revealed that the gene, designated Cc.rmt1, is disrupted by REMI mutagenesis and is responsible for the phenotypes in both mutants. Cc.rmt1 is predicted to encode a putative protein arginine methyltransferase, some homologs of which have been shown to be involved in the regulation of gene expression in eukaryotes.
The homobasidiomycete
Coprinopsis cinerea is a member of the fungi known as inky cap mushrooms, and its fruiting-body pileus autolyzes soon after completion of the development. During the last 3
h of ...the development, the pileus exhibits umbrella-like expansion: the pileal tissue is cracked at the base of each gill and then each gill tissue is split to form a V-shape, as seen in a cross section. We identified two
C. cinerea mutants defective in both pileus expansion and autolysis. The defects in both mutants are due to recessive mutations in a single gene, designated
exp1. The
exp1 gene is predicted to encode an HMG1/2-like protein with two HMG domains. The transcription of
exp1 is strongly induced in the pileus 3
h before pileus expansion. This result, together with the fact that the
exp1 mutations cause a specific developmental phenotype, suggest that Exp1 is a novel, transcriptional regulator controlling the final phase of fruiting-body morphogenesis.
► We disrupted ku70 and lig4 homologs in a model mushroom, Coprinopsis cinerea. ► The disruption of Cc.ku70 or Cc.lig4 greatly enhanced gene targeting. ► We verified by gene targeting that Cc.wc-2 is ...involved in perception of blue light.
Coprinopsis cinerea is a model for studies of sexual development in agaricomycetes (homobasidiomycetes). Efficient gene targeting should facilitate such studies, especially because increasing genome and transcriptome information is now available in C. cinerea. To estimate the frequency of gene disruption by homologous integration in this fungus, we tried to disrupt Cc.wc-2, which encodes a WC-2 homolog, a partner of the fungal blue-light photoreceptor, WC-1. Disruption of Cc.wc-2 did not occur when recipients (protoplasts) of the disrupting construct were prepared from asexual spores, oidia, from the wild type, 326, while it occurred when protoplasts were prepared from mycelial cells from the same strain, albeit at a low frequency (3%). Double-stranded RNA-mediated silencing of a ku70 homolog, named Cc.ku70, or the lig4 homolog Cc.lig4 more or less increased the frequency of Cc.wc-2 targeting. On the basis of these results, we disrupted Cc.ku70 using a Cc.lig4-silenced strain. We then disrupted Cc.lig4 using the Cc.ku70 disruptant. We found that the disruption of Cc.ku70 or Cc.lig4 greatly enhanced gene targeting. In addition, this study demonstrates that Cc.wc-2 is involved in blue light perception in this fungus.
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•C. cinerea hyphal knots formed simultaneously by light in glucose limited condition.•The fas1, cfs1, cfs2 and nod1 are highly transcribed at 1 h after light exposure.•Galectins, ...pheromone, laccase are upregulated 10 h after blue light exposure.•Glucose content affect expressions of those genes after blue light exposure.•Blue light would accelerate hyphal knot induction under nutrient limited condition.
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 fruiting-body primordium of Coprinopsis cinerea exhibits remarkable photomorphogenesis. Under a 12-h light/12-h dark regime, the primordium proceeds to the fruiting-body maturation phase in which ...the primordium successively undergoes basidiospore formation, stipe elongation and pileus expansion, resulting in the mature fruiting-body. In continuous darkness, however, the primordium never proceeds to the maturation phase: the pileus and stipe tissues at the upper part of the primordium remain rudimentary while the basal part of the primordium elongates, producing the etiolated “dark stipe” phenotype. In our previous studies, blind mutants, which produce dark stipes under light conditions that promote fruiting-body maturation in the wild-type, have been isolated, and two genes, dst1 and dst2, responsible for the mutant phenotype have been identified. In this study we show that the dst2-1 mutant exhibits a blind phenotype during asexual spore production in addition to that in fruiting-body photomorphogenesis. We also reveal that dst2 is predicted to encode a protein with a putative flavin adenine dinucleotide (FAD)-binding-4 domain. The two blind phenotypes, together with the existence of an FAD-binding domain in Dst2, suggest that Dst2 may play a role in perceiving blue light.
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