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The ink cap Coprinopsis cinerea is a model organism for studying fruiting body (mushroom) formation in homobasidiomycetes. Mutant screens and expression studies have implicated a number of genes in this developmental process. Functional analysis of these genes, however, is hampered by the lack of reliable reverse genetics tools for C. cinerea. Here, we report the applicability of gene targeting by RNA silencing for this organism. Efficient silencing of both an introduced GFP expression cassette and the endogenous cgl1 and cgl2 isogenes was achieved by expression of homologous hairpin RNAs. In latter case, silencing was the result of a hairpin construct containing solely cgl2 sequences, demonstrating the possibility of simultaneous silencing of whole gene families by a single construct. Expression of the hairpin RNAs reduced the mRNA levels of the target genes by at least 90%, as determined by quantitative real-time PCR. The reduced mRNA levels were accompanied by cytosine methylation of transcribed and nontranscribed DNA at both silencing and target loci in the case of constitutive high-level expression of the hairpin RNA but not in the case of transient expression. These results suggest the presence of both posttranscriptional and transcriptional gene silencing mechanisms in C. cinerea and demonstrate the applicability of targeted gene silencing as a powerful reverse genetics approach in this organism.The ink cap Coprinopsis cinerea is a model organism for studying fruiting body (mushroom) formation in homobasidiomycetes (reviewed in references 32 and 35). Mutant screens and expression studies have implicated a number of genes in this developmental process. An example for the latter case are the cgl1 and cgl2 isogenes, which code for two isogalectins that are highly induced during fruiting body formation (5). In addition, orthologues of genes involved in fruiting body formation in other fungi are revealed by the sequence of the C. cinerea genome (http://www.broad.mit.edu/annotation/fungi /coprinus_cinereus) and its annotation, which is in progress (http://fungal.genome.duke.edu/cgi-bin/gbrowse/ccin/). Functional analysis of C. cinerea genes, however, is hampered by the lack of reliable tools for gene targeting. Although homologous recombination seems to occur in C. cinerea (3), targeted gene knockouts appear difficult to achieve, possibly due to very efficient nonhomologous DNA end joining, as has been shown for the filamentous ascomycete Neurospora crassa (47).Recently, RNA-induced gene silencing (RNA silencing) has been emerging as a powerful tool for gene targeting in fungi, plants, and animals (reviewed in references 7, 11, and 13). This strategy exploits an endogenous gene regulatory mechanism of eukaryotic cells in which regulatory double-stranded RNAs (dsRNAs) interfere with homologous mRNA either by triggering its degradation or inhibiting its transcription or translation (see references 1 and 42 for recent reviews and specific references therein). For gene targeting, dsRNA homologous to the target gene...

Background:The enzyme generating free oligosaccharides (fOSs) in the lumen of the endoplasmic reticulum (ER) has been unidentified. Results: Oligosaccharyltransferase (OST), the N-glycosylating enzyme, hydrolyzes dolichol-linked oligosaccharides to release the fOSs. Conclusion: OST is responsible for the generation of fOSs in the ER lumen. Significance: This study provides a mechanistic insight into the formation of luminal fOSs in yeast.

Coprinopsis cinerea is a model organism for fruiting body development in homobasidiomycetes. Here, we focused on Nlinked oligosaccharides (NLO) of cell wall proteins in the hyphae of two developmental stages, vegetative mycelium and fruiting body. High mannose-type glycans were the most commonly found structures. In addition, we observed a novel glycan, predominantly present in fruiting body. This oligosaccharide structure was of the high mannose type with at least five mannoses and a bisecting ␣1-4 N-acetylglucosamine (GlcNAc) at the ␤-mannose of the N-glycan core. The transferase responsible for this modification, CcGnt1 (C. cinerea GlcNAc transferase 1), was identified and expressed in insect cells. In vitro activity of CcGnt1 was demonstrated. This novel glycosyltransferase belongs to the glycosyltransferase family 8 (GT8) and is predicted to be a type II membrane protein. Expression of the CcGnt1 locus was up-regulated in fruiting body, but down-regulation of expression by means of RNAi decreased the level of bisected NLO; however had no apparent effect on fruiting body formation.

The pathogenic fungus Cryptococcus neoformans synthesizes a complex family of glycosylinositolphosphoceramide (GIPC) structures. These glycosphingolipids (GSLs) consist of mannosylinositolphosphoceramide (MIPC) extended by β1-6-linked galactose, a unique structure that has to date only been identified in basidiomycetes. Further extension by up to five mannose residues and a branching xylose has been described. In this study, we identified and determined the gene structure of the enzyme Ggt1, which catalyzes the transfer of a galactose residue to MIPC. Deletion of the gene in C. neoformans resulted in complete loss of GIPCs containing galactose, a phenotype that could be restored by the episomal expression of Ggt1 in the deletion mutant. The entire annotated open reading frame, encoding a C-terminal GT31 galactosyltransferase domain and a large N-terminal domain of unknown function, was required for complementation. Notably, this gene does not encode a predicted signal sequence or transmembrane domain. The demonstration that Ggt1 is responsible for the transfer of a galactose residue to a GSL thus raises questions regarding the topology of this biosynthetic pathway and the function of the N-terminal domain. Phylogenetic analysis of the GGT1 gene shows conservation in hetero- and homobasidiomycetes but no homologs in ascomycetes or outside of the fungal kingdom.