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Identification, characterization, and biosynthesis of a novel N-glycan modification in the fruiting body of the basidiomycete Coprinopsis cinerea.

ABSTRACT: Coprinopsis cinerea is a model organism for fruiting body development in homobasidiomycetes. Here, we focused on N-linked 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 alpha1-4 N-acetylglucosamine (GlcNAc) at the beta-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.

SUBMITTER: Buser R

PROVIDER: S-EPMC2856279 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Identification, characterization, and biosynthesis of a novel N-glycan modification in the fruiting body of the basidiomycete Coprinopsis cinerea.

Buser Reto R Lazar Zbigniew Z Käser Sonja S Künzler Markus M Aebi Markus M

The Journal of biological chemistry 20100108 14

Coprinopsis cinerea is a model organism for fruiting body development in homobasidiomycetes. Here, we focused on N-linked 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 ...[more]

PMID: 20061575

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Project description:Coprinopsis cinerea is a model mushroom particularly suited for the study of fungal fruiting body development and the evolution of multicellularity in fungi. While microRNAs (miRNAs) have been extensively studied in animals and plants for their essential roles in post-transcriptional regulation of gene expression, miRNAs in fungi are less well characterized and their potential roles in controlling mushroom development remain unknown. To identify miRNA-like RNAs (milRNAs) in C. cinerea and explore their expression patterns during the early developmental transition of mushroom development, small RNA libraries of vegetative mycelium and primordium were generated and putative milRNA candidates were identified following the standards of miRNA prediction in animals and plants. Two out of 22 novel predicted milRNAs, cci-milR-12c and cci-milR-13e-5p, were validated by northern blot and stem-loop reverse transcription real-time PCR. Cci-milR-12c was differentially expressed whereas the expression levels of cci-milR-13e-5p were similar in the two developmental stages. Target prediction of the validated milRNAs resulted in genes associated with fruiting body development, including pheromone, hydrophobin, cytochrome P450, and protein kinase. Essential genes for miRNA biogenesis, including three coding for Dicer-like (DCL), one for Argonaute (AGO), one for AGO-like and one for quelling deficient-2 (QDE-2) proteins, were also identified in the C. cinerea genome. Phylogenetic analysis showed that the DCL and AGO proteins of C. cinerea were more closely related to those in other basidiomycetes and ascomycetes than to those in animals and plants. Taken together, our findings provided the first evidence for milRNAs in the model mushroom and their potential roles in regulating fruiting body development. New information on the evolutionary relationship of milRNA biogenesis proteins across kingdoms has also provided new insights for guiding further functional and evolutionary studies of miRNAs.

Project description:BackgroundThe transition from the vegetative mycelium to the primordium during fruiting body development is the most complex and critical developmental event in the life cycle of many basidiomycete fungi. Understanding the molecular mechanisms underlying this process has long been a goal of research on basidiomycetes. Large scale assessment of the expressed transcriptomes of these developmental stages will facilitate the generation of a more comprehensive picture of the mushroom fruiting process. In this study, we coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development.ResultsWe evaluated the expression of >3,000 genes in the two respective growth stages and discovered that almost one-third of these genes were preferentially expressed in either stage. This identified a significant turnover of the transcriptome during the course of fruiting body development. Additionally, we annotated more than 79,000 transcription start sites (TSSs) based on the transcriptomes of the mycelium and stage 1 primoridum stages. Patterns of enrichment based on gene annotations from the GO and KEGG databases indicated that various structural and functional protein families were uniquely employed in either stage and that during primordial growth, cellular metabolism is highly up-regulated. Various signaling pathways such as the cAMP-PKA, MAPK and TOR pathways were also identified as up-regulated, consistent with the model that sensing of nutrient levels and the environment are important in this developmental transition. More than 100 up-regulated genes were also found to be unique to mushroom forming basidiomycetes, highlighting the novelty of fruiting body development in the fungal kingdom.ConclusionsWe implicated a wealth of new candidate genes important to early stages of mushroom fruiting development, though their precise molecular functions and biological roles are not yet fully known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea.

Dataset Information

Identification, characterization, and biosynthesis of a novel N-glycan modification in the fruiting body of the basidiomycete Coprinopsis cinerea.

Publications

Identification, characterization, and biosynthesis of a novel N-glycan modification in the fruiting body of the basidiomycete Coprinopsis cinerea.

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