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Combining laser microdissection and RNA-seq to chart the transcriptional landscape of fungal development.

Teichert I, Wolff G, Kück U, Nowrousian M - BMC Genomics (2012)

Bottom Line: Fruiting bodies contain a number of cell types not found in vegetative mycelium, and these morphological differences are thought to be mediated by changes in gene expression.Our data revealed significant differences in gene expression between protoperithecia and non-reproductive mycelia, and showed that the transcription factor PRO1 is involved in the regulation of many genes expressed specifically in sexual structures.The LM/RNA-seq approach will also be relevant to other eukaryotic systems in which multicellular development is investigated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Bochum, Germany. ulrich.kueck@rub.de

ABSTRACT

Background: During sexual development, filamentous ascomycetes form complex, three-dimensional fruiting bodies for the protection and dispersal of sexual spores. Fruiting bodies contain a number of cell types not found in vegetative mycelium, and these morphological differences are thought to be mediated by changes in gene expression. However, little is known about the spatial distribution of gene expression in fungal development. Here, we used laser microdissection (LM) and RNA-seq to determine gene expression patterns in young fruiting bodies (protoperithecia) and non-reproductive mycelia of the ascomycete Sordaria macrospora.

Results: Quantitative analysis showed major differences in the gene expression patterns between protoperithecia and total mycelium. Among the genes strongly up-regulated in protoperithecia were the pheromone precursor genes ppg1 and ppg2. The up-regulation was confirmed by fluorescence microscopy of egfp expression under the control of ppg1 regulatory sequences. RNA-seq analysis of protoperithecia from the sterile mutant pro1 showed that many genes that are differentially regulated in these structures are under the genetic control of transcription factor PRO1.

Conclusions: We have generated transcriptional profiles of young fungal sexual structures using a combination of LM and RNA-seq. This allowed a high spatial resolution and sensitivity, and yielded a detailed picture of gene expression during development. Our data revealed significant differences in gene expression between protoperithecia and non-reproductive mycelia, and showed that the transcription factor PRO1 is involved in the regulation of many genes expressed specifically in sexual structures. The LM/RNA-seq approach will also be relevant to other eukaryotic systems in which multicellular development is investigated.

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Expression of putative sugartransporter genes. Eighty genes contained at least on Sugar_tr domain. Hierarchical clustering and heatmap generation of the log2 of fold ratios as determined by classic (C) and LOX (L) analysis were perfomed in R.
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Figure 3: Expression of putative sugartransporter genes. Eighty genes contained at least on Sugar_tr domain. Hierarchical clustering and heatmap generation of the log2 of fold ratios as determined by classic (C) and LOX (L) analysis were perfomed in R.

Mentions: Another hypothesis about fruiting body formation in filamentous fungi assumes that the non-reproductive mycelium first gathers nutrients until a stage of “competence” is reached when the production of fruiting bodies is energetically feasible, and that then the developing fruiting bodies are nurtured by the surrounding non-reproductive mycelium [3,46]. One might speculate that this process could require the transport of massive amounts of nutrients, including carbohydrates. Thus, we analyzed whether the expression of putative sugar transporters changed in the different samples (Figure 3). Of the 80 genes in the S. macrospora genome that contain at least on sugar transporter domain, more than 40% were significantly up- or down-regulated in at least one of the conditions investigated, thereby supporting the hypothesis that fruiting body morphogenesis is accompanied by a massive redistribution of nutrients.


Combining laser microdissection and RNA-seq to chart the transcriptional landscape of fungal development.

Teichert I, Wolff G, Kück U, Nowrousian M - BMC Genomics (2012)

Expression of putative sugartransporter genes. Eighty genes contained at least on Sugar_tr domain. Hierarchical clustering and heatmap generation of the log2 of fold ratios as determined by classic (C) and LOX (L) analysis were perfomed in R.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472292&req=5

Figure 3: Expression of putative sugartransporter genes. Eighty genes contained at least on Sugar_tr domain. Hierarchical clustering and heatmap generation of the log2 of fold ratios as determined by classic (C) and LOX (L) analysis were perfomed in R.
Mentions: Another hypothesis about fruiting body formation in filamentous fungi assumes that the non-reproductive mycelium first gathers nutrients until a stage of “competence” is reached when the production of fruiting bodies is energetically feasible, and that then the developing fruiting bodies are nurtured by the surrounding non-reproductive mycelium [3,46]. One might speculate that this process could require the transport of massive amounts of nutrients, including carbohydrates. Thus, we analyzed whether the expression of putative sugar transporters changed in the different samples (Figure 3). Of the 80 genes in the S. macrospora genome that contain at least on sugar transporter domain, more than 40% were significantly up- or down-regulated in at least one of the conditions investigated, thereby supporting the hypothesis that fruiting body morphogenesis is accompanied by a massive redistribution of nutrients.

Bottom Line: Fruiting bodies contain a number of cell types not found in vegetative mycelium, and these morphological differences are thought to be mediated by changes in gene expression.Our data revealed significant differences in gene expression between protoperithecia and non-reproductive mycelia, and showed that the transcription factor PRO1 is involved in the regulation of many genes expressed specifically in sexual structures.The LM/RNA-seq approach will also be relevant to other eukaryotic systems in which multicellular development is investigated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Bochum, Germany. ulrich.kueck@rub.de

ABSTRACT

Background: During sexual development, filamentous ascomycetes form complex, three-dimensional fruiting bodies for the protection and dispersal of sexual spores. Fruiting bodies contain a number of cell types not found in vegetative mycelium, and these morphological differences are thought to be mediated by changes in gene expression. However, little is known about the spatial distribution of gene expression in fungal development. Here, we used laser microdissection (LM) and RNA-seq to determine gene expression patterns in young fruiting bodies (protoperithecia) and non-reproductive mycelia of the ascomycete Sordaria macrospora.

Results: Quantitative analysis showed major differences in the gene expression patterns between protoperithecia and total mycelium. Among the genes strongly up-regulated in protoperithecia were the pheromone precursor genes ppg1 and ppg2. The up-regulation was confirmed by fluorescence microscopy of egfp expression under the control of ppg1 regulatory sequences. RNA-seq analysis of protoperithecia from the sterile mutant pro1 showed that many genes that are differentially regulated in these structures are under the genetic control of transcription factor PRO1.

Conclusions: We have generated transcriptional profiles of young fungal sexual structures using a combination of LM and RNA-seq. This allowed a high spatial resolution and sensitivity, and yielded a detailed picture of gene expression during development. Our data revealed significant differences in gene expression between protoperithecia and non-reproductive mycelia, and showed that the transcription factor PRO1 is involved in the regulation of many genes expressed specifically in sexual structures. The LM/RNA-seq approach will also be relevant to other eukaryotic systems in which multicellular development is investigated.

Show MeSH
Related in: MedlinePlus