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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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Genes that are differentially regulated during development and dependent on pro1for correct expression. Hierarchical clustering of the log2 of fold ratios as determined by classic (C) and LOX (L) analysis. Log2 ratios < −10 or > 10 were set to −10 and 10, respectively, for better scaling visibility. Hierarchical clustering and heatmap generation were performed in R.
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Figure 5: Genes that are differentially regulated during development and dependent on pro1for correct expression. Hierarchical clustering of the log2 of fold ratios as determined by classic (C) and LOX (L) analysis. Log2 ratios < −10 or > 10 were set to −10 and 10, respectively, for better scaling visibility. Hierarchical clustering and heatmap generation were performed in R.

Mentions: To determine which genes are directly or indirectly under the control of transcription factor PRO1 in developing protoperithecia, we looked in more detail at genes that are differentially regulated in wild-type protoperithecia compared to sexual mycelium and are also differentially regulated in pro1 protoperithecia compared to wild-type protoperithecia (Figure 5). This group contains a total of 423 genes, the majority of which are either upregulated in wild-type protoperithecia compared to sexual mycelium and downregulated in pro1 protoperithecia (115 genes) or the other way round (226 genes). For these genes, pro1 acts as an activator or repressor, respectively, during fruiting body formation. Only eight genes were up-regulated in wild-type protoperithecia compared to sexual mycelium and also up-regulated in pro1 protoperithecia compared to wild-type protoperithecia. Interestingly, six of these eight genes encode proteins that are predicted to be extracellular, including the pheromone genes ppg1 and ppg2 (Table 4). We already identified ppg1 and ppg2 as being up-regulated in sexual mycelium of mutant pro1 compared to the wild-type in a previous microarray analysis [10]; however, the spatial dimension of this differential expression was not yet known, and the combination of LM and RNA-seq now shows that protoperithecia-specific pheromone gene expression is regulated by the transcription factor gene pro1. Two other genes that are up-regulated in both comparisons are homologous to loosenin from the basidiomycete Bjerkandera adusta and fasciclin-like protein MoFLP1 from Magnaporthe grisea[48,49]. Both proteins have been implicated in cell-wall biogenesis/reorganization, and it is tempting to speculate that the corresponding S. macrospora proteins are involved in shaping the outer layers (perithecial wall) of the developing perithecium.


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)

Genes that are differentially regulated during development and dependent on pro1for correct expression. Hierarchical clustering of the log2 of fold ratios as determined by classic (C) and LOX (L) analysis. Log2 ratios < −10 or > 10 were set to −10 and 10, respectively, for better scaling visibility. Hierarchical clustering and heatmap generation were performed in R.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Genes that are differentially regulated during development and dependent on pro1for correct expression. Hierarchical clustering of the log2 of fold ratios as determined by classic (C) and LOX (L) analysis. Log2 ratios < −10 or > 10 were set to −10 and 10, respectively, for better scaling visibility. Hierarchical clustering and heatmap generation were performed in R.
Mentions: To determine which genes are directly or indirectly under the control of transcription factor PRO1 in developing protoperithecia, we looked in more detail at genes that are differentially regulated in wild-type protoperithecia compared to sexual mycelium and are also differentially regulated in pro1 protoperithecia compared to wild-type protoperithecia (Figure 5). This group contains a total of 423 genes, the majority of which are either upregulated in wild-type protoperithecia compared to sexual mycelium and downregulated in pro1 protoperithecia (115 genes) or the other way round (226 genes). For these genes, pro1 acts as an activator or repressor, respectively, during fruiting body formation. Only eight genes were up-regulated in wild-type protoperithecia compared to sexual mycelium and also up-regulated in pro1 protoperithecia compared to wild-type protoperithecia. Interestingly, six of these eight genes encode proteins that are predicted to be extracellular, including the pheromone genes ppg1 and ppg2 (Table 4). We already identified ppg1 and ppg2 as being up-regulated in sexual mycelium of mutant pro1 compared to the wild-type in a previous microarray analysis [10]; however, the spatial dimension of this differential expression was not yet known, and the combination of LM and RNA-seq now shows that protoperithecia-specific pheromone gene expression is regulated by the transcription factor gene pro1. Two other genes that are up-regulated in both comparisons are homologous to loosenin from the basidiomycete Bjerkandera adusta and fasciclin-like protein MoFLP1 from Magnaporthe grisea[48,49]. Both proteins have been implicated in cell-wall biogenesis/reorganization, and it is tempting to speculate that the corresponding S. macrospora proteins are involved in shaping the outer layers (perithecial wall) of the developing perithecium.

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