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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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Microscopic analysis of egfpexpression under the control of ppg1 regulatory regions. Plasmids pDS23 and pSNPTppg1 were transformed into the S. macrospora wild-type. Strain S106352 expresses egfp under the control of the Aspergillus nidulans gpd promoter and trpC terminator from pDS23 (black). Strains T71.1S29 and T75.10xfusS44 express egfp under the control of the S. macrospora ppg1 promoter and terminator regions from pSNPTppg1. Note that EGFP fluorescence under the control of ppg1 regulatory regions in protoperithecia was observed through a 6% neutral density filter because of very strong fluorescence, which is consistent with high ppg1 expression in these structures. GFP images are z projections of stacks spanning whole protoperithecia with a plane distance of 0.5 μm. DIC images show the middle plane of the corresponding stack. Scale bar, 10 μm.
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Figure 4: Microscopic analysis of egfpexpression under the control of ppg1 regulatory regions. Plasmids pDS23 and pSNPTppg1 were transformed into the S. macrospora wild-type. Strain S106352 expresses egfp under the control of the Aspergillus nidulans gpd promoter and trpC terminator from pDS23 (black). Strains T71.1S29 and T75.10xfusS44 express egfp under the control of the S. macrospora ppg1 promoter and terminator regions from pSNPTppg1. Note that EGFP fluorescence under the control of ppg1 regulatory regions in protoperithecia was observed through a 6% neutral density filter because of very strong fluorescence, which is consistent with high ppg1 expression in these structures. GFP images are z projections of stacks spanning whole protoperithecia with a plane distance of 0.5 μm. DIC images show the middle plane of the corresponding stack. Scale bar, 10 μm.

Mentions: We also analyzed if genes that were previously shown to be essential for perithecial development in S. macrospora or significantly upregulated during fruiting body formation are differentially regulated in protoperithecia compared to total mycelium ( Additional file 1 Figure S8). Interestingly, we found that both pheromone precursor genes ppg1 and ppg2 are strongly upregulated in protoperithecia compared to sexual or vegetative mycelium. The pheromones are required for full fertility [47]; however, where or when they act during the developmental cycle is not yet clear because S. macrospora is self-fertile (homothallic), and no obvious fertilization event that requires recognition of compatible partners by pheromones is necessary [27]. To address this question in more detail, we analyzed the expression of an egfp reporter gene under the control of the ppg1 upstream and downstream regulatory regions (Figure 4). No expression was observed in vegetative hyphae, in contrast to the expression of egfp from a control vector under the constitutive gpd promoter and trpC terminator from A. nidulans. EGFP fluorescence started to occur in ascogonia (female gametangia) and was strongest in young protoperithecia (diameter ≤ 30 μm). Interestingly, older protoperithecia (> 30 μm) exhibited a distinctly patchy expression pattern in the hyphae of the outer layers, whereas expression of the control vector led to a uniform fluorescence of protoperithecia. On the one hand, these data confirm the transcriptional up-regulation of ppg1 in protoperithecia as indicated by the RNA-seq analysis; and on the other hand, the microscopic analysis revealed a distinct expression pattern of ppg1 within protoperithecia.


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)

Microscopic analysis of egfpexpression under the control of ppg1 regulatory regions. Plasmids pDS23 and pSNPTppg1 were transformed into the S. macrospora wild-type. Strain S106352 expresses egfp under the control of the Aspergillus nidulans gpd promoter and trpC terminator from pDS23 (black). Strains T71.1S29 and T75.10xfusS44 express egfp under the control of the S. macrospora ppg1 promoter and terminator regions from pSNPTppg1. Note that EGFP fluorescence under the control of ppg1 regulatory regions in protoperithecia was observed through a 6% neutral density filter because of very strong fluorescence, which is consistent with high ppg1 expression in these structures. GFP images are z projections of stacks spanning whole protoperithecia with a plane distance of 0.5 μm. DIC images show the middle plane of the corresponding stack. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Microscopic analysis of egfpexpression under the control of ppg1 regulatory regions. Plasmids pDS23 and pSNPTppg1 were transformed into the S. macrospora wild-type. Strain S106352 expresses egfp under the control of the Aspergillus nidulans gpd promoter and trpC terminator from pDS23 (black). Strains T71.1S29 and T75.10xfusS44 express egfp under the control of the S. macrospora ppg1 promoter and terminator regions from pSNPTppg1. Note that EGFP fluorescence under the control of ppg1 regulatory regions in protoperithecia was observed through a 6% neutral density filter because of very strong fluorescence, which is consistent with high ppg1 expression in these structures. GFP images are z projections of stacks spanning whole protoperithecia with a plane distance of 0.5 μm. DIC images show the middle plane of the corresponding stack. Scale bar, 10 μm.
Mentions: We also analyzed if genes that were previously shown to be essential for perithecial development in S. macrospora or significantly upregulated during fruiting body formation are differentially regulated in protoperithecia compared to total mycelium ( Additional file 1 Figure S8). Interestingly, we found that both pheromone precursor genes ppg1 and ppg2 are strongly upregulated in protoperithecia compared to sexual or vegetative mycelium. The pheromones are required for full fertility [47]; however, where or when they act during the developmental cycle is not yet clear because S. macrospora is self-fertile (homothallic), and no obvious fertilization event that requires recognition of compatible partners by pheromones is necessary [27]. To address this question in more detail, we analyzed the expression of an egfp reporter gene under the control of the ppg1 upstream and downstream regulatory regions (Figure 4). No expression was observed in vegetative hyphae, in contrast to the expression of egfp from a control vector under the constitutive gpd promoter and trpC terminator from A. nidulans. EGFP fluorescence started to occur in ascogonia (female gametangia) and was strongest in young protoperithecia (diameter ≤ 30 μm). Interestingly, older protoperithecia (> 30 μm) exhibited a distinctly patchy expression pattern in the hyphae of the outer layers, whereas expression of the control vector led to a uniform fluorescence of protoperithecia. On the one hand, these data confirm the transcriptional up-regulation of ppg1 in protoperithecia as indicated by the RNA-seq analysis; and on the other hand, the microscopic analysis revealed a distinct expression pattern of ppg1 within protoperithecia.

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