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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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Laser microdissection of protoperithecia. Mycelia were grown on special membrane slides and fixed in ethanol. After drying of the slides, samples were covered with a glass slide (A) and visualized on an inverted microscope (B). Selected regions containing protoperithecia were cut with a UV laser through the microscope lens. To collect the cut out regions, the cap of a special collection tube was lowered onto the sample (C) where the membrane (with the sample attached) stuck to the cap and could be lifted off when the cap was raised again. Effective collection was indicated by corresponding holes in the samples (D).
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Figure 1: Laser microdissection of protoperithecia. Mycelia were grown on special membrane slides and fixed in ethanol. After drying of the slides, samples were covered with a glass slide (A) and visualized on an inverted microscope (B). Selected regions containing protoperithecia were cut with a UV laser through the microscope lens. To collect the cut out regions, the cap of a special collection tube was lowered onto the sample (C) where the membrane (with the sample attached) stuck to the cap and could be lifted off when the cap was raised again. Effective collection was indicated by corresponding holes in the samples (D).

Mentions: For LM, strains were grown directly on slides for fixation and dissection in situ. To allow protoperithecial development, slides had to be covered with a thin layer of agar that did not interfere with the laser sectioning. Samples were fixed in ethanol, and microdissection was performed with a CellCut Plus system (see Methods and Figure 1). Approximately 100–300 protoperithecia with a diameter of ~20 μm were collected from each slide, pooled in a collection tube, and RNA was extracted from the collected protoperithecia with the PicoPure kit. It was then tested whether transcripts of protein-coding genes could be detected in the protoperithecial RNA samples by quantitative real time PCR (qRT-PCR). Expression was detectable for several genes that were analyzed in microdissected samples from the wild-type; but the amount of RNA was not sufficient for RNA-seq analysis. Therefore, two rounds of linear RNA amplification were performed based on cDNA generation and in vitro transcription [37,38] to obtain polyA-tailed RNA in the amounts required for Illumina/Solexa library generation. This linear amplification method has been shown previously to preserve relative transcript amounts within samples and is used in many applications including target generation for microarray hybridization [39]. The amplified RNAs from microdissected protoperithecia from the wild-type as well as from mutant pro1 were used for RNA-seq analysis. The pro1 mutant lacks the gene for the transcription factor PRO1, which is essential for sexual development; thus, the mutant is able to form protoperithecia but not mature fruiting bodies [36,40]. Therefore, genes that are differentially regulated in pro1 protoperithecia compared to those of the wild-type are direct or indirect targets of PRO1, and some of these genes might be required for fruiting body formation.


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)

Laser microdissection of protoperithecia. Mycelia were grown on special membrane slides and fixed in ethanol. After drying of the slides, samples were covered with a glass slide (A) and visualized on an inverted microscope (B). Selected regions containing protoperithecia were cut with a UV laser through the microscope lens. To collect the cut out regions, the cap of a special collection tube was lowered onto the sample (C) where the membrane (with the sample attached) stuck to the cap and could be lifted off when the cap was raised again. Effective collection was indicated by corresponding holes in the samples (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Laser microdissection of protoperithecia. Mycelia were grown on special membrane slides and fixed in ethanol. After drying of the slides, samples were covered with a glass slide (A) and visualized on an inverted microscope (B). Selected regions containing protoperithecia were cut with a UV laser through the microscope lens. To collect the cut out regions, the cap of a special collection tube was lowered onto the sample (C) where the membrane (with the sample attached) stuck to the cap and could be lifted off when the cap was raised again. Effective collection was indicated by corresponding holes in the samples (D).
Mentions: For LM, strains were grown directly on slides for fixation and dissection in situ. To allow protoperithecial development, slides had to be covered with a thin layer of agar that did not interfere with the laser sectioning. Samples were fixed in ethanol, and microdissection was performed with a CellCut Plus system (see Methods and Figure 1). Approximately 100–300 protoperithecia with a diameter of ~20 μm were collected from each slide, pooled in a collection tube, and RNA was extracted from the collected protoperithecia with the PicoPure kit. It was then tested whether transcripts of protein-coding genes could be detected in the protoperithecial RNA samples by quantitative real time PCR (qRT-PCR). Expression was detectable for several genes that were analyzed in microdissected samples from the wild-type; but the amount of RNA was not sufficient for RNA-seq analysis. Therefore, two rounds of linear RNA amplification were performed based on cDNA generation and in vitro transcription [37,38] to obtain polyA-tailed RNA in the amounts required for Illumina/Solexa library generation. This linear amplification method has been shown previously to preserve relative transcript amounts within samples and is used in many applications including target generation for microarray hybridization [39]. The amplified RNAs from microdissected protoperithecia from the wild-type as well as from mutant pro1 were used for RNA-seq analysis. The pro1 mutant lacks the gene for the transcription factor PRO1, which is essential for sexual development; thus, the mutant is able to form protoperithecia but not mature fruiting bodies [36,40]. Therefore, genes that are differentially regulated in pro1 protoperithecia compared to those of the wild-type are direct or indirect targets of PRO1, and some of these genes might be required for fruiting body formation.

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