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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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Venn diagram of geneswith top 500 readcounts for each sample. Numbers of genes that are in the top 500 group for one or more or the four samples (vegetative mycelium, sexual mycelium, wild-type protoperithecia, pro1 protoperithecia) are given. In this analysis, only reads that map within 100 to 400 bases from the 3’ end of the mRNA were used to account for the 3’ bias in the microdissection samples and different mRNA lengths. An analysis using read counts for complete predicted mRNAs gave similar results (data not shown).
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Figure 6: Venn diagram of geneswith top 500 readcounts for each sample. Numbers of genes that are in the top 500 group for one or more or the four samples (vegetative mycelium, sexual mycelium, wild-type protoperithecia, pro1 protoperithecia) are given. In this analysis, only reads that map within 100 to 400 bases from the 3’ end of the mRNA were used to account for the 3’ bias in the microdissection samples and different mRNA lengths. An analysis using read counts for complete predicted mRNAs gave similar results (data not shown).

Mentions: Next, we investigated the transcripts that were most abundant in protoperithecia from the wild-type and mutant pro1, and whether there was a difference to the most abundant transcripts in sexual or vegetative mycelium. For this analysis, we counted reads that mapped to the 3’ end (100–400 nt from the 3’ end) of each predicted mRNA. This approach was chosen to account for the 3’ bias in the microdissected samples, and it generates numbers that are largely independent of transcript length. Read counts were normalized to the total number of counted reads in each sample, and the average read count from the two independent repetitions of each sample was used to determine the 500 genes in each of the four samples that had the highest number of reads (Figure 6, Additional file 3). The analysis showed that 104 genes were present in the top 500 in all four samples, and that sexual mycelium and vegetative mycelium, and protoperithecia from wild-type and pro1 had overlaps of 162 and 159 genes, respectively. In contrast, the number of common genes among the top 500 from the mycelial samples and the protoperithecial samples was much lower (Figure 6). This again indicates that the transcriptional landscapes of non-reproductive mycelia versus protoperithecia are rather different, and that overall transcription in sexual mycelium is driven by the non-reproductive hyphae that make up the majority of this sample.


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)

Venn diagram of geneswith top 500 readcounts for each sample. Numbers of genes that are in the top 500 group for one or more or the four samples (vegetative mycelium, sexual mycelium, wild-type protoperithecia, pro1 protoperithecia) are given. In this analysis, only reads that map within 100 to 400 bases from the 3’ end of the mRNA were used to account for the 3’ bias in the microdissection samples and different mRNA lengths. An analysis using read counts for complete predicted mRNAs gave similar results (data not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Venn diagram of geneswith top 500 readcounts for each sample. Numbers of genes that are in the top 500 group for one or more or the four samples (vegetative mycelium, sexual mycelium, wild-type protoperithecia, pro1 protoperithecia) are given. In this analysis, only reads that map within 100 to 400 bases from the 3’ end of the mRNA were used to account for the 3’ bias in the microdissection samples and different mRNA lengths. An analysis using read counts for complete predicted mRNAs gave similar results (data not shown).
Mentions: Next, we investigated the transcripts that were most abundant in protoperithecia from the wild-type and mutant pro1, and whether there was a difference to the most abundant transcripts in sexual or vegetative mycelium. For this analysis, we counted reads that mapped to the 3’ end (100–400 nt from the 3’ end) of each predicted mRNA. This approach was chosen to account for the 3’ bias in the microdissected samples, and it generates numbers that are largely independent of transcript length. Read counts were normalized to the total number of counted reads in each sample, and the average read count from the two independent repetitions of each sample was used to determine the 500 genes in each of the four samples that had the highest number of reads (Figure 6, Additional file 3). The analysis showed that 104 genes were present in the top 500 in all four samples, and that sexual mycelium and vegetative mycelium, and protoperithecia from wild-type and pro1 had overlaps of 162 and 159 genes, respectively. In contrast, the number of common genes among the top 500 from the mycelial samples and the protoperithecial samples was much lower (Figure 6). This again indicates that the transcriptional landscapes of non-reproductive mycelia versus protoperithecia are rather different, and that overall transcription in sexual mycelium is driven by the non-reproductive hyphae that make up the majority of this sample.

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