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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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MA-plots for gene expressiondata from different comparisons. Log2 of fold ratios (M) were plotted against the average read counts (A) for the respective locus tag. The ratios were from the LOX analysis, the plots from the classic analysis look similar (data not shown). The log2 of ratios in which the denominator was zero were set to 20, and the log2 of ratios in which the numerator was zero were set to −20.
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Figure 2: MA-plots for gene expressiondata from different comparisons. Log2 of fold ratios (M) were plotted against the average read counts (A) for the respective locus tag. The ratios were from the LOX analysis, the plots from the classic analysis look similar (data not shown). The log2 of ratios in which the denominator was zero were set to 20, and the log2 of ratios in which the numerator was zero were set to −20.

Mentions: MA-plots of gene expression comparing the different samples showed that sexual mycelium is much more similar to vegetative mycelium than to protoperithecia from the wild-type or mutant pro1, and that the mutant and wild-type protoperithecia differ strongly from each other with respect to gene expression (Figure 2, Table 3, Additional file 2). The largest numbers of differentially regulated genes were those that are downregulated in wild-type or pro1 protoperithecia compared to sexual mycelium; however, some of these genes might be false-positives due to not all 3’ UTRs being annotated yet. In those cases, genes in protoperithecia samples might appear to be not expressed because most of the reads map to the 3’ ends of the genes and would not be counted for a gene if the 3’ UTR is not annotated correctly. This hypothesis is supported by the fact that the percentage of genes with annotated 3’ UTRs is lower among the genes that appear to be down-regulated in protoperithecia (Table 3). However, this problem does not occur in the comparison of the two protoperithecial samples from the wild-type and mutant pro1, because any bias would concern both samples equally. Therefore, the high number of differentially expressed genes in the comparison of wild-type and pro1 protoperithecia with almost equal numbers of up- and down-regulated genes most likely represents true differences in gene expression. The same is true for genes that are upregulated in protoperithecia compared to sexual mycelium, because this can not be overestimated by missing 3’ UTRs. Therefore, even when not taking into account the high number of putatively down-regulated genes in protoperithecia versus total mycelium, the data indicate that the differences between wild-type and pro1 protoperithecia as well as between protoperithecia and total mycelium (sexual and vegetative) are much more pronounced than between the different mycelial samples. This finding is consistent with the hypothesis that the morphological changes that occur during fruiting body formation are mediated by drastic changes in gene expression at the level of transcription.


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)

MA-plots for gene expressiondata from different comparisons. Log2 of fold ratios (M) were plotted against the average read counts (A) for the respective locus tag. The ratios were from the LOX analysis, the plots from the classic analysis look similar (data not shown). The log2 of ratios in which the denominator was zero were set to 20, and the log2 of ratios in which the numerator was zero were set to −20.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: MA-plots for gene expressiondata from different comparisons. Log2 of fold ratios (M) were plotted against the average read counts (A) for the respective locus tag. The ratios were from the LOX analysis, the plots from the classic analysis look similar (data not shown). The log2 of ratios in which the denominator was zero were set to 20, and the log2 of ratios in which the numerator was zero were set to −20.
Mentions: MA-plots of gene expression comparing the different samples showed that sexual mycelium is much more similar to vegetative mycelium than to protoperithecia from the wild-type or mutant pro1, and that the mutant and wild-type protoperithecia differ strongly from each other with respect to gene expression (Figure 2, Table 3, Additional file 2). The largest numbers of differentially regulated genes were those that are downregulated in wild-type or pro1 protoperithecia compared to sexual mycelium; however, some of these genes might be false-positives due to not all 3’ UTRs being annotated yet. In those cases, genes in protoperithecia samples might appear to be not expressed because most of the reads map to the 3’ ends of the genes and would not be counted for a gene if the 3’ UTR is not annotated correctly. This hypothesis is supported by the fact that the percentage of genes with annotated 3’ UTRs is lower among the genes that appear to be down-regulated in protoperithecia (Table 3). However, this problem does not occur in the comparison of the two protoperithecial samples from the wild-type and mutant pro1, because any bias would concern both samples equally. Therefore, the high number of differentially expressed genes in the comparison of wild-type and pro1 protoperithecia with almost equal numbers of up- and down-regulated genes most likely represents true differences in gene expression. The same is true for genes that are upregulated in protoperithecia compared to sexual mycelium, because this can not be overestimated by missing 3’ UTRs. Therefore, even when not taking into account the high number of putatively down-regulated genes in protoperithecia versus total mycelium, the data indicate that the differences between wild-type and pro1 protoperithecia as well as between protoperithecia and total mycelium (sexual and vegetative) are much more pronounced than between the different mycelial samples. This finding is consistent with the hypothesis that the morphological changes that occur during fruiting body formation are mediated by drastic changes in gene expression at the level of transcription.

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