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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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Expression ratios for transcriptionfactors among the geneswith top 500 readcounts. (A) Expression ratios for the transcription factors among the 500 genes with the highest number of read counts in wild-type and mutant pro1 protoperithecia. Expression of these genes is largely independent of pro1. (B) Expression ratios for the transcription factors among the 500 genes with the highest number of read counts in wild-type protoperithecia but not pro1 protoperithecia. These genes are most likely dependent on pro1 for correct expression. Expression ratios in (A) and (B) are given as log2 values, and log2 ratios >1 and <−1 are indicated in red and blue, respectively. The genes in (A) are mostly not differentially expressed in pro1 protoperithecia compared to wild-type protoperithecia (indicated by the grey coloring), whereas the genes in (B) have a tendency towards down-regulation in pro1 protoperithecia, as expected for genes that are dependent on pro1 for correct expression. Protein domains were predicted with HMMER using the Hidden Markov models from the pfam database [74,75].
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Figure 7: Expression ratios for transcriptionfactors among the geneswith top 500 readcounts. (A) Expression ratios for the transcription factors among the 500 genes with the highest number of read counts in wild-type and mutant pro1 protoperithecia. Expression of these genes is largely independent of pro1. (B) Expression ratios for the transcription factors among the 500 genes with the highest number of read counts in wild-type protoperithecia but not pro1 protoperithecia. These genes are most likely dependent on pro1 for correct expression. Expression ratios in (A) and (B) are given as log2 values, and log2 ratios >1 and <−1 are indicated in red and blue, respectively. The genes in (A) are mostly not differentially expressed in pro1 protoperithecia compared to wild-type protoperithecia (indicated by the grey coloring), whereas the genes in (B) have a tendency towards down-regulation in pro1 protoperithecia, as expected for genes that are dependent on pro1 for correct expression. Protein domains were predicted with HMMER using the Hidden Markov models from the pfam database [74,75].

Mentions: We specifically analyzed whether any transcription factor genes are only present among the top 500 genes in wild-type protoperithecia or in wild-type and pro1 protoperithecia, but not in the mycelial samples. We found 14 putative transcription factors among the top 500 genes in both wild-type and mutant protoperithecia, and seven putative transcription factors among the top 500 genes in wild-type, but not pro1 protoperithecia (Figure 7). Analysis of the gene expression ratios showed that the first group of transcription factors is largely independent of pro1 (no difference in expression between pro1 and wild-type protoperithecia, Figure 7A), whereas the second group depends on pro1 for upregulation in protoperithecia (genes down-regulated in pro1 compared to wild-type protoperithecia, Figure 7B). Transcription factors that are strongly expressed in protoperithecia might be involved in regulating the expression of downstream genes that mediate fruiting body morphogenesis. Two of these transcription factors were already shown to be essential for fruiting body formation, namely mcm1 and pro44. Mutations in mcm1 or pro44 lead to sterility, and the corresponding mutants are able to produce protoperithecia, but not mature perithecia [28,54]. A comparison with homologous transcription factors from N. crassa and F. graminearum revealed that, out of the 21 transcription factors, knockout strains have been analyzed for 12 and 19 genes from N. crassa and F. graminearum, respectively, in large-scale knockout projects with these two organisms [55,56]. Of these deletion mutants, three showed defects in sexual development in N. crassa, and 12 in F. graminearum ( Additional file 1 Table S1). Homologs of pro44 were sterile in both species, and the corresponding homolog of Aspergillus nidulans was also shown to be essential for sexual development [57]. Thus, the transcription factors from this analysis might be promising candidates for further functional studies, especially those with developmental phenotypes in other filamentous fungi.


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)

Expression ratios for transcriptionfactors among the geneswith top 500 readcounts. (A) Expression ratios for the transcription factors among the 500 genes with the highest number of read counts in wild-type and mutant pro1 protoperithecia. Expression of these genes is largely independent of pro1. (B) Expression ratios for the transcription factors among the 500 genes with the highest number of read counts in wild-type protoperithecia but not pro1 protoperithecia. These genes are most likely dependent on pro1 for correct expression. Expression ratios in (A) and (B) are given as log2 values, and log2 ratios >1 and <−1 are indicated in red and blue, respectively. The genes in (A) are mostly not differentially expressed in pro1 protoperithecia compared to wild-type protoperithecia (indicated by the grey coloring), whereas the genes in (B) have a tendency towards down-regulation in pro1 protoperithecia, as expected for genes that are dependent on pro1 for correct expression. Protein domains were predicted with HMMER using the Hidden Markov models from the pfam database [74,75].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Expression ratios for transcriptionfactors among the geneswith top 500 readcounts. (A) Expression ratios for the transcription factors among the 500 genes with the highest number of read counts in wild-type and mutant pro1 protoperithecia. Expression of these genes is largely independent of pro1. (B) Expression ratios for the transcription factors among the 500 genes with the highest number of read counts in wild-type protoperithecia but not pro1 protoperithecia. These genes are most likely dependent on pro1 for correct expression. Expression ratios in (A) and (B) are given as log2 values, and log2 ratios >1 and <−1 are indicated in red and blue, respectively. The genes in (A) are mostly not differentially expressed in pro1 protoperithecia compared to wild-type protoperithecia (indicated by the grey coloring), whereas the genes in (B) have a tendency towards down-regulation in pro1 protoperithecia, as expected for genes that are dependent on pro1 for correct expression. Protein domains were predicted with HMMER using the Hidden Markov models from the pfam database [74,75].
Mentions: We specifically analyzed whether any transcription factor genes are only present among the top 500 genes in wild-type protoperithecia or in wild-type and pro1 protoperithecia, but not in the mycelial samples. We found 14 putative transcription factors among the top 500 genes in both wild-type and mutant protoperithecia, and seven putative transcription factors among the top 500 genes in wild-type, but not pro1 protoperithecia (Figure 7). Analysis of the gene expression ratios showed that the first group of transcription factors is largely independent of pro1 (no difference in expression between pro1 and wild-type protoperithecia, Figure 7A), whereas the second group depends on pro1 for upregulation in protoperithecia (genes down-regulated in pro1 compared to wild-type protoperithecia, Figure 7B). Transcription factors that are strongly expressed in protoperithecia might be involved in regulating the expression of downstream genes that mediate fruiting body morphogenesis. Two of these transcription factors were already shown to be essential for fruiting body formation, namely mcm1 and pro44. Mutations in mcm1 or pro44 lead to sterility, and the corresponding mutants are able to produce protoperithecia, but not mature perithecia [28,54]. A comparison with homologous transcription factors from N. crassa and F. graminearum revealed that, out of the 21 transcription factors, knockout strains have been analyzed for 12 and 19 genes from N. crassa and F. graminearum, respectively, in large-scale knockout projects with these two organisms [55,56]. Of these deletion mutants, three showed defects in sexual development in N. crassa, and 12 in F. graminearum ( Additional file 1 Table S1). Homologs of pro44 were sterile in both species, and the corresponding homolog of Aspergillus nidulans was also shown to be essential for sexual development [57]. Thus, the transcription factors from this analysis might be promising candidates for further functional studies, especially those with developmental phenotypes in other filamentous fungi.

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