Combining laser microdissection and RNA-seq to chart the transcriptional landscape of fungal development

Abstract

BackgroundDuring 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.ResultsQuantitative 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.ConclusionsWe 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.

New Insights Into the Roles of NADPH Oxidases in Sexual Development and Ascospore Germination in Sordaria macrospora

“…RNA-seq data show that both pheromone receptor genes pre1 and pre2 and the two mating type genes SmtA-1 and SmtA-3 are up-regulated in Δnox1 mycelia and protoperithecia. Similarly, genes for melanin biosynthesis (pks, teh, sdh, and tih) and two genes associated with fruiting body maturation, app and tap1, are down-regulated in Δnox1 mycelia and protoperithecia, similar to the situation in other pro mutants (Nowrousian et al , 2007Teichert et al 2012), whereas they only show minor expression changes in fertile mutants Δnox2 and Δste12 compared to wild type ( Figure 4C). These findings are in line with the proposed hypothesis of a common pro mutant-specific expression pattern.…”

“…Cultivation for ascospore germination assays and DNA extraction were performed as described previously (Nowrousian and Cebula 2005;Kamerewerd et al 2008;Teichert et al 2012). For rescue of fertility, strains were inoculated on filter paper covering solid BMM medium.…”

“…Differential expression was evaluated using DESeq (Anders and Huber 2010) and a method called "classical analysis." In this analysis, genes were grouped into five groups (0 to 4) containing genes that are not differentially expressed (group 0) to genes that are strongly and significantly differentially expressed (group 4) as described in Teichert et al (2012). Based on the DESseq and classical analysis, a consensus was calculated according to the following criteria for differential expression similar to what was described before ): a gene is described as differentially regulated if ratios in both DESeq and classical analysis are .4 or ,0.25, DESeq adjusted P-value # 0.1, and a gene in groups 1-4 in the classical analysis.…”

“…For protoperithecia isolation by laser microdissection (LM), the Δnox1 mutant was grown directly on slides for fixation and dissection in situ . RNA was isolated from protoperithecia with the Arcturus PicoPure kit (Applied Biosystems, Carlsbad, CA) and amplified in two linear amplification rounds using the TargetAmp 2-Round aRNA Amplification kit 2.0 (Epicentre Biotechnologies, Madison, WI) with modifications as described in Teichert et al (2012). For RNA isolation of total mycelia, strains were precultured for 2 days on solid BMM.…”

Biochemical and Molecular Analysis of Superoxide Dismutase in Sordaria fimicola and Aspergillus niger Collected from Different Environments

“…The 18S rRNA gene sequencing was used earlier for species identification in different studies [21]. For the amplification of the SOD-1 gene, a sequence of N. crassa (M58687.1) was used to design the primers because the genomic sequence of N. crassa was found most closely related to S. macrospora than any other sequenced filamentous fungi [22][23]. When SOD-1 gene sequences were aligned with the reference gene of the N. crassa (M58687.1), a total of 25 base substitutions were observed in the exonic region (Fig.…”