Global Gene Expression and Focused Knockout Analysis Reveals Genes Associated with Fungal Fruiting Body Development in Neurospora crassa

Abstract

Fungi can serve as highly tractable models for understanding genetic basis of sexual development in multicellular organisms. Applying a reverse-genetic approach to advance such a model, we used random and multitargeted primers to assay gene expression across perithecial development in Neurospora crassa. We found that functionally unclassified proteins accounted for most upregulated genes, whereas downregulated genes were enriched for diverse functions. Moreover, genes associated with developmental traits exhibited stage-specific peaks of expression. Expression increased significantly across sexual development for mating type gene mat a-1 and for mat A-1 specific pheromone precursor ccg-4. In addition, expression of a gene encoding a protein similar to zinc finger, stc1, was highly upregulated early in perithecial development, and a strain with a knockout of this gene exhibited arrest at the same developmental stage. A similar expression pattern was observed for genes in RNA silencing and signaling pathways, and strains with knockouts of these genes were also arrested at stages of perithecial development that paralleled their peak in expression. The observed stage specificity allowed us to correlate expression upregulation and developmental progression and to identify regulators of sexual development. Bayesian networks inferred from our expression data revealed previously known and new putative interactions between RNA silencing genes and pathways. Overall, our analysis provides a finescale transcriptomic landscape and novel inferences regarding the control of the multistage development process of sexual crossing and fruiting body development in N. crassa. S hifts in gene expression over the course of development have been attributed a primary role in the unfolding of the developmental program of animals and plants (1-4). However, the genomic basis of development in a third multicellular clade, the fungi, is arguably very different and the least well understood. Estimated as comprising 1.5 to 7.1 million species, fungi have deep evolutionary origins (5-7) and diverse body plans, ranging from highly reduced unicellular species such as microsporidia and yeasts to notoriously large hyphal mats that produce multicellular fruiting bodies such as mushrooms, which feature specialized cell types (8). To address multicellular fruiting body development from a reverse-genetic approach, model fungi can provide ideal systems, as they are easily manipulated, develop fruiting structures with a few well-characterized tissue types, and have relatively small genome sizes, so numerous fungal genomes have been sequenced.Neurospora crassa is a multicellular ascomycete fungus in the family Sordariomycetes, which has been used as a genetic model organism due to its simple filamentous asexual stage (9, 10), and which exhibits promise for revealing the molecular basis of the more complex sexual development of fungi. N. crassa has 28 morphologically distinct cell types, including 14 that are finely differentiated during the development of...

Genome‐wide identification of target genes of a mating‐type α‐domain transcription factor reveals functions beyond sexual development

“…For example, RMAexpress (http://rmaexpress.bmbolstad.com) analysis of normalized raw data obtained from microarray analysis using P. chrysogenum strain P2niaD18 revealed relative MAT1‐1‐1 expression levels of about 13.6% and 3.1% referred to actin ( Pc20g11630 ) and myosin ( Pc21g00710 ) expression levels, respectively (http://onlinelibrary.wiley.com/doi/10.1111/mmi.12987/suppinfo) (Böhm et al ., ). Furthermore, transcripts of mating‐type genes were reported to be barely detectable by Northern hybridization in Podospora anserina as well as RNA‐seq analysis in Neurospora crassa (Coppin and Debuchy, ; Wang et al ., ).…”

Genome-wide A-to-I RNA editing in fungi independent of ADAR enzymes

“…6A). Similar to PUK1, the transcription level of NCU03242 was relatively low, and A-to-I editing events were not observed in vegetative hyphae and other developmental stages in N. crassa (Wang et al 2014). Interestingly, the PUK1 ortholog in F. verticillioides (FVEG_ 01191) has a stop codon that is equivalent to the second of two tandem stop codons in its ORF.…”

“…6A). Comparative analysis of published RNA-seq data (Wang et al 2014) revealed that the A-to-I editing event at this site (UAGUGG to UGGUGG) also occurs in the transcripts of NCU03242 in perithecia (Fig. 6A).…”

Simple Genetic Tools to Study Fruiting Body Development in Fungi

“…Fruiting body mutants are now available for many fungi other than P. anserina, including Sordaria macrospora [27], N. crassa [28], A. nidulans [29] and Coprinopsis cinerea (formely Coprinus cinereus) [30,31] and could be analyzed using similar methods. They are complementary to the now more widespread large scale analyses of transcriptomes during fruiting body development as performed in the ascomycetes Fusarium graminearum and Fusarium verticilloides [32][33][34], N. crassa [35,36], S. macrospora [37], Pyronema confluens [38], Tuber melanosporum [39][40][41], Cordyceps militaris [42] and Ophiocordyceps sinensis [43], as well as in the basidiomycetes C. cinerea [44], Moniliophthora perniciosa [45], Agrocybe aegerita [46], Lentinula edodes [47,48], Auricularia polytricha [49] and Ganoderma lucidum [50].…”