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Myrtle V. Tools Request permission Export citation Add to favorites Track citation. Share Give access Share full text access. Share full text access. Please review our Terms and Conditions of Use and check box below to share full-text version of article. Abstract Paramecium tetraurelia, stock d, although completely homozygous, produces two kinds of genomically identical clones: N nondischarge clones incapable of trichocyst exocytosis discharge from intact cells in response to picric acid; and D discharge clones that do respond.

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1. Introduction

New Password. In cluster 1 Fig. Of the classified proteins we identified, most were involved in metabolism, cellular transport, cell cycle and DNA processing, protein with binding function, cellular communication and transcription. Genes in cluster 2 were mainly involved in metabolism, cellular transport, transport facilities and transport routes as well as proteins with binding function, while only 16 of the proteins in cluster 2 were not classified. In cluster 3, only 19 out of proteins were not classified, while the remaining proteins were mainly involved in metabolism, especially C-compound and carbohydrate metabolism.

Genes in cluster 3 also tended to be involved in cellular transport, including cation and heavy metal ion transport as well as proteins with binding functions, e. Cluster 4 contained proteins, which were involved mainly in metabolism, especially lipid, fatty acid and isoprenoid metabolism, cellular transport, and cell cycle including DNA restriction or modification. Overall, we observed an enrichment of genes involved in metabolic processes in early stages of perithecium development. Interestingly, in clusters 6—8 there appears to be a species-specific expression in later stages of the development.

Proteins of cluster 6 were mainly involved in transcriptional processes such as RNA synthesis, processing and modification, but also, cell cycle and DNA processing, as well as and metabolism. Cluster 7 contained genes involved in transcription, protein binding, cell type differentiation and cellular transport. In cluster 8 only 13 genes were not identified. The cluster mainly comprised proteins involved in cell differentiation, cellular transport and transcription. While in the early stages of the perithecium development, the fungi were observed to exhibit an enrichment of expressed genes involved in metabolic processes; in later stages of perithecium development, expressed genes from the ortholog set were enriched for genes involved in transcription, cell cycle, protein synthesis and cellular transport.

Crossing of the N. In order to identify genes that are required for the successful development of fruiting bodies, knockouts in N. Eight mutant phenotypes were observed that affected perithecium formation. Interestingly, development of NCU, encoding a HMG box high mobility group -containing protein was arrested after formation of protoperithecia on a fluffy white mycelium.

Putative functions of the remaining genes that affect fruiting body formation in N. However, the deletion of either gene NCU, a putative argonaute siRNA chaperone complex subunit which in fission yeast is required for histone H3 Lys9 H3-K9 methylation, heterochromatin, assembly and siRNA generation [1] , or NCU, a putative type-2 protein geranylgeranyltransferase subunit, caused the perithecium development to arrest at an early stage between 48 and 72 h Fig. In the perithecia arising from these KO strains, we observed no perithecial beak and found that the developing perithecia remained spherical, indicating an arrest in development.

Furthermore, the CA of these cultures was stained black along the mating zone, suggesting an increase in melanin biosynthesis Fig. Perithecia large, black and protoperithecia small, yellowish-gray to gray are indicated with black arrow heads. The expression patterns of the genes whose knockouts showed an impact on fruiting body formation were monitored over the time course of sexual development starting from mature protoperithecia before crossing until h, when wild-type cultures showed mature perithecia.

The genes NCU or exhibited a continuous increase in expression starting at 72 h Fig. The increased expression correlated with the arrest of the fruiting body formation in an early stage between 48 and 72 h in deletion mutants. Expression of NCU solid line and NCU dashed line , knockouts of which lead to early arrest of perithecial development at 48—72 h. Both genes exhibit a continuous increase of expression after 72 h.

Expression of NCU solid line and NCU dashed line , up-regulated during intermediate stages of perithecial development. Expression of NCU solid line and NCU dashed line , both changing significantly in late perithecial development. NCU encodes a putative secreted protein and has been described in Nectria haematococca as bacterial-type extracellular deoxyribonuclease [63]. NCU and were highly expressed between 48 and h, while expression of NCU peaked at 48 and 96 h after crossing.

NCU and showed an increased expression 96 h after crossing. NCU peaked at 48 h after crossing and remained fairly constant across the remainder of sexual development Fig. Although only protoperithecia were observed in mutant crosses, the culture medium for the knockout of NCU blackened, indicating the secretion of melanin, and implying that melanin biosynthesis remained functional despite the fact that no perithecia were formed.

Genes encoding enzymes in the melanin synthesis pathway showed a highly similar pattern across sexual development within each Neurospora species. While melanin synthesis genes were highly expressed in N. Further research is needed to understand function of melanin synthesis early sexual development in different Neurospora species. Recent studies have demonstrated that deletion of the ortholog of NCU in Podospora anserina and Fusarium graminearum had no effect on perithecium development [64] , [65].

However, an effect on the distribution of perithecia in P. The gene NCU belongs to cluster 5. The corresponding ortholog in N. The genes NCU, , , , , and belong to cluster 6. The expression of the ortholog NCU in N. Over the time course of sexual development, expression of this gene is very low. The gene NCU shows an increasing expression over the time course of the sexual development, peaking at 48 and 96 h.

In contrast, the corresponding ortholog in N. The expression of the corresponding ortholog in N. The expression of the gene NCU peaks between 48 and h in N. The gene NCU shows peaks in gene expression at 24 and 72 h, and an increase of expression from 96 h until the end of perithecium development. In contrast, in both N. The gene NCU belongs to cluster 8 and shows an increased gene expression between 24 and h in N. A similar pattern was observed for N. Key differences in gene expression patterns of meiosis-related genes between N. Many RNA processing genes and meiosis- and mitosis- specific genes have experienced multiple duplications.

Only a few single copy meiosis-specific gene homologs have been confirmed with our methods. Among these genes, we observed differences in gene expression between homothallic N. The meiotic chromosome segregation protein 3 homologs, N. In contrast, the expression of the ortholog of chromosome segregation protein 3 in N. Up-regulation of homologs of N. Similar up-regulation of asd-3 for N.

Except NCU and , all genes shown in Fig. A Expression of the N. C Expression of meiosis specific gene spo and its orthologs was up-regulated for all three species from 96 h after crossing, but the up-regulation started early for the N. D Expression of asm-1 ascus maturation in N. E Expression of N. Expression of N. To validate the linkage between the insertional mutation and the phenotype, we followed a strategy developed previously for N. Twelve to twenty single ascospore progeny displayed hygromycin resistance, and complete cosegregation of hygromycin resistance and identified phenotypes was observed for seven out of eight investigated genes Table S6.

No perithecia were produced in this mutant-wild type cross, preventing us from assessing segregation of the cross. Here, we compared the sexual development of three closely related Neurospora species, N. The comparison of gene expression levels across these species during fruiting body formation revealed eight genes that were shown to be crucial for the successful development of perithecia in N. We identified different expression patterns for meiosis-related genes between N.

Since meiosis gene sets and sporulation machinery are largely conserved in presence as well as sequence within these genomes, consistent differences among functionally related genes in these species would be strong evidence of dependent associations for the reconstruction of gene networks and thus for understanding the genetic basis of meiosis and sexual sporulation in Neurospora and ascomycetes.

Additionally, species-specific expression in later stages of development was observed when clustering the ortholog genes. Future efforts should expand analysis to identify a more complete set of genes involved in meiosis and sporulation based on genetics and reverse genetics on the model N. We distinguished eight gene clusters based on similarity of overall expression patterns across sexual development in three Neurospora species.

During early sexual development, genes involved in metabolism are enriched, and clusters that appear later during sexual development are enriched with genes involved in metabolism, energy, transcription, cell cycle, and DNA processing. The expression profiles of these clusters correlated with the morphological changes observed during sexual development: at first metabolic genes are upregulated, then transcription and cell cycle related genes are activated, and eventually protein synthesis and transport genes are transcribed to deliver proteins needed to their destination in order to form the complex three dimensional fruiting body.

Our study revealed eight genes that are required for the successful formation of fruiting bodies in N. Knockouts of two genes, NCU and resulted in the arrest of perithecium formation between 48 and 72 h after crossing Fig. In correlation with the gene expression data Fig. The developing perithecia for NCU a putative argonaute siRNA chaperone complex subunit and a putative type-2 protein geranylgeranyltransferase subunit remain round-shaped without formation of a beak. Six crosses of N. These results correlate with the gene expression observed over the time course of the sexual development in N.

Here, an up-regulation of gene expression during the later stages of the development was observed. Our observations indicate that there are several genes that are required for the successful formation of perithecia. We have used comparative transcriptomics as a tool to identify genes that are required for the successful formation of fruiting bodies.

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The impact of the deletion of the candidate genes was demonstrated in phenotypes observed in crosses exhibiting impaired perithecium formation at different stages of the development. Our findings shed light on the developmental process of fruiting body formation, evolution, and spore development in Neurospora species, thus establishing the foundation for future research particularly related to closely related pathogenic fungi. We suggest the potential utility of future research on the eight genes that we have discovered as essential contributors to fruit body development and as candidate genes for targets in the development of fungicides for control of plant pathogens.

Expression results of all genes of all three Neurospora species. LOX estimates the Level Of gene eXpression from high-throughput-expressed sequence datasets with multiple treatments or samples. The tables includes the corresponding upper and lower confidence intervals CI across developmental stages. Comparative gene expression analysis of N. The table includes the results from the calculations such as the differences of confidence intervals.

Comparison of species using the IF statement function. In each comparison the highest value across all time points for each gene was selected MAX and the maximum values were then sorted in descending order. Prioritization of the maximum value across the time course of the sexual development for each gene in speecies comparisons. Cosegregation of hygromycin resistance and identified phenotypes in KO strains of interest. We thank Dr. Performed the experiments: NL ZW.

Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Many fungi form complex three-dimensional fruiting bodies, within which the meiotic machinery for sexual spore production has been considered to be largely conserved over evolutionary time. Introduction Many ascomycete fungi sexually reproduce by forming three-dimensional fruiting bodies that produce their sexual spores ascospores in sacs called asci.

Materials and Methods 2. Strains and culture conditions Strains of complementary mating types mat a and mat A for N. Fixation and microscopy Perithecium development was monitored for all three Neurospora species with a stereomicroscope over the time course of the sexual development. Data acquisition and analysis The libraries were run on eight lanes of an Illumina Genome Analyzer, generating an average of 28 million single-end reads of 36 nucleotides each.

Assessing phenotypes of knock out mutants Knockout strains for the top candidate genes were compared to wild-type WT strains and screened for defects in fruiting body formation. Results 3.

Download: PPT. Figure 2. Key morphological characters of N. Figure 3. Comparative heat map of N. Figure 4. Phenotypes of perithecia from crosses in N. Table 1. Putative functions of genes whose deletion impacts fruiting body formation. Figure 5. Expression patterns of eight genes with impact on successful perithecium formation in N. Figure 6.

Comparative analysis of meiosis-related genes, exhibiting differences in gene expression between N. Discussion Here, we compared the sexual development of three closely related Neurospora species, N. Conclusions We have used comparative transcriptomics as a tool to identify genes that are required for the successful formation of fruiting bodies. Supporting Information. Table S1.

Table S2. Table S3. Table S4. Table S5. Table S6. Acknowledgments We thank Dr.

Guy Plunkett III, PhD

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