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Multidimensional regulation in transcriptome during sexual development in Neurospora crassa revealed with RNA sequencing

ABSTRACT: Fungi exhibit a huge diversity in morphology and ecology, and genetic basis of morphological development in sexual reproduction of multi-cellular fungi has not been fully investigated with genome-wide approaches. Model organism Neurospora crassa is the very first sequenced genome for multi-cellular fungi, and well-annotated genome and gene markers characterized for key morphological traits make N. crassa an ideal platform to investigate transcriptome and its regulation during sexual development. RNA sequencing with different priming strategies have been efficient to provide fine-scale transcriptome landscapes for a multiple-staged development process. RNA were sampled for eight time points cross perithecial development, including two points right before and after crossing, for which mat A protoperithecia were fertilized with mat a conidia. Transcription profiles for 9717 genes were achieved for all eight time-points using multi-targeting priming and additional 14 genes for five time-points using random hexmas priming. Majority of the genome showed continually up- or down- regulated expression patterns during the later perithecial development stages after 72 or 96 h. Functionally unclassified proteins were counted for most up-regulated genes, while genes were enriched with different functions for different down-regulation patterns. We observed significant increase in transcription for mat a-1 and for mat A-1 specific pheromone precursor ccg-4 during the perithecial development, and expression of genetic markers for morphological traits in perithecium, ascus, and ascospore, and for development traits in meiosis often showed multiple peaks during the experiment. We also observed different expression patterns in transcription for other transcription factors and for genes involved in Meiotic Silencing and Repeat Induced Point Mutation, and expression of a gene encoding a protein similar to stc-1 in fission yeast, was increased after crossing and reached almost 1000 fold changes at 144h. Different expression patterns were also observed for genes involved in heterokaryotic and vegetative incompatibility and for genes functioning in the heterotrimmeric G protein signalling systems and in the MAP kinase signalling pathways. By sequencing RNAs from different development stages with two priming strategies, this study provided a large amount of data to quantifiably describe the sophisticated transcriptomic landscape in pre-mating, crossing and sexual reproduction of N. crassa. As major expression patterns for genes in different function categories were recognized during this process, our results in general supported previous observation of expression patterns for genetic markers and regulatory genes/pathway. This study provided further evidence for functional correlation among genes involved in same function or pathways, which included mating type genes and pheromones, transcription factors, pigmentation, heterokaryotic incompatibility, singaling pathways, and RNA silencing. Besides, we also disclosed different expression patterns for the regulatory genes/pathways cross the whole sexual development, and functions of some of these genes were also suggested with phenotypic studies of knockouts. Understanding how these different regulatory elements and their function networks adjust genome-wide transcriptomic and proteomic expression will provide detailed insights about morphological development of tissue-differentiated perithecia in N. crassa.

ORGANISM(S): Neurospora crassa

PROVIDER: GSE41484 | GEO | 2013/10/10

SECONDARY ACCESSION(S): PRJNA177178

REPOSITORIES: GEO

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Project description:Fungi exhibit a huge diversity in morphology and ecology, and genetic basis of morphological development in sexual reproduction of multi-cellular fungi has not been fully investigated with genome-wide approaches. Model organism Neurospora crassa is the very first sequenced genome for multi-cellular fungi, and well-annotated genome and gene markers characterized for key morphological traits make N. crassa an ideal platform to investigate transcriptome and its regulation during sexual development. RNA sequencing with different priming strategies have been efficient to provide fine-scale transcriptome landscapes for a multiple-staged development process. RNA were sampled for eight time points cross perithecial development, including two points right before and after crossing, for which mat A protoperithecia were fertilized with mat a conidia. Transcription profiles for 9717 genes were achieved for all eight time-points using multi-targeting priming and additional 14 genes for five time-points using random hexmas priming. Majority of the genome showed continually up- or down- regulated expression patterns during the later perithecial development stages after 72 or 96 h. Functionally unclassified proteins were counted for most up-regulated genes, while genes were enriched with different functions for different down-regulation patterns. We observed significant increase in transcription for mat a-1 and for mat A-1 specific pheromone precursor ccg-4 during the perithecial development, and expression of genetic markers for morphological traits in perithecium, ascus, and ascospore, and for development traits in meiosis often showed multiple peaks during the experiment. We also observed different expression patterns in transcription for other transcription factors and for genes involved in Meiotic Silencing and Repeat Induced Point Mutation, and expression of a gene encoding a protein similar to stc-1 in fission yeast, was increased after crossing and reached almost 1000 fold changes at 144h. Different expression patterns were also observed for genes involved in heterokaryotic and vegetative incompatibility and for genes functioning in the heterotrimmeric G protein signalling systems and in the MAP kinase signalling pathways. By sequencing RNAs from different development stages with two priming strategies, this study provided a large amount of data to quantifiably describe the sophisticated transcriptomic landscape in pre-mating, crossing and sexual reproduction of N. crassa. As major expression patterns for genes in different function categories were recognized during this process, our results in general supported previous observation of expression patterns for genetic markers and regulatory genes/pathway. This study provided further evidence for functional correlation among genes involved in same function or pathways, which included mating type genes and pheromones, transcription factors, pigmentation, heterokaryotic incompatibility, singaling pathways, and RNA silencing. Besides, we also disclosed different expression patterns for the regulatory genes/pathways cross the whole sexual development, and functions of some of these genes were also suggested with phenotypic studies of knockouts. Understanding how these different regulatory elements and their function networks adjust genome-wide transcriptomic and proteomic expression will provide detailed insights about morphological development of tissue-differentiated perithecia in N. crassa. RNA were sampled for eight time points cross perithecial development, including two points right before and after crossing, for which mat A protoperithecia were fertilized with mat a conidia. Two priming MTP and N6 were used separately as technique replicates for the first strain synthesis during cDNA preparation of all samples. Total 16 cDNAs were sequenced

Project description:Fungal fruiting body size and form are influenced by the ecology of the species, including diverse environmental stimuli. Accordingly, nutritional resources available to the fungus during development can be vital to successful production of fruiting bodies. To investigate the effect of nutrition, perithecial development of N. crassa was induced on two different media, a sparsely nutritive Synthetic Crossing Medium (SCM) and a natural Carrot Agar (CA). Protoperithecia were collected before crossing, and perithecia were collected at 2, 24, 48, 72, 96, 120, and at full maturity 144 hours after crossing. No differences in fruiting body morphology were observed between the two media at any time point. A circuit of microarray hybridizations comparing cDNA from all neighboring stages was performed. For a majority of differentially expressed genes, expression was higher in SCM than in CA, and expression of core metabolic genes was particularly affected. Effects of nutrition were highest in magnitude before crossing, lowering in magnitude during early perithecial development. Interestingly, metabolic effects of the media were also large in magnitude during late perithecial development, at which stage the lower expression in CA presumably reflected the continued intake of diverse complex initial compounds, diminishing the need for expression of anabolic pathways. However, for genes with key regulatory roles in sexual development, including pheromone precursor ccg-4 and poi2, expression patterns were similar between treatments. When possible, a common nutritional environment is ideal for comparing transcriptional profiles between different fungi. Nevertheless, the observed consistency of the developmental program across media, despite considerable metabolic differentiation is reassuring. This result facilitates comparative studies that will require different nutritional resources for sexual development in different fungi. Neurospora crassa mat A FGSC#2489 and mat-a FGSC 4200: Eight perithecial developmental stages, 0hr, 2 hr, 24 hr, 48 hr, 72 hr, 96 hr, 120 hr, and 144 hr were sampled and compared

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Multidimensional regulation in transcriptome during sexual development in Neurospora crassa revealed with RNA sequencing

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