Differential impact of nutrition on developmental and metabolic gene expression during fruiting body development in Neurospora crassa.
ABSTRACT: 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 Neurospora crassa was induced on two different media, a chemically 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 h 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.
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
Project description: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 fine-scale transcriptomic landscape and novel inferences regarding the control of the multistage development process of sexual crossing and fruiting body development in N. crassa.
Project description:Sordaria fimicola, a coprophilous ascomycete, is a homothallic fungus that can undergo sexual differentiation with cellular and morphological changes followed by multicellular tissue development to complete its sexual cycle. In this study, we identified and characterized the blue-light photoreceptor gene in S. fimicola The S. fimicola white collar-1 photoreceptor (SfWC-1) contains light-oxygen-voltage-sensing (LOV), Per-Arnt-Sim (PAS), and other conserved domains and is homologous to the WC-1 blue-light photoreceptor of Neurospora crassa The LOV domain of Sfwc-1 was deleted by homologous recombination using Agrobacterium-mediated protoplast transformation. The Sfwc-1 ( ?lov ) mutant showed normal vegetative growth but produced less carotenoid pigment under illumination. The mutant showed delayed and less-pronounced fruiting-body formation, was defective in phototropism of the perithecial beaks, and lacked the fruiting-body zonation pattern compared with the wild type under the illumination condition. Gene expression analyses supported the light-induced functions of the Sfwc-1 gene in the physiology and developmental process of perithecial formation in S. fimicola Moreover, green fluorescent protein (GFP)-tagged SfWC-1 fluorescence signals were transiently strong upon light induction and prominently located inside the nuclei of living hyphae. Our studies focused on the putative blue-light photoreceptor in a model ascomycete and contribute to a better understanding of the photoregulatory functions and networks mediated by the evolutionarily conserved blue-light photoreceptors across diverse fungal phyla.IMPORTANCE Sordaria sp. has been a model for study of fruiting-body differentiation in fungi. Several environmental factors, including light, affect cellular and morphological changes during multicellular tissue development. Here, we created a light-oxygen-voltage-sensing (LOV) domain-deleted Sfwc-1 mutant to study blue-light photoresponses in Sordaria fimicola Phototropism and rhythmic zonation of perithecia were defective in the Sfwc-1 ( ?lov ) mutant. Moreover, fruiting-body development in the mutant was reduced and also significantly delayed. Gene expression analysis and subcellular localization study further revealed the light-induced differential gene expression and cellular responses upon light stimulation in S. fimicola.
Project description:Biosynthesis of the black perithecial pigment in the filamentous fungus Fusarium graminearum is dependent on the polyketide synthase PGL1 (oPKS3). A seven-membered PGL1 gene cluster was identified by over-expression of the cluster specific transcription factor pglR. Targeted gene replacement showed that PGL1, pglJ, pglM and pglV were essential for the production of the perithecial pigment. Over-expression of PGL1 resulted in the production of 6-O-demethyl-5-deoxybostrycoidin (1), 5-deoxybostrycoidin (2), and three novel compounds 5-deoxybostrycoidin anthrone (3), 6-O-demethyl-5-deoxybostrycoidin anthrone (4) and purpurfusarin (5). The novel dimeric bostrycoidin purpurfusarin (5) was found to inhibit the growth of Candida albicans with an IC50 of 8.0?+/-?1.9??M. The results show that Fusarium species with black perithecia have a previously undescribed form of 5-deoxybostrycoidin based melanin in their fruiting bodies.
Project description:MicroRNAs (miRNAs) are class of small RNA molecules with major impact on gene regulation. We analyzed the potential of miRNAs secreted from pre-implantation embryos into the embryonic culture media as biomarkers to predict successful pregnancy. Using microarray analysis, we profiled the miRNome of the 56 spent culture media (SCM) after embryos transfer and found a total of 621 miRNAs in the SCM. On average, we detected 163 miRNAs in SCM of samples with failed pregnancies, but only 149 SCM miRNAs of embryos leading to pregnancies. MiR-634 predicted an embryo transfer leading to a positive pregnancy with an accuracy of 71% and a sensitivity of 85%. Among the 621 miRNAs, 102 (16.4%) showed a differential expression between positive and negative outcome of pregnancy with miR-29c-3p as the most significantly differentially expressed miRNA. The number of extracellular vehicles was lower in SCM with positive outcomes (3.8?×?109/mL EVs), as compared to a negative outcome (7.35?×?109/mL EVs) possibly explaining the reduced number of miRNAs in the SCM associated with failed pregnancies. The analysis of the miRNome in the SCM of couples undergoing fertility treatment lays the ground towards development of biomarkers to predict successful pregnancy and towards understanding the role of embryonic miRNAs found in the SCM.
Project description:BACKGROUND: The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog. RESULTS: Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Deltatap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. CONCLUSION: The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora.
Project description:Neurospora intermedia is a heterothallic filamentous ascomycete. In this study we use microarray technology to study the difference in gene expression between vegetative growth and early reproductive development. Overall design: Neurospora intermedia FGSC#8882 mat-A and FGSC#8782 mat-a. Solid synthetic crossing medium (SCM) was used as a nutrient regime before sampling and processing. Two different conditions were sampled: vegetative mycelial tissue and young reproductive mycelial tissue.
Project description:Auricularia polytricha (Mont.) Sacc., a type of edible black-brown mushroom with a gelatinous and modality-specific fruiting body, is in high demand in Asia due to its nutritional and medicinal properties. Illumina Solexa sequenceing technology was used to generate very large transcript sequences from the mycelium and the mature fruiting body of A. polytricha for gene discovery and molecular marker development. De novo assembly generated 36,483 ESTs with an N50 length of 636 bp. A total of 28,108 ESTs demonstrated significant hits with known proteins in the nr database, and 94.03% of the annotated ESTs showed the greatest similarity to A. delicata, a related species of A. polytricha. Functional categorization of the Gene Ontology (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways revealed the conservation of genes involved in various biological processes in A. polytricha. Gene expression profile analysis indicated that a total of 2,057 ESTs were differentially expressed, including 1,020 ESTs that were up-regulated in the mycelium and 1,037 up-regulated in the fruiting body. Functional enrichment showed that the ESTs associated with biosynthesis, metabolism and assembly of proteins were more active in fruiting body development. The expression patterns of homologous transcription factors indicated that the molecular mechanisms of fruiting body formation and development were not exactly the same as for other agarics. Interestingly, an EST encoding tyrosinase was significantly up-regulated in the fruiting body, indicating that melanins accumulated during the processes of the formation of the black-brown color of the fruiting body in A. polytricha development. In addition, a total of 1,715 potential SSRs were detected in this transcriptome. The transcriptome analysis of A. polytricha provides valuable sequence resources and numerous molecular markers to facilitate further functional genomics studies and genetic researches on this fungus.
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:Lentinula edodes is a popular cultivated edible mushroom with high nutritional and medicinal value. To understand the regulation of gene expression in the dikaryotic mycelium and mature fruiting body in the commercially important Korean L. edodes strain, we first performed comparative transcriptomic analysis, using Illumina HiSeq platform. De novo assembly of these sequences revealed 11,675 representative transcripts in two different stages of L. edodes. A total of 9,092 unigenes were annotated and subjected to Gene Ontology, EuKaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Gene expression analysis revealed that 2,080 genes were differentially expressed, with 1,503 and 577 upregulated in the mycelium and a mature fruiting body, respectively. Analysis of 18 KEGG categories indicated that fruiting body-specific transcripts were significantly enriched in 'replication and repair' and 'transcription' pathways, which are important for premeiotic replication, karyogamy, and meiosis during maturation. We also searched for fruiting body-specific proteins such as aspartic protease, gamma-glutamyl transpeptidase, and cyclohexanone monooxygenase, which are involved in fruiting body maturation and isolation of functional substances. These transcriptomes will be useful in elucidating the molecular mechanisms of mature fruiting body development and beneficial properties, and contribute to the characterization of novel genes in L. edodes.