The ancestral levels of transcription and the evolution of sexual phenotypes in filamentous fungi.
ABSTRACT: Changes in gene expression have been hypothesized to play an important role in the evolution of divergent morphologies. To test this hypothesis in a model system, we examined differences in fruiting body morphology of five filamentous fungi in the Sordariomycetes, culturing them in a common garden environment and profiling genome-wide gene expression at five developmental stages. We reconstructed ancestral gene expression phenotypes, identifying genes with the largest evolved increases in gene expression across development. Conducting knockouts and performing phenotypic analysis in two divergent species typically demonstrated altered fruiting body development in the species that had evolved increased expression. Our evolutionary approach to finding relevant genes proved far more efficient than other gene deletion studies targeting whole genomes or gene families. Combining gene expression measurements with knockout phenotypes facilitated the refinement of Bayesian networks of the genes underlying fruiting body development, regulation of which is one of the least understood processes of multicellular development.
Project description:BACKGROUND:Chinese cordyceps, also known as Chinese caterpillar fungus (Ophiocordyceps sinensis, syn. Cordyceps sinensis), is of particular interest for its cryptic life cycle and economic and ecological importance. The large-scale artificial cultivation was succeeded recently after several decades of efforts and attempts. However, the induction of primordium, sexual development of O. sinensis and the molecular basis of its lifestyle still remain cryptic. RESULTS:The developmental transcriptomes were analyzed for six stages covering the whole developmental process, including hyphae (HY), sclerotium (ST), primordium (PR), young fruiting body (YF), developed fruiting body (DF) and mature fruiting body (MF), with a focus on the expression of sexual development-related genes. Principal component analysis revealed that the gene expression profiles at the stages of primordium formation and fruiting body development are more similar than those of the undifferentiated HY stage. The PR and MF stages grouped together, suggesting that primordium differentiation and sexual maturation have similar expression patterns. Many more DEGs were identified between the ST and HY stages, covering 47.5% of the O. sinensis genome, followed by the comparisons between the ST and PR stages. Using pairwise comparisons and weighted gene coexpression network analysis, modules of coexpressed genes and candidate hub genes for each developmental stage were identified. The four mating type loci genes expressed during primordium differentiation and sexual maturation; however, spatiotemporal specificity of gene expression indicated that they also expressed during the anamorphic HY stage. The four mating type genes were not coordinately expressed, suggesting they may have divergent roles. The expression of the four mating type genes was highest in the fertile part and lowest in the sclerotium of the MF stage, indicating that there is tissue specificity. Half of genes related to mating signaling showed as the highest expression in the ST stage, indicating fruiting was initiated in the ST stage. CONCLUSIONS:These results provide a new perspective to understanding of the key pathways and hub genes, and sexual development-related gene profile in the development of Chinese cordyceps. It will be helpful for underlying sexual reproduction, and add new information to existing models of fruiting body development in edible fungi.
Project description:Changes in gene expression are important for the landmark morphological events that occur during Myxococcus xanthus fruiting body development. Enhancer binding proteins (EBPs), which are transcriptional activators, play prominent roles in the coordinated expression of developmental genes. A mutation in the EBP gene nla4 affects the timing of fruiting body formation, the morphology of mature fruiting bodies, and the efficiency of sporulation. In this study, we showed that the nla4 mutant accumulates relatively low levels of the stringent nucleotide ppGpp. We also found that the nla4 mutant is defective for early developmental events and for vegetative growth, phenotypes that are consistent with a deficiency in ppGpp accumulation. Further studies revealed that nla4 cells produce relatively low levels of GTP, a precursor of RelA-dependent synthesis of (p)ppGpp. In addition, the normal expression patterns of all stringent response-associated genes tested, including the M. xanthus ppGpp synthetase gene relA, are altered in nla4 mutant cells. These findings indicate that Nla4 is part of regulatory pathway that is important for mounting a stringent response and for initiating fruiting body development.
Project description:Ophiocordyceps sinensis is a highly valued fungus that has been used as traditional Asian medicine. This fungus is one of the most important sources of income for the nomadic populations of the Tibetan Plateau. With global warming and excessive collection, the wild O. sinensis resources declined dramatically. The cultivation of O. sinensis hasn't been fully operational due to the unclear genetic basis of the fruiting body development. Here, our study conducted pairwise comparisons between transcriptomes acquired from different growth stages of O. sinensis including asexual mycelium (CM), developing fruiting body (DF) and mature fruiting body (FB). All RNA-Seq reads were aligned to the genome of O. sinensis CO18 prior to comparative analyses. Cluster analysis showed that the expression profiles of FB and DF were highly similar compared to CM. Alternative splicing analysis (AS) revealed that the stage-specific splicing genes may have important functions in the development of fruiting body. Functional enrichment analyses showed that differentially expressed genes (DEGs) were enriched in protein synthesis and baseline metabolism during fruiting body development, indicating that more protein and energy might be required for fruiting body development. In addition, some fruiting body development-associated genes impacted by ecological factors were up-regulated in FB samples, such as the nucleoside diphosphate kinase gene (ndk), ? subunit of the fatty acid synthase gene (cel-2) and the superoxide dismutase gene (sod). Moreover, the expression levels of several cytoskeletons genes were significantly altered during all these growth stages, suggesting that these genes play crucial roles in both vegetative growth and the fruiting body development. Quantitative PCR (qPCR) was used to validate the gene expression profile and the results supported the accuracy of the RNA-Seq and DEGs analysis. Our study offers a novel perspective to understand the underlying growth stage-specific molecular differences and the biology of O. sinensis fruiting body development.
Project description: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. Indeed, much of what we know about meiosis in plant and animal taxa has been deeply informed by studies of meiosis in Saccharomyces and Neurospora. Nevertheless, the genetic basis of fruiting body development and its regulation in relation to meiosis in fungi is barely known, even within the best studied multicellular fungal model Neurospora crassa. We characterized morphological development and genome-wide transcriptomics in the closely related species Neurospora crassa, Neurospora tetrasperma, and Neurospora discreta, across eight stages of sexual development. Despite diverse life histories within the genus, all three species produce vase-shaped perithecia. Transcriptome sequencing provided gene expression levels of orthologous genes among all three species. Expression of key meiosis genes and sporulation genes corresponded to known phenotypic and developmental differences among these Neurospora species during sexual development. We assembled a list of genes putatively relevant to the recent evolution of fruiting body development by sorting genes whose relative expression across developmental stages increased more in N. crassa relative to the other species. Then, in N. crassa, we characterized the phenotypes of fruiting bodies arising from crosses of homozygous knockout strains of the top genes. Eight N. crassa genes were found to be critical for the successful formation of perithecia. The absence of these genes in these crosses resulted in either no perithecium formation or in arrested development at an early stage. Our results provide insight into the genetic basis of Neurospora sexual reproduction, which is also of great importance with regard to other multicellular ascomycetes, including perithecium-forming pathogens, such as Claviceps purpurea, Ophiostoma ulmi, and Glomerella graminicola.
Project description:BACKGROUND:Fungal fruiting bodies are complex three-dimensional structures that are formed to protect and disperse the sexual spores. Their morphogenesis requires the concerted action of numerous genes; however, at the molecular level, the spatio-temporal sequence of events leading to the mature fruiting body is largely unknown. In previous studies, the transcription factor gene pro44 and the histone chaperone gene asf1 were shown to be essential for fruiting body formation in the ascomycete Sordaria macrospora. Both PRO44 and ASF1 are predicted to act on the regulation of gene expression in the nucleus, and mutants in both genes are blocked at the same stage of development. Thus, we hypothesized that PRO44 and ASF1 might be involved in similar aspects of transcriptional regulation. In this study, we characterized their roles in fruiting body development in more detail. RESULTS:The PRO44 protein forms homodimers, localizes to the nucleus, and is strongly expressed in the outer layers of the developing young fruiting body. Analysis of single and double mutants of asf1 and three other chromatin modifier genes, cac2, crc1, and rtt106, showed that only asf1 is essential for fruiting body formation whereas cac2 and rtt106 might have redundant functions in this process. RNA-seq analysis revealed distinct roles for asf1 and pro44 in sexual development, with asf1 acting as a suppressor of weakly expressed genes during morphogenesis. This is most likely not due to global mislocalization of nucleosomes as micrococcal nuclease-sequencing did not reveal differences in nucleosome spacing and positioning around transcriptional start sites between ?asf1 and the wild type. However, bisulfite sequencing revealed a decrease in DNA methylation in ?asf1, which might be a reason for the observed changes in gene expression. Transcriptome analysis of gene expression in young fruiting bodies showed that pro44 is required for correct expression of genes involved in extracellular metabolism. Deletion of the putative transcription factor gene asm2, which is downregulated in young fruiting bodies of ?pro44, results in defects during ascospore maturation. CONCLUSIONS:In summary, the results indicate distinct roles for the transcription factor PRO44 and the histone chaperone ASF1 in the regulation of sexual development in fungi.
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.
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:Metazoan embryogenesis is controlled by a limited number of signaling modules that are used repetitively at successive developmental stages. The development of social amoebas shows similar reiterated use of cAMP-mediated signaling. In the model Dictyostelium discoideum, secreted cAMP acting on 4 cAMP receptors (cARs1-4) coordinates cell movement during aggregation and fruiting body formation, and induces the expression of aggregation and sporulation genes at consecutive developmental stages. To identify hierarchy in the multiple roles of cAMP, we investigated cAR heterogeneity and function across the social amoeba phylogeny. The gene duplications that yielded cARs 2-4 occurred late in evolution. Many species have only a cAR1 ortholog that duplicated independently in the Polysphondylids and Acytostelids. Disruption of both cAR genes of Polysphondylium pallidum (Ppal) did not affect aggregation, but caused complete collapse of fruiting body morphogenesis. The stunted structures contained disorganized stalk cells, which supported a mass of cysts instead of spores; cAMP triggered spore gene expression in Ppal, but not in the cAR null mutant, explaining its sporulation defect. Encystation is the survival strategy of solitary amoebas, and lower taxa, like Ppal, can still encyst as single cells. Recent findings showed that intracellular cAMP accumulation suffices to trigger encystation, whereas it is a complementary requirement for sporulation. Combined, the data suggest that cAMP signaling in social amoebas evolved from cAMP-mediated encystation in solitary amoebas; cAMP secretion in aggregates prompted the starving cells to form spores and not cysts, and additionally organized fruiting body morphogenesis. cAMP-mediated aggregation was the most recent innovation.
Project description:BACKGROUND: The transition from the vegetative mycelium to the primordium during fruiting body development is the most complex and critical developmental event in the life cycle of many basidiomycete fungi. Understanding the molecular mechanisms underlying this process has long been a goal of research on basidiomycetes. Large scale assessment of the expressed transcriptomes of these developmental stages will facilitate the generation of a more comprehensive picture of the mushroom fruiting process. In this study, we coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development. RESULTS: We evaluated the expression of >3,000 genes in the two respective growth stages and discovered that almost one-third of these genes were preferentially expressed in either stage. This identified a significant turnover of the transcriptome during the course of fruiting body development. Additionally, we annotated more than 79,000 transcription start sites (TSSs) based on the transcriptomes of the mycelium and stage 1 primoridum stages. Patterns of enrichment based on gene annotations from the GO and KEGG databases indicated that various structural and functional protein families were uniquely employed in either stage and that during primordial growth, cellular metabolism is highly up-regulated. Various signaling pathways such as the cAMP-PKA, MAPK and TOR pathways were also identified as up-regulated, consistent with the model that sensing of nutrient levels and the environment are important in this developmental transition. More than 100 up-regulated genes were also found to be unique to mushroom forming basidiomycetes, highlighting the novelty of fruiting body development in the fungal kingdom. CONCLUSIONS: We implicated a wealth of new candidate genes important to early stages of mushroom fruiting development, though their precise molecular functions and biological roles are not yet fully known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea.
Project description:Bailinggu (Pleurotus tuoliensis) is a major, commercially cultivated mushroom and widely used for nutritional, medicinal, and industrial applications. Yet, the mushroom's genetic architecture and the molecular mechanisms underlying its formation are largely unknown. Here we performed comparative transcriptomic analysis during Bailinggu's mycelia, primordia, and fruiting body stages to identify genes regulating fruiting body development and develop EST-SSR markers assessing the genetic value of breeding materials. The stage-specific and differentially expressed unigenes (DEGs) involved in morphogenesis, primary carbohydrate metabolism, cold stimulation and blue-light response were identified using GO and KEGG databases. These unigenes might help Bailinggu adapt to genetic and environmental factors that influence fructification. The most pronounced change in gene expression occurred during the vegetative-to-reproductive transition, suggesting that is most active and key for Bailinggu development. We then developed 26 polymorphic and informative EST-SSR markers to assess the genetic diversity in 82 strains of Bailinggu breeding materials. These EST-SSRs exhibited high transferability in closely related species P. eryngii var. ferulae and var. eryngii. Genetic population structure analysis indicated that China's Bailinggu has low introgression with these two varieties and likely evolved independently. These findings provide new genes, SSR markers, and germplasm to enhance the breeding of commercially cultivated Bailinggu.