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Genomics and transcriptomics to study fruiting body development: An update
f u n g a l b i o l o g y r e v i e w s x x x ( 2 0 1 8 ) 1 e5
journal homepage: www.elsevier.com/locate/fbr
Review
Genomics and transcriptomics to study fruiting body development: An update Minou NOWROUSIAN* €t Bochum, 44780 Bochum, Germany € r Allgemeine und Molekulare Botanik, Ruhr-Universita Lehrstuhl fu
article info
abstract
Article history:
Fruiting bodies of asco- and basidiomycetes are complex three-dimensional structures that
Received 1 August 2017
protect and disperse the sexual spores. Their differentiation requires the concerted action
Received in revised form
of many genes, therefore "omics" techniques to analyze fungal genomes and gene expres-
24 January 2018
sion at a genome-wide level provide excellent means to gain insights into this differentia-
Accepted 23 February 2018
tion process. This review summarizes some recent examples of the use of “omics” techniques to study fruiting body morphogenesis. These include genome-centered ana-
Keywords:
lyses, and studies to analyze the regulation of gene expression including the analysis of
ChIP-seq
RNA editing as a novel layer in the regulation of gene expression during fruiting body
Fruiting body development
development in ascomycetes.
Genomics
ª 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
RNA editing RNA-seq Transcriptomics
1.
Introduction
The fruiting bodies of asco- and basidiomycetes are arguably the most complex multicellular structures that are produced by fungi. Fruiting bodies are the places for differentiation of sexual spores, and the fruiting body structures surrounding € es and the spores aid in their protection and dispersal (Ku € ggeler et al., 2006). Even though many molecular Liu, 2000, Po principles of the spatio-temporal regulation of fruiting body development remain to be discovered, much progress has been made in recent years, aided to a large degree by the application of “omics” techniques. This progress was reviewed some years ago (Nowrousian, 2014), but since then, a number of studies have been published using “omics” methods to gain further insights into the development of complex
multicellular structures in fungi. Therefore, this review will give an update on some recent examples in this area of research. For an overview of “omics” analyses of other aspects of the biology of filamentous fungi, the reader is referred to recent reviews covering different groups of species and fungal life styles (e.g. Kazan and Gardiner, 2017, Motaung et al., 2017, Muszkieta et al., 2013, Toh and Perlin, 2016, Wollenberg and Schirawski, 2014).
2. Genome-centered studies and “omics”related resources Since the advent of next-generation sequencing techniques, de novo sequencing of eukaryotic genomes is no longer
€ t Bochum, Universita € tsstr. 150, 44780 Bochum, Germany. Fax: þ49 € r Allgemeine und Molekulare Botanik, Ruhr-Universita * Lehrstuhl fu 234 3214184. E-mail address: [email protected] https://doi.org/10.1016/j.fbr.2018.02.004 1749-4613/ª 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Nowrousian, M., Genomics and transcriptomics to study fruiting body development: An update, Fungal Biology Reviews (2018), https://doi.org/10.1016/j.fbr.2018.02.004
2
restricted to few model organisms, but is feasible for a wide range of species and can be executed even by small research groups (Nowrousian, 2010). For filamentous fungi, among the first large-scale sequencing projects were the Broad Institute Fungal Genome Initiative as well as several fungal genome projects at the MIPS (Munich Information Center for Protein Sequences); however, many of the original databases are no longer actively maintained (see below). The 1000 Fungal Genomes Project (http://1000.fungalgenomes.org), a collaboration of an international research team with the JGI (Joint Genome Institute), is currently ongoing (Grigoriev et al., 2014). The aim of this project is to sequence at least two reference genomes from each of the more than 500 recognized families of fungi. The genome sequences and annotations are distributed to the research community through the JGI MycoCosm database, together with previously sequenced, publicly available fungal genome sequences. MycoCosm already holds more than 800 fungal genomes (http:// genome.jgi.doe.gov/fungi/fungi.info.html). Smaller genome databases have been established for selected groups of species (e.g. Dang et al., 2015, Kuan et al., 2016). Fungal genome databases will greatly aid future studies of fruiting body differentiation. For example, phylogenomics studies and the identification of orthologous genes and gene families will facilitate the analysis of evolutionary trajectories of gene expression and gene function at different developmental n and Koonin, 2013). Comparastages (Stajich, 2017, Gabaldo tive approaches are dependent on accurate gene models, and algorithms for gene annotation and functional predictions based on transcriptome data as well as comparative genomics are under constant development (e.g. Testa et al., 2015, Umemura et al., 2015, van der Burgt et al., 2014). A question related to the increased number of available genomes is how to provide stable access to the research community. Maintaining and improving databases and corresponding web sites requires funding, which is usually available only for finite amounts of time, as the recent shutdown of websites for Aspergillus and Neurospora genomic resources has made painfully plain to many researchers relying on these resources (Momany, 2016). Fortunately, these and many other species are now hosted by FungiDB, which not only provides genome data, but can also hold, for example, information about gene expression and mutant phenotypes where available (Stajich et al., 2012). It is possible for users to add knowledge about individual genes (e.g. about phenotypes, publications) in the form of user comments, and as manual annotation is still the best, but also among the most timeand therefore cost-intensive parts of data curation, researchers are encouraged to make use of this option to enrich the data available to the community. With respect to fruiting body formation, de novo sequencing and comparative analysis of genomes can yield insights into the evolution of complex multicellular structures. This aspect of genomics was recently demonstrated in an analysis of the genome of the Taphrinomycete Neolecta irregularis (Nguyen et al., 2017). The Taphrinomycetes are an early-branching group of ascomycetes, and while most Taphrinomycetes grow as unicellular yeasts, several genera have evolved complex multicellular structures, which in the case
M. Nowrousian
of Neolecta are reproductive structures resembling the fruiting bodies of filamentous ascomycetes (Pezizomycotina). Surprisingly, the 14.5 Mb Neolecta genome harbors only about 5500 genes, and therefore is more similar to the genomes of ascomycete yeasts than to the more complex filamentous ascomycetes with their larger and more gene-rich genomes. However, with respect to gene content and conservation, N. irregularis differs from the yeasts in that its genome contains homologs of about 1,000 genes that are lost or highly diverged in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae (Nguyen et al., 2017). In contrast, genes involved in hyphal fusion and hyphal pore gating are not generally conserved in N. irregularis, indicating that the evolution of complex multicellularity might have occurred independently in the Neolecta and filamentous ascomycete lineages, based on a core set of genes that could be recruited to this process (Nagy, 2017, Nguyen et al., 2017). Another application of genome sequencing is the identification of causative mutations in mutants generated by non-targeted mutagenesis, e.g. chemical or radiation-based mutagenesis approaches. Recent examples of sequenced mutants with defects in fruiting body formation include the identification of the novel developmental gene spd4 in Sordaria macrospora (Teichert et al., 2017b), and components of NOX (NADPH oxidase) complexes in S. macrospora and P. anserina (Dirschnabel et al., 2014, Lacaze et al., 2015). The noxD gene identified in P. anserina encodes a homolog of the mammalian p22phox protein, a member of NOX complexes in mammals for which no fungal homolog had been identified before (Aguirre and Lambeth, 2010, Marschall and Tudzynski, 2016). Its function in fruiting body formation might be conserved as the corresponding S. macrospora mutant is sterile, and a Botrytis cinerea mutant is unable to produce sclerotia, from which the fruiting bodies of B. cinerea are generated (Nowrousian et al., 2007, Siegmund et al., 2015). The availability of genome sequences has enabled largescale projects to generate deletion mutants in several fungi. With respect to fruiting body formation, especially largescale knockout studies in Neurospora crassa and Fusarium graminearum have been informative. The first large-scale deletion analysis of about 100 transcription factor genes of N. crassa was published in 2006, and a recent study complemented the first by analyzing 273 viable deletion mutants of the predicted 312 transcription factor genes of this species (Carrillo et al., 2017, Colot et al., 2006). F. graminearum contains more than twice the number of transcription factor genes than N. crassa, and the corresponding deletion mutants were studied in a large-scale knockout project (Son et al., 2011). In N. crassa, mutants in genes encoding catalytic subunits of serine/threonine and tyrosine protein phosphatases as well as G proteincoupled receptors were also analyzed already (Cabrera et al., 2015, Ghosh et al., 2014). In all studies, sexual development was one of the phenotypes that were scored during the analysis of mutant phenotypes. The resulting mutant strains that deviate from the wild-type phenotype with respect to fruiting body development make excellent candidates for indepth studies of the molecular functions of the corresponding genes.
Please cite this article in press as: Nowrousian, M., Genomics and transcriptomics to study fruiting body development: An update, Fungal Biology Reviews (2018), https://doi.org/10.1016/j.fbr.2018.02.004
Omics for the analysis of fruiting body development
3. “omics” to analyze the regulation of gene expression A combined approach of transcriptome and large-scale deletion analyses was performed to study potential target genes of the mating type proteins of F. graminearum (Kim et al., 2015). Genes that were differentially expressed in mating type mutants or strains overexpressing the mating type gene MAT1-2-1 were identified by microarray analysis, and 106 differentially expressed genes were further studied by generating the corresponding deletion strains. Of these, 25 showed non-wild type phenotypes during sexual development. A similar approach was taken by the groups of Frances Trail and Jeffrey Townsend based on RNA-seq data of a developmental time course of N. crassa (Wang et al., 2014). Among others, several genes involved in RNA silencing were upregulated during later stages of development, and deletion mutants of these genes showed phenotypes at stages corresponding to their peak expression. In 2017, the Trail/ Townsend groups took this approach one step further by additionally taking into account the evolutionary conservation or divergence of gene expression (Trail et al., 2017). In their study, the researchers predicted ancestral transcriptomes for the last common ancestors of two Fusarium and three Neurospora species. Based on these data, they identified genes that showed lineage-specific increase in expression during sexual development, and studied the functions of the corresponding genes in deletion mutants of N. crassa and F. graminearum. A high proportion of the analyzed mutants showed developmentspecific phenotypes in those lineages with increased expression compared to the predicted ancestral expression, thus confirming the validity of this approach. Apart from the identification of target genes for functional analysis, transcriptome data can also be used to study genome-wide changes in gene expression patterns during development. In metazoa, it was shown that the so-called phylotypic stage, a period in embryonic development during which animal morphology tends to be conserved, correlates with a period of conserved gene expression (Domazet-Loso and Tautz, 2010, Kalinka et al., 2010). Before and after the phylotypic stage, morphology and expression show greater divergence, and consequently this pattern is known as the morphological hourglass pattern. Even though fungi do not have an embryonic stage and evolved complex multicellular structures independently of animals (Knoll, 2011, Niklas, 2014), a recent study in the basidiomycete Coprinopsis cinerea indicated that a similar concept might be applicable in fungi (Cheng et al., 2015). In C. cinerea, the “young fruiting body” stage, which is an intermediate stage in the process leading from fruiting body initials to mature fruiting bodies, expressed the evolutionarily oldest transcriptome, indicating that it might be the analogue of the phylotypic stage in animals. Further studies with additional species are required to determine if a phylotypic stage with conserved morphology and gene expression exists in fungi, and if there is indeed an hourglass pattern of development in fungi. However, not all genome-wide expression patterns are conserved between animals and fungi. In metazoa, it was
3
found through the analysis of various transcriptome datasets that there is a distinct, double-peaked distribution of gene expression, categorizing genes in weakly and highly expressed genes, independent of species and cell type (Hebenstreit et al., 2011). However, an analysis of publicly available transcriptome data from six ascomycetes and one basidiomycete revealed distributions with one to three major peaks, and the number of peaks sometimes varied even within a single species depending on growth conditions (Nowrousian, 2013). Therefore, gene expression patterns in fungi are not generally bimodal. One possible explanation for this difference compared to metazoa might be that gene expression in fungi is highly dynamic, and fine-tuned at the level of transcription not only for individual genes, but also at a global level. Within fungi, a comparative study of gene expression between C. cinerea and the basidiomycetes Laccaria bicolor and Schizophyllum commune revealed conserved expression of about 70 orthologous genes in young fruiting body stages (Plaza et al., 2014). This finding confirms results from previous studies on basidiomycetes and ascomycetes indicating a certain degree of conservation of gene expression during sexual development (e.g. Gesing et al., 2013, Morin et al., 2012, Ohm et al., 2010, Sikhakolli et al., 2012, Traeger et al., 2013). Another focus of the analysis by Plaza and coworkers was on genes encoding defense proteins. Expression of these genes turned out to be tissue-specific, and correlated with the type of antagonists that the tissues were likely to encounter (Plaza et al., 2014). While transcriptomics methods have been used early on to study gene expression during sexual development in fungi (Nowrousian, 2014), other “omics” techniques such as ChIP (chromatin immunoprecipitation)-seq were only recently used to study fruiting body development. Examples include ChIP-seq analyses to identify target genes of the conserved developmental transcription factor PRO1 in S. macrospora and its ortholog ADV-1 in N. crassa (Dekhang et al., 2017, Steffens et al., 2016). For Penicillium chrysogenum, a fungus that was only recently shown to undergo sexual development € hm et al., 2013), ChIP-seq was used to identify target at all (Bo genes of the transcription factors MAT1-1-1 and VelvetA (Becker et al., 2015, 2016).
4. RNA editing: a novel layer in the regulation of gene expression during fruiting body development in ascomycetes While changes in transcript or protein levels that accompany sexual development have been studied for a long time, there are still many additional layers in the regulation of gene expression that have not been analyzed in detail and consequently are not known as factors that influence fruiting body formation. This includes, for example, alternative splicing or translational regulation by upstream ORFs and non-AUG start codons, processes that are known to occur in filamentous fungi, but have not been studied systematically with respect to multicellular development (Ivanov et al., 2017, Kempken, 2013). However, the availability of genome
Please cite this article in press as: Nowrousian, M., Genomics and transcriptomics to study fruiting body development: An update, Fungal Biology Reviews (2018), https://doi.org/10.1016/j.fbr.2018.02.004
4
M. Nowrousian
and genome-derived (e.g. transcriptome) data nowadays quickly extends discoveries made for single genes in a single species to a genome-wide scale and multiple species, thereby establishing regulatory principles. A case in point is the recent discovery of the role of RNA editing in fruiting body formation in ascomycetes by the group of Jin-Rong Xu (Liu et al., 2016). This phenomenon was found in an analysis of mutants of the F. graminearum puk1 gene, which encodes a protein kinase, and deletion of which leads to problems with ascospore morphogenesis and discharge. Unexpectedly, cloning of a puk1 cDNA led to the discovery that the transcript sequence contains two A-to-I (Adenosine to Inosine)-edited nucleotides, which changes two predicted stop codons in a (wrongly) predicted intron to amino acid-encoding codons in the transcript. Further analyses showed that only an edited PUK1 version is functional and able to complement the mutant phenotype. From this initial discovery, the researchers went on and performed RNA-seq with fruiting bodies and vegetative structures. Comparison of the transcriptome and genome data identified a striking 26,000 A-to-I RNA editing sites in fruiting body-derived transcripts, whereas less than 200 were identified in vegetative structures. More than 21,000 of the fruiting body-specific editing sites reside in coding sequences, and the majority of these are predicted to result in an amino acid change (Liu et al., 2016). A follow-up study of the amd1 gene encoding a major facilitator superfamily domain protein confirmed that editing of the corresponding transcript is necessary for its function in ascus maturation and ascospore discharge, thus confirming the important role of RNA editing in sexual development (Cao et al., 2017). Analysis of published transcriptome data of the related species Fusarium verticilloides (Sikhakolli et al., 2012) showed that A-to-I editing also occurs during sexual development of this species (Liu et al., 2016). The Fusarium species belong to the Sordariomycetes, but Ato-I editing during fruiting body development is not restricted to this group. The analysis of RNA-seq data from the Sordariomycete S. macrospora and the Pezizomycete Pyronema confluens showed that A-to-I editing occurs during fruiting body formation in both species, suggesting that this process might be evolutionary conserved in filamentous ascoymcetes (Teichert et al., 2017a). Interestingly, it might also be restricted to this latter group as an analysis of transcriptome data from the Taphrinomycete S. pombe and the basidiomycete Ganoderma lucidum did not reveal any preference for A-to-I editing during sexual development (Teichert et al., 2017a, Zhu et al., 2014). Further studies will be needed to unravel the extent and molecular mechanisms of RNA editing during fruiting body development in fungi.
5.
Conclusions
“omics” techniques allow discovery of genome-wide events including unexpected layers of gene expression regulation (e.g. RNA editing), and in combination with molecular biology (e.g. generation of deletion mutants) and genetic resources (e.g. libraries of non-targeted mutants) are a powerful means to unravel the still enigmatic principles of multicellular development in fungi.
Conflicts of interest None.
Acknowledgements € ck for support at the DepartI would like to thank Ulrich Ku ment of General and Molecular Botany, and the German Research Foundation for funding (DFG, grant NO407/7-1).
references
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Omics for the analysis of fruiting body development
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journal homepage: www.elsevier.com/locate/fbr
Review
Genomics and transcriptomics to study fruiting body development: An update Minou NOWROUSIAN* €t Bochum, 44780 Bochum, Germany € r Allgemeine und Molekulare Botanik, Ruhr-Universita Lehrstuhl fu
article info
abstract
Article history:
Fruiting bodies of asco- and basidiomycetes are complex three-dimensional structures that
Received 1 August 2017
protect and disperse the sexual spores. Their differentiation requires the concerted action
Received in revised form
of many genes, therefore "omics" techniques to analyze fungal genomes and gene expres-
24 January 2018
sion at a genome-wide level provide excellent means to gain insights into this differentia-
Accepted 23 February 2018
tion process. This review summarizes some recent examples of the use of “omics” techniques to study fruiting body morphogenesis. These include genome-centered ana-
Keywords:
lyses, and studies to analyze the regulation of gene expression including the analysis of
ChIP-seq
RNA editing as a novel layer in the regulation of gene expression during fruiting body
Fruiting body development
development in ascomycetes.
Genomics
ª 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
RNA editing RNA-seq Transcriptomics
1.
Introduction
The fruiting bodies of asco- and basidiomycetes are arguably the most complex multicellular structures that are produced by fungi. Fruiting bodies are the places for differentiation of sexual spores, and the fruiting body structures surrounding € es and the spores aid in their protection and dispersal (Ku € ggeler et al., 2006). Even though many molecular Liu, 2000, Po principles of the spatio-temporal regulation of fruiting body development remain to be discovered, much progress has been made in recent years, aided to a large degree by the application of “omics” techniques. This progress was reviewed some years ago (Nowrousian, 2014), but since then, a number of studies have been published using “omics” methods to gain further insights into the development of complex
multicellular structures in fungi. Therefore, this review will give an update on some recent examples in this area of research. For an overview of “omics” analyses of other aspects of the biology of filamentous fungi, the reader is referred to recent reviews covering different groups of species and fungal life styles (e.g. Kazan and Gardiner, 2017, Motaung et al., 2017, Muszkieta et al., 2013, Toh and Perlin, 2016, Wollenberg and Schirawski, 2014).
2. Genome-centered studies and “omics”related resources Since the advent of next-generation sequencing techniques, de novo sequencing of eukaryotic genomes is no longer
€ t Bochum, Universita € tsstr. 150, 44780 Bochum, Germany. Fax: þ49 € r Allgemeine und Molekulare Botanik, Ruhr-Universita * Lehrstuhl fu 234 3214184. E-mail address: [email protected] https://doi.org/10.1016/j.fbr.2018.02.004 1749-4613/ª 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Nowrousian, M., Genomics and transcriptomics to study fruiting body development: An update, Fungal Biology Reviews (2018), https://doi.org/10.1016/j.fbr.2018.02.004
2
restricted to few model organisms, but is feasible for a wide range of species and can be executed even by small research groups (Nowrousian, 2010). For filamentous fungi, among the first large-scale sequencing projects were the Broad Institute Fungal Genome Initiative as well as several fungal genome projects at the MIPS (Munich Information Center for Protein Sequences); however, many of the original databases are no longer actively maintained (see below). The 1000 Fungal Genomes Project (http://1000.fungalgenomes.org), a collaboration of an international research team with the JGI (Joint Genome Institute), is currently ongoing (Grigoriev et al., 2014). The aim of this project is to sequence at least two reference genomes from each of the more than 500 recognized families of fungi. The genome sequences and annotations are distributed to the research community through the JGI MycoCosm database, together with previously sequenced, publicly available fungal genome sequences. MycoCosm already holds more than 800 fungal genomes (http:// genome.jgi.doe.gov/fungi/fungi.info.html). Smaller genome databases have been established for selected groups of species (e.g. Dang et al., 2015, Kuan et al., 2016). Fungal genome databases will greatly aid future studies of fruiting body differentiation. For example, phylogenomics studies and the identification of orthologous genes and gene families will facilitate the analysis of evolutionary trajectories of gene expression and gene function at different developmental n and Koonin, 2013). Comparastages (Stajich, 2017, Gabaldo tive approaches are dependent on accurate gene models, and algorithms for gene annotation and functional predictions based on transcriptome data as well as comparative genomics are under constant development (e.g. Testa et al., 2015, Umemura et al., 2015, van der Burgt et al., 2014). A question related to the increased number of available genomes is how to provide stable access to the research community. Maintaining and improving databases and corresponding web sites requires funding, which is usually available only for finite amounts of time, as the recent shutdown of websites for Aspergillus and Neurospora genomic resources has made painfully plain to many researchers relying on these resources (Momany, 2016). Fortunately, these and many other species are now hosted by FungiDB, which not only provides genome data, but can also hold, for example, information about gene expression and mutant phenotypes where available (Stajich et al., 2012). It is possible for users to add knowledge about individual genes (e.g. about phenotypes, publications) in the form of user comments, and as manual annotation is still the best, but also among the most timeand therefore cost-intensive parts of data curation, researchers are encouraged to make use of this option to enrich the data available to the community. With respect to fruiting body formation, de novo sequencing and comparative analysis of genomes can yield insights into the evolution of complex multicellular structures. This aspect of genomics was recently demonstrated in an analysis of the genome of the Taphrinomycete Neolecta irregularis (Nguyen et al., 2017). The Taphrinomycetes are an early-branching group of ascomycetes, and while most Taphrinomycetes grow as unicellular yeasts, several genera have evolved complex multicellular structures, which in the case
M. Nowrousian
of Neolecta are reproductive structures resembling the fruiting bodies of filamentous ascomycetes (Pezizomycotina). Surprisingly, the 14.5 Mb Neolecta genome harbors only about 5500 genes, and therefore is more similar to the genomes of ascomycete yeasts than to the more complex filamentous ascomycetes with their larger and more gene-rich genomes. However, with respect to gene content and conservation, N. irregularis differs from the yeasts in that its genome contains homologs of about 1,000 genes that are lost or highly diverged in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae (Nguyen et al., 2017). In contrast, genes involved in hyphal fusion and hyphal pore gating are not generally conserved in N. irregularis, indicating that the evolution of complex multicellularity might have occurred independently in the Neolecta and filamentous ascomycete lineages, based on a core set of genes that could be recruited to this process (Nagy, 2017, Nguyen et al., 2017). Another application of genome sequencing is the identification of causative mutations in mutants generated by non-targeted mutagenesis, e.g. chemical or radiation-based mutagenesis approaches. Recent examples of sequenced mutants with defects in fruiting body formation include the identification of the novel developmental gene spd4 in Sordaria macrospora (Teichert et al., 2017b), and components of NOX (NADPH oxidase) complexes in S. macrospora and P. anserina (Dirschnabel et al., 2014, Lacaze et al., 2015). The noxD gene identified in P. anserina encodes a homolog of the mammalian p22phox protein, a member of NOX complexes in mammals for which no fungal homolog had been identified before (Aguirre and Lambeth, 2010, Marschall and Tudzynski, 2016). Its function in fruiting body formation might be conserved as the corresponding S. macrospora mutant is sterile, and a Botrytis cinerea mutant is unable to produce sclerotia, from which the fruiting bodies of B. cinerea are generated (Nowrousian et al., 2007, Siegmund et al., 2015). The availability of genome sequences has enabled largescale projects to generate deletion mutants in several fungi. With respect to fruiting body formation, especially largescale knockout studies in Neurospora crassa and Fusarium graminearum have been informative. The first large-scale deletion analysis of about 100 transcription factor genes of N. crassa was published in 2006, and a recent study complemented the first by analyzing 273 viable deletion mutants of the predicted 312 transcription factor genes of this species (Carrillo et al., 2017, Colot et al., 2006). F. graminearum contains more than twice the number of transcription factor genes than N. crassa, and the corresponding deletion mutants were studied in a large-scale knockout project (Son et al., 2011). In N. crassa, mutants in genes encoding catalytic subunits of serine/threonine and tyrosine protein phosphatases as well as G proteincoupled receptors were also analyzed already (Cabrera et al., 2015, Ghosh et al., 2014). In all studies, sexual development was one of the phenotypes that were scored during the analysis of mutant phenotypes. The resulting mutant strains that deviate from the wild-type phenotype with respect to fruiting body development make excellent candidates for indepth studies of the molecular functions of the corresponding genes.
Please cite this article in press as: Nowrousian, M., Genomics and transcriptomics to study fruiting body development: An update, Fungal Biology Reviews (2018), https://doi.org/10.1016/j.fbr.2018.02.004
Omics for the analysis of fruiting body development
3. “omics” to analyze the regulation of gene expression A combined approach of transcriptome and large-scale deletion analyses was performed to study potential target genes of the mating type proteins of F. graminearum (Kim et al., 2015). Genes that were differentially expressed in mating type mutants or strains overexpressing the mating type gene MAT1-2-1 were identified by microarray analysis, and 106 differentially expressed genes were further studied by generating the corresponding deletion strains. Of these, 25 showed non-wild type phenotypes during sexual development. A similar approach was taken by the groups of Frances Trail and Jeffrey Townsend based on RNA-seq data of a developmental time course of N. crassa (Wang et al., 2014). Among others, several genes involved in RNA silencing were upregulated during later stages of development, and deletion mutants of these genes showed phenotypes at stages corresponding to their peak expression. In 2017, the Trail/ Townsend groups took this approach one step further by additionally taking into account the evolutionary conservation or divergence of gene expression (Trail et al., 2017). In their study, the researchers predicted ancestral transcriptomes for the last common ancestors of two Fusarium and three Neurospora species. Based on these data, they identified genes that showed lineage-specific increase in expression during sexual development, and studied the functions of the corresponding genes in deletion mutants of N. crassa and F. graminearum. A high proportion of the analyzed mutants showed developmentspecific phenotypes in those lineages with increased expression compared to the predicted ancestral expression, thus confirming the validity of this approach. Apart from the identification of target genes for functional analysis, transcriptome data can also be used to study genome-wide changes in gene expression patterns during development. In metazoa, it was shown that the so-called phylotypic stage, a period in embryonic development during which animal morphology tends to be conserved, correlates with a period of conserved gene expression (Domazet-Loso and Tautz, 2010, Kalinka et al., 2010). Before and after the phylotypic stage, morphology and expression show greater divergence, and consequently this pattern is known as the morphological hourglass pattern. Even though fungi do not have an embryonic stage and evolved complex multicellular structures independently of animals (Knoll, 2011, Niklas, 2014), a recent study in the basidiomycete Coprinopsis cinerea indicated that a similar concept might be applicable in fungi (Cheng et al., 2015). In C. cinerea, the “young fruiting body” stage, which is an intermediate stage in the process leading from fruiting body initials to mature fruiting bodies, expressed the evolutionarily oldest transcriptome, indicating that it might be the analogue of the phylotypic stage in animals. Further studies with additional species are required to determine if a phylotypic stage with conserved morphology and gene expression exists in fungi, and if there is indeed an hourglass pattern of development in fungi. However, not all genome-wide expression patterns are conserved between animals and fungi. In metazoa, it was
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found through the analysis of various transcriptome datasets that there is a distinct, double-peaked distribution of gene expression, categorizing genes in weakly and highly expressed genes, independent of species and cell type (Hebenstreit et al., 2011). However, an analysis of publicly available transcriptome data from six ascomycetes and one basidiomycete revealed distributions with one to three major peaks, and the number of peaks sometimes varied even within a single species depending on growth conditions (Nowrousian, 2013). Therefore, gene expression patterns in fungi are not generally bimodal. One possible explanation for this difference compared to metazoa might be that gene expression in fungi is highly dynamic, and fine-tuned at the level of transcription not only for individual genes, but also at a global level. Within fungi, a comparative study of gene expression between C. cinerea and the basidiomycetes Laccaria bicolor and Schizophyllum commune revealed conserved expression of about 70 orthologous genes in young fruiting body stages (Plaza et al., 2014). This finding confirms results from previous studies on basidiomycetes and ascomycetes indicating a certain degree of conservation of gene expression during sexual development (e.g. Gesing et al., 2013, Morin et al., 2012, Ohm et al., 2010, Sikhakolli et al., 2012, Traeger et al., 2013). Another focus of the analysis by Plaza and coworkers was on genes encoding defense proteins. Expression of these genes turned out to be tissue-specific, and correlated with the type of antagonists that the tissues were likely to encounter (Plaza et al., 2014). While transcriptomics methods have been used early on to study gene expression during sexual development in fungi (Nowrousian, 2014), other “omics” techniques such as ChIP (chromatin immunoprecipitation)-seq were only recently used to study fruiting body development. Examples include ChIP-seq analyses to identify target genes of the conserved developmental transcription factor PRO1 in S. macrospora and its ortholog ADV-1 in N. crassa (Dekhang et al., 2017, Steffens et al., 2016). For Penicillium chrysogenum, a fungus that was only recently shown to undergo sexual development € hm et al., 2013), ChIP-seq was used to identify target at all (Bo genes of the transcription factors MAT1-1-1 and VelvetA (Becker et al., 2015, 2016).
4. RNA editing: a novel layer in the regulation of gene expression during fruiting body development in ascomycetes While changes in transcript or protein levels that accompany sexual development have been studied for a long time, there are still many additional layers in the regulation of gene expression that have not been analyzed in detail and consequently are not known as factors that influence fruiting body formation. This includes, for example, alternative splicing or translational regulation by upstream ORFs and non-AUG start codons, processes that are known to occur in filamentous fungi, but have not been studied systematically with respect to multicellular development (Ivanov et al., 2017, Kempken, 2013). However, the availability of genome
Please cite this article in press as: Nowrousian, M., Genomics and transcriptomics to study fruiting body development: An update, Fungal Biology Reviews (2018), https://doi.org/10.1016/j.fbr.2018.02.004
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M. Nowrousian
and genome-derived (e.g. transcriptome) data nowadays quickly extends discoveries made for single genes in a single species to a genome-wide scale and multiple species, thereby establishing regulatory principles. A case in point is the recent discovery of the role of RNA editing in fruiting body formation in ascomycetes by the group of Jin-Rong Xu (Liu et al., 2016). This phenomenon was found in an analysis of mutants of the F. graminearum puk1 gene, which encodes a protein kinase, and deletion of which leads to problems with ascospore morphogenesis and discharge. Unexpectedly, cloning of a puk1 cDNA led to the discovery that the transcript sequence contains two A-to-I (Adenosine to Inosine)-edited nucleotides, which changes two predicted stop codons in a (wrongly) predicted intron to amino acid-encoding codons in the transcript. Further analyses showed that only an edited PUK1 version is functional and able to complement the mutant phenotype. From this initial discovery, the researchers went on and performed RNA-seq with fruiting bodies and vegetative structures. Comparison of the transcriptome and genome data identified a striking 26,000 A-to-I RNA editing sites in fruiting body-derived transcripts, whereas less than 200 were identified in vegetative structures. More than 21,000 of the fruiting body-specific editing sites reside in coding sequences, and the majority of these are predicted to result in an amino acid change (Liu et al., 2016). A follow-up study of the amd1 gene encoding a major facilitator superfamily domain protein confirmed that editing of the corresponding transcript is necessary for its function in ascus maturation and ascospore discharge, thus confirming the important role of RNA editing in sexual development (Cao et al., 2017). Analysis of published transcriptome data of the related species Fusarium verticilloides (Sikhakolli et al., 2012) showed that A-to-I editing also occurs during sexual development of this species (Liu et al., 2016). The Fusarium species belong to the Sordariomycetes, but Ato-I editing during fruiting body development is not restricted to this group. The analysis of RNA-seq data from the Sordariomycete S. macrospora and the Pezizomycete Pyronema confluens showed that A-to-I editing occurs during fruiting body formation in both species, suggesting that this process might be evolutionary conserved in filamentous ascoymcetes (Teichert et al., 2017a). Interestingly, it might also be restricted to this latter group as an analysis of transcriptome data from the Taphrinomycete S. pombe and the basidiomycete Ganoderma lucidum did not reveal any preference for A-to-I editing during sexual development (Teichert et al., 2017a, Zhu et al., 2014). Further studies will be needed to unravel the extent and molecular mechanisms of RNA editing during fruiting body development in fungi.
5.
Conclusions
“omics” techniques allow discovery of genome-wide events including unexpected layers of gene expression regulation (e.g. RNA editing), and in combination with molecular biology (e.g. generation of deletion mutants) and genetic resources (e.g. libraries of non-targeted mutants) are a powerful means to unravel the still enigmatic principles of multicellular development in fungi.
Conflicts of interest None.
Acknowledgements € ck for support at the DepartI would like to thank Ulrich Ku ment of General and Molecular Botany, and the German Research Foundation for funding (DFG, grant NO407/7-1).
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