Using evolutionary genomics, transcriptomics, and systems biology to reveal gene networks underlying fungal development

Highlights

• Diverse model species contribute to research on the evolution of fungal development.

• Transcriptomics facilitates study of developmental evolution for non-model fungi.

• Evolution of expression underlying evolved phenotypes can guide functional analysis.

• Bayesian networks predict promising experiments using integrative systems biology.

• The future of fungal evo-devo lies with systems biology of developmental traits.

Abstract

Fungal model species have contributed to many aspects of modern biology, from biochemistry and cell biology to molecular genetics. Nevertheless, only a few genes associated with morphological development in fungi have been functionally characterized in terms of their genetic or molecular interactions. Evolutionary developmental biology in fungi faces challenges from a lack of fossil records and unresolved species phylogeny, to homoplasy associated with simple morphology. Traditionally, reductive approaches use genetic screens to reveal phenotypes from a large number of mutants; the efficiency of these approaches relies on profound prior knowledge of the genetics and biology of the designated development trait—knowledge which is often not available for even well-studied fungal model species. Reductive approaches become less efficient for the study of developmental traits that are regulated quantitatively by more than one gene via networks. Recent advances in genome-wide analysis performed in representative multicellular fungal models and non-models have greatly improved upon the traditional reductive approaches in fungal evo-devo research by providing clues for focused knockout strategies. In particular, genome-wide gene expression data across developmental processes of interest in multiple species can expedite the advancement of integrative synthetic and systems biology strategies to reveal regulatory networks underlying fungal development.

Introduction

“Morphology was studied because it was the material believed to be the most favorable for the elucidation of the problems of evolution…” (Bateson, 1922)

Developmental biology, one of the most longstanding and deeply examined subdisciplines in biology, reveals how complex life forms are constructed by the growth, repositioning, structure, and identity of cells and tissues. Examination of morphological development has been vigorous in almost every major organismal group, and knowledge of developmental biology has accumulated along with advances in cell biology, developmental genetics, and the systems biology. Nevertheless, we remain ignorant of some of the basic elements of how a body shape is planned and formed in response to genetic and environmental signals—even in well studied models.

Evolutionary biology and developmental biology have long been considered complementary disciplines, and their synthesis has recently been boosted by their merger into the exciting field of evolutionary developmental biology (a.k.a. Evo-Devo)—research that investigates the developmental processes and the evolution of those processes among different organisms (Hall, 2012). Among the oldest models in cell biology, fungi have contributed significantly to evolutionary developmental biology and in recent times continue to serve as highly tractable workhorses for genetics and genomics, and now systems biology revolution (Bennett and Arnold, 2001).

Fungi represent one of the most diverse organismal groups on earth in terms of their ecology, their ubiquity, and their manifold morphological forms. Among eukaryotes, fungi are relatively simple in terms of their cell and tissue types, along with high genome diversity accompanying their wide ecological distribution (Alexopoulos, 2007, Blackwell, 2011, Mohanta and Bae, 2015, Stajich et al., 2009, Taylor et al., 2017, Zeng et al., 2017). Fungal development encompasses a very wide range of complexity, ranging from unicellular yeasts that can be found in flowers and guts of beetles and wasps (Lachance et al., 2013, Stefanini et al., 2012), to the largest known fruiting body of a wood decaying fungus—weighing nearly 500 kg (Dai and Cui, 2011), to postfire fungi that blossom quickly and transiently after forest fires (Glassman et al., 2016), to the oldest known mycelium—over 1500 years in age and 15 ha in occupied area (Smith et al., 1992). In addition, morphological divergence, which in fungi commonly includes the evolution of morphologies of highly reduced complexity, can make it challenging to identify orthologous structures among even closely related lineages (Wang et al., 2016). Many higher fungi produce elaborate sexual reproductive structures predominantly for survival during their sexual life cycles, but during their asexual cycle these same fungi produce simple but vigorous structures that generate myriad, short lived asexual spores for rapid dispersal. Convergent and divergent evolution of phenotypes are not rare in fungi (Alexopoulos, 2007, Nagy et al., 2014, Shang et al., 2016, Torruella et al., 2015), often resulting from diverse ecological pressures on a limited set of available simple reduced morphologies. For example, many leaf endophytic fungi produce dark-colored, covered, small fruiting bodies, while their saprotrophic relatives produce bright-colored, exposed, large fruiting bodies (Ruibal et al., 2008, Wang et al., 2009). Like plants and animals, some fungi—despite their more limited palette of cell and tissue types—have evolved structures for specific functions that are not common to other fungi. Remarkable examples of these innovative morphologies include the specialized and diverse penetration structures (appressoria) present in many plant pathogenic fungi (Ames, 2017, Geoghegan et al., 2017, Mendgen et al., 1996), the mycorrhizal nutrient bridges that develop between mushroom-forming fungi and their associated plants (Bravo et al., 2017, Iwaniuk and Błaszkowski, 2014, Jiang et al., 2017, Marks, 2012), differentiation of wood decay models among higher fungi (Floudas et al., 2012, Hibbett and Donoghue, 2001, Nagy et al., 2016), and the trapping rings, nets and adhesive hyphae unique to the nematode-trapping fungi in the Orbilliomycetes (Hyde et al., 2014).

Fungal species have supplied model systems that have advanced multiple disciplines. Many of the fungi selected and developed as model systems share similar features: fast growth, short life cycle, manipulability in a laboratory setting, and distinctive morphology facilitating identification. Because these traits are not unusual in fungal species, the model species are often also of direct economic importance. The best-known and most well-studied experimental fungal model species are the yeasts Saccharomyces cerevisiae (bread and wine yeast) and Schizosaccharomyces pombe (Egel, 2013, Rose, 1981), which are important tools in molecular genetics, as well as human associated Candida species (Ene et al., 2016, Prasad, 2017). As single-celled eukaryotes, these yeasts serve as powerful model systems for the investigation of numerous fundamental principles and mechanisms, including but not limited to cell-cycle control, mitosis and meiosis tool kits, genome organization, epigenetics and epigenomics, DNA recombination and repair, signal transduction, population genetics, and the aging process (Boynton et al., 2017, Caudy et al., 2017, Egel, 2013, Fuchs and Quasem, 2014, Salehzadeh-Yazdi et al., 2014, Tsubouchi, 2006). A global genetic interaction network based on S. cerevisiae provides a wiring diagram of cellular function (Costanzo et al., 2016). Yeasts also are models for developmental biology, especially for cell-to-cell communication, cell polarity, and budding and mating processes, although the scope of their contributions has been restricted to cellular development (Drubin, 1991, Liti, 2014, Tomičić and Raspor, 2017, Winters and Chiang, 2016). Representatives of long-time multicellular fungal models for developmental biology include the ascomycetous species Aspergillus nidulans (Braus et al., 2002, Croft, 1966, Seo, 2005, Timberlake, 1993) and Neurospora crassa (Aramayo and Selker, 2013, Davis, 2000, Davis and Perkins, 2002, Mitchell, 1955, Selker, 2017) and their closely related species, and basidiomycetous species including Schizophellum commune (Casselton and Kües, 2007, Essig, 1922, Wessels, 1989), Coprinopsis cinerea (Plaza et al., 2014), and Coprinus comatus (Casselton and Kües, 2007, Junjie-Yuan and Pingping-Li, 2010, Winterboer and Eicker, 1983). Among these multicellular models, N. crassa was the first one whose genome was sequenced, a tribute to its longstanding position as a model for genetics, beginning with its role in the formulation of the “one gene–one enzyme” hypothesis proposed by Edward Tatum and George Wells Beadle (1941), who—for this insight—won the Nobel Prize in Physiology or Medicine in 1958 (Davis and Perkins, 2002). Unlike yeasts and Aspergillus species, N. crassa has little importance in medical, agriculture and food industries—although it is consumed as ontjom (Beuchat, 1976), and spurred by its model status, some have investigated its potential in biofuel development and biotechnology (Benz et al., 2014, Roche et al., 2014, Seibert et al., 2016, Tian et al., 2009). Studies of the developmental biology of N. crassa have been mainly focused on asexual development, although over many years, numerous sexual development- and meiosis-related genes have been characterized (Borkovich et al., 2004, Iyer et al., 2009, Krystofova and Borkovich, 2006, Lehr et al., 2014, Springer, 1993, Springer and Yanofsky, 1992, Stajich, 2014, Vigfusson and Weijer, 1972, Wang et al., 2012, Wang et al., 2014).

Compared with most plant or animal models, the genomes of fungal model species are comparatively small and simple. Nevertheless, they retain genes for most of the core metabolism, conserved pathways, and cellular developmental regulatory mechanisms in eukaryotes (Brown, 2006). For research at the cellular development level, yeast definitely exemplifies “a model for all eukaryotic biology derives from the facility with which the relation between gene structure and protein function can be established” (Botstein and Fink, 1988, Botstein and Fink, 2011). Early genetics in yeast models reaped benefits from approaches pioneered to generate biochemical mutants in Neurospora, which had been a model to study nitrogen, sulfur, and phosphate metabolism for eukaryotes seven decades ago (Beadle and Tatum, 1941), and more recently a model for understanding recombination, differentiation (Borkovich et al., 2004), silencing and DNA methylation (Honda et al., 2016), morphogenesis and cell biology (Kronholm et al., 2016), and circadian rhythms (Borkovich et al., 2004, Dunlap, 2008). Circadian rhythm studies in N. crassa resulted in the cloning of the first clock genes in the early 1980s (Dunlap et al., 2007a, Dunlap et al., 2007b). Hall and Rosbash later identified the period gene from fruit flies, work that led to a recent Nobel prize shared by Hall, Rosbach, and Young. Discovery of the fungal circadian system occurred more than a decade prior to the cloning of the first human clock genes (Antoch et al., 1997). With the traditional gene by gene approach, a community of hard-working Neurospora geneticists contributed knowledge of 1100 genes with phenotypes over 60 years, before the recent high-throughput approach that produced 11,000 knockout strains providing a means to knowledge of function in over 9600 genes (Collopy et al., 2010). Recent studies on Neurospora and Aspergillus shed light on how internal clock-related oscillations and asexual development are regulated in response to environmental signals in fungi (Dasgupta et al., 2015, Hong et al., 2014, Montenegro-Montero et al., 2015, Rokas, 2013). Fungal models have also been used to understand how sex may have evolved in eukaryotes (Heitman, 2015). Many of the core methods of genomics and systems biology applied to eukaryotic systems were first developed from fungal genetics, including physical mapping, pulsed field gel electrophoresis, knockout collections, signature tagged mutagenesis, genome assembly methods, transcriptomic profiling, proteomic profiling, and genome-scale identification of protein-DNA interactions (Kück, 2013).

Other fungal models have also been examined to illuminate fungal multicellular developmental biology. For example, whether an “hourglass” model of the conservation of expression of developmentally-related genes appears in fungi like it appears in other eukaryotes (Drost et al., 2015, Kalinka et al., 2010, Ninova et al., 2014, Quint et al., 2012) was investigated using the model mushroom-forming fungus C. cinerea (Cheng et al., 2015). Aspergillus species have been intensively studied for their asexual growth—mainly because secondary metabolic products, such as mycotoxins, antibiotics and citric acids, are enriched during late asexual development (Adams and Yu, 1998, Bayram et al., 2016, Garzia et al., 2013, Keller, 2006, Yin et al., 2013). Some fungi do not form fruiting bodies in laboratory conditions, implying that unknown environmental factors are involved in the growth and development in these fungi. For instance, metagenomic evidence suggests that the composition of bacterial communities is associated with the differentiation and development of sexual structures in black truffle fungus (Antony-Babu et al., 2014). Once the genome sequencing of N. crassa was complete (Galagan et al., 2003), genome sequences of Aspergillus species (Galagan et al., 2005, Machida et al., 2005, Nierman et al., 2005, Ronning et al., 2005, Yu et al., 2005), Magnaporthe oryzae (Dean et al., 2005), and Fusarium graminearum (Cuomo et al., 2007) quickly followed. These core filamentous ascomycete species established a jumping board to study previously intractable species and elucidate host–pathogen interactions in both human and plants, development of infection-associated structures, and hyphal development and sporulation, both as conidia and sexual fruiting bodies. These advances in core filamentous species enabled study of development and sporulation in the obligately biotrophic pathogens such as rusts (Cuomo et al., 2007, Yin et al., 2015) and powdery mildews, and to determine the adaptive genome evolution of Malessizia, the human skin pathogen in the Ustilaginomycotina, a subphylum of predominantly plant smut fungi (Wu et al., 2015). Study of fungal development has led to the realization that regulation of secondary metabolite production and development are correlated (Keller et al., 2005), and the global regulator complex, VelB/VeA/LaeA, that coordinates light signaling with development also regulates secondary metabolism through chromatin remodeling mediated by LaeA (Bayram et al., 2016). Very recently, regulators of the stages of asexual sporulation have been shown to also regulate the genes for secondary metabolites that accumulate during those stages of development (Lind et al., 2017).