Tools for live-cell imaging of cytoskeletal and nuclear behavior in the unconventional yeast, Aureobasidium pullulans.

Aureobasidium pullulans is a ubiquitous fungus with a wide variety of morphologies and growth modes including "typical" single-budding yeast, and interestingly, larger multinucleate yeast than can make multiple buds in a single cell cycle. The study of A. pullulans promises to uncover novel cell biology, but currently tools are lacking to achieve this goal. Here, we describe initial components of a cell biology toolkit for A. pullulans, which is used to express and image fluorescent probes for nuclei as well as components of the cytoskeleton. These tools allowed live-cell imaging of the multinucleate and multibudding cycles, revealing highly synchronous mitoses in multinucleate yeast that occur in a semiopen manner with an intact but permeable nuclear envelope. These findings open the door to using this ubiquitous polyextremotolerant fungus as a model for evolutionary cell biology.

Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

A guide to Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus.

Chytrid fungi play key ecological roles in aquatic ecosystems, and some species cause a devastating skin disease in frogs and salamanders. Additionally, chytrids occupy a unique phylogenetic position- sister to the well-studied Dikarya (the group including yeasts, sac fungi, and mushrooms) and related to animals- making chytrids useful for answering important evolutionary questions. Despite their importance, little is known about the basic cell biology of chytrids. A major barrier to understanding chytrid biology has been a lack of genetic tools with which to test molecular hypotheses. Medina and colleagues recently developed a protocol for Agrobacterium -mediated transformation of Spizellomyces punctatus. In this manuscript, we describe the general procedure including planning steps and expected results. We also provide in-depth, step-by-step protocols and video guides for performing the entirety of this transformation procedure on protocols.io (dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v1).

Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology.

Chytrids are early-diverging fungi that share features with animals that have been lost in most other fungi. They hold promise as a system to study fungal and animal evolution, but we lack genetic tools for hypothesis testing. Here, we generated transgenic lines of the chytrid Spizellomyces punctatus, and used fluorescence microscopy to explore chytrid cell biology and development during its life cycle. We show that the chytrid undergoes multiple rounds of synchronous nuclear division, followed by cellularization, to create and release many daughter 'zoospores'. The zoospores, akin to animal cells, crawl using actin-mediated cell migration. After forming a cell wall, polymerized actin reorganizes into fungal-like cortical patches and cables that extend into hyphal-like structures. Actin perinuclear shells form each cell cycle and polygonal territories emerge during cellularization. This work makes Spizellomyces a genetically tractable model for comparative cell biology and understanding the evolution of fungi and early eukaryotes.

Chytrid fungi.

Medina and Buchler provide an introduction to chytrid fungi, an early diverging fungal lineage exhibiting characteristics found in both animals and fungi.

Evolutionary innovation, fungal cell biology, and the lateral gene transfer of a viral KilA-N domain.

Fungi are found in diverse ecological niches as primary decomposers, mutualists, or parasites of plants and animals. Although animals and fungi share a common ancestor, fungi dramatically diversified their life cycle, cell biology, and metabolism as they evolved and colonized new niches. This review focuses on a family of fungal transcription factors (Swi4/Mbp1, APSES, Xbp1, Bqt4) derived from the lateral gene transfer of a KilA-N domain commonly found in prokaryotic and eukaryotic DNA viruses. These virus-derived fungal regulators play central roles in cell cycle, morphogenesis, sexual differentiation, and quiescence. We consider the possible origins of KilA-N and how this viral DNA binding domain came to be intimately associated with fungal processes.

The evolution of a G1/S transcriptional network in yeasts.

The G1-to-S cell cycle transition is promoted by the periodic expression of a large set of genes. In Saccharomyces cerevisiae G1/S gene expression is regulated by two transcription factor (TF) complexes, the MBF and SBF, which bind to specific DNA sequences, the MCB and SCB, respectively. Despite extensive research little is known regarding the evolution of the G1/S transcription regulation including the co-evolution of the DNA binding domains with their respective DNA binding sequences. We have recently examined the co-evolution of the G1/S TF specificity through the systematic generation and examination of chimeric Mbp1/Swi4 TFs containing different orthologue DNA binding domains in S. cerevisiae (Hendler et al. in PLoS Genet 13:e1006778. doi: 10.1371/journal.pgen.1006778 , 2017). Here, we review the co-evolution of G1/S transcriptional network and discuss the evolutionary dynamics and specificity of the MBF-MCB and SBF-SCB interactions in different fungal species.

Gene duplication and co-evolution of G1/S transcription factor specificity in fungi are essential for optimizing cell fitness.

Transcriptional regulatory networks play a central role in optimizing cell survival. How DNA binding domains and cis-regulatory DNA binding sequences have co-evolved to allow the expansion of transcriptional networks and how this contributes to cellular fitness remains unclear. Here we experimentally explore how the complex G1/S transcriptional network evolved in the budding yeast Saccharomyces cerevisiae by examining different chimeric transcription factor (TF) complexes. Over 200 G1/S genes are regulated by either one of the two TF complexes, SBF and MBF, which bind to specific DNA binding sequences, SCB and MCB, respectively. The difference in size and complexity of the G1/S transcriptional network across yeast species makes it well suited to investigate how TF paralogs (SBF and MBF) and DNA binding sequences (SCB and MCB) co-evolved after gene duplication to rewire and expand the network of G1/S target genes. Our data suggests that whilst SBF is the likely ancestral regulatory complex, the ancestral DNA binding element is more MCB-like. G1/S network expansion took place by both cis- and trans- co-evolutionary changes in closely related but distinct regulatory sequences. Replacement of the endogenous SBF DNA-binding domain (DBD) with that from more distantly related fungi leads to a contraction of the SBF-regulated G1/S network in budding yeast, which also correlates with increased defects in cell growth, cell size, and proliferation.

Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family.

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi.

Although cell cycle control is an ancient, conserved, and essential process, some core animal and fungal cell cycle regulators share no more sequence identity than non-homologous proteins. Here, we show that evolution along the fungal lineage was punctuated by the early acquisition and entrainment of the SBF transcription factor through horizontal gene transfer. Cell cycle evolution in the fungal ancestor then proceeded through a hybrid network containing both SBF and its ancestral animal counterpart E2F, which is still maintained in many basal fungi. We hypothesize that a virally-derived SBF may have initially hijacked cell cycle control by activating transcription via the cis-regulatory elements targeted by the ancestral cell cycle regulator E2F, much like extant viral oncogenes. Consistent with this hypothesis, we show that SBF can regulate promoters with E2F binding sites in budding yeast.

Complex history of the amphibian-killing chytrid fungus revealed with genome resequencing data.

Understanding the evolutionary history of microbial pathogens is critical for mitigating the impacts of emerging infectious diseases on economically and ecologically important host species. We used a genome resequencing approach to resolve the evolutionary history of an important microbial pathogen, the chytrid Batrachochytrium dendrobatidis (Bd), which has been implicated in amphibian declines worldwide. We sequenced the genomes of 29 isolates of Bd from around the world, with an emphasis on North, Central, and South America because of the devastating effect that Bd has had on amphibian populations in the New World. We found a substantial amount of evolutionary complexity in Bd with deep phylogenetic diversity that predates observed global amphibian declines. By investigating the entire genome, we found that even the most recently evolved Bd clade (termed the global panzootic lineage) contained more genetic variation than previously reported. We also found dramatic differences among isolates and among genomic regions in chromosomal copy number and patterns of heterozygosity, suggesting complex and heterogeneous genome dynamics. Finally, we report evidence for selection acting on the Bd genome, supporting the hypothesis that protease genes are important in evolutionary transitions in this group. Bd is considered an emerging pathogen because of its recent effects on amphibians, but our data indicate that it has a complex evolutionary history that predates recent disease outbreaks. Therefore, it is important to consider the contemporary effects of Bd in a broader evolutionary context and identify specific mechanisms that may have led to shifts in virulence in this system.

Parallel molecular evolution in an herbivore community.

Numerous insects have independently evolved the ability to feed on plants that produce toxic secondary compounds called cardenolides and can sequester these compounds for use in their defense. We surveyed the protein target for cardenolides, the alpha subunit of the sodium pump, Na(+),K(+)-ATPase (ATPα), in 14 species that feed on cardenolide-producing plants and 15 outgroups spanning three insect orders. Despite the large number of potential targets for modulating cardenolide sensitivity, amino acid substitutions associated with host-plant specialization are highly clustered, with many parallel substitutions. Additionally, we document four independent duplications of ATPα with convergent tissue-specific expression patterns. We find that unique substitutions are disproportionately associated with recent duplications relative to parallel substitutions. Together, these findings support the hypothesis that adaptation tends to take evolutionary paths that minimize negative pleiotropy.

Surviving chytridiomycosis: differential anti-Batrachochytrium dendrobatidis activity in bacterial isolates from three lowland species of Atelopus.

In the Neotropics, almost every species of the stream-dwelling harlequin toads (genus Atelopus) have experienced catastrophic declines. The persistence of lowland species of Atelopus could be explained by the lower growth rate of Batrachochytrium dendrobatidis (Bd) at temperatures above 25 °C. We tested the complementary hypothesis that the toads' skin bacterial microbiota acts as a protective barrier against the pathogen, perhaps delaying or impeding the symptomatic phase of chytridiomycosis. We isolated 148 cultivable bacterial strains from three lowland Atelopus species and quantified the anti-Bd activity through antagonism assays. Twenty-six percent (38 strains representing 12 species) of the bacteria inhibited Bd growth and just two of them were shared among the toad species sampled in different localities. Interestingly, the strongest anti-Bd activity was measured in bacteria isolated from A. elegans, the only species that tested positive for the pathogen. The cutaneous bacterial microbiota is thus likely a fitness-enhancing trait that may (adaptation) or not (exaptation) have appeared because of natural selection mediated by chytridiomycosis. Our findings reveal bacterial strains for development of local probiotic treatments against chytridiomycosis and also shed light on the mechanisms behind the frog-bacteria-pathogen interaction.

Reconstructing the fungal tree of life using phylogenomics and a preliminary investigation of the distribution of yeast prion-like proteins in the fungal kingdom.

We have used three independent phylogenomic approaches (concatenated alignments, single-, and multi-gene supertrees) to reconstruct the fungal tree of life (FTOL) using publicly available fungal genomes. This is the first time multi-gene families have been used in fungal supertree reconstruction and permits us to use up to 66% of the 1,001,217 genes in our fungal database. Our analyses show that different phylogenomic datasets derived from varying clustering criteria and alignment orientation do not have a major effect on phylogenomic supertree reconstruction. Overall the resultant phylogenomic trees are relatively congruent with one another and successfully recover the major fungal phyla, subphyla and classes. We find that where incongruences do occur, the inferences are usually poorly supported. Within the Ascomycota phylum, our phylogenies reconstruct monophyletic Saccharomycotina and Pezizomycotina subphyla clades and infer a sister group relationship between these to the exclusion of the Taphrinomycotina. Within the Pezizomycotina subphylum, all three phylogenies infer a sister group relationship between the Leotiomycetes and Sordariomycetes classes. However, there is conflict regarding the relationships with the Dothideomycetes and Eurotiomycetes classes. Within the Basidiomycota phylum, supertrees derived from single- and multi-gene families infer a sister group relationship between the Pucciniomycotina and Agaricomycotina subphyla while the concatenated phylogeny infers a poorly supported relationship between the Agaricomycotina and Ustilagomycotina. The reconstruction of a robust FTOL is important for future fungal comparative analyses. We illustrate this point by performing a preliminary investigation into the phyletic distribution of yeast prion-like proteins in the fungal kingdom.