Determination and inference of eukaryotic transcription factor sequence specificity.

Transcription factor (TF) DNA sequence preferences direct their regulatory activity, but are currently known for only ∼1% of eukaryotic TFs. Broadly sampling DNA-binding domain (DBD) types from multiple eukaryotic clades, we determined DNA sequence preferences for >1,000 TFs encompassing 54 different DBD classes from 131 diverse eukaryotes. We find that closely related DBDs almost always have very similar DNA sequence preferences, enabling inference of motifs for ∼34% of the ∼170,000 known or predicted eukaryotic TFs. Sequences matching both measured and inferred motifs are enriched in chromatin immunoprecipitation sequencing (ChIP-seq) peaks and upstream of transcription start sites in diverse eukaryotic lineages. SNPs defining expression quantitative trait loci in Arabidopsis promoters are also enriched for predicted TF binding sites. Importantly, our motif "library" can be used to identify specific TFs whose binding may be altered by human disease risk alleles. These data present a powerful resource for mapping transcriptional networks across eukaryotes.

Transcriptional rewiring of an evolutionarily conserved circadian clock.

Circadian clocks temporally coordinate daily organismal biology over the 24-h cycle. Their molecular design, preserved between fungi and animals, is based on a core-oscillator composed of a one-step transcriptional-translational-negative-feedback-loop (TTFL). To test whether this evolutionarily conserved TTFL architecture is the only plausible way for achieving a functional circadian clock, we adopted a transcriptional rewiring approach, artificially co-opting regulators of the circadian output pathways into the core-oscillator. Herein we describe one of these semi-synthetic clocks which maintains all basic circadian features but, notably, it also exhibits new attributes such as a "lights-on timer" logic, where clock phase is fixed at the end of the night. Our findings indicate that fundamental circadian properties such as period, phase and temperature compensation are differentially regulated by transcriptional and posttranslational aspects of the clockworks.

An Out-of-Patagonia migration explains the worldwide diversity and distribution of Saccharomyces eubayanus lineages.

Population-level sampling and whole-genome sequences of different individuals allow one to identify signatures of hybridization, gene flow and potential molecular mechanisms of environmental responses. Here, we report the isolation of 160 Saccharomyces eubayanus strains, the cryotolerant ancestor of lager yeast, from ten sampling sites in Patagonia along 2,000 km of Nothofagus forests. Frequency of S. eubayanus isolates was higher towards southern and colder regions, demonstrating the cryotolerant nature of the species. We sequenced the genome of 82 strains and, together with 23 available genomes, performed a comprehensive phylogenetic analysis. Our results revealed the presence of five different lineages together with dozens of admixed strains. Various analytical methods reveal evidence of gene flow and historical admixture between lineages from Patagonia and Holarctic regions, suggesting the co-occurrence of these ancestral populations. Analysis of the genetic contribution to the admixed genomes revealed a Patagonian genetic origin of the admixed strains, even for those located in the North Hemisphere. Overall, the Patagonian lineages, particularly the southern populations, showed a greater global genetic diversity compared to Holarctic and Chinese lineages, in agreement with a higher abundance in Patagonia. Thus, our results are consistent with a likely colonization of the species from peripheral glacial refugia from South Patagonia. Furthermore, fermentative capacity and maltose consumption resulted negatively correlated with latitude, indicating better fermentative performance in northern populations. Our genome analysis, together with previous reports in the sister species S. uvarum suggests that a S. eubayanus ancestor was adapted to the harsh environmental conditions of Patagonia, a region that provides the ecological conditions for the diversification of these ancestral lineages.

Genome-Wide Characterization of Light-Regulated Gene Expression in Botrytis cinerea Reveals Underlying Complex Photobiology.

Botrytis cinerea is a necrotrophic fungus characterized mainly by its wide host range of infected plants. The deletion of the white-collar-1 gene (bcwcl1), which encodes for a blue-light receptor/transcription factor, causes a decrease in virulence, particularly when assays are conducted in the presence of light or photocycles. However, despite ample characterization, the extent of the light-modulated transcriptional responses regulated by BcWCL1 remains unknown. In this study, pathogen and pathogen:host RNA-seq analyses, conducted during non-infective in vitro plate growth and when infecting Arabidopsis thaliana leaves, respectively, informed on the global gene expression patterns after a 60 min light pulse on the wild-type B05.10 or ∆bcwcl1 B. cinerea strains. The results revealed a complex fungal photobiology, where the mutant did not react to the light pulse during its interaction with the plant. Indeed, when infecting Arabidopsis, no photoreceptor-encoding genes were upregulated upon the light pulse in the ∆bcwcl1 mutant. Differentially expressed genes (DEGs) in B. cinerea under non-infecting conditions were predominantly related to decreased energy production in response to the light pulse. In contrast, DEGs during infection significantly differ in the B05.10 strain and the ∆bcwcl1 mutant. Upon illumination at 24 h post-infection in planta, a decrease in the B. cinerea virulence-associated transcripts was observed. Accordingly, after a light pulse, biological functions associated with plant defense appear enriched among light-repressed genes in fungus-infected plants. Taken together, our results show the main transcriptomic differences between wild-type B. cinerea B05.10 and ∆bcwcl1 after a 60 min light pulse when growing saprophytically on a Petri dish and necrotrophically over A. thaliana.

Daily magnesium fluxes regulate cellular timekeeping and energy balance.

Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behaviour and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes. A fundamental knowledge gap exists, however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg(2+)]i, which act as a cell-autonomous timekeeping component to determine key clock properties both in a human cell line and in a unicellular alga that diverged from each other more than 1 billion years ago. Given the essential role of Mg(2+) as a cofactor for ATP, a functional consequence of [Mg(2+)]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg(2+) availability has potential to impact upon many of the cell's more than 600 MgATP-dependent enzymes and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR is regulated through [Mg(2+)]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease.

The rise and shine of yeast optogenetics.

Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared with traditional chemically inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost, and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, and protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast.

Conserved RNA helicase FRH acts nonenzymatically to support the intrinsically disordered neurospora clock protein FRQ.

Protein conformation dictates a great deal of protein function. A class of naturally unstructured proteins, termed intrinsically disordered proteins (IDPs), demonstrates that flexibility in structure can be as important mechanistically as rigid structure. At the core of the circadian transcription/translation feedback loop in Neurospora crassa is the protein FREQUENCY (FRQ), shown here shown to share many characteristics of IDPs. FRQ in turn binds to FREQUENCY-Interacting RNA Helicase (FRH), whose clock function has been assumed to relate to its predicted helicase function. However, mutational analyses reveal that the helicase function of FRH is not essential for the clock, and a region of FRH distinct from the helicase region is essential for stabilizing FRQ against rapid degradation via a pathway distinct from its typical ubiquitin-mediated turnover. These data lead to the hypothesis that FRQ is an IDP and that FRH acts nonenzymatically, stabilizing FRQ to enable proper clock circuitry/function.

Modular and Molecular Optimization of a LOV (Light-Oxygen-Voltage)-Based Optogenetic Switch in Yeast.

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light-oxygen-voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call "HAP-LOV", displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

The Clock Keeps on Ticking: Emerging Roles for Circadian Regulation in the Control of Fungal Physiology and Pathogenesis.

Tic-tac, tic-tac, the sound of time is familiar to us, yet, it also silently shapes daily biological processes conferring 24-hour rhythms in, among others, cellular and systemic signaling, gene expression, and metabolism. Indeed, circadian clocks are molecular machines that permit temporal control of a variety of processes in individuals, with a close to 24-hour period, optimizing cellular dynamics in synchrony with daily environmental cycles. For over three decades, the molecular bases of these clocks have been extensively described in the filamentous fungus Neurospora crassa, yet, there have been few molecular studies in fungi other than Neurospora, despite evidence of rhythmic phenomena in many fungal species, including pathogenic ones. This chapter will revise the mechanisms underlying clock regulation in the model fungus N. crassa, as well as recent findings obtained in several fungi. In particular, this chapter will review the effect of circadian regulation of virulence and organismal interactions, focusing on the phytopathogen Botrytis cinerea, as well as several entomopathogenic fungi, including the behavior-manipulating species Ophiocordyceps kimflemingiae and Entomophthora muscae. Finally, this review will comment current efforts in the study of mammalian pathogenic fungi, while highlighting recent circadian lessons from parasites such as Trypanosoma and Plasmodium. The clock keeps on ticking, whether we can hear it or not.

Indirect monitoring of TORC1 signalling pathway reveals molecular diversity among different yeast strains.

Saccharomyces cerevisiae is the main species responsible for the alcoholic fermentation in wine production. One of the main problems in this process is the deficiency of nitrogen sources in the grape must, which can lead to stuck or sluggish fermentations. Currently, yeast nitrogen consumption and metabolism are under active inquiry, with emphasis on the study of the TORC1 signalling pathway, given its central role responding to nitrogen availability and influencing growth and cell metabolism. However, the mechanism by which different nitrogen sources activates TORC1 is not completely understood. Existing methods to evaluate TORC1 activation by nitrogen sources are time-consuming, making difficult the analyses of large numbers of strains. In this work, a new indirect method for monitoring TORC1 pathway was developed on the basis of the luciferase reporter gene controlled by the promoter region of RPL26A gene, a gene known to be expressed upon TORC1 activation. The method was tested in strains representative of the clean lineages described so far in S. cerevisiae. The activation of the TORC1 pathway by a proline-to-glutamine upshift was indirectly evaluated using our system and the traditional direct methods based on immunoblot (Sch9 and Rps6 phosphorylation). Regardless of the different molecular readouts obtained with both methodologies, the general results showed a wide phenotypic variation between the representative strains analysed. Altogether, this easy-to-use assay opens the possibility to study the molecular basis for the differential TORC1 pathway activation, allowing to interrogate a larger number of strains in the context of nitrogen metabolism phenotypic differences.

Circadian clocks and the regulation of virulence in fungi: Getting up to speed.

You cannot escape time. Therefore, it seems wise to learn how to keep track of it and use it to your advantage. Circadian clocks are molecular circuits that allow organisms to temporally coordinate a plethora of processes, including gene expression, with a close to 24h rhythm, optimizing cellular function in synchrony with daily environmental cycles. The molecular bases of these clocks have been extensively studied in the fungus Neurospora crassa, providing a detailed molecular description. Surprisingly, there is scarce molecular information of clocks in fungi other than Neurospora, despite the existence of rhythmic phenomena in many fungal species, including pathogenic ones. This review will comment on the overall importance of clocks, what is known in Neurospora and what has been described in other fungi including new insights on the evolution of fungal clock components. The molecular description of the circadian system of the phytopathogenic fungus Botrytis cinerea will be revisited, as well as time-of-the-day variation in host-pathogen interaction dynamics, utilizing an Arabidopsis-Botrytis system, including also what is known regarding circadian regulation of defense mechanisms in the Arabidopsis thaliana plant model. Finally, this review will mention how little is known about circadian regulation of human pathogenic fungi, commenting on potential future directions and the overall perspective of fungal circadian studies.

A circadian oscillator in the fungus Botrytis cinerea regulates virulence when infecting Arabidopsis thaliana.

The circadian clock of the plant model Arabidopsis thaliana modulates defense mechanisms impacting plant-pathogen interactions. Nevertheless, the effect of clock regulation on pathogenic traits has not been explored in detail. Moreover, molecular description of clocks in pathogenic fungi--or fungi in general other than the model ascomycete Neurospora crassa--has been neglected, leaving this type of question largely unaddressed. We sought to characterize, therefore, the circadian system of the plant pathogen Botrytis cinerea to assess if such oscillatory machinery can modulate its virulence potential. Herein, we show the existence of a functional clock in B. cinerea, which shares similar components and circuitry with the Neurospora circadian system, although we found that its core negative clock element FREQUENCY (BcFRQ1) serves additional roles, suggesting extracircadian functions for this protein. We observe that the lesions produced by this necrotrophic fungus on Arabidopsis leaves are smaller when the interaction between these two organisms occurs at dawn. Remarkably, this effect does not depend solely on the plant clock, but instead largely relies on the pathogen circadian system. Genetic disruption of the B. cinerea oscillator by mutation, overexpression of BcFRQ1, or by suppression of its rhythmicity by constant light, abrogates circadian regulation of fungal virulence. By conducting experiments with out-of-phase light:dark cycles, we confirm that indeed, it is the fungal clock that plays the main role in defining the outcome of the Arabidopsis-Botrytis interaction, providing to our knowledge the first evidence of a microbial clock modulating pathogenic traits at specific times of the day.

Optogenetic switches for light-controlled gene expression in yeast.

Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.

Circadian rhythms synchronize mitosis in Neurospora crassa.

The cell cycle and the circadian clock communicate with each other, resulting in circadian-gated cell division cycles. Alterations in this network may lead to diseases such as cancer. Therefore, it is critical to identify molecular components that connect these two oscillators. However, molecular mechanisms between the clock and the cell cycle remain largely unknown. A model filamentous fungus, Neurospora crassa, is a multinucleate system used to elucidate molecular mechanisms of circadian rhythms, but not used to investigate the molecular coupling between these two oscillators. In this report, we show that a conserved coupling between the circadian clock and the cell cycle exists via serine/threonine protein kinase-29 (STK-29), the Neurospora homolog of mammalian WEE1 kinase. Based on this finding, we established a mathematical model that predicts circadian oscillations of cell cycle components and circadian clock-dependent synchronized nuclear divisions. We experimentally demonstrate that G1 and G2 cyclins, CLN-1 and CLB-1, respectively, oscillate in a circadian manner with bioluminescence reporters. The oscillations of clb-1 and stk-29 gene expression are abolished in a circadian arrhythmic frq(ko) mutant. Additionally, we show the light-induced phase shifts of a core circadian component, frq, as well as the gene expression of the cell cycle components clb-1 and stk-29, which may alter the timing of divisions. We then used a histone hH1-GFP reporter to observe nuclear divisions over time, and show that a large number of nuclear divisions occur in the evening. Our findings demonstrate the circadian clock-dependent molecular dynamics of cell cycle components that result in synchronized nuclear divisions in Neurospora.

Analysis of the Phlebiopsis gigantea genome, transcriptome and secretome provides insight into its pioneer colonization strategies of wood.

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

RIM15 antagonistic pleiotropy is responsible for differences in fermentation and stress response kinetics in budding yeast.

Different natural yeast populations have faced dissimilar selective pressures due to the heterogeneous fermentation substrates available around the world; this increases the genetic and phenotypic diversity in Saccharomyces cerevisiae In this context, we expect prominent differences between isolates when exposed to a particular condition, such as wine or sake musts. To better comprehend the mechanisms underlying niche adaptation between two S. cerevisiae isolates obtained from wine and sake fermentation processes, we evaluated fermentative and fungicide resistance phenotypes and identify the molecular origin of such adaptive variation. Multiple regions were associated with fermentation rate under different nitrogen conditions and fungicide resistance, with a single QTL co-localizing in all traits. Analysis around this region identified RIM15 as the causative locus driving fungicide sensitivity, together with efficient nitrogen utilization and glycerol production in the wine strain. A null RIM15 variant confers a greater fermentation rate through the utilization of available glucose instead of its storage. However, this variant has a detrimental effect on fungicide resistance since complex sugars are not synthesized and transported into the membrane. Together, our results reveal the antagonist pleiotropic nature of a RIM15 null variant, positively affecting a series of fermentation related phenotypes, but apparently detrimental in the wild.

bcpmr1 encodes a P-type Ca(2+)/Mn(2+)-ATPase mediating cell-wall integrity and virulence in the phytopathogen Botrytis cinerea.

The cell wall of fungi is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. These usually contain both N- and O-linked oligosaccharides, coupled to the proteins by stepwise addition of mannose residues by mannosyltransferases in the endoplasmic reticulum and the Golgi apparatus. In yeast, an essential luminal cofactor for these mannosyltransferases is Mn(2+) provided by the Ca(2+)/Mn(2+)-ATPase known as Pmr1. In this study, we have identified and characterized the Botrytis cinerea pmr1 gene, the closest homolog of yeast PMR1. We hypothesized that bcpmr1 also encodes a Ca(2+)/Mn(2+)-ATPase that plays an important role in the protein glycosylation pathway. Phenotypic analysis showed that bcpmr1 null mutants displayed a significant reduction in conidial production, radial growth and diameter of sclerotia. Significant alterations in hyphal cell wall composition were observed including a 83% decrease of mannan levels and an increase in the amount of chitin and glucan. These changes were accompanied by a hypersensitivity to cell wall-perturbing agents such as Calcofluor white, Congo red and zymolyase. Importantly, the Δbcpmr1 mutant showed reduced virulence in tomato (leafs and fruits) and apple (fruits) and reduced biofilm formation. Together, our results highlight the importance of bcpmr1 for protein glycosylation, cell wall structure and virulence of B. cinerea.

Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis.

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn(2+). Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

Environmental responses and the control of iron homeostasis in fungal systems.

Organisms need to actively respond to changes in the environment and, particularly under diverse conditions, they ought to ensure access to nutrients. Among micronutrients, iron is a key component of several enzymes and participates in a variety of cellular processes. Iron deprivation therefore poses a serious challenge to both unicellular and multicellular individuals. Nevertheless, excess of this metal is toxic, compromising cell function and viability. Thus, it is not surprising that organisms have evolved sophisticated mechanisms to tightly regulate cellular iron levels. In the last decade, major advances have been achieved in the molecular understanding of how fungi respond to changing iron concentrations. Moreover, this metal has been recognized as an important element impacting pathogenic and saprophytic fungal lifestyles. An interconnected transcriptional negative feedback loop has been described as central in the regulation of genes encoding for iron uptake and utilization components in fungi. The observation that light, oxygen, or nutrients can also impact the expression of some of these elements suggests that additional environmental inputs-besides iron levels-may as well modulate the machinery underpinning iron homeostasis. This review highlights some of the latest findings associated with iron-regulated processes in fungi and revisits the increasing transcriptional complexity involved in the control of this metal homeostasis. In addition, we present the first in silico evidence of genes encoding for putative ferritins in zygomycetes and chytrids, as well as other ferritin-like sequences widespread among fungi, which raises interesting questions relative to iron storage in this particular group of organisms.