Light reception of Phycomyces revisited: several white collar proteins confer blue- and red-light sensitivity and control dynamic range and adaptation.

The giant-fruiting body, sporangiophore, of the fungus Phycomyces blakesleeanus grows toward near-UV/blue-light (phototropism). The blue-light photoreceptor, MadA, should contain FAD bound to the LOV domain, and forms a complex with MadB. Both proteins are homologs of white collar proteins WC-1 and WC-2 from the fungus Neurospora crassa and should be localized in nuclei, where they function as a light-sensitive transcription factor complex. The photoreceptor properties of two further Wc proteins, WcoA and WcoB, remain unclear because of lack of mutants. We propose that WcoA and/or WcoB play essential roles in photoreception by enlarging the dynamic range that help explain complex stimulus-response relationships. Even though red light does not elicit photo-movement or -differentiation in Phycomyces, it affects the effectiveness of blue light which indicates an underlying photochromic receptor. Protein sequence searches show that other fungal red-light receptors are absent in Phycomyces. The solution to the red-light riddle is thus sought in the ability of Wc complexes to generate after blue-light irradiation a neutral flavosemiquinone radical that absorbs red light and functions as primary photochemical signal. Phototropism requires Ras-GAP (MadC) as part of the signal transduction cascade and, we propose, to allocate photoreceptors in the plasmalemma of the growing zone, which allows for receptor dichroism, range adjustment and contrast recognition for spatial orientation. Phototropic signal chains must entail transduction networks between Wc receptors and small G-proteins and their associated Ras-GAP and Ras-GEF proteins. The interactions among these proteins should occur in trans-Golgi vesicles and the plasmalemma of the growing zone.

Symmetric and asymmetric DNA N6-adenine methylation regulates different biological responses in Mucorales.

DNA N6-adenine methylation (6mA) has recently gained importance as an epigenetic modification in eukaryotes. Its function in lineages with high levels, such as early-diverging fungi (EDF), is of particular interest. Here, we investigated the biological significance and evolutionary implications of 6mA in EDF, which exhibit divergent evolutionary patterns in 6mA usage. The analysis of two Mucorales species displaying extreme 6mA usage reveals that species with high 6mA levels show symmetric methylation enriched in highly expressed genes. In contrast, species with low 6mA levels show mostly asymmetric 6mA. Interestingly, transcriptomic regulation throughout development and in response to environmental cues is associated with changes in the 6mA landscape. Furthermore, we identify an EDF-specific methyltransferase, likely originated from endosymbiotic bacteria, as responsible for asymmetric methylation, while an MTA-70 methylation complex performs symmetric methylation. The distinct phenotypes observed in the corresponding mutants reinforced the critical role of both types of 6mA in EDF.

Transcriptional Regulation by the Velvet Protein VE-1 during Asexual Development in the Fungus Neurospora crassa.

Asexual reproduction in fungi facilitates the dispersal and colonization of new substrates and, in pathogenic fungi, allows infection of plants and animals. The velvet complex is a fungus-specific protein complex that participates in the regulation of gene expression in response to environmental signals like light, as well as developmental processes, pathogenesis, and secondary metabolism. The velvet complex in the fungus Neurospora crassa is composed of three proteins, VE-1, VE-2, and LAE-1. Mutations in ve-1 or ve-2, but not in lae-1, led to shorter heights of aerial tissue, a mixture of aerial hyphae and developing macroconidia, and increased microconidiation when they were combined with mutations in the transcription factor gene fl. VE-2 and LAE-1 were detected during vegetative growth and conidiation, unlike VE-1, which was mostly observed in samples obtained from submerged vegetative hyphae. We propose that VE-1 is the limiting component of the velvet complex during conidiation and has a major role in the transcriptional regulation of conidiation. Characterization of the role of VE-1 during mycelial growth and asexual development (conidiation) by transcriptome sequencing (RNA-seq) experiments allowed the identification of a set of genes regulated by VE-1 that participate in the regulation of conidiation, most notably the transcription factor genes vib-1 and fl. We propose that VE-1 and VE-2 regulate the development of aerial tissue and the balance between macro- and microconidiation in coordination with FL and VIB-1. IMPORTANCE Most fungi disperse in nature and infect new hosts by producing vegetative spores or conidia during asexual development. This is a process that is regulated by environmental signals like light and the availability of nutrients. A protein complex, the velvet complex, participates in the integration of environmental signals to regulate conidiation. We have found that a key component of this complex in the fungus Neurospora crassa, VE-1, has a major role in the regulation of transcription during conidiation. VE-1 regulates a large number of genes, including the genes for the transcription factors FL and VIB-1. Our results will help to understand how environmental signals are integrated in the fungal cell to regulate development.

Light regulates the degradation of the regulatory protein VE-1 in the fungus Neurospora crassa.

BACKGROUND: Fungi use light as an environmental signal to regulate developmental transitions that are key aspects of their biological cycles and that are also relevant for their dispersal and infectivity as plant or animal pathogens. In addition, light regulates the accumulation of photoprotective pigments, like carotenoids, and other secondary metabolites. Most fungal light responses occur after changes in gene transcription and we describe here a novel effect of light in the regulation of degradation of VE-1, a key component of the velvet complex, in the model fungus Neurospora crassa. The velvet complex is a fungal-specific protein complex that coordinates fungal development, secondary metabolism, and light regulation by interacting with other regulators and photoreceptors and modifying gene expression. RESULTS: We have characterized the role of VE-1 during conidiation in N. crassa. In vegetative mycelia, VE-1 is localized in the cytoplasm and nuclei and is required for light-dependent transcription but does not interact with the photoreceptor and transcription factor WC-1. VE-1 is more stable in light than in darkness during asexual development (conidiation). We have shown that this light effect requires the blue-light photoreceptor WC-1. We have characterized the role of the proteasome, the COP9 signalosome (CSN), and the adaptor component of cullin-RING ubiquitin ligases, FWD-1, in the degradation of VE-1. CONCLUSIONS: We propose that this new effect of light allows the fungal cell to adapt quickly to changes in light exposure by promoting the accumulation of VE-1 for the regulation of genes that participate in the biosynthesis of photoprotective pigments.

Photobiology of the keystone genus Metarhizium.

Metarhizium fungi are soil-inhabiting ascomycetes which are saprotrophs, symbionts of plants, pathogens of insects, and participate in other trophic/ecological interactions, thereby performing multiple essential ecosystem services. Metarhizium species are used to control insect pests of crop plants and insects that act as vectors of human and animal diseases. To fulfil their functions in the environment and as biocontrol agents, these fungi must endure cellular stresses imposed by the environment, one of the most potent of which is solar ultraviolet (UV) radiation. Here, we examine the cellular stress biology of Metarhizium species in context of their photobiology, showing how photobiology facilitates key aspects of their ecology as keystone microbes and as mycoinsectides. The biophysical basis of UV-induced damage to Metarhizium, and mechanistic basis of molecular and cellular responses to effect damage repair, are discussed and interpreted in relation to the solar radiation received on Earth. We analyse the interplay between UV and visible light and how the latter increases cellular tolerance to the former via expression of a photolyase gene. By integrating current knowledge, we propose the mechanism through which Metarhizium species use the visible fraction of (low-UV) early-morning light to mitigate potentially lethal damage from intense UV radiation later in the day. We also show how this mechanism could increase Metarhizium environmental persistence and improve its bioinsecticide performance. We discuss the finding that visible light modulates stress biology in the context of further work needed on Metarhizium ecology in natural and agricultural ecosystems, and as keystone microbes that provide essential services within Earth's biosphere.

The DASH-type Cryptochrome from the Fungus Mucor circinelloides Is a Canonical CPD-Photolyase.

Cryptochromes and photolyases are blue-light photoreceptors and DNA-repair enzymes, respectively, with conserved domains and a common ancestry [1-3]. Photolyases use UV-A and blue light to repair lesions in DNA caused by UV radiation, photoreactivation, although cryptochromes have specialized roles ranging from the regulation of photomorphogenesis in plants, to clock function in animals [4-7]. A group of cryptochromes (cry-DASH) [8] from bacteria, plants, and animals has been shown to repair in vitro cyclobutane pyrimidine dimers (CPDs) in single-stranded DNA (ssDNA), but not in double-stranded DNA (dsDNA) [9]. Cry-DASH are evolutionary related to 6-4 photolyases and animal cryptochromes, but their biological role has remained elusive. The analysis of several crystal structures of members of the cryptochrome and photolyase family (CPF) allowed the identification of structural and functional similarities between photolyases and cryptochromes [8, 10-12] and led to the proposal that the absence of dsDNA repair activity in cry-DASH is due to the lack of an efficient flipping of the lesion into the catalytic pocket [13]. However, in the fungus Phycomyces blakesleeanus, cry-DASH has been shown to be capable of repairing CPD lesions in dsDNA as a bona fide photolyase [14]. Here, we show that cry-DASH of a related fungus, Mucor circinelloides, not only repairs CPDs in dsDNA in vitro but is the enzyme responsible for photoreactivation in vivo. A structural model of the M. circinelloides cry-DASH suggests that the capacity to repair lesions in dsDNA is an evolutionary adaptation from an ancestor that only had the capacity to repair lesions in ssDNA.

Outcome of blue, green, red, and white light on Metarhizium robertsii during mycelial growth on conidial stress tolerance and gene expression.

Fungi sense light and utilize it as a source of environmental information to prepare against many stressful conditions in nature. In this study, Metarhizium robertsii was grown on: 1) potato dextrose agar medium (PDA) in the dark (control); 2) under nutritive stress in the dark; and 3) PDA under continuous (A) white light; (B) blue light lower irradiance = LI; (C) blue light higher irradiance = HI; (D) green light; and (E) red light. Conidia produced under these treatments were tested against osmotic stress and UV radiation. In addition, a suite of genes usually involved in different stress responses were selected to study their expression patterns. Conidia produced under nutritive stress in the dark were the most tolerant to both osmotic stress and UV radiation, and the majority of their stress- and virulence-related genes were up-regulated. For osmotic stress tolerance, conidia produced under white, blue LI, and blue HI lights were the second most tolerant, followed by conidia produced under green light. Conidia produced under red light were the least tolerant to osmotic stress and less tolerant than conidia produced on PDA medium in the dark. For UV tolerance, conidia produced under blue light LI were the second most tolerant to UV radiation, followed by the UV tolerances of conidia produced under white light. Conidia produced under blue HI, green, and red lights were the least UV tolerant and less tolerant than conidia produced in the dark. The superoxide dismutases (sod1 and sod2), photolyases (6-4phr and CPDphr), trehalose-phosphate synthase (tps), and protease (pr1) genes were highly up-regulated under white light condition, suggesting a potential role of these proteins in stress protection as well as virulence after fungal exposure to visible spectrum components.

The Third International Symposium on Fungal Stress - ISFUS.

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.

Light regulates a Phycomyces blakesleeanus gene family similar to the carotenogenic repressor gene of Mucor circinelloides.

The transcription of about 5-10 % of the genes in Phycomyces blakesleeanus is regulated by light. Among the most up-regulated, we have identified four genes, crgA-D, with similarity to crgA of Mucor circinelloides, a gene encoding a repressor of light-inducible carotenogenesis. The four proteins have the same structure with two RING RING Finger domains and a LON domain, suggesting that they could act as ubiquitin ligases, as their M. circinelloides homolog. The expression of these genes is induced by light with different thresholds as in other Mucoromycotina fungi like Blakeslea trispora and M. circinelloides. Only the P. blakesleeanus crgD gene could restore the wild type phenotype in a M. circinelloides null crgA mutant suggesting that P. blakesleeanus crgD is the functional homolog of crgA in M. circinelloides. Despite their sequence similarity it is possible that the P. blakesleeanus Crg proteins do not participate in the regulation of beta-carotene biosynthesis since none of the carotene-overproducing mutants of P. blakesleeanus had mutations in any of the crg genes. Our results provide further support of the differences in the regulation of the biosynthesis of beta-carotene in these two Mucoromycotina fungi.

Conidiation in Neurospora crassa: vegetative reproduction by a model fungus.

Asexual development, conidiation, in the filamentous fungus Neurospora crassa is a simple developmental process that starts with the growth of aerial hyphae. Then, the formation of constrictions and subsequent maturation gives rise to the mature conidia that are easily dispersed by air currents. Conidiation is regulated by environmental factors such as light, aeration and nutrient limitation, and by the circadian clock. Different regulatory proteins acting at different stages of conidiation have been described. The role of transcription factors such as FL, and components of signal transduction pathways such as the cAMP phosphodiesterase ACON-2 suggest a complex interplay between differential transcription and signal transduction pathways. Comparisons between the molecular basis of conidiation in N. crassa and other filamentous fungi will help to identify common regulatory elements.

Light in the Fungal World: From Photoreception to Gene Transcription and Beyond.

Fungi see light of different colors by using photoreceptors such as the White Collar proteins and cryptochromes for blue light, opsins for green light, and phytochromes for red light. Light regulates fungal development, promotes the accumulation of protective pigments and proteins, and regulates tropic growth. The White Collar complex (WCC) is a photoreceptor and a transcription factor that is responsible for regulating transcription after exposure to blue light. In Neurospora crassa, light promotes the interaction of WCCs and their binding to the promoters to activate transcription. In Aspergillus nidulans, the WCC and the phytochrome interact to coordinate gene transcription and other responses, but the contribution of these photoreceptors to fungal photobiology varies across fungal species. Ultimately, the effect of light on fungal biology is the result of the coordinated transcriptional regulation and activation of signal transduction pathways.

Control of Development, Secondary Metabolism and Light-Dependent Carotenoid Biosynthesis by the Velvet Complex of Neurospora crassa.

Neurospora crassa is an established reference organism to investigate carotene biosynthesis and light regulation. However, there is little evidence of its capacity to produce secondary metabolites. Here, we report the role of the fungal-specific regulatory velvet complexes in development and secondary metabolism (SM) in N. crassa Three velvet proteins VE-1, VE-2, VOS-1, and a putative methyltransferase LAE-1 show light-independent nucleocytoplasmic localization. Two distinct velvet complexes, a heterotrimeric VE-1/VE-2/LAE-1 and a heterodimeric VE-2/VOS-1 are found in vivo The heterotrimer-complex, which positively regulates sexual development and represses asexual sporulation, suppresses siderophore coprogen production under iron starvation conditions. The VE-1/VE-2 heterodimer controls carotene production. VE-1 regulates the expression of >15% of the whole genome, comprising mainly regulatory and developmental features. We also studied intergenera functions of the velvet complex through complementation of Aspergillus nidulans veA, velB, laeA, vosA mutants with their N. crassa orthologs ve-1, ve-2, lae-1, and vos-1, respectively. Expression of VE-1 and VE-2 in A. nidulans successfully substitutes the developmental and SM functions of VeA and VelB by forming two functional chimeric velvet complexes in vivo, VelB/VE-1/LaeA and VE-2/VeA/LaeA, respectively. Reciprocally, expression of veA restores the phenotypes of the N. crassa ve-1 mutant. All N. crassa velvet proteins heterologously expressed in A. nidulans are localized to the nuclear fraction independent of light. These data highlight the conservation of the complex formation in N. crassa and A. nidulans However, they also underline the intergenera similarities and differences of velvet roles according to different life styles, niches and ontogenetic processes.

Measurement of Phototropism of the Sporangiophore of Phycomyces blakesleeanus.

The giant sporangiophore, fruiting body, of the fungus Phycomyces blakesleeanus is a single cell that grows guided by several environmental signals, including light. The phototropic response has been investigated in detail. Three proteins, the components of a photoreceptor and transcription factor complex and a regulator of the signal transduction protein Ras, participate in the signal transduction pathway. We describe the basic methods for characterizing phototropic bending and the correlated elongation and rotation responses of the sporangiophore.

The second International Symposium on Fungal Stress: ISFUS.

The topic of 'fungal stress' is central to many important disciplines, including medical mycology, chronobiology, plant and insect pathology, industrial microbiology, material sciences, and astrobiology. The International Symposium on Fungal Stress (ISFUS) brought together researchers, who study fungal stress in a variety of fields. The second ISFUS was held in May 8-11 2017 in Goiania, Goiás, Brazil and hosted by the Instituto de Patologia Tropical e Saúde Pública at the Universidade Federal de Goiás. It was supported by grants from CAPES and FAPEG. Twenty-seven speakers from 15 countries presented their research related to fungal stress biology. The Symposium was divided into seven topics: 1. Fungal biology in extreme environments; 2. Stress mechanisms and responses in fungi: molecular biology, biochemistry, biophysics, and cellular biology; 3. Fungal photobiology in the context of stress; 4. Role of stress in fungal pathogenesis; 5. Fungal stress and bioremediation; 6. Fungal stress in agriculture and forestry; and 7. Fungal stress in industrial applications. This article provides an overview of the science presented and discussed at ISFUS-2017.

Glucose sensing and light regulation: A mutation in the glucose sensor RCO-3 modifies photoadaptation in Neurospora crassa.

Light regulates fungal gene transcription transiently leading to photoadaptation. In the ascomycete Neurospora crassa photoadaptation is mediated by interactions between a light-regulated transcription factor complex, the white collar complex, and the small photoreceptor VVD. Other proteins, like the RCO-1/RCM-1 repressor complex participate indirectly in photoadaptation. We show that RCO-3, a protein with high similarity to glucose transporters, is needed for photoadaptation. The mutation in rco-3 modifies the transcriptional response to light of several genes and leads to changes in photoadaptation without significantly changing the amount and regulation of WC-1. The mutation in rco-3, however, does not modify the capacity of the circadian clock to be reset by light. Our results add support to the proposal that there is a connection between glucose sensing and light regulation in Neurospora and that the fungus integrates different environmental signals to regulate transcription.

Transcriptional basis of enhanced photoinduction of carotenoid biosynthesis at low temperature in the fungus Neurospora crassa.

Stimulation by light of carotenoid biosynthesis in the mycelia of the fungus Neurospora crassa starts with transient transcriptional induction of the structural genes of the pathway triggered by the White Collar photoreceptor complex. Most studies on this process were carried out under standard growth conditions, but photoinduced carotenoid accumulation is more efficient if the fungus is incubated at low temperatures, from 6 to 12 °C. We have investigated the transcriptional photoresponse at 8 °C of the genes for proteins that participate in the carotenoid pathway. Exposure to light pulses of different light intensities revealed higher sensitivity if the mycelia were subsequently incubated at 8 °C compared to 30 °C. Illumination of precooled mycelia resulted in delayed kinetics of mRNA accumulation for the structural genes, and high mRNA accumulation for a longer time. Additionally, after a light pulse, stronger reduction in mRNAs for carotenoid genes was observed at 30 °C compared to 8 °C. A similar pattern was found for mRNAs of the photoreceptor genes wc-1 and vvd, the latter involved in photoadaptation. These results suggest that the increased efficiency in carotenoid photoinduction at low temperature is due to the higher mRNA levels of the structural genes under these conditions.

A Ras GTPase associated protein is involved in the phototropic and circadian photobiology responses in fungi.

Light is an environmental signal perceived by most eukaryotic organisms and that can have major impacts on their growth and development. The MadC protein in the fungus Phycomyces blakesleeanus (Mucoromycotina) has been postulated to form part of the photosensory input for phototropism of the fruiting body sporangiophores, but the madC gene has remained unidentified since the 1960s when madC mutants were first isolated. In this study the madC gene was identified by positional cloning. All madC mutant strains contain loss-of-function point mutations within a gene predicted to encode a GTPase activating protein (GAP) for Ras. The madC gene complements the Saccharomyces cerevisiae Ras-GAP ira1 mutant and the encoded MadC protein interacts with P. blakesleeanus Ras homologs in yeast two-hybrid assays, indicating that MadC is a regulator of Ras signaling. Deletion of the homolog in the filamentous ascomycete Neurospora crassa affects the circadian clock output, yielding a pattern of asexual conidiation similar to a ras-1 mutant that is used in circadian studies in N. crassa. Thus, MadC is unlikely to be a photosensor, yet is a fundamental link in the photoresponses from blue light perceived by the conserved White Collar complex with Ras signaling in two distantly-related filamentous fungal species.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

The Complexity of Fungal Vision.

Life, as we know it, would not be possible without light. Light is not only a primary source of energy, but also an important source of information for many organisms. To sense light, only a few photoreceptor systems have developed during evolution. They are all based on an organic molecule with conjugated double bonds that allows energy transfer from visible (or UV) light to its cognate protein to translate the primary physical photoresponse to cell-biological actions. The three main classes of receptors are flavin-based blue-light, retinal-based green-light (such as rhodopsin), and linear tetrapyrrole-based red-light sensors. Light not only controls the behavior of motile organisms, but is also important for many sessile microorganisms including fungi. In fungi, light controls developmental decisions and physiological adaptations as well as the circadian clock. Although all major classes of photoreceptors are found in fungi, a good level of understanding of the signaling processes at the molecular level is limited to some model fungi. However, current knowledge suggests a complex interplay between light perception systems, which goes far beyond the simple sensing of light and dark. In this article we focus on recent results in several fungi, which suggest a strong link between light-sensing and stress-activated mitogen-activated protein kinases.