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Lineage-specific genes (LSGs) have long been postulated to play roles in the establishment of genetic barriers to intercrossing and speciation. However, there is a lack of working hypotheses as to how they might play that role. In the genome ofNeurospora crassa, most of the 670NeurosporaLSGs that are aggregated adjacent to the telomeres are clustered with 61% of the HET-domain genes, which regulate self-recognition and define vegetative incompatibility groups. Among the 342 LSGs that are dynamically expressed during both asexual and sexual phases, 64% were detectable on unusual carbon sources such as furfural and HMF—wildfire-produced chemicals that are a strong inducer of sexual development. Expression of a significant portion of the LSGs was sensitive to light and temperature, factors that regulate the switch from asexual to sexual reproduction. Furthermore, expression of the LSGs was significantly affected in the knockouts ofadv-1andpp-1that regulate hyphal communication, and expression of more than one quarter of the LSGs was affected by perturbation of the mating locus. Accordingly, we propose a gene-by-environment interaction model encouraging further investigation of the roles of LSGs and HET-domain genes in speciation inNeurospora. This gene-by-environment interaction model emphasizes the roles of the LSGs in response to genetic and environmental factors, leading to the regulation of the switch from the asexual growth and fusion, such that vegetative incompatibility governed by allorecognition promotes allelic homogeneity, sexual reproduction, and outbreeding, whereas VI repression and meiotic recombination promotes allelic polymorphism.

The filamentous mycoparasitic fungus Trichoderma asperelloides (Hypocreales, Ascomycota, Dikarya) strain T 203 was isolated from soil in Israel by the Ilan Chet group in the 1980s. As it has been the subject of laboratory, greenhouse, and field experiments and has been incorporated into commercial agricultural preparations, its genome has been sequenced and analyzed.

Impairment of theNeurospora crassaCOT-1 kinase results in defects in hyphal polarity. Some of these effects are partially suppressed by inactivation of gul-1 (encoding an mRNA-binding protein involved in translational regulation). Here, we report on the transcriptional profiling of cot-1 inactivation and demonstrate that gul-1 affects transcript abundance of multiple genes in the COT-1 pathway, including processes such as cell wall remodeling, nitrogen and amino acid metabolism. The GUL-1 protein itself was found to be distributed within the entire hyphal cell, along with a clear presence of aggregates that traffic within the cytoplasm. Live imaging of GUL-1-GFP demonstrated that GUL-1 transport is microtubule-dependent. Cellular stress, as imposed by the presence of the cell wall biosynthesis inhibitor Nikkomycin Z or by nitrogen limitation, resulted in a 2-3-fold increase of GUL-1 aggregate association with nuclei. Taken together, this study demonstrates that GUL-1 affects multiple processes, its function is stress-related and linked with cellular traffic and nuclear association.

Coral associated fungi are widespread, highly diverse and are part and parcel of the coral holobiont. To study how environmental conditions prevailing near the coral-host may affect fungal diversity, the culturable (isolated on potato dextrose agar) mycobiome associated with Acropora loripes colonies was seasonally sampled along a depth gradient in the Gulf of Aqaba. Fragments were sampled from both apparently healthy coral colonies as well as those exhibiting observable lesions. Based on phylogenetic analysis of 197 fungal sequences, Ascomycota were the most prevalent (91.9%). The abundance of fungi increased with increasing water depth, where corals sampled at 25 m yielded up to 70% more fungal colony forming units (CFUs) than those isolated at 6 m. Fungal diversity at 25 m was also markedly higher, with over 2-fold more fungal families represented. Diversity was also higher in lesioned coral samples, when compared to apparently healthy colonies. In winter, concurrent with water column mixing and increased levels of available nutrients, at the shallow depths, Saccharomytacea and Sporidiobolacea were more prevalent, while in spring and fall Trichocomacea (overall, the most prevalent family isolated throughout this study) were the most abundant taxa isolated at these depths as well as at deeper sampling sites. Our results highlight the dynamic nature of the culturable coral mycobiome and its sensitivity to environmental conditions and coral health.

Species of Didymellaceae have a cosmopolitan distribution and are geographically widespread, occurring in diverse ecosystems. The family includes several important plant pathogenic fungi associated with fruit, leaf, stem and root diseases on a wide variety of hosts, as well as endophytic, saprobic and clinically relevant species. The Didymellaceae was recently revised based on morphological and phylogenetic analyses of ex-type strains subjected to DNA sequencing of partial gene data of the LSU, ITS, rpb2 and tub2 loci. Several poly- and paraphyletic genera, including Ascochyta, Didymella and Phoma were redefined, along with the introduction of new genera. In the present study, a global collection of 1 124 Didymellaceae strains from 92 countries, 121 plant families and 55 other substrates, including air, coral, human tissues, house dust, fungi, insects, soil, and water were examined via multi-locus phylogenetic analyses and detailed morphological comparisons, representing the broadest sampling of Didymellaceae to date. Among these, 97 isolates representing seven new genera, 40 new species and 21 new combinations were newly introduced in Didymellaceae. In addition, six epitypes and six neotypes were designated to stabilise the taxonomy and use of older names. A robust, multi-locus reference phylogenetic tree of Didymellaceae was generated. In addition, rpb2 was revealed as the most effective locus for the identification of Didymellaceae at species level, and is proposed as a secondary DNA marker for the family.

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 Sao Jose dos Campos, Sao 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.

Background During the process of bioethanol production, cellulose is hydrolyzed into its monomeric soluble units. For efficient hydrolysis, a chemical and/or mechanical pretreatment step is required. Such pretreatment is designed to increase enzymatic digestibility of the cellulose chains inter alia by de-crystallization of the cellulose chains and by removing barriers, such as lignin from the plant cell wall. Biological pretreatment, in which lignin is decomposed or modified by white-rot fungi, has also been considered. One disadvantage in biological pretreatment, however, is the consumption of the cellulose by the fungus. Thus, fungal species that attack lignin with only minimal cellulose loss are advantageous. The secretomes of white-rot fungi contain carbohydrate-active enzymes (CAZymes) including lignin-modifying enzymes. Thus, modification of secretome composition can alter the ratio of lignin/cellulose degradation. Results Pleurotus ostreatus PC9 was genetically modified to either overexpress or eliminate (by gene replacement) the transcriptional regulator CRE1, known to act as a repressor in the process of carbon catabolite repression. The cre1-overexpressing transformant demonstrated lower secreted cellulolytic activity and slightly increased selectivity (based on the chemical composition of pretreated wheat straw), whereas the knockout transformant demonstrated increased cellulolytic activity and significantly reduced residual cellulose, thereby displaying lower selectivity. Pretreatment of wheat straw using the wild-type PC9 resulted in 2.8-fold higher yields of soluble sugar compared to untreated wheat straw. The overexpression transformant showed similar yields (2.6-fold), but the knockout transformant exhibited lower yields (1.2-fold) of soluble sugar. Based on proteomic secretome analysis, production of numerous CAZymes was affected by modification of the expression level of cre1. Conclusions The gene cre1 functions as a regulator for expression of fungal CAZymes active against plant cell wall lignocelluloses, hence altering the substrate preference of the fungi tested. While the cre1 knockout resulted in a less efficient biological pretreatment, i.e., less saccharification of the treated biomass, the converse manipulation of cre1 (overexpression) failed to improve efficiency. Despite the inverse nature of the two genetic alterations, the expected “mirror image” (i.e., opposite regulatory response) was not observed, indicating that the secretion level of CAZymes, was not exclusively dependent on CRE1 activity. Electronic supplementary material The online version of this article (10.1186/s13068-018-1209-6) contains supplementary material, which is available to authorized users.

Fourteen Trichoderma (Hypocreales) species were identified during a survey of the genus in South Africa. These include T. afroharzianum, T. asperelloides, T. asperellum, T. atrobrunneum, T. atroviride, T. camerunense, T. gamsii, T. hamatum, T. koningii, T. koningiopsis, T. saturnisporum, T. spirale, T. virens, and T. viride. Ten of these species were not known to occur in South Africa prior to this investigation. Five additional species were novel and are described here as T. beinartii, T. caeruleimontis, T. chetii, T. restrictum, and T. undulatum. These novel Trichoderma species display morphological traits that are typical of the genus. Based on molecular identification using calmodulin, endochitinase, nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2), RNA polymerase II subunit B, and translation elongation factor 1-α gene sequence data, T. beinartii, T. caeruleimontis, and T. chetii were found to belong to the Longibrachiatum clade, whereas T. restrictum is a member of the Hamatum clade. Trichoderma undulatum occupies a distinct lineage distantly related to other Trichoderma species. Strains of T. beinartii and T. chetii were isolated previously in Hawaii and Israel; however, T. caeruleimontis, T. restrictum, and T. undulatum are so far known only from South Africa.

The Neurospora crassa Mps One Binder (MOB) proteins MOB2A and MOB2B physically interact with the Nuclear Dbf2 Related (NDR) kinase COT1 and have been shown to have overlapping functions in various aspects of asexual development. Here, we identified two N. crassa MOB2A residues, Tyr117 and Tyr119, which are potentially phosphorylated. Using phosphomimetic mob-2a mutants we have been able to establish that apart from their previously described roles, MOB2A/B are involved in additional developmental processes. Enhanced conidial germination, accompanied by conidial agglutination, in the phosphomimetic mutants indicated that MOB2A is a negative regulator of germination. Thick-section imaging of perithecia revealed slow maturation and a lack of asci alignment in the mutant strains demonstrating a role for MOB2A in sexual development. We demonstrate that even though MOB2A and MOB2B have some overlapping functions, MOB2B cannot compensate for the roles MOB2A has in conidiation and germination. Altering Tyr residues 117 and 119 impaired the physical interactions between MOB2A and COT1, most likely contributing to some of the observed effects. As cot-1 and the phosphomimetic mutants share an extragenic suppressor (gul-1), we concluded that at least some of the effects imposed by altering Tyr117 and Tyr119 are mediated by the NDR kinase.

Marine sponges harbor a diverse array of microorganisms and the composition of the microbial community has been suggested to be linked to holo-biont health. Most of the attention concerning sponge mycobiomes has been given to sponges present in shallow depths. Here, we describe the presence of 146 culturable mycobiome taxa isolated from mesophotic niche (100 m depth)-inhabiting samples of Agelas oroides, in the Mediterranean Sea. We identify some potential in vitro interactions between several A. oroides-associated fungi and show that sponge meso-hyl extract, but not its predominantly collagen-rich part, is sufficient to support hyphal growth. We demonstrate that changes in the diversity of culturable mycobiome constituents occur following sponge transplantation from its original mesophotic habitat to shallow (10 m) waters, where historically (60 years ago) this species was found. We conclude that among the 30 fungal genera identified as associated with A. oroides, Aspergillus, Penicillium and Trichoderma constitute the core mycobiome of A. oroides, and that they persist even when the sponge is transplanted to a suboptimal environment, indicative of the presence of constant, as well as dynamic, components of the sponge mycobiome. Other genera seemed more depth-related and appeared or disappeared upon host’s transfer from 100 to 10 m.

Small secreted proteins (SSPs), along with lignocellulose degrading enzymes, are integral components of the secretome of Pleurotus ostreatus, a white rot fungus. In this study, we identified 3 genes (ssp1, 2 and 3) encoding proteins that are annotated as SSPs and that exhibited of ~4,500- fold expression, 24 hr following exposure to the toxic compound 5-hydroxymethylfurfural (HMF). Homologues to genes encoding these SSPs are present in the genomes of other basidiomycete fungi, however the role of SSPs is not yet understood. SSPs, aryl-alcohol oxidases (AAO) and the intracellular aryl-alcohol dehydrogenases (AAD) were also produced after exposure to other aryl-alcohols, known substrates and inducers of AAOs, and during idiophase (after the onset of secondary metabolism). A knockdown strain of ssp1 exhibited reduced production of AAO-and AAD-encoding genes after HMF exposure. Conversely, a strain overexpressing ssp1 exhibited elevated expression of genes encoding AAOs and ADD, resulting in a 3-fold increase in enzymatic activity of AAOs, as well as increased expression and protein abundance of versatile peroxidase 1, which directly degrades lignin. We propose that in addition to symbionts and pathogens, SSPs also have roles in saprophytes and function in P. ostreatus as components of the ligninolytic system.

We used the photoacid 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) that converts blue photons to acidic protons in water, with an efficiency of close to 100%, and determined that this treatment conferred changes to colony morphology of the plant pathogen Colletotrichum gloeosporioides. The time elapsed until hyphal collapse is noticed depends on both the laser intensity in mW/cm2, and the concentration of HPTS in the Agar hydrogel. The time elapsed until hyphal collapse is noticed varies by only ±8% at HPTS concentrations of 500μM and at lower concentrations of HPTS the variance increases as the inverse of the concentration. We found that the effect on C. gloeosporioides was photoacid concentration and irradiation dose dependent. In the presence of 500μM of HPTS within the agar hydrogel-based medium, hyphae collapsed after 37±3.5min of irradiation at 405nm at an intensity of 25mW/cm2. We propose two mechanisms for such photo-alteration of C. gloeosporioides. One is based on the pH drop in the extracellular environment by the photo-protolytic process that the photoacid molecule undergoes. The second mechanism is based on an intracellular mechanism in which there is an uptake of HPTS into the interior of the fungus. We suggest that both mechanisms for photo-alteration which we found in this study may occur in plants during fungal infection.

Finding intracellular pathways and molecules that can prevent the proliferation of colon cancer cells can provide significant bases for developing treatments for this disease. Ostreolysin (Oly) is a protein found in the mushroom Pleurotus ostreatus, and we have produced a recombinant version of this protein (rOly). We measured the viability of several colon cancer cells treated with rOly. Xenografts and syngeneic colon cancer cells were injected into in vivo mouse models, which were then treated with this recombinant protein. rOly treatment induced a significant reduction in viability of human and mouse colon cancer cells. In contrast, there was no reduction in the viability of normal epithelial cells from the small intestine. In the search for cellular targets of rOly, we showed that it enhances the anti-proliferative activity of drugs targeting cellular tubulin. This was accompanied by a reduction in the weight and volume of tumours in mice injected with rOly as compared to their respective control mice in two in vivo models. Our results advance the functional understanding of rOly as a potential anti-cancer treatment associated with pro-apoptotic activities preferentially targeting colon cancer cells.

Background Biofuels derived from lignocellulosic biomass are a viable alternative to fossil fuels required for transportation. Following plant biomass pretreatment, the furan derivative furfural is present at concentrations which are inhibitory to yeasts. Detoxification of furfural is thus important for efficient fermentation. Here, we searched for new genetic attributes in the fungus Neurospora crassa that may be linked to furfural tolerance. The fact that furfural is involved in the natural process of sexual spore germination of N. crassa and that this fungus is highly amenable to genetic manipulations makes it a rational candidate for this study. Results Both hypothesis-based and unbiased (random promotor mutagenesis) approaches were performed to identify N. crassa genes associated with the response to furfural. Changes in the transcriptional profile following exposure to furfural revealed that the affected processes were, overall, similar to those observed in Saccharomyces cerevisiae. N. crassa was more tolerant (by ~ 30%) to furfural when carboxymethyl cellulose was the main carbon source as opposed to sucrose, indicative of a link between carbohydrate metabolism and furfural tolerance. We also observed increased tolerance in a Δcre-1 mutant (CRE-1 is a key transcription factor that regulates the ability of fungi to utilize non-preferred carbon sources). In addition, analysis of aldehyde dehydrogenase mutants showed that ahd-2 (NCU00378) was involved in tolerance to furfural as well as the predicted membrane transporter NCU05580 (flr-1), a homolog of FLR1 in S. cerevisiae. Further to the rational screening, an unbiased approach revealed additional genes whose inactivation conferred increased tolerance to furfural: (i) NCU02488, which affected the abundance of the non-anchored cell wall protein NCW-1 (NCU05137), and (ii) the zinc finger protein NCU01407. Conclusions We identified attributes in N. crassa associated with tolerance or degradation of furfural, using complementary research approaches. The manipulation of the genes involved in furan sensitivity can provide a means for improving the production of biofuel producing strains. Similar research approaches can be utilized in N. crassa and other filamentous fungi to identify additional attributes relevant to other furans or toxic chemicals.

The function of small secreted proteins (SSPs) in saprotrophic fungi is, for the most part, unknown. The white-rot mushroom Pleurotus ostreatus produces considerable amounts of SSPs at the onset of secondary metabolism, during colony development, and in response to chemical compounds such as 5-hydroxymethylfurfural and aryl alcohols. Genetic manipulation of Ssp1, by knockdown (KDssp1) or overexpression (OEssp1), indicated that they are, in fact, involved in the regulation of the ligninolytic system. To elucidate their potential involvement in fungal development, quantitative secretome analysis was performed during the trophophase and the idiophase and at a transition point between the two growth phases. The mutations conferred a time shift in the secretion and expression patterns: OEssp1 preceded the entrance to idiophase and secondary metabolism, while KDssp1 was delayed. This was also correlated with expression patterns of selected genes. The KDssp1 colony aged at a slower pace, accompanied by a slower decline in biomass over time. In contrast, the OEssp1 strain exhibited severe lysis and aging of the colony at the same time point. These phenomena were accompanied by variations in yellow pigment production, characteristic of entrance of the wild type into idiophase. The pigment was produced earlier and in a larger amount in the OEssp1 strain and was absent from the KDssp1 strain. Furthermore, the dikaryon harboring OEssp1 exhibited a delay in the initiation of fruiting body formation as well as earlier aging. We propose that Ssp1 might function as a part of the fungal communication network and regulate the pattern of fungal development and metabolism in P. ostreatus. IMPORTANCE Small secreted proteins (SSPs) are common in fungal saprotrophs, but their roles remain elusive. As such, they comprise part of a gene pool which may be involved in governing fungal lifestyles not limited to symbiosis and pathogenicity, in which they are commonly referred to as “effectors.” We propose that Ssp1 in the white-rot fungus Pleurotus ostreatus regulates the transition from primary to secondary metabolism, development, aging, and fruiting body initiation. Our observations uncover a novel regulatory role of effector-like SSPs in a saprotroph, suggesting that they may act in fungal communication as well as in response to environmental cues. The presence of Ssp1 homologues in other fungal species supports a common potential role in environmental sensing and fungal development.

Fungal spores germinate and undergo vegetative growth, leading to either asexual or sexual reproductive dispersal. Previous research has indicated that among developmental regulatory genes, expression is conserved across nutritional environments, whereas pathways for carbon and nitrogen metabolism appear highly responsive—perhaps to accommodate differential nutritive processing. To comprehensively investigate conidial germination and the adaptive life history decision-making underlying these two modes of reproduction, we profiled transcription of Neurospora crassa germinating on two media: synthetic Bird medium, designed to promote asexual reproduction; and a natural maple sap medium, on which both asexual reproduction and sexual reproduction manifest. A later start to germination but faster development was observed on synthetic medium. Metabolic genes exhibited altered expression in response to nutrients—at least 34% of the genes in the genome were significantly downregulated during the first two stages of conidial germination on synthetic medium. Knockouts of genes exhibiting differential expression across development altered germination and growth rates, as well as in one case causing abnormal germination. A consensus Bayesian network of these genes indicated especially tight integration of environmental sensing, asexual and sexual development, and nitrogen metabolism on a natural medium, suggesting that in natural environments, a more dynamic and tentative balance of asexual and sexual development may be typical of N. crassa colonies. IMPORTANCE One of the most remarkable successes of life is its ability to flourish in response to temporally and spatially varying environments. Fungi occupy diverse ecosystems, and their sensitivity to these environmental changes often drives major fungal life history decisions, including the major switch from vegetative growth to asexual or sexual reproduction. Spore germination comprises the first and simplest stage of vegetative growth. We examined the dependence of this early life history on the nutritional environment using genome-wide transcriptomics. We demonstrated that for developmental regulatory genes, expression was generally conserved across nutritional environments, whereas metabolic gene expression was highly labile. The level of activation of developmental genes did depend on current nutrient conditions, as did the modularity of metabolic and developmental response network interactions. This knowledge is critical to the development of future technologies that could manipulate fungal growth for medical, agricultural, or industrial purposes.

Terrestrial fungi play critical roles in nutrient cycling and food webs and can shape macroorganism communities as parasites and mutualists. Although estimates for the number of fungal species on the planet range from 1.5 to over 5 million, likely fewer than 10% of fungi have been identified so far., Terrestrial fungi play critical roles in nutrient cycling and food webs and can shape macroorganism communities as parasites and mutualists. Although estimates for the number of fungal species on the planet range from 1.5 to over 5 million, likely fewer than 10% of fungi have been identified so far. To date, a relatively small percentage of described species are associated with marine environments, with ∼1,100 species retrieved exclusively from the marine environment. Nevertheless, fungi have been found in nearly every marine habitat explored, from the surface of the ocean to kilometers below ocean sediments. Fungi are hypothesized to contribute to phytoplankton population cycles and the biological carbon pump and are active in the chemistry of marine sediments. Many fungi have been identified as commensals or pathogens of marine animals (e.g., corals and sponges), plants, and algae. Despite their varied roles, remarkably little is known about the diversity of this major branch of eukaryotic life in marine ecosystems or their ecological functions. This perspective emerges from a Marine Fungi Workshop held in May 2018 at the Marine Biological Laboratory in Woods Hole, MA. We present the state of knowledge as well as the multitude of open questions regarding the diversity and function of fungi in the marine biosphere and geochemical cycles.

The Neurospora crassa Mps One Binder (MOB) proteins MOB2A and MOB2B physically interact with the Nuclear Dbf2 Related (NDR) kinase COT1 and have been shown to have overlapping functions in various aspects of asexual development. Here, we identified two N. crassa MOB2A residues, Tyr117 and Tyr119, which are potentially phosphorylated. Using phosphomimetic mob-2a mutants we have been able to establish that apart from their previously described roles, MOB2A/B are involved in additional developmental processes. Enhanced conidial germination, accompanied by conidial agglutination, in the phosphomimetic mutants indicated that MOB2A is a negative regulator of germination. Thick-section imaging of perithecia revealed slow maturation and a lack of asci alignment in the mutant strains demonstrating a role for MOB2A in sexual development. We demonstrate that even though MOB2A and MOB2B have some overlapping functions, MOB2B cannot compensate for the roles MOB2A has in conidiation and germination. Altering Tyr residues 117 and 119 impaired the physical interactions between MOB2A and COT1, most likely contributing to some of the observed effects. As cot-1 and the phosphomimetic mutants share an extragenic suppressor (gul-1), we concluded that at least some of the effects imposed by altering Tyr117 and Tyr119 are mediated by the NDR kinase.

Author(s): Feldman, Daria; Kowbel, David J; Glass, N Louise; Yarden, Oded; Hadar, Yitzhak | Abstract: A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Plants and fungi have had many years of friendly and not-so friendly competition for resources and quality of life. As a result, diverse pathosystems evolved numerous strategies, coupled with the emergence of multifaceted pathogenic and saprophytic lifestyles. We discuss fungal lifestyle classifications and how the views associated with certain fungal pathogens, particularly necrotophs, are changing as we learn more about the complexities of their interactions with a given host plant. We discuss the physiological events leading to the transition from biotrophy to necrotrophy in hemi-biotrophs, and conclude that both the control of plant immune responses and the need for a more efficient mode of nutrient acquisition are possible triggers for the transition to necrotrophy. Based on recent findings, we focus on the polyphagous plant pathogen Sclerotinia sclerotiorum. Rather than overwhelming plant foes, S. sclerotiorum has evolved clever means to compromise host recognition and establish disease, resulting in a broad and immensely successful pathogenic lifestyle. The tactics used by this fungus to achieve pathogenic success are being clarified. We propose that the hemi-biotrophic lifestyle may be more temporally and spatially complex than currently depicted, and that combining lifestyle attributes with damage response curves that consider the contribution of both the fungus and the host to pathogenesis, may provide a more holistic manner to view plant pathogens.

Fungal models have been used, for nearly a century, to answer fundamental questions relevant to the fungal kingdom and beyond and have also provided major contributions for the success of the general fungal research community. Cadres of scientists that study a model organism develop a strong ethos of sharing, derived from communal efforts which, in turn, also contribute to the education of future researchers. There is an increasing trend in preferred funding of research which is problem-driven in contrast to that which is just curiosity-driven. Securing resources for research that does not require practical deliverables is one way of circumventing the slow, unplanned, erosion of support for curiosity-driven fungal research. The role of model fungi as proven, long-term, powerful, engines of scientific insights should not be neglected or abandoned. Rather, they should be continuously celebrated.

COT1 is the founding member of the highly conserved nuclear Dbf2-related (NDR) Ser/Thr kinase family and plays a role in the regulation of polar growth and development in Neurospora crassa and other fungi. Changes in COT1 phosphorylation state have been shown to affect hyphal elongation, branching, and conidiation. The function of NDR protein kinases has been shown to be regulated by type 2A protein phosphatases (PP2As). PP2As are heterotrimers comprised of a catalytic and scaffolding protein along with an interchangeable regulatory subunit involved in determining substrate specificity. Inactivation of the N. crassa PP2A regulatory subunits rgb-1 and b56 conferred severe hyphal growth defects. Partial suppression of defects observed in the rgb-1RIP strain (but not in the Δb56 mutant) was observed in cot-1 phosphomimetic mutants, demonstrating that altering COT1 phosphorylation state can bypass, at least in part, the requirement of a functional RGB1 subunit. The functional fusion proteins RGB1::GFP and B56::GFP predominantly localized to hyphal tips and septa, respectively, indicating that their primary activity is in different cellular locations. COT1 protein forms exhibited a hyperphosphorylated gel migration pattern in an rgb-1RIP mutant background, similar to that observed when the fungus was cultured in the presence of the PP2A inhibitor cantharidin. COT1 was hypophosphorylated in a Δb56 mutant background, suggesting that this regulatory subunit may be involved in determining COT1 phosphorylation state, yet in an indirect manner. Reciprocal co-immunoprecipitation analyses, using tagged COT1, PPH1, RGB1, and B56 subunits established that these proteins physically interact. Taken together, our data determine the presence of a functional and physical link between PP2A and COT1 and show that two of the PP2A regulatory subunits interact with the kinase and determine COT1 phosphorylation state.

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 .

Mushrooms of the genus Pleurotus are comprised of cultivated edible ligninolytic fungi with medicinal properties and a wide array of biotechnological and environmental applications. Like other white-rot fungi (WRF), they are able to grow on a variety of lignocellulosic biomass substrates and degrade both natural and anthropogenic aromatic compounds. This is due to the presence of the non-specific oxidative enzymatic systems, which are mainly consisted of lacasses, versatile peroxidases (VPs), and short manganese peroxidases (short-MnPs). Additional, less studied, peroxidase are dye-decolorizing peroxidases (DyPs) and heme-thiolate peroxidases (HTPs). During the past two decades, substantial information has accumulated concerning the biochemistry, structure and function of the Pleurotus ligninolytic peroxidases, which are considered to play a key role in many biodegradation processes. The production of these enzymes is dependent on growth media composition, pH, and temperature as well as the growth phase of the fungus. Mn(2+) concentration differentially affects the expression of the different genes. It also severs as a preferred substrate for these preoxidases. Recently, sequencing of the Pleurotus ostreatus genome was completed, and a comprehensive picture of the ligninolytic peroxidase gene family, consisting of three VPs and six short-MnPs, has been established. Similar enzymes were also discovered and studied in other Pleurotus species. In addition, progress has been made in the development of molecular tools for targeted gene replacement, RNAi-based gene silencing and overexpression of genes of interest. These advances increase the fundamental understanding of the ligninolytic system and provide the opportunity for harnessing the unique attributes of these WRF for applied purposes.

Fusarium root and stem rot caused by the fungus Fusarium oxysporum f. sp. radicis-cucumerinum is a major disease in greenhouse cucumbers. Over the past decade, the disease has been documented in melon greenhouses in Greece, and recently it has been sporadically recorded in greenhouse melons in Israel. Variations in disease response were found among 41 melon accessions artificially inoculated with the pathogen: 10 accessions were highly susceptible (90–100% mortality), 23 exhibited an intermediate response (20–86%) and eight were resistant (0–4%). Two melon accessions – HEM (highly resistant) and TAD (partially resistant) – were crossed with the susceptible accession DUL. The responses of the three accessions and F1 crosses between the resistant and susceptible parents were evaluated. HEM contributed higher resistance to the F1 hybrid than TAD. Roots of susceptible and resistant accessions were 100 and 79% colonized, respectively, following artificial inoculation. However, only susceptible plants showed colonization of the upper plant tissues. Microscopic evaluation of cross sections taken from the crown region of the susceptible DUL revealed profuse fungal growth in the intercellular spaces of the parenchyma and in xylem vessels. In the resistant cultivar HEM, very little fungal growth was detected in the intercellular spaces of the parenchyma, and none in the xylem or any other vascular tissue. Finding resistant accessions may create an opportunity to study the genetics of resistance inheritance and to develop molecular markers that will facilitate breeding resistant melon cultivars.

The manganese peroxidase gene family (mnps) is a part of the ligninolytic system of Pleurotus ostreatus. This gene family is comprised of nine members, mnp1-9, encoding short manganese peroxidases (short-MnPs) or versatile peroxidases (VPs). We show that unlike in Mn(2+)-amended glucose-peptone (GP) medium, where redundancy among mnps was reported, in Mn(2+)-deficient GP medium mnp4 [encoding versatile peroxidase isoenzyme 4 (VP4)] has a key and nonredundant function. The abundance of mnps transcripts at time points corresponding to the tropophase (active growth), early idiophase, and idiophase indicates that mnp4 is the predominantly expressed mnp gene and that its relative predominance is dependent on the age of the culture. In this medium, azo dye, Orange II (OII) decolorization occurs only during the idiophase and a Δmnp4 strain showed a drastic reduction in this decolorization. Three degradation metabolites were identified by liquid chromatography-mass spectroscopy (LC-MS), indicating both asymmetric and symmetric enzymatic cleavage of the azo-bond. In addition, the culture filtrate of Δmnp4 showed negligible values of oxidation capability of four typical VP substrates: Mn(2+), 2,6-dimethoxyphenol, phenol red, and Reactive Black 5 (RB5), compared to the wild-type strain PC9. We concluded that under Mn(2+)-deficient GP culture, VP4 (encoded by mnp4) is the main active ligninolytic enzyme able to oxidize Mn(2+) as well as high and low redox potential aromatic substrate, including dyes. Furthermore, other VPs/MnPs do not compensate for the lack of VP4 activity.

Reactive oxygen species (ROS) are essential for pathogenic development of Sclerotinia sclerotiorum . A key question for S. sclerotiorum and many other pathogens concerns how fungi tolerate/dampen the oxidative environment during growth and pathogenesis. Regulatory components of oxidative stress include both enzymatic and non-enzymatic antioxidants. Catalases are a ubiquitous family of enzymes that play an important role in the enzymatic detoxification of ROS by converting hydrogen peroxide (H 2 O 2 ) to water and molecular oxygen. The genome of the omnivorous pathogen S. sclerotiorum contains seven predicted catalase genes. In this study we evaluate and functionally characterize the type A catalase ( Scat1 ) in S. sclerotiorum , whose expression is highly induced during host infection. Insertional inactivation of Scat1 (Δ Scat1 ) resulted in hyperbranching of hyphae accompanied by slower growth and smaller sclerotia. Δ Scat1 strains were attenuated in pathogenicity and rendered the fungus hypersensitive to Sodium dodecyl sulfate (SDS), as well as to osmotic and salt stresses. Unexpectedly, Δ Scat1 exhibited increased tolerance to H 2 O 2 , suggesting that although a member of the catalase family, generally associated with amelioration of oxidative stress, Scat1 is probably not required for detoxification of this oxygen species and presumably has different function(s). Δ Scat1 strains had a 2-fold decrease in ergosterol content, and overall lower sterol levels compared to the wild-type strain. These observations are consistent with increased resistance to the polyene drugs amphotericin-B and nystatin. Taken together, our results suggest Scat1 is involved in modulation of ROS in a manner that deviates from the detoxification of H 2 O 2 , alters membrane integrity and contributes to the pathogenic success of S. sclerotiorum .