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.

Impairment of the Neurospora crassa Nuclear DBF2-related kinase-encoding gene cot-1 results in pleiotropic effects, including abnormally thick hyphal cell walls and septa. An increase in the transcript abundance of genes encoding chitin and glucan synthases and the chitinase gh18-5, but not the cell wall integrity pathway transcription factor rlm-1, accompany the phenotypic changes observed. Deletion of chs-5 or chs-7 in a cot-1 background results in a reduction of hyperbranching frequency characteristic of the cot-1 parent. gul-1 (a homologue of the yeast SSD1 gene) encodes a translational regulator and has been shown to partially suppress cot-1. We demonstrate that the high expression levels of the cell wall remodeling genes analyzed is curbed, and reaches near wild type levels, when gul-1 is inactivated. This is accompanied by morphological changes that include reduced cell wall thickness and restoration of normal chitin levels. We conclude that gul-1 is a mediator of cell wall remodeling within the cot-1 pathway.

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.

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 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 PP2A regulatory subunits and conferred severe hyphal growth defects. Partial suppression of defects observed in the strain (but not in the Δ mutant) was observed in 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 mutant background, similar to that observed when the fungus was cultured in the presence of the PP2A inhibitor cantharidin. COT1 was hypophosphorylated in a Δ 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.

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.

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.

ABSTRACTUnderstanding the chemical interactions of common allergens in urban environments may help to decipher the general increase in susceptibility to allergies observed in recent decades. In this study, asexual conidia of the allergenic mold Aspergillus fumigatus were exposed to air pollution under natural (ambient) and controlled (laboratory) conditions. The allergenic activity was measured using two immunoassays and supported by a protein mass spectrometry analysis. The allergenicity of the conidia was found to increase by 2–5 fold compared to the control for short exposure times of up to 12h (accumulated exposure of about 50ppb NO2 and 750ppb O3), possibly due to nitration. At higher exposure times, the allergenicity increase lessened due to protein deamidation. These results indicate that during the first 12h of exposure, the allergenic potency of the fungal allergen A. fumigatus in polluted urban environments is expected to increase. Additional work is needed in order to determine if this behavior occurs for other allergens.

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.

Although Mn2+ is the most abundant substrate of versatile peroxidases (VPs), repression of Pleurotus ostreatus vp1 expression occurred in Mn2+-sufficient medium. This seems to be a biological contradiction. The aim of this study was to explore the mechanism of direct oxidation by VP1 under Mn2+-deficient conditions, as it was found to be the predominant enzyme during fungal growth in the presence of synthetic and natural substrates. The native VP1 was purified and characterized using three substrates, Mn2+, Orange II (OII), and Reactive Black 5 (RB5), each oxidized by a different active site in the enzyme. While the pH optimum for Mn2+ oxidation is 5, the optimum pH for direct oxidation of both dyes was found to be 3. Indeed, effective in vivo decolorization occurred in media without addition of Mn2+ only under acidic conditions. We have determined that Mn2+ inhibits in vitro the direct oxidation of both OII and RB5 while RB5 stabilizes both Mn2+ and OII oxidation. Furthermore, OII was found to inhibit the oxidation of both Mn2+ and RB5. In addition, we could demonstrate that VP1 can cleave OII in two different modes. Under Mn2+-mediated oxidation conditions, VP1 was able to cleave the azo bond only in asymmetric mode, while under the optimum conditions for direct oxidation (absence of Mn2+ at pH 3) both symmetric and asymmetric cleavages occurred. We concluded that the oxidation mechanism of aromatic compounds by VP1 is controlled by Mn2+ and pH levels both in the growth medium and in the reaction mixture. IMPORTANCE VP1 is a member of the ligninolytic heme peroxidase gene family of the white rot fungus Pleurotus ostreatus and plays a fundamental role in biodegradation. This enzyme exhibits a versatile nature, as it can oxidize different substrates under altered environmental conditions. VPs are highly interesting enzymes due to the fact that they contain unique active sites that are responsible for direct oxidation of various aromatic compounds, including lignin, in addition to the well-known Mn2+ binding active site. This study demonstrates the limits of versatility of P. ostreatus VP1, which harbors multiple active sites, exhibiting a broad range of enzymatic activities, but they perform differently under distinct conditions. The versatility of P. ostreatus and its enzymes is an advantageous factor in the fungal ability to adapt to changing environments. This trait expands the possibilities for the potential utilization of P. ostreatus and other white rot fungi.