Hydrophobin Gene Research Articles

Transcriptional Responses of Beauveria bassiana Blastospores Cultured Under Varying Glucose Concentrations.

Culturing the entomopathogenic fungus, Beauveria bassiana, under high glucose concentrations coupled with high aeration results in a fungal developmental shift from hyphal growth to mostly blastospores (yeast-like cells). The underlying molecular mechanisms involved in this shift remain elusive. A systematic transcriptome analysis of the differential gene expression was preformed to uncover the fungal transcriptomic response to osmotic and oxidative stresses associated with the resulting high blastospore yield. Differential gene expression was compared under moderate (10% w/v) and high (20% w/v) glucose concentrations daily for three days. The RNAseq-based transcriptomic results depicted a higher proportion of downregulated genes when the fungus was grown under 20% glucose than 10%. Additional experiments explored a broader glucose range (4, 8, 12, 16, 20% w/v) with phenotype assessment and qRT-PCR transcript abundance measurements of selected genes. Antioxidant, calcium transport, conidiation, and osmosensor-related genes were highly upregulated in higher glucose titers (16-20%) compared to growth in lower glucose (4-6%) concentrations. The class 1 hydrophobin gene (Hyd1) was highly expressed throughout the culturing. Hyd1 is known to be involved in spore coat rodlet layer assembly, and indicates that blastospores or another cell type containing hydrophobin 1 is expressed in the haemocoel during the infection process. Furthermore, we found implications of the HOG signaling pathway with upregulation of homologous genes Ssk2 and Hog1 for all fermentation time points under hyperosmotic medium (20% glucose). These findings expand our knowledge of the molecular mechanisms behind blastospore development and may help facilitate large-scale industrial production of B. bassiana blastospores for pest control applications.

Identification of hydrophobin genes and their physiological functions related to growth and development in Pleurotus ostreatus

Hydrophobins are small secreted proteins with important physiological functions and potential applications. Here, Pleurotus ostreatus hydrophobin genes were systematically analyzed: they were characterized, classified, and their expression profiles and gene functions were explored. In total, 40 P. ostreatus hydrophobin genes were found and showed genetic diversity, of which 15 were newly identified. The hydrophobin protein sequences were diverse but all contained eight cysteine residues with a conserved spacing pattern, and 33 of them were class I hydrophobins. The expression profile analyses showed that Vmh3 and Hydph20 were abundant in monokaryotic and dikaryotic mycelia, whereas Hydph17, Po.hyd16, Hydph8 were specifically expressed in monokaryotic mycelia and Po.hyd10 were specific in dikaryotic mycelia. Furthermore, Vmh3, Hydph20, Po.hyd7, and Po.hyd10 were abundant when dikaryotic mycelia cultivated on PDA, which are different from on substrate (Vmh2, Vmh3, Hydph7, Po.hyd3, Po.hyd7, Po.hyd9); Hydph12, POH1, and Po.hyd4 can be induced by natural light and cold stimulation during development from mycelia to primordia; Vmh3, FBH1, Hydph12, Po.hyd1–Po.hyd5, and Po.hyd8 were highly expressed in primordia and young fruiting bodies; Hydph12, Po.hyd1, Po.hyd4, and Po.hyd5 were specifically expressed in pilei. In addition, RNAi transformants of FBH1 exhibited slower growth rates and had fewer primordia and fruiting bodies, which suggests FBH1 affects the growth rate and primordia formation of P. ostreatus. Therefore, P. ostreatus hydrophobin genes belong to a large family and are temporally and spatially expressed to meet the developmental needs of mushroom.

Characterisation and comparative analysis of hydrophobin isolated from Pleurotus floridanus (PfH)

Hydrophobins are a class of small cysteine rich surface active proteins produced exclusively by filamentous fungi. It forms a nano layer in the cell-water interface, thereby protecting the emerging fungal hyphae from surrounding water. Even though hydrophobins have similar functions in fungi, they share less sequence similarity. In the current study, we made a comparative study of the hydrophobin produced by the mushroom Pleurotus floridanus (PfH). Mushroom P. floridanus was cultured in PD broth. The hydrophobin was purified by foam fractionation and characterized in terms of molecular weight, solubility and glycosylation. In the RP-HPLC analysis, the hydrophobin eluted at a retention time of 45.56 min. The molecular weight of the PfH was found to be 13.52 kDa by MALDI-TOF MS and the LC-MS/MS showed no similar sequence in MASCOT database. The hydrophobin gene of P. floridanus was amplified using custom-designed primers and the BLAST analysis showed 80% sequence similarity with the Vmh2-1 gene of Pleurotus ostreatus. The sequence was translated into protein using ExPASy, secondary and tertiary structure predictions were carried out using Jpred4 and Phyre2. The tertiary structure showed 91.5% similarity with the HYD1 hydrophobin of Schizophyllum commune. A comparative study of PfH with Vmh2-1 and HYD1 was performed using bioinformatics tools. Hydrophobic cluster analysis revealed that three of these proteins have uniformity in terms of amphiphilic and non-amphiphilic α-helices, whereas PfH has a unique proline clustering. Physicochemical analysis by ProtParam revealed that PfH shares similar properties with HYD1 and Vmh2-1, which can be correlated with its function.

G\u03b13 subunit Thga3 positively regulates conidiation, mycoparasitism, chitinase activity, and hydrophobicity of Trichoderma harzianum

Heterotrimeric G-proteins are key elements of signal transduction pathways, which participate in regulating multiple biological processes in fungi including growth, conidiation, antagonism, and mycoparasitism. Among G protein subunits, Gα3 showed diverse regulatory functions in different fungi. In this study, we cloned a Gα3 subunit coding gene thga3 from T. harzianum Th33 that can antagonize Rhizoctonia solani and some other plant pathogenic fungi. A thga3 deletion strain Δthga3 was generated using the double-crossover homologous recombination strategy, and Rthga3 was generated by transforming thga3-expressing vector into the protoplasts of Δthga3 by the PEG/CaCl2-mediated method. The biological characteristics of wild-type Th33, Δthga3 and Rthga3 were evaluated. Compared with wild-type Th33, Δthga3 showed 15%, 94%, and 23% decrease in hyphal growth, conidia yield, and chitinase activity, respectively, and Δthga3 showed lower antagonistic and mycoparasitism abilities, while there were no significant differences between wild-type Th33 and Rthga3. The hyphal surface hydrophobicity of Δthga3 significantly decreased compared with those of the wild-type Th33 and Rthga3. qRT-PCR analysis revealed that transcript abundance of the hydrophobin gene (tha_09745) of Δthga3 decreased by 80% compared with that of wild-type Th33 and Rthga3. The results showed that thga3 positively regulates the growth, conidiation, hydrophobicity, chitinase activities, and mycoparasitism of Th33 towards R. solani. We hence deduced that the expression level of Tha_09745 is correlated to the hyphal hydrophobicity of Th33 and therefore affects the other biological characteristics of Th33. The findings of this report provide a foundation for elucidating the G-protein signal regulatory mechanisms of fungi.

Molecular Identification of Aspergillus Fumigatus Complex from Lung Transplant Recipients Using Multi Locus Sequencing Analysis (MLSA)

Aspergillus fumigatus is responsible for Invasive aspergillosis (IA). The role of other closely related species such as Aspergillus lentulus and Aspergillus viridinutans which are members of the section Fumigati (A. fumigatus complex) in the setting of pre-emptive therapy in lung transplant recipients is not well defined, and may be associated with poor clinical response. A total of 99 A. fumigatus isolates that were identified by the mycology laboratory at Mount Sinai Hospital (MSH) using conventional phenotypic procedures were genotyped using Multilocus Sequencing Analysis (MLSA) of the β-tubulin (benA), hydrophobin (rodA), and calmodulin (cmd) gene. These isolates were originally isolated from BAL or sputum specimens collected from immunocompromised patients. Of the 99 isolates, 71, 12, and 16 were isolated from post-lung transplant recipients (LRT), pre-transplant recipients, and non-lung transplants, respectively. 98 isolates (99.8%) carried the DNA sequences of the aforementioned genes, which were identical to those of A. fumigatus reference strains in the GeneBank, and thus clustered in the same clade with A. fumigatus. Only one variant isolate from a case of disseminated infection was identified and clustered with the same clade as the fumigati reference species; A. thermomutatus. Four reference strains were processed along with our isolates in this study to ensure the sensitivity and specificity of the assays. The MLSA data analysis revealed that A. fumigatus were the only strains recovered in our population of lung transplant recipients. The MLSA results correlated very well with those obtained by conventional mycological procedures. A. fumigatus appears to be the main causative agent of Invasive aspergillosis in our LTRs in the setting of pre-emptive therapy. Other fumigati species were not detected in LTRs.

Stress Reshapes the Physiological Response of Halophile Fungi to Salinity.

(1) Background: Mechanisms of cellular and molecular adaptation of fungi to salinity have been commonly drawn from halotolerant strains and few studies in basidiomycete fungi. These studies have been conducted in settings where cells are subjected to stress, either hypo- or hyperosmotic, which can be a confounding factor in describing physiological mechanisms related to salinity. (2) Methods: We have studied transcriptomic changes in Aspergillus sydowii, a halophilic species, when growing in three different salinity conditions (No NaCl, 0.5 M, and 2.0 M NaCl). (3) Results: In this fungus, major physiological modifications occur under high salinity (2.0 M NaCl) and not when cultured under optimal conditions (0.5 M NaCl), suggesting that most of the mechanisms described for halophilic growth are a consequence of saline stress response and not an adaptation to saline conditions. Cell wall modifications occur exclusively at extreme salinity, with an increase in cell wall thickness and lamellar structure, which seem to involve a decrease in chitin content and an augmented content of alfa and beta-glucans. Additionally, three hydrophobin genes were differentially expressed under hypo- or hyperosmotic stress but not when the fungus grows optimally. Regarding compatible solutes, glycerol is the main compound accumulated in salt stress conditions, whereas trehalose is accumulated in the absence of salt. (4) Conclusions: Physiological responses to salinity vary greatly between optimal and high salt concentrations and are not a simple graded effect as the salt concentration increases. Our results highlight the influence of stress in reshaping the response of extremophiles to environmental challenges.

The Methylcitrate Cycle is Required for Development and Virulence in the Rice Blast Fungus Pyricularia oryzae.

The methylcitrate cycle metabolizes propionyl-CoA, a toxic metabolite, into pyruvate. Pyricularia oryzae (syn. Magnaporthe oryzae) is a phytopathogenic fungus that causes a destructive blast disease in rice and wheat. We characterized the essential roles of the methylcitrate cycle in the development and virulence of P. oryzae using functional genomics. In P. oryzae, the transcript levels of MCS1 and MCL1, which encode a 2-methylcitrate synthase and a 2-methylisocitrate lyase, respectively, were upregulated during appressorium formation and when grown on propionyl-CoA-producing carbon sources. We found that deletion of MCS1 and MCL1 inhibited fungal growth on media containing both glucose and propionate, and media using propionate or propionyl-CoA-producing amino acids (valine, isoleucine, methionine, and threonine) as the sole carbon or nitrogen sources. The Δmcs1 mutant formed sparse aerial hyphae and did not produce conidia on complete medium (CM), while the Δmcl1 mutant showed decreased conidiation. The aerial mycelium of Δmcs1 displayed a lowered NAD+/NADH ratio, reduced nitric oxide content, and downregulated transcription of hydrophobin genes. Δmcl1 showed reduced appressorium turgor, severely delayed plant penetration, and weakened virulence. Addition of acetate recovered the growth of the wild type and Δmcs1 on medium containing both glucose and propionate and recovered the conidiation of both Δmcs1 and Δmcl1 on CM by reducing propionyl-CoA formation. Deletion of MCL1 together with ICL1, an isocitrate lyase gene in the glyoxylate cycle, greatly reduced the mutant's virulence as compared with the single-gene deletion mutants (Δicl1 and Δmcl1). This experimental evidence provides important information about the role of the methylcitrate cycle in development and virulence of P. oryzae by detoxification of propionyl-CoA and 2-methylisocitrate.

A hydrophobin gene, Hyd9, plays an important role in the formation of aerial hyphae and primordia in Flammulina filiformis

Flammulina filiformis is an edible fungus that is largely cultivated and widely consumed around the world. The quantity and quality of the primordia, which gives rise to the fruiting body, affects its production efficiency. Hydrophobins are involved in the formation of the fruiting body of macrofungi. However, functional verification of the hydrophobin genes is limited to date. In this study, we used gene silencing and overexpression analyses to investigate the function of one F. filiformis hydrophobin gene (Hyd9) during the development of the fruiting body. The Hyd9-silenced transformants exhibited sparse aerial hyphae, resulting in fewer primordia and fruiting bodies. In contrast, the Hyd9 overexpression strain displayed denser aerial hyphae and more primordia. The phenotypes of these transgenic lines strongly suggested that Hyd9 plays an important role in the formation of aerial hyphal knots (the primary stage of primordia) and primordia in F. filiformis. These results will be beneficial for developing more efficient methods to induce primordia formation in F. filiformis and other commercially valuable mushrooms.

Effect of gene copy number and chaperone coexpression on recombinant hydrophobin HFBI biosurfactant production in Pichia pastoris.

Hydrophobins are small highly surface-active fungal proteins with potential as biosurfactants in a wide array of applications. However, practical implementation of hydrophobins at large scale has been hindered by low recombinant yields. In this study, the effects of increasing hydrophobin gene copy number and overexpressing endoplasmic reticulum resident chaperone proteins Kar2p, Pdi1p, and Ero1p were explored as a means to enhance recombinant yields of the class II hydrophobin HFBI in the eukaryotic expression host Pichia pastoris. One-, 2-, and 3-copy-HFBI strains were attained using an in vitro multimer ligation approach, with strains displaying copy number stability following subsequent transformations as measured by quantitative polymerase chain reaction. Increasing HFBI copy number alone had no effect on increasing HFBI secretion, but increasing copy number in concert with chaperone overexpression synergistically increased HFBI secretion. Overexpression of PDI1 or ERO1 caused insignificant changes in HFBI secretion in 1- and 2-copy strains, but a statistically significant HFBI secretion increase in 3-copy strain. KAR2 overexpression consistently resulted in enhanced HFBI secretion in all copy number strains, with 3-copy-HFBI secreting 22±1.6 fold more than the 1-copy-HFBI/no chaperone strain. The highest increase was seen in 3-copy-HFBI/Ero1p overexpressing strain with 30±4.0 fold increase in HFBI secretion over 1-copy-HFBI/no chaperone strain. This corresponded to an expression level of approximately 330 mg/L HFBI in the 5 ml small-scale format used in this study.

Hydrophobin HFBII-4 from Trichoderma asperellum induces antifungal resistance in poplar.

Herein, the class II hydrophobin gene HFBII-4 was cloned from the biocontrol agent Trichoderma asperellum ACCC30536 and recombinant rHFBII-4 was expressed in Pichia pastoris GS115. Treatment of Populus davidiana × P. alba var. pyramidalis (PdPap poplar) with rHFBII-4 altered the expression levels of genes in the auxin, salicylic acid (SA), and jasmonic acid (JA) signal transduction pathways. Polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) enzyme activities were induced with rHFBII-4. Evans Blue and nitro blue tetrazolium (NBT) staining indicated that cell membrane permeability and reactive oxygen species were lower in the leaves of plants treated with rHFBII-4. The chlorophyll content was higher than that of control at 2-5days after treatment. Furthermore, poplar seedlings were inoculated with Alternaria alternata, disease symptoms were observed. The diseased area was smaller in leaves induced with rHFBII-4 compared with control. In summary, rHFBII-4 enhances resistance to A. alternata.

Influence of O2 and CO2on fungal development and hydrophobin expression during the interaction between Heterobasidion parviporum and Phlebiopsis gigantea

AbstractHeterobasidion annosum is a very destructive fungal pathogen causing massive losses on forest trees in the northern hemisphere. Tree infection usually takes place through the cut surface after felling. Phlebiopsis gigantea is commonly used as a biological control agent, applied directly after cutting on the stump surface. We investigated the interaction between both fungi in different gas compositions with lowered O2 (0.1% and 0.4%) and enhanced CO2 (15%) concentrations, imitating conditions as present on the wood surface and inside living wood tissue. When grown under 0.1% O2 and 15% CO2, fungal development of both species was altered, growth rate of P. giganteawas decreased and of H. parviporum slightly increased. Low O2 and high CO2 induced the formation of highly hydrophilic tissue with impaired conidia formation and germination capacity. In H. parviporum,this effect was more striking after 7 dpi with 45% reduced vitality, in P. gigantea after 14 dpi with 89% vitality reduction. These observations suggest that entering the tree through the cut surface is accompanied by altering fungal tissue to be more hydrophilic with a concurrent reduction of conidia formation and germination. Expression of the hydrophobin genes HAH1 and HAH2 of H. parviporum at ambient air decreased significantly in response to fungal interaction with P. gigantea. Gene expression was also influenced by O2 and CO2 content. Reduction of O2to 0.4% led to significantly decreased transcription rate of both HAH1 and HAH2 in single and dual cultures.

Comparative transcriptome analysis identified candidate genes involved in mycelium browning in Lentinula edodes

BackgroundLentinula edodes is one of the most popular edible mushroom species in the world and contains useful medicinal components, such as lentinan. The light-induced formation of brown film on the vegetative mycelial tissues of L. edodes is an important process for ensuring the quantity and quality of this edible mushroom. To understand the molecular mechanisms underlying this critical developmental process in L. edodes, we characterized the morphological phenotypic changes in a strain, Chamaram, associated with abnormal brown film formation and compared its genome-wide transcriptional features.ResultsIn the present study, we performed genome-wide transcriptome analyses of different vegetative mycelium growth phenotypes, namely, early white, normal brown, and defective dark yellow partial brown films phenotypes which were exposed to different light conditions. The analysis revealed the identification of clusters of genes specific to the light-induced brown film phenotypes. These genes were significantly associated with light sensing via photoreceptors such as FMN- and FAD-bindings, signal transduction by kinases and GPCRs, melanogenesis via activation of tyrosinases, and cell wall degradation by glucanases, chitinases, and laccases, which suggests these processes are involved in the formation of mycelial browning in L. edodes. Interestingly, hydrophobin genes such as SC1 and SC3 exhibited divergent expression levels in the normal and abnormal brown mycelial films, indicating the ability of these genes to act in fruiting body initiation and formation of dikaryotic mycelia. Furthermore, we identified the up-regulation of glycoside hydrolase domain-containing genes in the normal brown film but not in the abnormal film phenotype, suggesting that cell wall degradation in the normal brown film phenotype is crucial in the developmental processes related to the initiation and formation of fruiting bodies.ConclusionsThis study systematically analysed the expression patterns of light-induced browning-related genes in L. edodes. Our findings provide information for further investigations of browning formation mechanisms in L. edodes and a foundation for future L. edodes breeding.

Detection of Talaromyces macrosporus and Talaromyces trachyspermus by a PCR assay targeting the hydrophobin gene.

Talaromyces species are typical fungi capable of producing the heat-resistant ascospores responsible for the spoilage of processed food products. Hydrophobins, which are unique to fungi, are small secreted proteins that form amphipathic layers on the outer surface of fungal cell walls. In this study, species-specific primer sets for detecting and identifying Talaromyces macrosporus and Talaromyces trachyspermus were designed based on hydrophobin gene sequences. A conventional polymerase chain reaction (PCR) assay using these primer sets produced species-specific amplicons for T. macrosporus and T. trachyspermus. The detection limit for each primer set was 100pg template DNA. This assay also detected fungal DNA extracted from blueberries inoculated with T. macrosporus. Other heat-resistant fungi, including Byssochlamys, Neosartorya and Talaromyces species, which cause food spoilage, were not detected in PCR amplifications with these primer sets. Furthermore, a conventional PCR assay using a crude DNA extract as the template also yielded amplicons specific to T. macrosporus and T. trachyspermus. The simple and rapid PCR assay described herein is highly species-specific and can reliably detect T. macrosporus and T. trachyspermus, suggesting it may be relevant for the food and beverage industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The heat-resistant ascospores of Talaromyces macrosporus and Talaromyces trachyspermus are responsible for food spoilage after pasteurization. Traditional methods for detecting fungal contamination based on morphological characteristics are time-consuming and exhibit low sensitivity and specificity. In this study, a conventional polymerase chain reaction (PCR) assay based on hydrophobin gene sequences was developed for the specific detection of T. macrosporus and T. trachyspermus. This detection method was simple, rapid and highly specific. These results suggest that the conventional PCR assay developed in this study may be useful for detecting T. macrosporus and T. trachyspermus in raw materials and processed food products.

The regulator of G-protein signalling Thn1 links pheromone response to volatile production in Schizophyllum commune.

The regulator of G-protein signalling, Thn1, is involved in sexual development through pheromone signalling in the mushroom forming basidiomycete Schizophyllum commune affecting hyphal morphology and mating interactions. Thn1 plays a key role in coordinating sesquiterpene production, pheromone response and sexual development. The gene thn1 is transcriptionally regulated in response to mating with a role in clamp cell development and hydrophobin gene transcription. Further, it negatively regulates cAMP signalling and secondary metabolism. Disruption of thn1 affects dikaryotization by reducing clamp fusion and development with predominant non-fused pseudoclamps. Enhanced protein kinase A (PKA) activities in Δthn1 strains indicate that Thn1 regulates pheromone signalling by de-activating G-protein α subunits, which control cAMP-dependent PKA. The repressed formation of aerial hyphae could be linked to a reduced metabolic activity and to a transcriptional down-regulation of hyd6 and sc3 hydrophobin genes. Thn1 was also shown to be necessary for the biosynthesis of sesquiterpenes and an altered spectrum of sesquiterpenes in Δthn1 is linked to transcriptional up-regulation of biosynthesis genes. Proteome analysis indicated changes in cytoskeletal structure affecting actin localization, linking the major regulator Thn1 to growth and development of S. commune. The results support a role for Thn1 in G-protein signalling connecting development and secondary metabolism.

Disruption of gul-1 decreased the culture viscosity and improved protein secretion in the filamentous fungus Neurospora crassa

BackgroundThe cellulolytic fungus Neurospora crassa is considered a potential host for enzyme and bioethanol production. However, large scale applications are hindered by its filamentous growth. Although previous investigations have shown that mycelial morphology in submerged culture can be controlled by altering physical factors, there is little knowledge available about the potential for morphology control by genetic modification.ResultsIn this study, we screened morphological mutants in the filamentous fungus N. crassa. Of the 90 morphological mutants screened, 14 mutants exhibited considerably higher viscosity compared with that of the wild type strain, and only two mutants showed low-viscosity morphologies in submerged culture. We observed that disruption of gul-1 (NCU01197), which encodes an mRNA binding protein involved in cell wall remodeling, caused pellet formation as the fermentation progressed, and resulted in the most significant decrease in viscosity of culture broth. Moreover, over-expression of gul-1 caused dramatically increased viscosity, suggesting that the gul-1 had an important function in mycelial morphology during submerged cultivation. Transcriptional profiling showed that expression of genes encoding eight GPI-anchored cell wall proteins was lowered in Δgul-1 while expression of genes associated with two non-anchored cell wall proteins was elevated. Meanwhile, the expression levels of two hydrophobin genes were also significantly altered. These results suggested that GUL-1 affected the transcription of cell wall-related genes, thereby influencing cell wall structure and mycelial morphology. Additionally, the deletion of gul-1 caused increased protein secretion, probably due to a defect in cell wall integrity, suggesting this as an alternative strategy of strain improvement for enzyme production. To confirm practical applications, deletion of gul-1 in the hyper-cellulase producing strain (∆ncw-1∆Ncap3m) significantly reduced the viscosity of culture broth.ConclusionsUsing the model filamentous fungus N. crassa, genes that affect mycelial morphology in submerged culture were explored through systematic screening of morphological mutants. Disrupting several candidate genes altered viscosities in submerged culture. This work provides an example for controlling fungal morphology in submerged fermentation by genetic engineering, and will be beneficial for industrial fungal strain improvement.

Hydrophobin gene deletion and environmental growth conditions impact mechanical properties of mycelium by affecting the density of the material

Filamentous fungi colonize substrates by forming a mycelium. This network of hyphae can be used as a bio-based material. Here, we assessed the impact of environmental growth conditions and deletion of the hydrophobin gene sc3 on material properties of the mycelium of the mushroom forming fungus Schizophyllum commune. Thermogravimetric analysis showed that Δsc3 mycelium retained more water with increasing temperature when compared to the wild type. The Young’s modulus (E) of the mycelium ranged between 438 and 913 MPa when the wild type strain was grown in the dark or in the light at low or high CO2 levels. This was accompanied by a maximum tensile strength (σ) of 5.1–9.6 MPa. In contrast, E and σ of the Δsc3 strain were 3–4- fold higher with values of 1237–2727 MPa and 15.6–40.4 MPa, respectively. These values correlated with mycelium density, while no differences in chemical composition of the mycelia were observed as shown by ATR-FTIR. Together, genetic modification and environmental growth conditions impact mechanical properties of the mycelium by affecting the density of the mycelium. As a result, mechanical properties of wild type mycelium were similar to those of natural materials, while those of Δsc3 were more similar to thermoplastics.

Hydrophobins contribute to root colonization and stress responses in the rhizosphere-competent insect pathogenic fungus Beauveria bassiana.

The hyd1/hyd2 hydrophobins are important constituents of the conidial cell wall of the insect pathogenic fungus Beauveria bassiana. This fungus can also form intimate associations with several plant species. Here, we show that inactivation of two Class I hydrophobin genes, hyd1 or hyd2, significantly decreases the interaction of B. bassiana with bean roots. Curiously, the ∆hyd1/∆hyd2 double mutant was less impaired in root association than Δhyd1 or Δhyd2. Loss of hyd genes affected growth rate, conidiation ability and oosporein production. Expression patterns for genes involved in conidiation, cell wall integrity, insect virulence, signal transduction, adhesion, hydrophobicity and oosporein production were screened in the deletion mutants grown in different conditions. Repression of the major MAP-Kinase signal transduction pathways (Slt2 MAPK pathway) was observed that was more pronounced in the single versus double hyd mutants under certain conditions. The ∆hyd1/∆hyd2 double mutant showed up-regulation of the Hog1 MAPK and the Msn2 transcription factor under certain conditions when compared to the wild-type or single hyd mutants. The expression of the bad2 adhesin and the oosporein polyketide synthase 9 gene was severely reduced in all of the mutants. On the other hand, fewer changes were observed in the expression of key conidiation and cell wall integrity genes in hyd mutants compared to wild-type. Taken together, the data from this study indicated pleiotropic consequences of deletion of hyd1 and hyd2 on signalling and stress pathways as well as the ability of the fungus to form stable associations with plant roots.

3,5-Dicaffeoylquinic Acid Disperses Aspergillus Fumigatus Biofilm and Enhances Fungicidal Efficacy of Voriconazole and Amphotericin B.

BackgroundThe aim of this study was to evaluate the dispersal effects of 3,5-dicaffeoylquinic acid (3,5-DCQA) against the preformed biofilm of Aspergillus fumigatus and to investigate its potential mechanism.Material/MethodsAspergillus fumigatus biofilms of laboratory strain AF293 and clinical strain GXMU04 were generated in 24- or 96-well polystyrene microtiter plates in vitro. Crystal violet assay and XTT reduction assay were performed to evaluate the effects of 3,5-DCQA on biofilm biomass, extracellular matrix, and metabolic activity alteration of cells in biofilms. Real-time PCR was performed to quantify the expression of hydrophobin genes. The cytotoxicity of 3,5-DCQA on human erythrocytes was evaluated by a hemolytic assay.ResultsThe results indicated that 3,5-DCQA in subminimum inhibitory concentrations (256 to 1024 mg/L) elicited optimal A. fumigatus biofilm dispersion activity and improved the efficacy of VRC and AMB in minimal fungicidal concentrations (MFCs) to combat fungal cells embedded in biofilms. The results of scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM) revealed 3,5-DCQA facilitated the entry of antifungal agents into the A. fumigatus biofilm through eliminating the hydrophobic extracellular matrix (ECM) without affecting fungal growth. Real-time PCR indicated that 3,5-DCQA down-regulated the expression of hydrophobin genes. Hemolytic assay confirmed that 3,5-DCQA exhibited a low cytotoxicity against human erythrocytes.ConclusionsSubminimum inhibitory concentrations of 3,5-DCQA can disperse A. fumigatus biofilm and enhance fungicidal efficacy of VRC and AMB through down-regulating expression of the hydrophobin genes. The study indicated the anti-biofilm potential of 3,5-DCQA for the management of A. fumigatus biofilm-associated infection.

CHARACTERISTICS AND FUNCTIONS OF HYDROPHOBINS AND THEIR USE IN MANIFOLD INDUSTRIES

Abstract Hydrophobins are surface active proteins produced by filamentous fungi. They have a role in fungal growth and their life cycle. Although proteins with similar properties are being found in prokaryotic organisms as well. Hydrophobins are characterized by a specific arrangement of cysteine residues, which form four disulfide bridges in the amino acid sequence. This construction gives hydrophobins hydrophobic properties. These proteins are able to assemble spontaneously into amphipathic monolayers at hydrophobic-hydrophilic interfaces. The unique properties of hydrophobins make them more and more popular with regard to their potential application in industry. New ways of use hydrophobins in various branches of the economy are being developed. Hydrophobins are already widely used in the food industry, pharmaceutical industry, but also in molecular biology. 1. Introduction. 2. Classification of hydrophobins. 3. Structure of hydrophobin genes and proteins. 4. Formation of hydrophobin film. 5. Production, secretion and formation of hydrophobins in the natural environment. 6. Properties of hydrophobins. 7. The use of hydrophobins in various fields. 8. Manufacturing of hydrophobins. 9. Summary

Cloning and heterologous expression of a hydrophobin gene Ltr.hyd from the tiger milk mushroom Lentinus tuber-regium in yeast-like cells of Tremella fuciformis

Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier.