Neurospora Crassa Research Articles

Magic-angle spinning NMR spectral editing of polysaccharides in whole cells using the DREAM scheme

Most bacterial, plant and fungal cells possess at their surface a protective layer called the cell wall, conferring strength, plasticity and rigidity to withstand the osmotic pressure. This molecular barrier is crucial for pathogenic microorganisms, as it protects the cell from the local environment and often constitutes the first structural component encountered in the host-pathogen interaction. In pathogenic molds and yeasts, the cell wall constitutes the main target for the development of clinically-relevant antifungal drugs. In the past decade, solid-state NMR has emerged as a powerful analytical technique to investigate the molecular organization of microbial cell walls in the context of intact cells. 13C NMR chemical shift is an exquisite source of information to identify the polysaccharides present in the cell wall, and two-dimensional 13C–13C correlation experiments provide an efficient tool to rapidly access the polysaccharide composition in whole cells. Here we investigate the use of the adiabatic DREAM (for dipolar recoupling enhancement through amplitude modulation) recoupling scheme to improve solid-state NMR analysis of polysaccharides in intact cells. We demonstrate the advantages of two-dimensional 13C–13C experiments using the DREAM recoupling scheme. We report the spectral editing of polysaccharide signals by varying the radio-frequency carrier position. We provide practical considerations for the implementation of DREAM experiments to characterize polysaccharides in whole cells. We demonstrate the approach on intact fungal cells of Neurospora crassa and Aspergillus fumigatus, a model and a pathogenic filamentous fungus, respectively. The approach could be envisioned to efficiently reduce the spectral crowding of more complex cell surfaces, such as cell wall and peptidoglycan in bacteria.

The First Observation of the Filamentous Fungus Neurospora crassa Growing in the Roots of the Grass Brachypodium distachyon.

The model organism Neurospora crassa has been cultivated in laboratories since the 1920s and its saprotrophic lifestyle has been established for decades. However, beyond their role as saprotrophs, fungi engage in intricate relationships with plants, showcasing diverse connections ranging from mutualistic to pathogenic. Although N. crassa has been extensively investigated under laboratory conditions, its ecological characteristics remain largely unknown. In contrast, Brachypodium distachyon, a sweet grass closely related to significant crops, demonstrates remarkable ecological flexibility and participates in a variety of fungal interactions, encompassing both mutualistic and harmful associations. Through a comprehensive microscopic analysis using electron, fluorescence, and confocal laser scanning microscopy, we discovered a novel endophytic interaction between N. crassa and B. distachyon roots, where fungal hyphae not only thrive in the apoplastic space and vascular bundle but also may colonize plant root cells. This new and so far hidden trait of one of the most important fungal model organisms greatly enhances our view of N. crassa, opening new perspectives concerning the fungus' ecological role. In addition, we present a new tool for studying plant-fungus interspecies communication, combining two well-established model systems, which improves our possibilities of experimental design on the molecular level.

Cellobionate production from sodium hydroxide pretreated wheat straw by engineered Neurospora crassa HL10

This study investigated cellobionate production from a lignocellulosic substrate using Neurospora crassa HL10. Utilizing NaOH-pretreated wheat straw as the substrate obviated the need for an exogenous redox mediator addition, as lignin contained in the pretreated wheat served as a natural mediator. The low laccase production by N. crassa HL10 on pretreated wheat straw caused slow cellobionate production, and exogenous laccase addition accelerated the process. Cycloheximide induced substantial laccase production in N. crassa HL10, enabling the strain to yield approximately 57 mM cellobionate from pretreated wheat straw (equivalent to 20 g/L cellulose), shortening the conversion time from 8 to 6 days. About 92% of the cellulose contained in the pretreated wheat straw is converted to cellobionate. In contrast to existing methods requiring pure cellobiose or cellulase enzymes, this process efficiently converts a low-cost feedstock into cellobionate at a high yield without enzyme or redox mediator supplementation.

The CRZ-1 transcription factor binds to specific sequences in the promoters of al-2, cao-2, and ylo-1 to regulate the expression of these carotenoid biosynthesis genes in Neurospora crassa

The CRZ-1 transcription factor binds to specific sequences in the promoters of al-2, cao-2, and ylo-1 to regulate the expression of these carotenoid biosynthesis genes in Neurospora crassa

Crecimiento y producción de invertasa por Neurospora crassa en cultivos sumergido y sólido

Filamentous fungi are widely used in industry to produce enzymes, organic acids, and secondary metabolites. Neurospora crassa has gained attention due to its flexibility for easy genetic manipulation, fast growth, and non-pathogenic characteristics. This study evaluated the growth of N. crassa and invertase production in submerged culture (SC) and solid-state culture (SSC) using pine sawdust (SSC-PS) and polyurethane foam (SSC-PUF) as supports. Modified Vogel's medium with initial sucrose concentrations of 1.5%, 3%, and 5% was used. The specific and maximum CO2 production rates were higher in SSC than SC, particularly with SSC-PS. Protein and invertase production were higher in SSC, with SSC-PS demonstrating the highest sucrose concentration as the inducer and carbon source. The water-holding capacity (WHC) of SSC-PUF was approximately 25 times higher than that of SSC-PS, facilitating a more productive process. SSC-PUF enables higher biomass growth, protein, and invertase production than SC. Moreover, using inert supports, such as PUF, allows the correct substrates and product assessment without interferences due to the chemical composition and heterogeneity of conventional agro-industrial by-products, such as PS.

The clock in growing hyphae and their synchronization in Neurospora crassa

Utilizing a microfluidic chip with serpentine channels, we inoculated the chip with an agar plug with Neurospora crassa mycelium and successfully captured individual hyphae in channels. For the first time, we report the presence of an autonomous clock in hyphae. Fluorescence of a mCherry reporter gene driven by a clock-controlled gene-2 promoter (ccg-2p) was measured simultaneously along hyphae every half an hour for at least 6 days. We entrained single hyphae to light over a wide range of day lengths, including 6,12, 24, and 36 h days. Hyphae tracked in individual serpentine channels were highly synchronized (K = 0.60-0.78). Furthermore, hyphae also displayed temperature compensation properties, where the oscillation period was stable over a physiological range of temperatures from 24 °C to 30 °C (Q10 = 1.00-1.10). A Clock Tube Model developed could mimic hyphal growth observed in the serpentine chip and provides a mechanism for the stable banding patterns seen in race tubes at the macroscopic scale and synchronization through molecules riding the growth wave in the device.

A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa.

Genome organization is essential for proper function, including gene expression. In metazoan genome organization, chromatin loops and Topologically Associated Domains (TADs) facilitate local gene clustering, while chromosomes form distinct nuclear territories characterized by compartmentalization of silent heterochromatin at the nuclear periphery and active euchromatin in the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation, where heterochromatic centromeres and telomeres independently cluster at the nuclear membrane, while interspersed heterochromatic loci in Neurospora aggregate across megabases of linear genomic distance for forming TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. Here, we examined the genome organization of a Neurospora strain harboring a ~47.4 kilobase facultative (temporarily silent) heterochromatic region deletion, as well as the genome organization of a strain deleted of a 110.6 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion had little effect on local chromatin structure, the constitutive heterochromatin deletion altered local TAD-like structures, gene expression, and the predicted 3D genome structure by qualitatively repositioning genes into the nucleus center. Our work elucidates the role of individual heterochromatic regions for genome organization and function.

Signatures of transposon-mediated genome inflation, host specialization, and photoentrainment in Entomophthora muscae and allied entomophthoralean fungi

Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. In this manuscript, we present a genome for a laboratory-tractable Entomophthora muscae isolate that infects fruit flies. Our E. muscae assembly is 1.03 Gb, consists of 7810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with recent datasets from entomophthoralean fungi, we show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity. In addition, we find that E. muscae and its closest allies possess genes that are likely homologs to the blue-light sensor white-collar 1, a Neurospora crassa gene that has a well-established role in maintaining circadian rhythms. We uncover evidence that E. muscae diverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent with E. muscae’s species-specific, biotrophic lifestyle. Finally, we offer a head-to-head comparison of morphological and molecular data for species within the E. muscae species complex that support the need for taxonomic revision within this group. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi.

Signatures of transposon-mediated genome inflation, host specialization, and photoentrainment in Entomophthora muscae and allied entomophthoralean fungi.

Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. In this manuscript, we present a genome for a laboratory-tractable Entomophthora muscae isolate that infects fruit flies. Our E. muscae assembly is 1.03 Gb, consists of 7810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with recent datasets from entomophthoralean fungi, we show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity. In addition, we find that E. muscae and its closest allies possess genes that are likely homologs to the blue-light sensor white-collar 1, a Neurospora crassa gene that has a well-established role in maintaining circadian rhythms. We uncover evidence that E. muscae diverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent with E. muscae's species-specific, biotrophic lifestyle. Finally, we offer a head-to-head comparison of morphological and molecular data for species within the E. muscae species complex that support the need for taxonomic revision within this group. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi.

Neurospora sp. Mediated Synthesis of Naringenin for the Production of Bioactive Nanomaterials.

The application of Neurospora sp., a fungus that commonly thrives on complex agricultural and plant wastes, has proven successful in utilizing citrus peel waste as a source of naringin. A UV-Vis spectrophotometric method proved the biotransformation of naringin, with an absorption maximum (λmax) observed at 310 nm for the biotransformed product, naringenin (NAR). Further verification of the conversion of naringin was provided through thin layer chromatography (TLC). The Neurospora crassa mediated biotransformation of naringin to NAR was utilized for the rapid (within 5 min) synthesis of silver (Ag) and gold (Au) nanoconjugates using sunlight to accelerate the reaction. The synthesized NAR-nano Ag and NAR-nano Au conjugates exhibited monodispersed spherical and spherical as well as polygonal shaped particles, respectively. Both of the nanoconjugates showed average particle sizes of less than 90 nm from TEM analysis. The NAR-Ag and NAR-Au nanoconjugates displayed potential enhancement of the antimicrobial activities, including antibacterial and nematicidal properties over either standalone NAR or Ag or Au NPs. This study reveals the potential of naringinase-producing Neurospora sp. for transforming naringin into NAR. Additionally, the resulting NAR-Ag and NAR-Au nanoconjugates showed promise as sustainable antibiotics and biochemical nematicides.

Isolation and Molecular Identification of Endophytic Fungi Associated with Brown Algae for Inhibiting Escherichia coli ESBL

Background: The marine environment is the main source of research on natural products in the future. In addition, marine microorganisms have been identified as a natural source capable of developing new antibiotic compounds, including controlling urinary tract infections caused by Escherichia coli ESBL. Purpose: This study aims to isolate, select, and test the potential of brown macroalgae endophytic fungi (Phaeophyceae) collected from the coastal waters of Sanur, Bali Province. Methods: Three types of brown algae were collected from Sanur Beach and their endophytic fungi were isolated using PDA media. Antibacterial activity was determined by measuring the inhibition zone and determining the inhibition category. The selected isolates were cultured and the phytochemical profile was determined qualitatively. In addition, molecular identification using the Internal Transcribed Spacer (ITS) primer set and comparison with the GenBank (NCBI) database were carried out in this study. Results: The results showed that 10 isolates were successfully isolated from three types of brown macroalgae. It was found that isolates T1, S1, and P3 had the highest inhibition in the very strong category. There were variations in mycelial weight, pH value, and inhibition shown by the culture filtrate of the three endophytic fungal isolates against Escherichia coli ESBL. Isolate S1 had the highest phytochemical profile, namely alkaloids, triterpenoids, saponins, and phenolics. The three endophytic fungal isolates showed isolate T1 (Phlebiopsis magnicystidiata) (MT5617191), isolate P3 (Neurospora crassa strain RT3M) (MT1028551), and isolate S1 (Peniophora sp.) (MH2680421). Conclusion: The results of this study provide initial information regarding the potential of bioprospecting brown macroalgal endophytic fungi as a source of new antibiotics against Escherichia coli ESBL.

Improved consolidated bioprocessing for itaconic acid production by simultaneous optimization of cellulase and metabolic pathway of Neurospora crassa

Lignocellulose was directly used in itaconic acid production by a model filamentous fungus Neurospora crassa. The promoters of two clock control genes and cellobiohydrolase 1 gene were selected for heterologous genes expression by evaluating different types of promoters. The effect of overexpression of different cellulase was compared, and it was found that expression of cellobiohydrolase 2 from Trichoderma reesei increased the filter paper activity by 2 times, the cellobiohydrolase activity by 4.5 times, and that the itaconic acid titer was also significantly improved. A bidirectional cis-aconitic acid accumulation strategy was established by constructing the reverse glyoxylate shunt and expressing the transporter MTTA, which increased itaconic acid production to 637.2 mg/L. The simultaneous optimization of cellulase and metabolic pathway was more conducive to the improvement of cellulase activity than that of cellulase alone, so as to further increase itaconic acid production. Finally, through the combination of fermentation by optimized strains and medium optimization, the titers of itaconic acid using Avicel and corn stover as substrate were 1165.1 mg/L and 871.3 mg/L, respectively. The results prove the potential of the consolidated bioprocessing that directly converts lignocellulose to itaconic acid by a model cellulase synthesizing strain.

Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ’s interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant “blocks” within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Cleavage-independent activation of ancient eukaryotic gasdermins and structural mechanisms.

Gasdermins (GSDMs) are pore-forming proteins that execute pyroptosis for immune defense. GSDMs are two-domain proteins activated by proteolytic removal of the inhibitory domain. In this work, we report two types of cleavage-independent GSDM activation. First, TrichoGSDM, a pore-forming domain-only protein from the basal metazoan Trichoplax adhaerens, is a disulfides-linked autoinhibited dimer activated by reduction of the disulfides. The cryo-electron microscopy (cryo-EM) structure illustrates the assembly mechanism for the 44-mer TrichoGSDM pore. Second, RCD-1-1 and RCD-1-2, encoded by the polymorphic regulator of cell death-1 (rcd-1) gene in filamentous fungus Neurospora crassa, are also pore-forming domain-only GSDMs. RCD-1-1 and RCD-1-2, when encountering each other, form pores and cause pyroptosis, underlying allorecognition in Neurospora. The cryo-EM structure reveals a pore of 11 RCD-1-1/RCD-1-2 heterodimers and a heterodimerization-triggered pore assembly mechanism. This study shows mechanistic diversities in GSDM activation and indicates versatile functions of GSDMs.

The regulatory network of the White Collar complex during early mushroom development in Schizophyllum commune

Blue light is an important signal for fungal development. In the mushroom-forming basidiomycete Schizophyllum commune, blue light is detected by the White Collar complex, which consists of WC-1 and WC-2. Most of our knowledge on this complex is derived from the ascomycete Neurospora crassa, where both WC-1 and WC-2 contain GATA zinc-finger transcription factor domains. In basidiomycetes, WC-1 is truncated and does not contain a transcription factor domain, but both WC-1 and WC-2 are still important for development. We show that dimerization of WC-1 and WC-2 happens independent of light in S. commune, but that induction by light is required for promoter binding by the White Collar complex. Furthermore, the White Collar complex is a promoter of transcription, but binding of the complex alone is not always sufficient to initiate transcription. For its function, the White Collar complex associates directly with the promoters of structural genes involved in mushroom development, like hydrophobins, but also promotes the expression of other transcription factors that play a role in mushroom development.

High-Throughput Screening Method Using Escherichia coli Keio Mutants for Assessing Primary Damage Mechanism of Antimicrobials.

The Escherichia coli Keio mutant collection has been a tool for assessing the role of specific genes and determining their role in E. coli physiology and uncovering novel functions. In this work, specific mutants in the DNA repair pathways and oxidative stress response were evaluated to identify the primary targets of silver nanoparticles (NPs) and their mechanism of action. The results presented in this work suggest that NPs mainly target DNA via double-strand breaks and base modifications since the recA, uvrC, mutL, and nfo mutants rendered the most susceptible phenotype, rather than involving the oxidative stress response. Concomitantly, during the establishment of the control conditions for each mutant, the katG and sodA mutants showed a hypersensitive phenotype to mitomycin C, an alkylating agent. Thus, we propose that KatG catalase plays a key role as a cellular chaperone, as reported previously for the filamentous fungus Neurospora crassa, a large subunit catalase. The Keio collection mutants may also be a key tool for assessing the resistance mechanism to metallic NPs by using their potential to identify novel pathways involved in the resistance to NPs.

Induction of mutation in Neurospora Crassa using neem (Azadirachta Indica A. Juss) leaf extract

Filamentous fungus has been used as a significant source of biotechnological applications. Neurospora crassa, a type of red bread mold, has been well recognized as a model system in fundamental scientific investigations. It can be effectively utilized as a valuable resource for molecular tools, and many mutations are available. Furthermore, N. crassa exhibits rapid growth and has no harmful properties. These characteristics indicate a significant, although unexplored, capacity of this fungus for use in biotechnological endeavors. The present investigation aimed to induce morphological changes in the N. crassa wild type by employing leaf extracts derived from Azadirachta indica A. Juss. To determine the mutagenic and growth-inhibitory effects of A. indica leaves, morphological mutants were identified and individually examined. Six morphological mutants such as albino (al), cauliflower (clf), conidial band (co.band), ropy light (ro.lig), dirty (dir), and ropy (ro) were obtained from the conidia of the wild-type N. crassa Ema (5297) strain, treated with A. indica A. Juss. leaf extracts. These mutants were compared to the wild-type, natural form of the organism in terms of their morphology, radial growth, and reproduction ability. Apparent variations were observed when a comparative study of Ema and the selected morphological mutants was undertaken. Linkage among the selected mutants in their specific linkage group was determined. Ropy, dirty, and albino were all linked to leu-3 (linkage group I), conidial-band and ropy light to trp-1 (linkage group III), and cauliflower to trp-4 (linkage group VII). Complementation was not observed in similar morphological N. crassa mutants. Dhaka Univ. J. Biol. Sci. 33(1): 79-98, 2024 (January)

Encapsulation of Trichoderma reesei and Neurospora crassa in calcium alginate capsules for fungi-mediated self-healing concrete: An explorative study on the protocol, survival of the organisms and CaCO3 biomineralization

Concrete, a predominant building material, is known for its material degradation due to crack formation. The associated durability concerns initiated the use of microorganisms to self-heal concrete cracks by promoting CaCO3 precipitation on their cell walls. Filamentous fungi grow in mycelial networks that provide abundant nucleation sites for the precipitation. As demonstrated by the research on bacteria, protection of the microorganism in the concrete mix is crucial to safeguard its survival. In this work, an encapsulation method based on sodium alginate and calcium lactate was identified in the bacteria-based literature and adapted to the fungi Trichoderma reesei and Neurospora crassa. Results showed spore survival after the encapsulation procedure, however survival was negated when immersed in concrete-relevant conditions. Simultaneously, biomass formation and CaCO3 biomineralization were quantified in liquid media. This study features possibilities to optimize the encapsulation procedure and aids the identification of optimal biomineralization conditions.

Antagonistic Effect of Endophytic Fungi Isolated from Nerium olender against Rhizoctonia solani

Objective: To investigate the population of endophytic fungi accompanying to Nerium olender plant. Materials and Methods: The study was conducted on three different sites in two governorates for three consecutive months with five samples from each site. The identification of the fungi was carried out using microscopic and molecular methods while the isolation, identification of fungal pathogen and preparation of aqueous extract were also successfully done. Results: 14 species of fungi (Penicillium notatum, Rhizopus nigricans, Rhizopus stolonifera, Fusarium Solani, Fusarium oxysporum, Alternaria sp., Cladosporium sp .and Curvularia sp.), while the other sex species were identified by DNA investigation method and where (Aspergillus spp. (40%), Penicillium spp. (20%), Rhizopus spp. (12%), Fusarium spp. (10%), Alternaria sp. (4%), Mucor circinelloides (4%), Neurospora Crassa (3%), Cladosporium sp. (1%) and Curvularia sp. (1%). Conclusions: The highest percentage of inhibition was for fungi Aspergillus Flavus, Penicillium commune and Mucor circinelloides which amounted to 100% and from the third day of growth until the completion of the control dish, then Aspergillus niger and Rhizopus stolonifera, with a 90% on the seventh day of growth, and Penicillium commune, Rhizopus nigricans with a rate ranging between 82-68%, while the rest of the fungi showed an antagonistic effect, but with rates less than 50%.

Steve Brown's legacy: Tools to study the individual human molecular circadian clock and its regulation.

Since the discovery of the genetic origin of the circadian clock in Drosophila melanogaster by Konopka and Benzer in 1971, most of the research about the regulation of the molecular circadian clock relies on laboratory models. Additional models such as Cyanobacteria, Neurospora crassa, Arabidopsis and rodents helped chronobiologists to describe the species-specific molecular clocks and their regulation. However, the lack of tools and the difficulty to access biological samples somehow excluded human from this research landscape outside behavioural research. Among many other impressive achievements, Steve Brown provided to the community of chronobiologists new tools and strategies to study the individual human circadian clock and its regulation.