Functions and mechanisms of A-to-I RNA editing in filamentous ascomycetes.

Although lack of ADAR (adenosine deaminase acting on RNA) orthologs, genome-wide A-to-I editing occurs specifically during sexual reproduction in a number of filamentous ascomycetes, including Fusarium graminearum and Neurospora crassa. Unlike ADAR-mediated editing in animals, fungal A-to-I editing has a strong preference for hairpin loops and U at -1 position, which leads to frequent editing of UAG and UAA stop codons. Majority of RNA editing events in fungi are in the coding region and cause amino acid changes. Some of these editing events have been experimentally characterized for providing heterozygote and adaptive advantages in F. graminearum. Recent studies showed that FgTad2 and FgTad3, 2 ADAT (adenosine deaminase acting on tRNA) enzymes that normally catalyze the editing of A34 in the anticodon of tRNA during vegetative growth mediate A-to-I mRNA editing during sexual reproduction. Stage specificity of RNA editing is conferred by stage-specific expression of short transcript isoforms of FgTAD2 and FgTAD3 as well as cofactors such as AME1 and FIP5 that facilitate the editing of mRNA in perithecia. Taken together, fungal A-to-I RNA editing during sexual reproduction is catalyzed by ADATs and it has the same sequence and structural preferences with editing of A34 in tRNA.

Phylogenetic and Structure-Function Analyses of ENA ATPases: A Case Study of the ENA1 Protein from the Fungus Neurospora crassa.

ENA transporters are a group of P-type ATPases that are characterized by actively moving Na+ or K+ out of the cell against their concentration gradient. The existence of these transporters was initially attributed to some fungi, although more recently they have also been identified in mosses, liverworts, and some protozoa. Given the current increase in the number of organisms whose genomes are completely sequenced, we set out to expand our knowledge about the existence of ENA in organisms belonging to other phylogenetic groups. For that, a hidden Markov model profile was constructed to identify homologous sequences to ENA proteins in protein databases. This analysis allowed us to identify the existence of ENA-type ATPases in the most primitive groups of fungi, as well as in other eukaryotic organisms not described so far. In addition, this study has allowed the identification of a possible new group of P-ATPases, initially proposed as ENA but which maintain phylogenetic distances with these proteins. Finally, this work has also addressed this study of the structure of ENA proteins, which remained unknown due to the lack of crystallographic data. For this purpose, a 3D structure prediction of the NcENA1 protein of the fungus Neurospora crassa was performed using AlphaFold2 software v2.3.1. From this structure, the electrostatic potential of the protein was analyzed. With all these data, the protein regions and the amino acids involved in the transport of Na+ or K+ ions across the membrane were proposed for the first time. Targeted mutagenesis of some of these residues has confirmed their relevant participation in the transport function of ENA proteins.

The Neurospora crassa molybdate transporter: Characterizing a novel transporter homologous to the plant MOT1 family.

Molybdenum (Mo) is an essential element for animals, plants, and fungi. To achieve biological activity in eukaryotes, Mo must be complexed into the molybdenum cofactor (Moco). Cells are known to take up Mo in the form of the oxyanion molybdate. However, molybdate transporters are scarcely characterized in the fungal kingdom. In plants and algae, molybdate is imported into the cell via two families of molybdate transporters (MOT), MOT1 and MOT2. For the filamentous fungus Neurospora crassa, a sequence homologous to the MOT1 family was previously annotated. Here we report a characterization of this molybdate-related transporter, encoded by the ncmot-1 gene. We found that the deletion of ncmot-1 leads to an accumulation of total Mo within the mycelium and a roughly 51 % higher tolerance against high molybdate levels when grown on ammonium medium. The localization of a GFP tagged NcMOT-1 was identified among the vacuolar membrane. Thereby, we propose NcMOT-1 as an exporter, transporting molybdate out of the vacuole into the cytoplasm. Lastly, the heterologous expression of NcMOT-1 in Saccharomyces cerevisiae verifies the functionality of this protein as a MOT. Our results open the way towards understanding molybdate transport as part of Mo homeostasis and Moco-biosynthesis in fungi.

Neurospora crassa is a potential source of anti-cancer agents against breast cancer.

Fungi are an excellent source of pharmaceuticals including anti-tumor agents. Neurospora crassa generates metabolites with diverse structural classes, however, its potential as an anti-tumor agent source has not been explored. The purpose of this study aimed to investigate the potential of Neurospora crassa mixture against breast cancer. The in vitro T-47D and MDA-MB-231 experiments showed that N. crassa mixture at the concentrations of both 1.7 and 0.85 µg/ml significantly inhibited tumor cell proliferation, migration and invasion, and 3D spheroid formation. However, the inhibition rates of MCF-10A ranged 10-20% at concentrations of 0.85 and 1.7 µg/ml. The mixture at the concentration of 0.85 µg/ml could significantly downregulate the expressions of transcription factors of E2F1 and E2F3, cancer stem cell-related genes of LIN28, HIWI, and CD133, and onco-lncRNA HOTAIR, and increase CASP3 activity in either T-47D or MDA-MD-231 breast cancer cell lines. In vivo breast cancer C3H mouse model results showed that N. crassa mixture significantly inhibited tumor growth. These findings suggest that N. crassa contains an antitumor component(s) against breast cancer invasiveness, which may inhibit the self-renewal and differentiation of breast cancer stem cells possibly by downregulating cancer stem cell-associated and/or transcription factor genes and oncogenes, and promoting apoptosis.

The genome organization of Neurospora crassa at high resolution uncovers principles of fungal chromosome topology.

The eukaryotic genome must be precisely organized for its proper function, as genome topology impacts transcriptional regulation, cell division, replication, and repair, among other essential processes. Disruptions to human genome topology can lead to diseases, including cancer. The advent of chromosome conformation capture with high-throughput sequencing (Hi-C) to assess genome organization has revolutionized the study of nuclear genome topology; Hi-C has elucidated numerous genomic structures, including chromosomal territories, active/silent chromatin compartments, Topologically Associated Domains, and chromatin loops. While low-resolution heatmaps can provide important insights into chromosomal level contacts, high-resolution Hi-C datasets are required to reveal folding principles of individual genes. Of particular interest are high-resolution chromosome conformation datasets of organisms modeling the human genome. Here, we report the genome topology of the fungal model organism Neurospora crassa at a high resolution. Our composite Hi-C dataset, which merges 2 independent datasets generated with restriction enzymes that monitor euchromatin (DpnII) and heterochromatin (MseI), along with our DpnII/MseI double digest dataset, provide exquisite detail for both the conformation of entire chromosomes and the folding of chromatin at the resolution of individual genes. Within constitutive heterochromatin, we observe strong yet stochastic internal contacts, while euchromatin enriched with either activating or repressive histone post-translational modifications associates with constitutive heterochromatic regions, suggesting intercompartment contacts form to regulate transcription. Consistent with this, a strain with compromised heterochromatin experiences numerous changes in gene expression. Our high-resolution Neurospora Hi-C datasets are outstanding resources to the fungal community and provide valuable insights into higher organism genome topology.

Versatile cell-based assay for measuring DNA alkylation damage and its repair.

DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.

Discovery of fungal surface NADases predominantly present in pathogenic species.

Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host's NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.

IMITATION SWITCH is required for normal chromatin structure and gene repression in PRC2 target domains.

Polycomb Group (PcG) proteins are part of an epigenetic cell memory system that plays essential roles in multicellular development, stem cell biology, X chromosome inactivation, and cancer. In animals, plants, and many fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to assemble transcriptionally repressed facultative heterochromatin. PRC2 is structurally and functionally conserved in the model fungus Neurospora crassa, and recent work in this organism has generated insights into PRC2 control and function. To identify components of the facultative heterochromatin pathway, we performed a targeted screen of Neurospora deletion strains lacking individual ATP-dependent chromatin remodeling enzymes. We found the Neurospora homolog of IMITATION SWITCH (ISW) is critical for normal transcriptional repression, nucleosome organization, and establishment of typical histone methylation patterns in facultative heterochromatin domains. We also found that stable interaction between PRC2 and chromatin depends on ISW. A functional ISW ATPase domain is required for gene repression and normal H3K27 methylation. ISW homologs interact with accessory proteins to form multiple complexes with distinct functions. Using proteomics and molecular approaches, we identified three distinct Neurospora ISW-containing complexes. A triple mutant lacking three ISW accessory factors and disrupting multiple ISW complexes led to widespread up-regulation of PRC2 target genes and altered H3K27 methylation patterns, similar to an ISW-deficient strain. Taken together, our data show that ISW is a key component of the facultative heterochromatin pathway in Neurospora, and that distinct ISW complexes perform an apparently overlapping role to regulate chromatin structure and gene repression at PRC2 target domains.

The msh1 gene is responsible for short life span mutant natural death and functions to maintain mitochondrial DNA integrity.

Wild-type filamentous fungus Neurospora crassa continues to grow its hyphae for a very lengthy period of time (>2 years), whereas mutations at the natural death (nd) locus shorten life span (approximately 20 days). By positional cloning based on heat augmented mutagen sensitivity of the nd strain, we identified a nonsense mutation in the msh1 gene, an eukaryotic homolog of bacterial MutS, and this mutation resulted in encoding non-functional polypeptide. By tagging with GFP, subcellular localization of the MSH1 protein in the mitochondria was observed, and knock out of the msh1 gene caused severe growth deficiency accompanying mitochondrial DNA (mtDNA) aberrations such as large-scale mtDNA deletions and rearrangements as seen in the nd strain. These results suggested that MSH1 may maintain mtDNA integrity. Thus, loss of function compromises mtDNA, leading to the acceleration of cellular aging.

RNA Imaging with RNase-Inactivated Csy4 in Plants and Filamentous Fungi.

Subcellular localizations of RNAs can be imaged in vivo with genetically encoded reporters consisting of a sequence-specific RNA-binding protein (RBP) fused to a fluorescent protein. Several such reporter systems have been described based on RBPs that recognize RNA stem-loops. Here we describe RNA tagging for imaging with an inactive mutant of the bacterial endonuclease Csy4, which has a significantly higher affinity for its cognate stem-loop than alternative systems. This property allows for sensitive imaging with only few tandem copies of the target stem-loop inserted into the RNA of interest.

Adaptation of codon usage to tRNA I34 modification controls translation kinetics and proteome landscape.

Codon usage bias is a universal feature of all genomes and plays an important role in regulating protein expression levels. Modification of adenosine to inosine at the tRNA anticodon wobble position (I34) by adenosine deaminases (ADATs) is observed in all eukaryotes and has been proposed to explain the correlation between codon usage and tRNA pool. However, how the tRNA pool is affected by I34 modification to influence codon usage-dependent gene expression is unclear. Using Neurospora crassa as a model system, by combining molecular, biochemical and bioinformatics analyses, we show that silencing of adat2 expression severely impaired the I34 modification levels for the ADAT-related tRNAs, resulting in major ADAT-related tRNA profile changes and reprogramming of translation elongation kinetics on ADAT-related codons. adat2 silencing also caused genome-wide codon usage-biased ribosome pausing on mRNAs and proteome landscape changes, leading to selective translational repression or induction of different mRNAs. The induced expression of CPC-1, the Neurospora ortholog of yeast GCN4p, mediates the transcriptional response after adat2 silencing and amino acid starvation. Together, our results demonstrate that the tRNA I34 modification by ADAT plays a major role in driving codon usage-biased translation to shape proteome landscape.

Production of plant-derived anticancer precursor glucoraphanin in chromosomally engineered Escherichia coli.

Glucoraphanin is a methionine-derived glucosinolate that imparts numerous health-benefits with broad bioactivity. Low amounts in plant tissues and high cost of extraction have limited the production of glucoraphanin. Metabolic engineering in heterologous microorganisms is an attractive approach to achieve efficient production of valuable natural products. In this study, a microbial fermentation process for glucoraphanin production was demonstrated. The engineered bacterial strain stably expressed 10 allogeneic enzymes in E. coli chromosome, including nine heterologous genes from Arabidopsis and Brassica and one from fungus Neurospora crassa, which could produce the specialized glucosinolate compound glucoraphanin with a titer of 0.675 µg/L by fermentation from glucose. The cofactor supplements and individual gene overexpression for glucoraphanin production were also investigated. This work highlights the possibility of supplying specialized plant glucosinolates by microbial fermentation process, instead of chemical extraction. Additionally, the limiting step enzyme, UDP-glucose-thiohydroximate glucosyltransferase, identified in this study also laid a foundation for further optimizing the glucoraphanin-producing cell factory.

A transmission ratio distortion and the 'max-4' ascus phenotype: Do both reflect the same Bateson-Dobzhansky-Muller Incompatibility emerging during trans-species introgression of translocations in Neurospora?

The T(EB4)Nta, T(IBj5)Nta, and T(B362i)NtA strains were constructed by introgressing the insertional translocations EB4, IBj5, and B362i from Neurospora crassa into the related species N. tetrasperma. The progeny from crosses of T(IBj5)Nta and T(B362i)NtA with opposite mating-type derivatives of the standard N. tetrasperma strain 85 exhibited a unique and unprecedented transmission ratio distortion (TRD) that disfavored homokaryons produced following alternate segregation relative to those produced following adjacent-1 segregation. The TRD was not evident among the [mat A + mat a] dikaryons produced following either segregation. Further, crosses of the T(IBj5)Nta and T(B362i)NtA strains with the Eight spore (E) mutant showed an unusual ascus phenotype called 'max-4'. We propose that the TRD and the max-4 phenotype are manifestations of the same Bateson-Dobzhansky-Muller incompatibility (BDMI). Since the TRD selects against 2/3 of the homokaryotic progeny from each introgression cross, the BDMI would have enriched for the dikaryotic progeny in the viable ascospores, and thus, paradoxically, facilitated the introgressions.

Ergothioneine production with Aspergillus oryzae.

To establish a reliable and practical ergothioneine (ERG) supply, we employed fermentative ERG production using Aspergillus oryzae, a fungus used for food production. We heterologously overexpressed the egt-1 and -2 genes of Neurospora crassa in A. oryzae and succeeded in producing ERG (231.0 mg/kg of media, which was 20 times higher than the wild type). Abbreviations: ERG: ergothioneine; HER: hercynine; Cys-HER: hercynylcysteine-sulfoxide; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine; l-His: l-histidine; l-Cys: l-cysteine; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry.

A conditional silencing suppression system for transient expression.

RNA silencing is a powerful tool deployed by plants against viral infection and abnormal gene expression. Plant viruses have evolved a suite of silencing suppressors for counter-defense, which are also widely used to boost transcript and protein accumulation in transient assays. However, only wild type silencing suppressor proteins have been reported to date. Here we demonstrate that P0 of Potato leafroll virus (PLRV), PLP0, can be split into two proteins that only show silencing suppression activity upon co-expression. We cloned each of these proteins in two different constructs and transiently co-infiltrated them in N. benthamiana leaves. We expressed a fluorescent protein from one of the vectors and observed that cells expressing both halves of PLP0 suppressed gene silencing. Further, we showed that Q system of Neurospora crassa, based on co-expression of a transcription activator and inhibitor, is functional in agroinfiltrated leaves of N. benthamiana. Q system combined with the split PLP0 system showed very tight co-expression of Q system's transcriptional activator and inhibitor. Altogether, our experiments demonstrate a functioning conditional silencing suppressor system and its potential as a powerful tool for transient expression in N. benthamiana leaves, as well as the application of the Q system in plants.

mus-52 disruption and metabolic regulation in Neurospora crassa: Transcriptional responses to extracellular phosphate availability.

Advances in the understanding of molecular systems depend on specific tools like the disruption of genes to produce strains with the desired characteristics. The disruption of any mutagen sensitive (mus) genes in the model fungus Neurospora crassa, i.e. mus-51, mus-52, or mus-53, orthologous to the human genes KU70, KU80, and LIG4, respectively, provides efficient tools for gene targeting. Accordingly, we used RNA-sequencing and reverse transcription-quantitative polymerase chain reaction amplification techniques to evaluate the effects of mus-52 deletion in N. crassa gene transcriptional modulation, and thus, infer its influence regarding metabolic response to extracellular availability of inorganic phosphate (Pi). Notably, the absence of MUS-52 affected the transcription of a vast number of genes, highlighting the expression of those coding for transcription factors, kinases, circadian clocks, oxi-reduction balance, and membrane- and nucleolus-related proteins. These findings may provide insights toward the KU molecular mechanisms, which have been related to telomere maintenance, apoptosis, DNA replication, and gene transcription regulation, as well as associated human conditions including immune system disorders, cancer, and aging.

The cell free protein synthesis system from the model filamentous fungus Neurospora crassa.

Cell-free protein synthesis (CFPS) can be used in many applications to produce polypeptides and to analyze mechanisms of mRNA translation. Here we describe how to make and use a CPFS system from the model filamentous fungus Neurospora crassa. The extensive genetic resources available in this system provide capacities to exploit robust CFPS for understanding translational control. Included are procedures for the growth and harvesting of cells, the preparation of cell-free extracts that serve as the source of the translational machinery in the CFPS and the preparation of synthetic mRNA to program the CFPS. Methods to accomplish cell-free translation and analyze protein synthesis, and to map positions of ribosomes on mRNAs by toeprinting, are described.

A-to-I RNA editing is developmentally regulated and generally adaptive for sexual reproduction in Neurospora crassa.

Although fungi lack adenosine deaminase acting on RNA (ADAR) enzymes, adenosine to inosine (A-to-I) RNA editing was reported recently in Fusarium graminearum during sexual reproduction. In this study, we profiled the A-to-I editing landscape and characterized its functional and adaptive properties in the model filamentous fungus Neurospora crassa A total of 40,677 A-to-I editing sites were identified, and approximately half of them displayed stage-specific editing or editing levels at different sexual stages. RNA-sequencing analysis with the Δstc-1 and Δsad-1 mutants confirmed A-to-I editing occurred before ascus development but became more prevalent during ascosporogenesis. Besides fungal-specific sequence and secondary structure preference, 63.5% of A-to-I editing sites were in the coding regions and 81.3% of them resulted in nonsynonymous recoding, resulting in a significant increase in the proteome complexity. Many genes involved in RNA silencing, DNA methylation, and histone modifications had extensive recoding, including sad-1, sms-3, qde-1, and dim-2. Fifty pseudogenes harbor premature stop codons that require A-to-I editing to encode full-length proteins. Unlike in humans, nonsynonymous editing events in N. crassa are generally beneficial and favored by positive selection. Almost half of the nonsynonymous editing sites in N. crassa are conserved and edited in Neurospora tetrasperma Furthermore, hundreds of them are conserved in F. graminearum and had higher editing levels. Two unknown genes with editing sites conserved between Neurospora and Fusarium were experimentally shown to be important for ascosporogenesis. This study comprehensively analyzed A-to-I editing in N. crassa and showed that RNA editing is stage-specific and generally adaptive, and may be functionally related to repeat induced point mutation and meiotic silencing by unpaired DNA.

Revealing Hidden Conformational Space of LOV Protein VIVID Through Rigid Residue Scan Simulations.

VIVID(VVD) protein is a Light-Oxygen-Voltage(LOV) domain in circadian clock system. Upon blue light activation, a covalent bond is formed between VVD residue Cys108 and its cofactor flavin adenine dinucleotide(FAD), and prompts VVD switching from Dark state to Light state with significant conformational deviation. However, the mechanism of this local environment initiated global protein conformational change remains elusive. We employed a recently developed computational approach, rigid residue scan(RRS), to systematically probe the impact of the internal degrees of freedom in each amino acid residue of VVD on its overall dynamics by applying rigid body constraint on each residue in molecular dynamics simulations. Key residues were identified with distinctive impacts on Dark and Light states, respectively. All the simulations display wide range of distribution on a two-dimensional(2D) plot upon structural root-mean-square deviations(RMSD) from either Dark or Light state. Clustering analysis of the 2D RMSD distribution leads to 15 representative structures with drastically different conformation of N-terminus, which is also a key difference between Dark and Light states of VVD. Further principle component analyses(PCA) of RRS simulations agree with the observation of distinctive impact from individual residues on Dark and Light states.

Establishment of CRISPR/Cas9 in Alternaria alternata.

The filamentous fungus Alternaria alternata is a potent producer of many secondary metabolites, some of which like alternariol or alternariol-methyl ether are toxic and/or cancerogenic. Many Alternaria species do not only cause post-harvest losses of food and feed, but are aggressive plant pathogens. Despite the great economic importance and the large number of research groups working with the fungus, the molecular toolbox is rather underdeveloped. Gene deletions often result in heterokaryotic strains and therefore, gene-function analyses are rather tedious. In addition, A. alternata lacks a sexual cycle and classical genetic approaches cannot be combined with molecular biological methods. Here, we show that CRISPR/Cas9 can be efficiently used for gene inactivation. Two genes of the melanin biosynthesis pathway, pksA and brm2, were chosen as targets. Several white mutants were obtained after several rounds of strain purification through protoplast regeneration or spore inoculation. Mutation of the genes was due to deletions from 1bp to 1.5kbp. The CRISPR/Cas9 system was also used to inactivate the orotidine-5-phosphate decarboxylase gene pyrG to create a uracil-auxotrophic strain. The strain was counter-selected with fluor-orotic acid and could be re-transformed with pyrG from Aspergillus fumigatus and pyr-4 from Neurospora crassa. In order to test the functioning of GFP, the fluorescent protein was fused to a nuclear localization signal derived from the StuA transcription factor of Aspergillus nidulans. After transformation bright nuclei were visible.