A rapid PCR-based method to determine the Neurospora crassa mating type.

So far mating type determination in Neurospora crassa requires test crosses with strains of known mating type. We present a simple, quick, and reliable polymerase chain reaction-based method for mating type determination in N. crassa.

Insights into Taxol® biosynthesis by endophytic fungi.

There have been two hundred reports that endophytic fungi produce Taxol®, but its production yield is often rather low. Although considerable efforts have been made to increase Taxol/taxanes production in fungi by manipulating cocultures, mutagenesis, genome shuffles, and gene overexpression, little is known about the molecular signatures of Taxol biosynthesis and its regulation. It is known that some fungi have orthologs of the Taxol biosynthetic pathway, but the overall architecture of this pathway is unknown. A biosynthetic putative gene homology approach, combined with genomics and transcriptomics analysis, revealed that a few genes for metabolite residues may be located on dispensable chromosomes. This review explores a number of crucial topics (i) finding biosynthetic pathway genes using precursors, elicitors, and inhibitors; (ii) orthologs of the Taxol biosynthetic pathway for rate-limiting genes/enzymes; and (iii) genomics and transcriptomics can be used to accurately predict biosynthetic putative genes and regulators. This provides promising targets for future genetic engineering approaches to produce fungal Taxol and precursors. KEY POINTS: • A recent trend in predicting Taxol biosynthetic pathway from endophytic fungi. • Understanding the Taxol biosynthetic pathway and related enzymes in fungi. • The genetic evidence and formation of taxane from endophytic fungi.

PRECOCIOUS1 (POCO1), a mitochondrial pentatricopeptide repeat protein affects flowering time in Arabidopsis thaliana.

Flowering is a vital developmental shift in plants from vegetative to reproductive phase. The timing of this shift is regulated by various linked genetic pathways including environmental cues and internal regulation. Here we report a role for an Arabidopsis gene, AT1G15480, which encodes a P-class pentatricopeptide repeat (PPR) protein, affecting flowering time. We show that AT1G15480 is localized to mitochondria. An AT1G15480 T-DNA insertion line exhibits an early-flowering phenotype, which is quite a rare phenotype among PPR mutants. The early-flowering phenotype was observed under both long and short days compared with wild type plants. Genetic complementation confirmed the observed phenotype. We therefore named the PPR protein PRECOCIOUS1 (POCO1). poco1 plants showed lower respiration, ATP content and higher accumulation of superoxide. Importantly, the quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that the expression of FLOWERING LOCUS C (FLC), which is a key floral repressor, was strongly downregulated in the poco1. Likewise, the expression level of the FLC positive regulator ABSCISIC ACID-INSENSITIVE 5 (ABI5) was reduced in the poco1. Consistent with the qRT-PCR results, poco1 plants showed reduced sensitivity to abscisic acid compared with wild type with respect to primary root growth and days to flowering. Furthermore, the poco1 mutation enhances the sensitivity to drought stress. Further analysis showed that POCO1 affects mitochondrial RNA editing. Taken together, our data demonstrate a remarkable function of POCO1 in flowering time and the abscisic acid signalling pathway.

Transcriptomic analysis of poco1, a mitochondrial pentatricopeptide repeat protein mutant in Arabidopsis thaliana.

BACKGROUND: Flowering is a crucial stage during plant development. Plants may respond to unfavorable conditions by accelerating reproductive processes like flowering. In a recent study, we showed that PRECOCIOUS1 (POCO1) is a mitochondrial pentatricopeptide repeat (PPR) protein involved in flowering time and abscisic acid (ABA) signaling in Arabidopsis thaliana. Here, we use RNA-seq data to investigate global gene expression alteration in the poco1 mutant. RESULTS: RNA-seq analysis was performed during different developmental stages for wild-type and poco1 plants. The most profound differences in gene expression were found when wild-type and poco1 plants of the same developmental stage were compared. Coverage analysis confirmed the T-DNA insertion in POCO1, which was concomitant with truncated transcripts. Many biological processes were found to be enriched. Several flowering-related genes such as FLOWERING LOCUS T (FT), which may be involved in the early-flowering phenotype of poco1, were differentially regulated. Numerous ABA-associated genes, including the core components of ABA signaling such as ABA receptors, protein phosphatases, protein kinases, and ABA-responsive element (ABRE) binding proteins (AREBs)/ABRE-binding factors (ABFs) as well as important genes for stomatal function, were mostly down-regulated in poco1. Drought and oxidative stress-related genes, including ABA-induced stress genes, were differentially regulated. RNA-seq analysis also uncovered differentially regulated genes encoding various classes of transcription factors and genes involved in cellular signaling. Furthermore, the expression of stress-associated nuclear genes encoding mitochondrial proteins (NGEMPs) was found to be altered in poco1. Redox-related genes were affected, suggesting that the redox state in poco1 might be altered. CONCLUSION: The identification of various enriched biological processes indicates that complex regulatory mechanisms underlie poco1 development. Differentially regulated genes associated with flowering may contribute to the early-flowering phenotype of poco1. Our data suggest the involvement of POCO1 in the early ABA signaling process. The down-regulation of many ABA-related genes suggests an association of poco1 mutation with the ABA signaling deficiency. This condition further affects the expression of many stress-related, especially drought-associated genes in poco1, consistent with the drought sensitivity of poco1. poco1 mutation also affects the expression of genes associated with the cellular regulation, redox, and mitochondrial perturbation.

Genomics- and Metabolomics-Based Investigation of the Deep-Sea Sediment-Derived Yeast, Rhodotorula mucilaginosa 50-3-19/20B.

Red yeasts of the genus Rhodotorula are of great interest to the biotechnological industry due to their ability to produce valuable natural products, such as lipids and carotenoids with potential applications as surfactants, food additives, and pharmaceuticals. Herein, we explored the biosynthetic potential of R. mucilaginosa 50-3-19/20B collected from the Mid-Atlantic Ridge using modern genomics and untargeted metabolomics tools. R. mucilaginosa 50-3-19/20B exhibited anticancer activity when grown on PDA medium, while antimicrobial activity was observed when cultured on WSP-30 medium. Applying the bioactive molecular networking approach, the anticancer activity was linked to glycolipids, namely polyol esters of fatty acid (PEFA) derivatives. We purified four PEFAs (1-4) and the known methyl-2-hydroxy-3-(1H-indol-2-yl)propanoate (5). Their structures were deduced from NMR and HR-MS/MS spectra, but 1-5 showed no anticancer activity in their pure form. Illumina-based genome sequencing, de novo assembly and standard biosynthetic gene cluster (BGC) analyses were used to illustrate key components of the PEFA biosynthetic pathway. The fatty acid producing BGC3 was identified to be capable of producing precursors of PEFAs. Some Rhodotorula strains are able to convert inulin into high-yielding PEFA and cell lipid using a native exo-inulinase enzyme. The genomic locus for an exo-inulinase enzyme (g1629.t1), which plays an instrumental role in the PEFA production via the mannitol biosynthesis pathway was identified. This is the first untargeted metabolomics study on R. mucilaginosa providing new genomic insights into PEFA biosynthesis.

An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

The genome and development-dependent transcriptomes of Pyronema confluens: a window into fungal evolution.

Fungi are a large group of eukaryotes found in nearly all ecosystems. More than 250 fungal genomes have already been sequenced, greatly improving our understanding of fungal evolution, physiology, and development. However, for the Pezizomycetes, an early-diverging lineage of filamentous ascomycetes, there is so far only one genome available, namely that of the black truffle, Tuber melanosporum, a mycorrhizal species with unusual subterranean fruiting bodies. To help close the sequence gap among basal filamentous ascomycetes, and to allow conclusions about the evolution of fungal development, we sequenced the genome and assayed transcriptomes during development of Pyronema confluens, a saprobic Pezizomycete with a typical apothecium as fruiting body. With a size of 50 Mb and ~13,400 protein-coding genes, the genome is more characteristic of higher filamentous ascomycetes than the large, repeat-rich truffle genome; however, some typical features are different in the P. confluens lineage, e.g. the genomic environment of the mating type genes that is conserved in higher filamentous ascomycetes, but only partly conserved in P. confluens. On the other hand, P. confluens has a full complement of fungal photoreceptors, and expression studies indicate that light perception might be similar to distantly related ascomycetes and, thus, represent a basic feature of filamentous ascomycetes. Analysis of spliced RNA-seq sequence reads allowed the detection of natural antisense transcripts for 281 genes. The P. confluens genome contains an unusually high number of predicted orphan genes, many of which are upregulated during sexual development, consistent with the idea of rapid evolution of sex-associated genes. Comparative transcriptomics identified the transcription factor gene pro44 that is upregulated during development in P. confluens and the Sordariomycete Sordaria macrospora. The P. confluens pro44 gene (PCON_06721) was used to complement the S. macrospora pro44 deletion mutant, showing functional conservation of this developmental regulator.

Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis.

Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1), a polyketide synthase (PKS2), a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster.

A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa.

As a widely used eukaryotic model organism, Neurospora crassa offers advantages in genetic studies due to its diverse biology and rapid growth. Traditional genetic manipulation methods, such as homologous recombination, require a considerable amount of time and effort. In this study, we present an easy-to-use CRIPSR/Cas9 system for N. crassa, in which the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. Our approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process. Using cyclosporin-resistant-1 (csr-1) as a selectable marker gene, we achieved 100% editing efficiency under selection conditions. Furthermore, we successfully edited the non-selectable gene N-acylethanolamine amidohydrolase-2 (naa-2), demonstrating the versatility of the system. Combining gRNAs targeting csr-1 and naa-2 simultaneously increased the probability of finding mutants carrying the non-selectable mutation. The system is not only user-friendly but also effective, providing a rapid and efficient method for generating loss-of-function mutants in N. crassa compared to traditional methods.

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.

Characterization of bud emergence 46 (BEM46) protein: sequence, structural, phylogenetic and subcellular localization analyses.

The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/ß-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved "known unknown" eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional analyses. The evolutionary history of BEM46 proteins is characterized by exonic indels in lineage specific manner.

Alternative splicing in ascomycetes.

Alternative splicing is a complex and regulated process, which results in mRNA with different coding capacities from a single gene. Extend and types of alternative splicing vary greatly among eukaryotes. In this review, I focus on alternative splicing in ascomycetes, which in general have significant lower extend of alternative splicing than mammals. Yeast-like species have low numbers of introns and consequently alternative splicing is lower compared to filamentous fungi. Several examples from single studies as well as from genomic scale analysis are presented, including a survey of alternative splicing in Neurospora crassa. Another focus is regulation by riboswitch RNA and alternative splicing in a heterologous system, along with putative protein factors involved in regulation.

De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

Complementation of an Eisosomal Yeast pil1 Mutant and Characteristics of Eisosomal Distribution in Hyphae of Neurospora crassa Germinating from Two Different Spore Types.

Eisosomes are plasma-membrane-associated protein complexes of fungi and algae involved in various cellular processes. The eisosome composition of the budding yeast is well described, but there is a limited number of studies only about eisosomes in filamentous fungi. In our study, we examined the Neurospora crassa LSP-1 protein (NcLSP1). By complementing a Saccharomyces cerevisiae Δpil1 mutant strain with nclsp1, we show the functional homology of the NcLSP1 to yeast PIL1 rather than to yeast LSP1 and hereby confirm that the NcLSP1 is an eisosomal core protein and suitable eisosomal marker. The subsequent cloning and expression of the nclsp1::trfp reporter gene construct in N. crassa allowed for a systematical investigation of the characteristics of eisosome formation and distribution in different developmental stages. In N. crassa, the hyphae germinating from sexual and asexual spores are morphologically identical and have been historically recognized as the same type of cells. Here, we demonstrate the structural differences on the cellular level between the hyphae germinating from sexual and asexual spores.

The P-type pentatricopeptide repeat protein DWEORG1 is a non-previously reported rPPR protein of Arabidopsis mitochondria.

Gene expression in plant mitochondria is mainly regulated by nuclear-encoded proteins on a post-transcriptional level. Pentatricopeptide repeat (PPR) proteins play a major role by participating in mRNA stability, splicing, RNA editing, and translation initiation. PPR proteins were also shown to be part of the mitochondrial ribosome (rPPR proteins), which may act as regulators of gene expression in plants. In this study, we focus on a mitochondrial-located P-type PPR protein-DWEORG1-from Arabidopsis thaliana. Its abundance in mitochondria is high, and it has a similar expression pattern as rPPR proteins. Mutant dweorg1 plants exhibit a slow-growth phenotype. Using ribosome profiling, a decrease in translation efficiency for cox2, rps4, rpl5, and ccmFN2 was observed in dweorg1 mutants, correlating with a reduced accumulation of the Cox2 protein in these plants. In addition, the mitochondrial rRNA levels are significantly reduced in dweorg1 compared with the wild type. DWEORG1 co-migrates with the ribosomal proteins Rps4 and Rpl16 in sucrose gradients, suggesting an association of DWEORG1 with the mitoribosome. Collectively, this data suggests that DWEORG1 encodes a novel rPPR protein that is needed for the translation of cox2, rps4, rpl5, and ccmFN2 and provides a stabilizing function for mitochondrial ribosomes.

Suitability of Vader for transposon-mediated mutagenesis in Aspergillus niger.

The filamentous fungus Aspergillus niger is widely used in biotechnological applications. Strain CBS513.88 is known to harbor 21 copies of the nonautonomous transposon Vader. Upon selection of chlorate-resistant A. niger colonies, one Vader copy was found integrated in the nirA gene. This copy was used for vector construction and development of a transposon-tagging method. Vader showed an excision frequency of about 1 in 2.2 × 10(5) conidiospores. A total of 95 of 97 colonies analyzed exhibited an excision event at the DNA level, and Vader footprints were found. By employing thermal asymmetric interlaced (TAIL)-PCR, the reintegration sites of 21 independent excision events were determined. All reintegration events occurred within or very close to genes. Therefore, this method can be used for transposon mutagenesis in A. niger.

Expression of ribonuclease A and ribonuclease N1 in the filamentous fungus Neurospora crassa.

In this study, we investigated the ability of the fungus Neurospora crassa to produce and secrete two ribonucleases: the heterologous bovine RNase A and the endogenous RNase N(1). A set of expression vectors was constructed, each consisting of an RNase A open reading frame under the control of a specific promoter and each with a specific terminator. N. crassa transformants were analyzed at the transcriptional and protein levels. Irrespective of the promoter used, all transformants showed an RNase A-specific transcript in northern hybridization, but transcriptional strengths differed significantly. The strongest transcription was detected in transformants under the control of the cfp promoter. Western blot analysis and ELISA assays of selected transformants showed an effective secretion up to 356 ng/mL of recombinant RNase A protein. However, the highest ribonuclease activity could be detected in transformants carrying the endogenous RNase N(1) under the control of the ccg1 promoter. Expression and secretion of RNase N(1) thus represent an alternative to recombinant expression of RNase A protein. In conclusion, we have created a viable expression system for expression of homologous and heterologous proteins in N. crassa.

The Composition and the Structure of MCC/Eisosomes in Neurospora crassa.

MCC/eisosomes are protein-organized domains in the plasma membrane of fungi and algae. However, the composition and function(s) of MCC/eisosomes in the filamentous fungus Neurospora crassa were previously unknown. To identify proteins that localize to MCC/eisosomes in N. crassa, we isolated proteins that co-purified with the core MCC/eisosome protein LSP-1, which was tagged with GFP. Proteins that co-fractionated with LSP-1:GFP were then identified by mass spectrometry. Eighteen proteins were GFP-tagged and used to identify six proteins that highly colocalized with the MCC/eisosome marker LSP-1:RFP, while five other proteins showed partial overlap with MCC/eisosomes. Seven of these proteins showed amino acid sequence homology with proteins known to localize to MCC/eisosomes in the yeast Saccharomyces cerevisiae. However, homologs of three proteins known to localize to MCC/eisosomes in S. cerevisiae (Can1, Pkh1/2, and Fhn1) were not found to colocalize with MCC/eisosome proteins in N. crassa by fluorescence microscopy. Interestingly, one new eisosome protein (glutamine-fructose-6-phosphate aminotransferase, gene ID: NCU07366) was detected in our studies. These findings demonstrate that there are interspecies differences of the protein composition of MCC/eisosomes. To gain further insight, molecular modeling and bioinformatics analysis of the identified proteins were used to propose the organization of MCC/eisosomes in N. crassa. A model will be discussed for how the broad range of functions predicted for the proteins localized to MCC/eisosomes, including cell wall synthesis, response and signaling, transmembrane transport, and actin organization, suggests that MCC/eisosomes act as organizing centers in the plasma membrane.

RNA processing in plant mitochondria is independent of transcription.

We analyzed the ability of plant mitochondria to process introduced RNA. Arabidopsis thaliana cox2 transcripts were synthesized in vitro. The in vitro transcribed mRNA was electroporated into maize and cauliflower mitochondria and incubated in organello. RNA was isolated and RT-PCR was carried out to analyze RNA processing. Our data indicate that cox2 transcripts introduced into isolated plant mitochondria are processed completely. This is the first report of in organello editing of introduced transcripts. We also found that none of the transcription, translation, or respiration inhibitors we used influenced RNA splicing or RNA editing of the cox2 transcript. Thus, our data also demonstrate that plant mitochondrial RNA processing may be independent of both transcription and respiratory regulation.