Terricoxanthones A-E, unprecedented dihydropyran-containing dimeric xanthones from the endophytic fungus Neurospora terricola HDF-Br-2 associated with the vulnerable conifer Pseudotsuga gaussenii.

An investigation on the secondary metabolites from a rice culture broth of the endophytic fungus Neurospora terricola HDF-Br-2 derived from the vulnerable conifer Pseudotsuga gaussenii led to the isolation and characterization of 34 structurally diverse polyketides (1-34). Seven of them are previously undescribed, including five unprecedented dihydropyran-containing (terricoxanthones A-E, 1-5, resp.) and one rare tetrahydrofuran-containing (terricoxanthone F, 6) dimeric xanthones. The structures were elucidated by spectroscopic methods and single-crystal X-ray diffraction analyses. Terricoxanthones each were obtained as a racemic mixture. Their plausible biosynthetic relationships were briefly proposed. Compounds 6, aspergillusone A (8), and alatinone (27) displayed considerable inhibition against Candida albicans with MIC values of 8-16 µg/mL. 4-Hydroxyvertixanthone (12) and 27 exhibited significant inhibitory activities against Staphylococcus aureus, with MIC values of 4-8 µg/mL. Furthermore, compounds 8 and 27 could disrupt biofilm of S. aureus and C. albicans at 128 µg/mL. The findings not only extend the skeletons of xanthone dimers and contribute to the diversity of metabolites of endophytes associated with the endangered Chinese conifer P. gaussenii, but could further reveal the important role of protecting plant species diversity in support of chemical diversity and potential sources of new therapeutics.

Recombination hotspots in Neurospora crassa controlled by idiomorphic sequences and meiotic silencing.

Genes regulating recombination in specific chromosomal intervals of Neurospora crassa were described in the 1960s, but the mechanism is still unknown. For each of the rec-1, rec-2, and rec-3 genes, a single copy of the putative dominant allele, for example, rec-2SL found in St Lawrence OR74 A wild type, reduces recombination in chromosomal regions specific to that gene. However, when we sequenced the recessive allele, rec-2LG (derived from the Lindegren 1A wild type), we found that a 10 kb region in rec-2SL strains was replaced by a 2.7 kb unrelated sequence, making the "alleles" idiomorphs. When we introduced sad-1, a mutant lacking the RNA-dependent RNA polymerase that silences unpaired coding regions during meiosis into crosses heterozygous rec-2SL/rec-2LG, it increased recombination, indicating that meiotic silencing of a gene promoting recombination is responsible for dominant suppression of recombination. Consistent with this, mutation of rec-2LG by Repeat-Induced Point mutation generated an allele with multiple stop codons in the predicted rec-2 gene, which does not promote recombination and is recessive to rec-2LG. Sad-1 also relieves suppression of recombination in relevant target regions, in crosses heterozygous for rec-1 alleles and in crosses heterozygous for rec-3 alleles. We conclude that for all 3 known rec genes, 1 allele appears dominant only because meiotic silencing prevents the product of the active, "recessive," allele from stimulating recombination during meiosis. In addition, the proposed amino acid sequence of REC-2 suggests that regulation of recombination in Neurospora differs from any currently known mechanism.

Lineage-specific genes are clustered with HET-domain genes and respond to environmental and genetic manipulations regulating reproduction in Neurospora.

Lineage-specific genes (LSGs) have long been postulated to play roles in the establishment of genetic barriers to intercrossing and speciation. In the genome of Neurospora crassa, most of the 670 Neurospora LSGs that are aggregated adjacent to the telomeres are clustered with 61% of the HET-domain genes, some of which regulate self-recognition and define vegetative incompatibility groups. In contrast, the LSG-encoding proteins possess few to no domains that would help to identify potential functional roles. Possible functional roles of LSGs were further assessed by performing transcriptomic profiling in genetic mutants and in response to environmental alterations, as well as examining gene knockouts for phenotypes. Among the 342 LSGs that are dynamically expressed during both asexual and sexual phases, 64% were detectable on unusual carbon sources such as furfural, a wildfire-produced chemical that is a strong inducer of sexual development, and the structurally-related furan 5-hydroxymethyl furfural (HMF). Expression of a significant portion of the LSGs was sensitive to light and temperature, factors that also regulate the switch from asexual to sexual reproduction. Furthermore, expression of the LSGs was significantly affected in the knockouts of adv-1 and pp-1 that regulate hyphal communication, and expression of more than one quarter of the LSGs was affected by perturbation of the mating locus. These observations encouraged further investigation of the roles of clustered lineage-specific and HET-domain genes in ecology and reproduction regulation in Neurospora, especially the regulation of the switch from the asexual growth to sexual reproduction, in response to dramatic environmental conditions changes.

Sensing of H2O2-induced oxidative stress by the UPF factor complex is crucial for activation of catalase-3 expression in Neurospora.

UPF-1-UPF-2-UPF-3 complex-orchestrated nonsense-mediated mRNA decay (NMD) is a well-characterized eukaryotic cellular surveillance mechanism that not only degrades aberrant transcripts to protect the integrity of the transcriptome but also eliminates normal transcripts to facilitate appropriate cellular responses to physiological and environmental changes. Here, we describe the multifaceted regulatory roles of the Neurospora crassa UPF complex in catalase-3 (cat-3) gene expression, which is essential for scavenging H2O2-induced oxidative stress. First, losing UPF proteins markedly slowed down the decay rate of cat-3 mRNA. Second, UPF proteins indirectly attenuated the transcriptional activity of cat-3 gene by boosting the decay of cpc-1 and ngf-1 mRNAs, which encode a well-studied transcription factor and a histone acetyltransferase, respectively. Further study showed that under oxidative stress condition, UPF proteins were degraded, followed by increased CPC-1 and NGF-1 activity, finally activating cat-3 expression to resist oxidative stress. Together, our data illustrate a sophisticated regulatory network of the cat-3 gene mediated by the UPF complex under physiological and H2O2-induced oxidative stress conditions.

Transcription activator WCC recruits deacetylase HDA3 to control transcription dynamics and bursting in Neurospora.

RNA polymerase II initiates transcription either randomly or in bursts. We examined the light-dependent transcriptional activator White Collar Complex (WCC) of Neurospora to characterize the transcriptional dynamics of the strong vivid (vvd) promoter and the weaker frequency (frq) promoter. We show that WCC is not only an activator but also represses transcription by recruiting histone deacetylase 3 (HDA3). Our data suggest that bursts of frq transcription are governed by a long-lived refractory state established and maintained by WCC and HDA3 at the core promoter, whereas transcription of vvd is determined by WCC binding dynamics at an upstream activating sequence. Thus, in addition to stochastic binding of transcription factors, transcription factor-mediated repression may also influence transcriptional bursting.

Permissiveness and competition within and between Neurospora crassa syncytia.

A multinucleate syncytium is a common growth form in filamentous fungi. Comprehensive functions of the syncytial state remain unknown, but it likely allows for a wide range of adaptations to enable filamentous fungi to coordinate growth, reproduction, responses to the environment, and to distribute nuclear and cytoplasmic elements across a colony. Indeed, the underlying mechanistic details of how syncytia regulate cellular and molecular processes spatiotemporally across a colony are largely unexplored. Here, we implemented a strategy to analyze the relative fitness of different nuclear populations in syncytia of Neurospora crassa, including nuclei with loss-of-function mutations in essential genes, based on production of multinucleate asexual spores using flow cytometry of pairings between strains with differentially fluorescently tagged nuclear histones. The distribution of homokaryotic and heterokaryotic asexual spores in pairings was assessed between different auxotrophic and morphological mutants, as well as with strains that were defective in somatic cell fusion or were heterokaryon incompatible. Mutant nuclei were compartmentalized into both homokaryotic and heterokaryotic asexual spores, a type of bet hedging for maintenance and evolution of mutational events, despite disadvantages to the syncytium. However, in pairings between strains that were blocked in somatic cell fusion or were heterokaryon incompatible, we observed a "winner-takes-all" phenotype, where asexual spores originating from paired strains were predominantly one genotype. These data indicate that syncytial fungal cells are permissive and tolerate a wide array of nuclear functionality, but that cells/colonies that are unable to cooperate via syncytia formation actively compete for resources.

A case of Neurospora sitophila causing PD peritonitis.

We describe a rare case of fungal peritoneal dialysis (PD) peritonitis caused by the ascomycete fungus Neurospora sitophila (N. sitophila). The patient had little response to initial antibiotics and PD catheter removal was necessary for source control. The fungal biomarker ß-d-glucan (BDG) was positive prior to N. sitophila being cultured and remained positive for 6 months after discharge. Use of BDG early in the assessment of PD peritonitis may reduce time to definitive therapy in fungal peritonitis.

Installing the neurospora carotenoid pathway in plants enables cytosolic formation of provitamin A and its sequestration in lipid droplets.

Vitamin A deficiency remains a severe global health issue, which creates a need to biofortify crops with provitamin A carotenoids (PACs). Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored. Here, we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves, Arabidopsis seeds, and citrus callus cells, using a fungal (Neurospora crassa) carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs, including ß-carotene. This strategy led to the accumulation of significant amounts of phytoene and γ- and ß-carotene, in addition to fungal, health-promoting carotenes with 13 conjugated double bonds, such as the PAC torulene, in the cytosol. Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production. Engineered carotenes accumulate in cytosolic lipid droplets (CLDs), which represent a novel sequestering sink for storing these pigments in plant cytosol. Importantly, ß-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidial ß-carotene. Moreover, engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of ß-apocarotenoids, including retinal, the aldehyde corresponding to vitamin A. Collectively, our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues, especially in lipid-storing seeds, and thus paves the way for further optimization of carotenoid biofortification in crops.

Identifying the gluc-1 and gluc-2 mutations in Neurospora crassa by genome resequencing.

Genome resequencing is an efficient strategy for associating mutant phenotypes with physical genomic loci (Baker 2009). A pilot study of this approach demonstrated that the Neurospora crassa genetic map was critical in narrowing the possible candidate mutations in a strain to a small number in a limited, defined region of the genome (McCluskey et al. 2011). In this study, we utilize a resequencing strategy to identify the mutations underlying the gluc-1 and gluc-2 genes in N. crassa.

Meiotic drive is associated with sexual incompatibility in Neurospora.

Evolution of Bateson-Dobzhansky-Muller (BDM) incompatibilities is thought to represent a key step in the formation of separate species. They are incompatible alleles that have evolved in separate populations and are exposed in hybrid offspring as hybrid sterility or lethality. In this study, we reveal a previously unconsidered mechanism promoting the formation of BDM incompatibilities, meiotic drive. Theoretical studies have evaluated the role that meiotic drive, the phenomenon whereby selfish elements bias their transmission to progeny at ratios above 50:50, plays in speciation, and have mostly concluded that drive could not result in speciation on its own. Using the model fungus Neurospora, we demonstrate that the large meiotic drive haplotypes, Sk-2 and Sk-3, contain putative sexual incompatibilities. Our experiments revealed that although crosses between Neurospora intermedia and Neurospora metzenbergii produce viable progeny at appreciable rates, when strains of N. intermedia carry Sk-2 or Sk-3 the proportion of viable progeny drops substantially. Additionally, it appears that Sk-2 and Sk-3 have accumulated different incompatibility phenotypes, consistent with their independent evolutionary history. This research illustrates how meiotic drive can contribute to reproductive isolation between populations, and thereby speciation.

On the Mechanistic Basis of Killer Meiotic Drive in Fungi.

Spore killers are specific genetic elements in fungi that kill sexual spores that do not contain them. A range of studies in the last few years have provided the long-awaited first insights into the molecular mechanistic aspects of spore killing in different fungal models, including both yeast-forming and filamentous Ascomycota. Here we describe these recent advances, focusing on the wtf system in the fission yeast Schizosaccharomyces pombe; the Sk spore killers of Neurospora species; and two spore-killer systems in Podospora anserina, Spok and [Het-s]. The spore killers appear thus far mechanistically unrelated. They can involve large genomic rearrangements but most often rely on the action of just a single gene. Data gathered so far show that the protein domains involved in the killing and resistance processes differ among the systems and are not homologous. The emerging picture sketched by these studies is thus one of great mechanistic and evolutionary diversity of elements that cheat during meiosis and are thereby preferentially inherited over sexual generations.

Cryptochrome 1 as a state variable of the circadian clockwork of the suprachiasmatic nucleus: Evidence from translational switching.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal clock driving circadian rhythms of physiology and behavior that adapt mammals to environmental cycles. Disruption of SCN-dependent rhythms compromises health, and so understanding SCN time keeping will inform management of diseases associated with modern lifestyles. SCN time keeping is a self-sustaining transcriptional/translational delayed feedback loop (TTFL), whereby negative regulators inhibit their own transcription. Formally, the SCN clock is viewed as a limit-cycle oscillator, the simplest being a trajectory of successive phases that progresses through two-dimensional space defined by two state variables mapped along their respective axes. The TTFL motif is readily compatible with limit-cycle models, and in Neurospora and Drosophila the negative regulators Frequency (FRQ) and Period (Per) have been identified as state variables of their respective TTFLs. The identity of state variables of the SCN oscillator is, however, less clear. Experimental identification of state variables requires reversible and temporally specific control over their abundance. Translational switching (ts) provides this, the expression of a protein of interest relying on the provision of a noncanonical amino acid. We show that the negative regulator Cryptochrome 1 (CRY1) fulfills criteria defining a state variable: ts-CRY1 dose-dependently and reversibly suppresses the baseline, amplitude, and period of SCN rhythms, and its acute withdrawal releases the TTFL to oscillate from a defined phase. Its effect also depends on its temporal pattern of expression, although constitutive ts-CRY1 sustained (albeit less stable) oscillations. We conclude that CRY1 has properties of a state variable, but may operate among several state variables within a multidimensional limit cycle.

Nuclear-specific gene expression in heterokaryons of the filamentous ascomycete Neurospora tetrasperma.

Heterokaryosis is a system in which genetically distinct nuclei coexist within the same cytoplasm. While heterokaryosis dominates the life cycle of many fungal species, the transcriptomic changes associated with the transition from homokaryosis to heterokaryosis is not well understood. Here, we analyse gene expression profiles of homokaryons and heterokaryons from three phylogenetically and reproductively isolated lineages of the filamentous ascomycete Neurospora tetrasperma. We show that heterokaryons are transcriptionally distinct from homokaryons in the sexual stage of development, but not in the vegetative stage, suggesting that the phenotypic switch to fertility in heterokaryons is associated with major changes in gene expression. Heterokaryon expression is predominantly defined by additive effects of its two nuclear components. Furthermore, allele-specific expression analysis of heterokaryons with varying nuclear ratios show patterns of expression ratios strongly dependent on nuclear ratios in the vegetative stage. By contrast, in the sexual stage, strong deviations of expression ratios indicate a co-regulation of nuclear gene expression in all three lineages. Taken together, our results show two levels of expression control: additive effects suggest a nuclear level of expression, whereas co-regulation of gene expression indicate a heterokaryon level of control.

Neurospora casein kinase 1a recruits the circadian clock protein FRQ via the C-terminal lobe of its kinase domain.

Timing by the circadian clock of Neurospora is associated with hyperphosphorylation of frequency (FRQ), which depends on anchoring casein kinase 1a (CK1a) to FRQ. It is not known how CK1a is anchored so that approximately 100 sites in FRQ can be targeted. Here, we identified two regions in CK1a, p1 and p2, that are required for anchoring to FRQ. Mutation of p1 or p2 impairs progressive hyperphosphorylation of FRQ. A p1-mutated strain is viable but its circadian clock is non-functional, whereas a p2-mutated strain is non-viable. Our data suggest that p1 and potentially also p2 in CK1a provide an interface for interaction with FRQ. Anchoring via p1-p2 leaves the active site of CK1a accessible for phosphorylation of FRQ at multiple sites.

Characterizing the gene-environment interaction underlying natural morphological variation in Neurospora crassa conidiophores using high-throughput phenomics and transcriptomics.

Neurospora crassa propagates through dissemination of conidia, which develop through specialized structures called conidiophores. Recent work has identified striking variation in conidiophore morphology, using a wild population collection from Louisiana, United States of America to classify 3 distinct phenotypes: Wild-Type, Wrap, and Bulky. Little is known about the impact of these phenotypes on sporulation or germination later in the N. crassa life cycle, or about the genetic variation that underlies them. In this study, we show that conidiophore morphology likely affects colonization capacity of wild N. crassa isolates through both sporulation distance and germination on different carbon sources. We generated and crossed homokaryotic strains belonging to each phenotypic group to more robustly fit a model for and estimate heritability of the complex trait, conidiophore architecture. Our fitted model suggests at least 3 genes and 2 epistatic interactions contribute to conidiophore phenotype, which has an estimated heritability of 0.47. To uncover genes contributing to these phenotypes, we performed RNA-sequencing on mycelia and conidiophores of strains representing each of the 3 phenotypes. Our results show that the Bulky strain had a distinct transcriptional profile from that of Wild-Type and Wrap, exhibiting differential expression patterns in clock-controlled genes (ccgs), the conidiation-specific gene con-6, and genes implicated in metabolism and communication. Combined, these results present novel ecological impacts of and differential gene expression underlying natural conidiophore morphological variation, a complex trait that has not yet been thoroughly explored.

Inoculation of Neurospora sp. for improving ammonia production during thermophilic composting of organic sludge.

Recent attempts have been made to develop a thermophilic composting process for organic sludge to not only produce organic fertilizers and soil conditioners, but to also utilize the generated ammonia gas to produce high value-added algae. The hydrolysis of organic nitrogen in sludge is a bottleneck in ammonia conversion, and its improvement is a major challenge. The present study aimed to elucidate the effects of inoculated Neurospora sp. on organic matter decomposition and ammonia conversion during thermophilic composting of two organic sludge types: anaerobic digestion sludge and shrimp pond sludge. A laboratory-scale sludge composting experiment was conducted with a 6-day pretreatment period at 30 °C with Neurospora sp., followed by a 10-day thermophilic composting period at 50 °C by inoculating the bacterial community. The final organic matter decomposition was significantly higher in the sludge pretreated with Neurospora sp. than in the untreated sludge. Correspondingly, the amount of non-dissolved nitrogen was also markedly reduced by pretreatment, and the ammonia conversion rate was notably improved. Five enzymes exhibiting high activity only during the pretreatment period were identified, while no or low activity was observed during the subsequent thermophilic composting period, suggesting the involvement of these enzymes in the degradation of hardly degradable fractions, such as bacterial cells. The bacterial community analysis and its function prediction suggested the contribution of Bacillaceae in the degradation of easily degradable organic matter, but the entire bacterial community was highly incapable in degrading the hardly degradable fraction. To conclude, this study is the first to demonstrate that Neurospora sp. decomposes those organic nitrogen fractions that require a long time to be decomposed by the bacterial community during thermophilic composting.

Structure of the translating Neurospora ribosome arrested by cycloheximide.

Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX's mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.

Codon usage and protein length-dependent feedback from translation elongation regulates translation initiation and elongation speed.

Essential cellular functions require efficient production of many large proteins but synthesis of large proteins encounters many obstacles in cells. Translational control is mostly known to be regulated at the initiation step. Whether translation elongation process can feedback to regulate initiation efficiency is unclear. Codon usage bias, a universal feature of all genomes, plays an important role in determining gene expression levels. Here, we discovered that there is a conserved but codon usage-dependent genome-wide negative correlation between protein abundance and CDS length. The codon usage effects on protein expression and ribosome flux on mRNAs are influenced by CDS length; optimal codon usage preferentially promotes production of large proteins. Translation of mRNAs with long CDS and non-optimal codon usage preferentially induces phosphorylation of initiation factor eIF2α, which inhibits translation initiation efficiency. Deletion of the eIF2α kinase CPC-3 (GCN2 homolog) in Neurospora preferentially up-regulates large proteins encoded by non-optimal codons. Surprisingly, CPC-3 also inhibits translation elongation rate in a codon usage and CDS length-dependent manner, resulting in slow elongation rates for long CDS mRNAs. Together, these results revealed a codon usage and CDS length-dependent feedback mechanism from translation elongation to regulate both translation initiation and elongation kinetics.

Revisiting the Neurospora crassa mitochondrial genome.

The mitochondrial genome of Neurospora crassa has been less studied than its nuclear counterpart, yet it holds great potential for understanding the diversity and evolution of this important fungus. Here we describe a new mitochondrial DNA (mtDNA) complete sequence of a N. crassa wild type strain. The genome with 64 839 bp revealed 21 protein-coding genes and several hypothetical open reading frames with no significant homology to any described gene. Five large repetitive regions were identified across the genome, including partial or complete genes. The largest repeated region holds a partial nd2 section that was also detected in Neurospora intermedia, suggesting a rearrangement that occurred before the N. crassa speciation. Interestingly, N. crassa has a palindrome adjacent to the partial nd2 repeated region possibly related to the genomic rearrangement, which is absent in N. intermedia. Finally, we compared the sequences of the three available N. crassa complete mtDNAs and found low levels of intraspecific variability. Most differences among strains were due to small indels in noncoding regions. The revisiting of the N. crassa mtDNA forms the basis for future studies on mitochondrial genome organization and variability.

Starch and protein recovery from brewer's spent grain using hydrothermal pretreatment and their conversion to edible filamentous fungi - A brewery biorefinery concept.

This study aimed at recovering a highly concentrated starch and protein stream from the brewer's spent grain (BSG). The effect of pretreatment temperature and retention time on the solubilization of starch and protein; and the generation of fermentation inhibitors were studied. Then, the application of recovered streams for fungal cultivation was evaluated using different edible fungi Aspergillus oryzae, Neurospora intermedia, and Rhizopus delemar. The hydrothermal pretreatment resulted in the highest solubilized starch concentration, 43 g/L, corresponding to 83% solubilization of initial BSG starch content. The highest protein concentration was 27 g/L (48% solubilization of initial BSG protein content). Cultivation with Neurospora intermedia on the recovered streams from the two best pretreatment conditions, 140 ℃ for 4 h and 180 ℃ for 30 min, resulted in pure fungal biomass with the highest protein content 59.62% and 50.42% w/w, respectively. Finally, a brewery biorefinery was proposed for the valorization of BSG.