Search

© 2018 Pomraning et al. The yeast Yarrowia lipolytica undergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All the smooth mutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5'-CCCCT-3') and upregulation of genes with cell cycle box (5'-ACGCG-3') motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p in Saccharomyces cerevisiae. We confirmed that the Y. lipolytica msn2 (Ylmsn2) ortholog is required for hyphal growth and found that overexpression of Ylmsn2 enables hyphal growth in smooth strains. The cell cycle box is bound by the Mbp1p/Swi6p complex in S. cerevisiae to regulate G1-to-S phase progression. We found that overexpression of either the Ylmbp1 or Ylswi6 homologs decreased hyphal growth and that deletion of either Ylmbp1 or Ylswi6 promotes hyphal growth in smooth strains. A second forward genetic screen for reversion to hyphal growth was performed with the smooth-33 mutant to identify additional genetic factors regulating hyphal growth in Y. lipolytica. Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Ylchk1 and Ylnik1 and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Ylssk2, Ylpbs2, and Ylhog1. Together, these results demonstrate that Y. lipolytica transitions to hyphal growth in response to stress through multiple signaling pathways.

Understanding fungal lipid biology and metabolism is critical for antifungal target discovery as lipids play central roles in cellular processes. Nuances in lipid structural differences can significantly impact their functions, making it necessary to characterize lipids in detail to understand their roles in these complex systems. In particular, lipid double bond (DB) locations are an important component of lipid structure that can only be determined using a few specialized analytical techniques. Ozone-induced dissociation mass spectrometry (OzID-MS) is one such technique that uses ozone to break lipid DBs, producing pairs of characteristic fragments that allow the determination of DB positions. In this work, we apply OzID-MS and LipidOz software to analyze the complex lipids of Saccharomyces cerevisiae yeast strains transformed with different fatty acid desaturases from Histoplasma capsulatum to determine the specific unsaturated lipids produced. The automated data analysis in LipidOz made the determination of DB positions from this large dataset more practical, but manual verification for all targets was still time-consuming. The DL model reduces manual involvement in data analysis, but since it was trained using mammalian lipid extracts, the prediction accuracy on yeast-derived data was reduced. We addressed both shortcomings by retraining the DL model to act as a pre-filter to prioritize targets for automated analysis, providing confident manually verified results but requiring less computational time and manual effort. Our workflow resulted in the determination of detailed DB positions and enzymatic specificity., (© 2024 Battelle Memorial Institute. Journal of Mass Spectrometry published by John Wiley & Sons Ltd.)

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae , and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L -1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2μ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L -1 nerolidol and ∼0.96 g L -1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2μ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

The dynamics of microbial processes are difficult to study in natural soil, owing to the small spatial scales on which microorganisms operate and to the opacity and chemical complexity of the soil habitat. To circumvent these challenges, we have created a 3D-bioprinted habitat that mimics aspects of natural soil aggregates while providing a chemically defined and translucent alternative culturing method for soil microorganisms. Our Synthetic Soil Aggregates (SSAs) retain the porosity, permeability, and patchy resource distribution of natural soil aggregates-parameters that are expected to influence emergent microbial community interactions. We demonstrate the printability and viability of several different microorganisms within SSAs and show how the SSAs can be integrated into a multi-omics workflow for single SSA resolution genomics, metabolomics, proteomics, lipidomics, and biogeochemical assays. We study the impact of the structured habitat on the distribution of a model co-culture microbial community and find that it is significantly different from the spatial organization of the same community in liquid culture, indicating a potential for SSAs to reproduce naturally occurring emergent community phenotypes. The SSAs have the potential as a tool to help researchers quantify microbial scale processes in situ and achieve high-resolution data from the interplay between environmental properties and microbial ecology.

Using a legacy of genetic mutants of Neurospora crassa , paired with resequencing efforts through JGI, we have identified the gene responsible for the 'scumbo' mutant. This early morphological mutant was described as "Irregular flat, spreading growth with knobby protrusions and abnormal conidiation, but no free conidia. Mycelium usually appears yellowish rather than orange. Female fertile." (Perkins, Radford et al. 2000). Our further investigation has found new insights into the identity and associated functions of scumbo as a ceramide C9 methyltransferase, previously annotated as "similar to cyclopropane-fatty-acyl-phospholipidsynthase", encoded by the gene NCU07859. This enzyme performs a fungal-specific methyl modification of glycosyl-ceramides and has implications for membrane homeostasis and hyphal polarity in filamentous fungi., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Lipids play a fundamental role in fungal cell biology, being essential cell membrane components and major targets of antifungal drugs. A deeper knowledge of lipid metabolism is key for developing new drugs and a better understanding of fungal pathogenesis. Here, we built a comprehensive map of the Histoplasma capsulatum lipid metabolic pathway by incorporating proteomic and lipidomic analyses. We performed genetic complementation and overexpression of H. capsulatum genes in Saccharomyces cerevisiae to validate reactions identified in the map and to determine enzymes responsible for catalyzing orphan reactions. The map led to the identification of both the fatty acid desaturation and the sphingolipid biosynthesis pathways as targets for drug development. We found that the sphingolipid biosynthesis inhibitor myriocin, the fatty acid desaturase inhibitor thiocarlide, and the fatty acid analog 10-thiastearic acid inhibit H. capsulatum growth in nanomolar to low-micromolar concentrations. These compounds also reduced the intracellular infection in an alveolar macrophage cell line. Overall, this lipid metabolic map revealed pathways that can be targeted for drug development. IMPORTANCE It is estimated that 150 people die per hour due to the insufficient therapeutic treatments to combat fungal infections. A major hurdle to developing antifungal therapies is the scarce knowledge on the fungal metabolic pathways and mechanisms of virulence. In this context, fungal lipid metabolism is an excellent candidate for developing drugs due to its essential roles in cellular scaffolds, energy storage, and signaling transductors. Here, we provide a detailed map of Histoplasma capsulatum lipid metabolism. The map revealed points of this fungus lipid metabolism that can be targeted for developing antifungal drugs.

Optimizing the metabolism of microbial cell factories for yields and titers is a critical step for economically viable production of bioproducts and biofuels. In this process, tuning the expression of individual enzymes to obtain the desired pathway flux is a challenging step, in which data from separate multiomics techniques must be integrated with existing biological knowledge to determine where changes should be made. Following a design-build-test-learn strategy, building on recent advances in Bayesian metabolic control analysis, we identify key enzymes in the oleaginous yeast Yarrowia lipolytica that correlate with the production of itaconate by integrating a metabolic model with multiomics measurements. To this extent, we quantify the uncertainty for a variety of key parameters, known as flux control coefficients (FCCs), needed to improve the bioproduction of target metabolites and statistically obtain key correlations between the measured enzymes and boundary flux. Based on the top five significant FCCs and five correlated enzymes, our results show phosphoglycerate mutase, acetyl-CoA synthetase (ACSm), carbonic anhydrase (HCO3E), pyrophosphatase (PPAm), and homoserine dehydrogenase (HSDxi) enzymes in rate-limiting reactions that can lead to increased itaconic acid production.

Extracellular vesicles (EVs) are lipid bilayer structures released by organisms from all kingdoms of life. The diverse biogenesis pathways of EVs result in a wide variety of physical properties and functions across different organisms. Fungal EVs were first described in 2007 and different omics approaches have been fundamental to understand their composition, biogenesis, and function. In this review, we discuss the role of omics in elucidating fungal EVs biology. Transcriptomics, proteomics, metabolomics, and lipidomics have each enabled the molecular characterization of fungal EVs, providing evidence that these structures serve a wide array of functions, ranging from key carriers of cell wall biosynthetic machinery to virulence factors. Omics in combination with genetic approaches have been instrumental in determining both biogenesis and cargo loading into EVs. We also discuss how omics technologies are being employed to elucidate the role of EVs in antifungal resistance, disease biomarkers, and their potential use as vaccines. Finally, we review recent advances in analytical technology and multi-omic integration tools, which will help to address key knowledge gaps in EVs biology and translate basic research information into urgently needed clinical applications such as diagnostics, and immuno- and chemotherapies to fungal infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zamith-Miranda, Peres da Silva, Couvillion, Bredeweg, Burnet, Coelho, Camacho, Nimrichter, Puccia, Almeida, Casadevall, Rodrigues, Alves, Nosanchuk and Nakayasu.)

Pathway localization by fluorophore or epitope tagging can be accomplished through a multi-staged DNA construct and confirmation process, to generate a series of successfully tagged protein targets. Prerequisite conditions for this process in Y. lipolytica are auxotrophic selection (leu2 or ura3), impaired non-homologous end joining by deletion or impairment of ku70, and plasmids or gene pieces for epitope-selection cassette construction. The general approach for gene tagging can work for C- or N-terminal tags. Gene overexpression from an episomal plasmid can be accomplished through transcript amplification and cloning. C-terminal tagging allows expression of a gene-GFP fusion to be regulated from the endogenous promoter. The epitope-selection cassette also includes a constitutive or highly expressed promoter driving the auxotrophic or other selectable marker gene such as one conferring antifungal or antibiotic resistance. Strains for pathway localization utilize overlap PCR, PEG-based transformation, and a fast DNA preparation for rapid colony screening. Successful transformants can be used for pathway localization and condition-specific response analysis.

Light-sheet microscopy enables considerable speed and phototoxicity gains, while quantitative-phase imaging confers label-free recognition of cells and organelles, and quantifies their number-density that, thermodynamically, is more representative of metabolism than size. Here, we report the fusion of these two imaging modalities onto a standard inverted microscope that retains compatibility with microfluidics and open-source software for image acquisition and processing. An accelerating Airy-beam light-sheet critically enabled imaging areas that were greater by more than one order of magnitude than a Gaussian beam illumination and matched exactly those of quantitative-phase imaging. Using this integrative imaging system, we performed a demonstrative multivariate investigation of live-cells in microfluidics that unmasked that cellular noise can affect the compartmental localization of metabolic reactions. We detail the design, assembly, and performance of the integrative imaging system, and discuss potential applications in biotechnology and evolutionary biology.

Antibodies play a central role in host immunity by directly inactivating or recognizing an invading pathogen to enhance different immune responses to combat the invader. However, the cellular responses of pathogens to the presence of antibodies are not well-characterized. Here, we used different mass spectrometry techniques to study the cellular responses of the pathogenic fungus Histoplasma capsulatum to monoclonal antibodies (mAb) against HSP60, the surface protein involved in infection. A proteomic analysis of H. capsulatum yeast cells revealed that mAb binding regulates a variety of metabolic and signaling pathways, including fatty acid metabolism, sterol metabolism, MAPK signaling and ubiquitin-mediated proteolysis. The regulation of the fatty acid metabolism was accompanied by increases in the level of polyunsaturated fatty acids, which further augmented the degree of unsaturated lipids in H. capsulatum 's membranes and energy storage lipids, such as triacylglycerols, phosphatidylcholines, phosphatidylethanolamines and phosphatidylinositols. MAb treatment also regulated sterol metabolism by increasing the levels of cholesterol and ergosterol in the cells. We also showed that global changes in the lipid profiles resulted in an increased susceptibility of H. capsulatum to the ergosterol-targeting drug amphotericin B. Overall, our data showed that mAb induction of global changes in the composition of H. capsulatum membranes can potentially impact antifungal treatment during histoplasmosis.

Candida auris is a recently described pathogenic fungus that is causing invasive outbreaks on all continents. The fungus is of high concern given the numbers of multidrug-resistant strains that have been isolated in distinct sites across the globe. The fact that its diagnosis is still problematic suggests that the spreading of the pathogen remains underestimated. Notably, the molecular mechanisms of virulence and antifungal resistance employed by this new species are largely unknown. In the present work, we compared two clinical isolates of C. auris with distinct drug susceptibility profiles and a Candida albicans reference strain using a multi-omics approach. Our results show that, despite the distinct drug resistance profile, both C. auris isolates appear to be very similar, albeit with a few notable differences. However, compared to C. albicans both C. auris isolates have major differences regarding their carbon utilization and downstream lipid and protein content, suggesting a multifactorial mechanism of drug resistance. The molecular profile displayed by C. auris helps to explain the antifungal resistance and virulence phenotypes of this new emerging pathogen. IMPORTANCE Candida auris was first described in Japan in 2009 and has now been the cause of significant outbreaks across the globe. The high number of isolates that are resistant to one or more antifungals, as well as the high mortality rates from patients with bloodstream infections, has attracted the attention of the medical mycology, infectious disease, and public health communities to this pathogenic fungus. In the current work, we performed a broad multi-omics approach on two clinical isolates isolated in New York, the most affected area in the United States and found that the omic profile of C. auris differs significantly from C. albicans In addition to our insights into C. auris carbon utilization and lipid and protein content, we believe that the availability of these data will enhance our ability to combat this rapidly emerging pathogenic yeast., (Copyright © 2019 Zamith-Miranda et al.)

Modern live-cell imaging approaches permit real-time visualization of biological processes, yet limitations exist for unicellular organism isolation, culturing, and long-term imaging that preclude fully understanding how cells sense and respond to environmental perturbations and the link between single-cell variability and whole-population dynamics. Here, we present a microfluidic platform that provides fine control over the local environment with the capacity to replace media components at any experimental time point, and provides both perfused and compartmentalized cultivation conditions depending on the valve configuration. The functionality and flexibility of the platform were validated using both bacteria and yeast having different sizes, motility, and growth media. The demonstrated ability to track the growth and dynamics of both motile and non-motile prokaryotic and eukaryotic organisms emphasizes the versatility of the devices, which should enable studies in bioenergy and environmental research.

Fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeast Yarrowia lipolytica , growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism in Y. lipolytica . Deletion of the GATA transcription factor genes gzf3 and gzf2 resulted in nitrogen source-specific growth defects and greater accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion of gzf1 , which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion of gzf3 results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, while gzf2 is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressor mig1 , including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism. IMPORTANCE Nitrogen source is commonly used to control lipid production in industrial fungi. Here we identified regulators of nitrogen catabolite repression in the oleaginous yeast Y. lipolytica to determine how the nitrogen source regulates lipid metabolism. We show that disruption of both activators and repressors of nitrogen catabolite repression leads to increased lipid accumulation via activation of carbon catabolite repression through an as yet uncharacterized method.

In Neurospora crassa , blocking the function of the standard mitochondrial electron transport chain results in the induction of an alternative oxidase (AOX). AOX transfers electrons directly from ubiquinol to molecular oxygen. AOX serves as a model of retrograde regulation since it is encoded by a nuclear gene that is regulated in response to signals from mitochondria. The N. crassa transcription factors AOD2 and AOD5 are necessary for the expression of the AOX gene. To gain insight into the mechanism by which these factors function, and to determine if they have roles in the expression of additional genes in N. crassa , we constructed strains expressing only tagged versions of the proteins. Cell fractionation experiments showed that both proteins are localized to the nucleus under both AOX inducing and noninducing conditions. Furthermore, chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) analysis revealed that the proteins are bound to the promoter region of the AOX gene under both conditions. ChIP-seq also showed that the transcription factors bind to the upstream regions of a number of genes that are involved in energy production and metabolism. Dependence on AOD2 and AOD5 for the expression of several of these genes was verified by quantitative PCR. The majority of ChIP-seq peaks observed were enriched for both AOD2 and AOD5. However, we also observed occasional sites where one factor appeared to bind preferentially. The most striking of these was a conserved sequence that bound large amounts of AOD2 but little AOD5. This sequence was found within a 310 bp repeat unit that occurs at several locations in the genome., (Copyright © 2017 Qi et al.)

Light and the circadian clock have a profound effect on the biology of organisms through the regulation of large sets of genes. Toward understanding how light and the circadian clock regulate gene expression, we used genome-wide approaches to identify the direct and indirect targets of the light-responsive and clock-controlled transcription factor ADV-1 in Neurospora crassa A large proportion of ADV-1 targets were found to be light- and/or clock-controlled, and enriched for genes involved in development, metabolism, cell growth, and cell fusion. We show that ADV-1 is necessary for transducing light and/or temporal information to its immediate downstream targets, including controlling rhythms in genes critical to somatic cell fusion. However, while ADV-1 targets are altered in predictable ways in Δadv-1 cells in response to light, this is not always the case for rhythmic target gene expression. These data suggest that a complex regulatory network downstream of ADV-1 functions to generate distinct temporal dynamics of target gene expression relative to the central clock mechanism., (Copyright © 2017 Dekhang et al.)

Background: Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain., Results: We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica . To complement these strains, we constructed plasmids with Y. lipolytica -optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the " Yarrowia lipolytica Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells., Conclusions: These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica . This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products.

The global push toward an efficient and economical biobased economy has driven research to develop more cost-effective applications for the entirety of plant biomass, including lignocellulosic crops. As discussed elsewhere (Karlsson M, Atanasova L, Funck Jensen D, Zeilinger S, in Heitman J et al. [ed], Tuberculosis and the Tubercle Bacillus, 2nd ed, in press), significant progress has been made in the use of polysaccharide fractions from lignocellulose, cellulose, and various hemicellulose types. However, developing processes for use of the lignin fraction has been more challenging. In this chapter, we discuss characteristics of lignolytic enzymes and the fungi that produce them as well as potential and current uses of lignin-derived products.

Background: Yarrowia lipolytica is an oleaginous ascomycete yeast that stores lipids in response to limitation of nitrogen. While the enzymatic pathways responsible for neutral lipid accumulation in Y. lipolytica are well characterized, regulation of these pathways has received little attention. We therefore sought to characterize the response to nitrogen limitation at system-wide levels, including the proteome, phosphoproteome and metabolome, to better understand how this organism regulates and controls lipid metabolism and to identify targets that may be manipulated to improve lipid yield., Results: We found that ribosome structural genes are down-regulated under nitrogen limitation, during which nitrogen containing compounds (alanine, putrescine, spermidine and urea) are depleted and sugar alcohols and TCA cycle intermediates accumulate (citrate, fumarate and malate). We identified 1219 novel phosphorylation sites in Y. lipolytica, 133 of which change in their abundance during nitrogen limitation. Regulatory proteins, including kinases and DNA binding proteins, are particularly enriched for phosphorylation. Within lipid synthesis pathways, we found that ATP-citrate lyase, acetyl-CoA carboxylase and lecithin cholesterol acyl transferase are phosphorylated during nitrogen limitation while many of the proteins involved in β-oxidation are down-regulated, suggesting that storage lipid accumulation may be regulated by phosphorylation of key enzymes. Further, we identified short DNA elements that associate specific transcription factor families with up- and down-regulated genes., Conclusions: Integration of metabolome, proteome and phosphoproteome data identifies lipid accumulation in response to nitrogen limitation as a two-fold result of increased production of acetyl-CoA from excess citrate and decreased capacity for β-oxidation.

Interrogating polarized growth is technologically challenging due to extensive cellular branching and uncontrollable environmental conditions in conventional assays. Here we present a robust and high-performance microfluidic system that enables observations of polarized growth with enhanced temporal and spatial control over prolonged periods. The system has built-in tunability and versatility to accommodate a variety of scientific applications requiring precisely controlled environments. Using the model filamentous fungus, Neurospora crassa, our microfluidic system enabled direct visualization and analysis of cellular heterogeneity in a clonal fungal cell population, nuclear distribution and dynamics at the subhyphal level, and quantitative dynamics of gene expression with single hyphal compartment resolution in response to carbon source starvation and exchange. Although the microfluidic device is demonstrated on filamentous fungi, the technology is immediately extensible to a wide array of other biosystems that exhibit similar polarized cell growth, with applications ranging from bioenergy production to human health.

Yarrowia lipolytica is an oleaginous ascomycete yeast that accumulates large amounts of lipids and has potential as a biofuel producing organism. Despite a growing scientific literature focused on lipid production by Y. lipolytica, there remain significant knowledge gaps regarding the key biological processes involved. We applied a combination of metabolomic and lipidomic profiling approaches as well as microscopic techniques to identify and characterize the key pathways involved in de novo lipid accumulation from glucose in batch cultured, wild-type Y. lipolytica. We found that lipids accumulated rapidly and peaked at 48 hours during the five day experiment, concurrent with a shift in amino acid metabolism. We also report that exhaustion of extracellular sugars coincided with thickening of the cell wall, suggesting that genes involved in cell wall biogenesis may be a useful target for improving the efficiency of lipid producing yeast strains.

The filamentous fungus Neurospora crassa responds to light in complex ways. To thoroughly study the transcriptional response of this organism to light, RNA-seq was used to analyze capped and polyadenylated mRNA prepared from mycelium grown for 24 hr in the dark and then exposed to light for 0 (control) 15, 60, 120, and 240 min. More than three-quarters of all defined protein coding genes (79%) were expressed in these cells. The increased sensitivity of RNA-seq compared with previous microarray studies revealed that the RNA levels for 31% of expressed genes were affected two-fold or more by exposure to light. Additionally, a large class of mRNAs, enriched for transcripts specifying products involved in rRNA metabolism, showed decreased expression in response to light, indicating a heretofore undocumented effect of light on this pathway. Based on measured changes in mRNA levels, light generally increases cellular metabolism and at the same time causes significant oxidative stress to the organism. To deal with this stress, protective photopigments are made, antioxidants are produced, and genes involved in ribosome biogenesis are transiently repressed., (Copyright © 2014 Wu et al.)

Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane-associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.

High-throughput sequencing (HTS) has quickly become a valuable tool for comparative genetics and genomics and is now regularly carried out in laboratories that are not connected to large sequencing centers. Here we describe an updated version of our protocol for constructing single- and paired-end Illumina sequencing libraries, beginning with purified genomic DNA. The present protocol can also be used for "multiplexing," i.e. the analysis of several samples in a single flowcell lane by generating "barcoded" or "indexed" Illumina sequencing libraries in a way that is independent from Illumina-supported methods. To analyze sequencing results, we suggest several independent approaches but end users should be aware that this is a quickly evolving field and that currently many alignment (or "mapping") and counting algorithms are being developed and tested.

Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.