CNTN4 modulates neural elongation through interplay with APP.

The neuronal cell adhesion molecule contactin-4 (CNTN4) is genetically associated with autism spectrum disorder (ASD) and other psychiatric disorders. Cntn4-deficient mouse models have previously shown that CNTN4 plays important roles in axon guidance and synaptic plasticity in the hippocampus. However, the pathogenesis and functional role of CNTN4 in the cortex has not yet been investigated. Our study found a reduction in cortical thickness in the motor cortex of Cntn4 -/- mice, but cortical cell migration and differentiation were unaffected. Significant morphological changes were observed in neurons in the M1 region of the motor cortex, indicating that CNTN4 is also involved in the morphology and spine density of neurons in the motor cortex. Furthermore, mass spectrometry analysis identified an interaction partner for CNTN4, confirming an interaction between CNTN4 and amyloid-precursor protein (APP). Knockout human cells for CNTN4 and/or APP revealed a relationship between CNTN4 and APP. This study demonstrates that CNTN4 contributes to cortical development and that binding and interplay with APP controls neural elongation. This is an important finding for understanding the physiological function of APP, a key protein for Alzheimer's disease. The binding between CNTN4 and APP, which is involved in neurodevelopment, is essential for healthy nerve outgrowth.

The α-(1,3)-glucan synthase gene agsE impacts the secretome of Aspergillus niger.

Aspergillus niger is widely used as a cell factory for the industrial production of enzymes. Previously, it was shown that deletion of α-1-3 glucan synthase genes results in smaller micro-colonies in liquid cultures of Aspergillus nidulans. Also, it has been shown that small wild-type Aspergillus niger micro-colonies secrete more protein than large mirco-colonies. We here assessed whether deletion of the agsC or agsE α-1-3 glucan synthase genes results in smaller A. niger micro-colonies and whether this is accompanied by a change in protein secretion. Biomass formation was not affected in the deletion strains but pH of the culture medium had changed from 5.2 in the case of the wild-type to 4.6 and 6.4 for ΔagsC and ΔagsE, respectively. The diameter of the ΔagsC micro-colonies was not affected in liquid cultures. In contrast, diameter of the ΔagsE micro-colonies was reduced from 3304 ± 338 µm to 1229 ± 113 µm. Moreover, the ΔagsE secretome was affected with 54 and 36 unique proteins with a predicted signal peptide in the culture medium of MA234.1 and the ΔagsE, respectively. Results show that these strains have complementary cellulase activity and thus may have complementary activity on plant biomass degradation. Together, α-1-3 glucan synthesis (in)directly impacts protein secretion in A. niger.

Heterogeneity in Spore Aggregation and Germination Results in Different Sized, Cooperative Microcolonies in an Aspergillus niger Culture.

The fungus Aspergillus niger is among the most abundant fungi in the world and is widely used as a cell factory for protein and metabolite production. This fungus forms asexual spores called conidia that are used for dispersal. Notably, part of the spores and germlings aggregate in an aqueous environment. The aggregated conidia/germlings give rise to large microcolonies, while the nonaggregated spores/germlings result in small microcolonies. Here, it is shown that small microcolonies release a larger variety and quantity of secreted proteins compared to large microcolonies. Yet, the secretome of large microcolonies has complementary cellulase activity with that of the small microcolonies. Also, large microcolonies are more resistant to heat and oxidative stress compared to small microcolonies, which is partly explained by the presence of nongerminated spores in the core of the large microcolonies. Together, it is proposed that heterogeneity in germination and aggregation has evolved to form a population of different sized A. niger microcolonies, thereby increasing stress survival and producing a meta-secretome more optimally suited to degrade complex substrates. IMPORTANCE Aspergillus niger can form microcolonies of different size due to partial aggregation of spores and germlings. So far, this heterogeneity was considered a negative trait by the industry. We here, however, show that heterogeneity in size within a population of microcolonies is beneficial for food degradation and stress survival. This functional heterogeneity is not only of interest for the industry to make blends of enzymes (e.g., for biofuel or bioplastic production) but could also play a role in nature for effective nutrient cycling and survival of the fungus.

Sbk2, a Newly Discovered Atrium-Enriched Regulator of Sarcomere Integrity.

BACKGROUND: Heart development relies on tight spatiotemporal control of cardiac gene expression. Genes involved in this intricate process have been identified using animals and pluripotent stem cell-based models of cardio(myo)genesis. Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal line of conditionally immortalized neonatal rat atrial myocytes (NRAMs), which allows toggling between proliferative and differentiated (ie, excitable and contractile) phenotypes in a synchronized and homogenous manner. METHODS: In this study, the unique properties of conditionally immortalized NRAMs (iAMs) were exploited to identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation. RESULTS: Transcriptome analysis of iAM-1 cells at different stages during one cycle of differentiation and subsequent dedifferentiation identified ≈13 000 transcripts, of which the dynamic changes in expression upon cardiomyogenic differentiation mostly opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many with known (lineage-specific) functions in cardiac muscle formation. Filtering for cardiac-enriched low-abundance transcripts, identified multiple genes with an uncharacterized role during cardio(myo)genesis including Sbk2 (SH3 domain binding kinase family member 2). Sbk2 encodes an evolutionarily conserved putative serine/threonine protein kinase, whose expression is strongly up- and downregulated during iAM-1 cell differentiation and dedifferentiation, respectively. In neonatal and adult rats, the protein is muscle-specific, highly atrium-enriched, and localized around the A-band of cardiac sarcomeres. Knockdown of Sbk2 expression caused loss of sarcomeric organization in NRAMs, iAMs and their human counterparts, consistent with a decrease in sarcomeric gene expression as evinced by transcriptome and proteome analyses. Interestingly, co-immunoprecipitation using Sbk2 as bait identified possible interaction partners with diverse cellular functions (translation, intracellular trafficking, cytoskeletal organization, chromatin modification, sarcomere formation). CONCLUSIONS: iAM-1 cells are a relevant and suitable model to identify (lowly expressed) genes with a hitherto unidentified role in cardiomyocyte differentiation as exemplified by Sbk2: a regulator of atrial sarcomerogenesis.

Ribociclib Induces Broad Chemotherapy Resistance and EGFR Dependency in ESR1 Wildtype and Mutant Breast Cancer.

While endocrine therapy is highly effective for the treatment of oestrogen receptor-α (ERα)-positive breast cancer, a significant number of patients will eventually experience disease progression and develop treatment-resistant, metastatic cancer. The majority of resistant tumours remain dependent on ERα-action, with activating ESR1 gene mutations occurring in 15-40% of advanced cancers. Therefore, there is an urgent need to discover novel effective therapies that can eradicate cancer cells with aberrant ERα and to understand the cellular response underlying their action. Here, we evaluate the response of MCF7-derived, CRISPR-Cas9-generated cell lines expressing mutant ERα (Y537S) to a large number of drugs. We report sensitivity to numerous clinically approved inhibitors, including CDK4/6 inhibitor ribociclib, which is a standard-of-care therapy in the treatment of metastatic ERα-positive breast cancer and currently under evaluation in the neoadjuvant setting. Ribociclib treatment induces senescence in both wildtype and mutant ERα breast cancer models and leads to a broad-range drug tolerance. Strikingly, viability of cells undergoing ribociclib-induced cellular senescence is maintained via engagement of EGFR signalling, which may be therapeutically exploited in both wildtype and mutant ERα-positive breast cancer. Our study highlights a wide-spread reduction in sensitivity to anti-cancer drugs accompanied with an acquired vulnerability to EGFR inhibitors following CDK4/6 inhibitor treatment.

Multistep mechanism of G-quadruplex resolution during DNA replication.

G-quadruplex (or G4) structures form in guanine-rich DNA sequences and threaten genome stability when not properly resolved. G4 unwinding occurs during S phase via an unknown mechanism. Using Xenopus egg extracts, we define a three-step G4 unwinding mechanism that acts during DNA replication. First, the replicative helicase composed of Cdc45, MCM2-7 and GINS (CMG) stalls at a leading strand G4 structure. Second, the DEAH-box helicase 36 (DHX36) mediates bypass of the CMG past the intact G4 structure, allowing approach of the leading strand to the G4. Third, G4 structure unwinding by the Fanconi anemia complementation group J helicase (FANCJ) enables DNA polymerase to synthesize past the G4 motif. A G4 on the lagging strand template does not stall CMG but still requires DNA replication for unwinding. DHX36 and FANCJ have partially redundant roles, conferring pathway robustness. This previously unknown genome maintenance pathway promotes faithful G4 replication, thereby avoiding genome instability.

Proteomics and Phosphoproteomics Profiling of Drug-Addicted BRAFi-Resistant Melanoma Cells.

Acquired resistance to MAPK inhibitors limits the clinical efficacy in melanoma treatment. We and others have recently shown that BRAF inhibitor (BRAFi)-resistant melanoma cells can develop a dependency on the therapeutic drugs to which they have acquired resistance, creating a vulnerability for these cells that can potentially be exploited in cancer treatment. In drug-addicted melanoma cells, it was shown that this induction of cell death was preceded by a specific ERK2-dependent phenotype switch; however, the underlying molecular mechanisms are largely lacking. To increase the molecular understanding of this drug dependency, we applied a mass spectrometry-based proteomic approach on BRAFi-resistant BRAFMUT 451Lu cells, in which ERK1, ERK2, and JUNB were silenced separately using CRISPR-Cas9. Inactivation of ERK2 and, to a lesser extent, JUNB prevents drug addiction in these melanoma cells, while, conversely, knockout of ERK1 fails to reverse this phenotype, showing a response similar to that of control cells. Our analysis reveals that ERK2 and JUNB share comparable proteome responses dominated by reactivation of cell division. Importantly, we find that EMT activation in drug-addicted melanoma cells upon drug withdrawal is affected by silencing ERK2 but not ERK1. Moreover, transcription factor (regulator) enrichment shows that PIR acts as an effector of ERK2 and phosphoproteome analysis reveals that silencing of ERK2 but not ERK1 leads to amplification of GSK3 kinase activity. Our results depict possible mechanisms of drug addiction in melanoma, which may provide a guide for therapeutic strategies in drug-resistant melanoma.

Dynamic remodelling of the human host cell proteome and phosphoproteome upon enterovirus infection.

The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.

FlbA-Regulated Gene rpnR Is Involved in Stress Resistance and Impacts Protein Secretion when Aspergillus niger Is Grown on Xylose.

Proteins are secreted throughout the mycelium of Aspergillus niger except for the sporulating zone. A link between sporulation and repression of protein secretion was underlined by the finding that inactivation of the sporulation gene flbA results in mycelial colonies that secrete proteins throughout the colony. However, ΔflbA strain hyphae also lyse and have thinner cell walls. This pleiotropic phenotype is associated with differential expression of 36 predicted transcription factor genes, one of which, rpnR, was inactivated in this study. Sporulation, biomass, and secretome complexity were not affected in the ΔrpnR deletion strain of the fungus. In contrast, ribosomal subunit expression and protein secretion into the medium were reduced when A. niger was grown on xylose. Moreover, the ΔrpnR strain showed decreased resistance to H2O2 and the proteotoxic stress-inducing agent dithiothreitol. Taking the data together, RpnR is involved in proteotoxic stress resistance and impacts protein secretion when A. niger is grown on xylose.IMPORTANCEAspergillus niger secretes a large amount and diversity of industrially relevant enzymes into the culture medium. This makes the fungus a widely used industrial cell factory. For instance, carbohydrate-active enzymes of A. niger are used in biofuel production from lignocellulosic feedstock. These enzymes represent a major cost factor in this process. Higher production yields could substantially reduce these costs and therefore contribute to a more sustainable economy and less dependence on fossil fuels. Enzyme secretion is inhibited in A. niger by asexual reproduction. The sporulation protein FlbA is involved in this process by impacting the expression of 36 predicted transcription factor genes. Here, we show that one of these predicted transcriptional regulators, RpnR, regulates protein secretion and proteotoxic stress resistance. The gene is thus an interesting target to improve enzyme production in A. niger.

In vivo phosphoproteomics reveals kinase activity profiles that predict treatment outcome in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, we use high-throughput phosphoproteomics to build a functional TNBC taxonomy. A cluster of 159 phosphosites is upregulated in relapsed cases of a training set (n = 34 patients), with 11 hyperactive kinases accounting for this phosphoprofile. A mass-spectrometry-to-immunohistochemistry translation step, assessing 2 independent validation sets, reveals 6 kinases with preserved independent prognostic value. The kinases split the validation set into two patterns: one without hyperactive kinases being associated with a >90% relapse-free rate, and the other one showing ≥1 hyperactive kinase and being associated with an up to 9.5-fold higher relapse risk. Each kinase pattern encompasses different mutational patterns, simplifying mutation-based taxonomy. Drug regimens designed based on these 6 kinases show promising antitumour activity in TNBC cell lines and patient-derived xenografts. In summary, the present study elucidates phosphosites and kinases implicated in TNBC and suggests a target-based clinical classification system for TNBC.

The physiology of Agaricus bisporus in semi-commercial compost cultivation appears to be highly conserved among unrelated isolates.

The white button mushroom Agaricus bisporus is one of the most widely produced edible fungus with a great economical value. Its commercial cultivation process is often performed on wheat straw and animal manure based compost that mainly contains lignocellulosic material as a source of carbon and nutrients for the mushroom production. As a large portion of compost carbohydrates are left unused in the current mushroom cultivation process, the aim of this work was to study wild-type A. bisporus strains for their potential to convert the components that are poorly utilized by the commercial strain A15. We therefore focused our analysis on the stages where the fungus is producing fruiting bodies. Growth profiling was used to identify A. bisporus strains with different abilities to use plant biomass derived polysaccharides, as well as to transport and metabolize the corresponding monomeric sugars. Six wild-type isolates with diverse growth profiles were compared for mushroom production to A15 strain in semi-commercial cultivation conditions. Transcriptome and proteome analyses of the three most interesting wild-type strains and A15 indicated that the unrelated A. bisporus strains degrade and convert plant biomass polymers in a highly similar manner. This was also supported by the chemical content of the compost during the mushroom production process. Our study therefore reveals a highly conserved physiology for unrelated strains of this species during growth in compost.

Cultivation of Podospora anserina on soybean hulls results in an efficient enzyme cocktail for plant biomass hydrolysis.

The coprophilic ascomycete fungus Podospora anserina was cultivated on three different plant biomasses, i.e. cotton seed hulls (CSH), soybean hulls (SBH) and acid-pretreated wheat straw (WS) for four days, and the potential of the produced enzyme mixtures was compared in the enzymatic saccharification of the corresponding lignocellulose feedstocks. The enzyme cocktail P. anserina produced after three days of growth on SBH showed superior capacity to release reducing sugars from all tested plant biomass feedstocks compared to the enzyme mixtures from CSH and WS cultures. Detailed proteomics analysis of the culture supernatants revealed that SBH contained the most diverse set of enzymes targeted on plant cell wall polymers and was particularly abundant in xylan, mannan and pectin acting enzymes. The importance of lytic polysaccharide monooxygenases (LPMOs) in plant biomass deconstruction was supported by identification of 20 out of 33 AA9 LPMOs in the SBH cultures. The results highlight the suitability of P. anserina as a source of plant cell wall degrading enzymes for biotechnological applications and the importance of selecting the most optimal substrate for the production of enzyme mixtures.

Toward an Optimized Workflow for Middle-Down Proteomics.

Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < Mw < 30 kDa), respectively, termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates, we explored the use of the proteases Asp-N and Glu-C and a nonenzymatic acid induced cleavage. To increase the depth of the proteome, a strong cation exchange (SCX) separation, carefully tuned to improve the separation of longer peptides, combined with reversed phase-liquid chromatography (RP-LC) using columns packed with material possessing a larger pore size, was used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques, including higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer combined with higher-energy collision dissociation (EThcD), for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, among other things, the enhanced analysis of (co-occurring) post-translational modifications.

Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 µg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 µg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.

Association of Cell Adhesion Molecules Contactin-6 and Latrophilin-1 Regulates Neuronal Apoptosis.

In view of important neurobiological functions of the cell adhesion molecule contactin-6 (Cntn6) that have emerged from studies on null-mutant mice and autism spectrum disorders patients, we set out to examine pathways underlying functions of Cntn6 using a proteomics approach. We identified the cell adhesion GPCR latrophilin-1 (Lphn1, a.k.a. CIRL1/CL, ADGRL1) as a binding partner for Cntn6 forming together a heteromeric cis-complex. Lphn1 expression in cultured neurons caused reduction in neurite outgrowth and increase in apoptosis, which was rescued by coexpression of Cntn6. In cultured neurons derived from Cntn6-/- mice, Lphn1 knockdown reduced apoptosis, suggesting that the observed apoptosis was Lphn1-dependent. In line with these data, the number of apoptotic cells was increased in the cortex of Cntn6-/- mice compared to wild-type littermate controls. These results show that Cntn6 can modulate the activity of Lphn1 by direct binding and suggests that Cntn6 may prevent apoptosis thereby impinging on neurodevelopment.

Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions.

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5ß and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.

Assessment of SRM, MRM(3) , and DIA for the targeted analysis of phosphorylation dynamics in non-small cell lung cancer.

Hypothesis-driven MS-based targeted proteomics has gained great popularity in a relatively short timespan. Next to the widely established selected reaction monitoring (SRM) workflow, data-independent acquisition (DIA), also referred to as sequential window acquisition of all theoretical spectra (SWATH) was introduced as a high-throughput targeted proteomics method. DIA facilitates increased proteome coverage, however, does not yet reach the sensitivity obtained with SRM. Therefore, a well-informed method selection is crucial for designing a successful targeted proteomics experiment. This is especially the case when targeting less conventional peptides such as those that contain PTMs, as these peptides do not always adhere to the optimal fragmentation considerations for targeted assays. Here, we provide insight into the performance of DIA, SRM, and MRM cubed (MRM(3) ) in the analysis of phosphorylation dynamics throughout the phosphoinositide 3-kinase mechanistic target of rapamycin (PI3K-mTOR) and mitogen-activated protein kinase (MAPK) signaling network. We observe indeed that DIA is less sensitive when compared to SRM, however demonstrates increased flexibility, by postanalysis selection of alternative phosphopeptide precursors. Additionally, we demonstrate the added benefit of MRM(3) , allowing the quantification of two poorly accessible phosphosites. In total, targeted proteomics enabled the quantification of 42 PI3K-mTOR and MAPK phosphosites, gaining a so far unachieved in-depth view mTOR signaling events linked to tyrosine kinase inhibitor resistance in non-small cell lung cancer.

Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry.

We describe an integrated workflow that robustly identifies cross-links from endogenous protein complexes in human cellular lysates. Our approach is based on the application of mass spectrometry (MS)-cleavable cross-linkers, sequential collision-induced dissociation (CID)-tandem MS (MS/MS) and electron-transfer dissociation (ETD)-MS/MS acquisitions, and a dedicated search engine, XlinkX, which allows rapid cross-link identification against a complete human proteome database. This approach allowed us to detect 2,179 unique cross-links (1,665 intraprotein cross-links at a 5% false discovery rate (FDR) and 514 interprotein cross-links at 1% FDR) in HeLa cell lysates. We validated the confidence of our cross-linking results by using a target-decoy strategy and mapping the observed cross-link distances onto existing high-resolution structures. Our data provided new structural information about many protein assemblies and captured dynamic interactions of the ribosome in contact with different elongation factors.

Spatial differentiation of gene expression in Aspergillus niger colony grown for sugar beet pulp utilization.

Degradation of plant biomass to fermentable sugars is of critical importance for the use of plant materials for biofuels. Filamentous fungi are ubiquitous organisms and major plant biomass degraders. Single colonies of some fungal species can colonize massive areas as large as five soccer stadia. During growth, the mycelium encounters heterogeneous carbon sources. Here we assessed whether substrate heterogeneity is a major determinant of spatial gene expression in colonies of Aspergillus niger. We analyzed whole-genome gene expression in five concentric zones of 5-day-old colonies utilizing sugar beet pulp as a complex carbon source. Growth, protein production and secretion occurred throughout the colony. Genes involved in carbon catabolism were expressed uniformly from the centre to the periphery whereas genes encoding plant biomass degrading enzymes and nitrate utilization were expressed differentially across the colony. A combined adaptive response of carbon-catabolism and enzyme production to locally available monosaccharides was observed. Finally, our results demonstrate that A. niger employs different enzymatic tools to adapt its metabolism as it colonizes complex environments.

Uncovering the abilities of Agaricus bisporus to degrade plant biomass throughout its life cycle.

The economically important edible basidiomycete mushroom Agaricus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and contributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A. bisporus grows in the controlled environment in commercial production facilities and utilizes its substrate. Using transcriptomics and proteomics, we showed that changes in plant biomass degradation by A. bisporus occur throughout its life cycle. Ligninolytic genes were only highly expressed during the spawning stage day 16. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exo-proteome analysis. Our data also support a model of sequential lignocellulose degradation by wood-decaying fungi proposed in previous studies, concluding that lignin is degraded at the initial stage of growth in compost and is not modified after the spawning stage. The observed differences in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the number of mushrooms during the second flush.