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.

Probing Cell-Surface Interactions in Fungal Cell Walls by High-Resolution 1 H-Detected Solid-State NMR Spectroscopy.

Solid-state NMR (ssNMR) spectroscopy facilitates the non-destructive characterization of structurally heterogeneous biomolecules in their native setting, for example, comprising proteins, lipids and polysaccharides. Here we demonstrate the utility of high and ultra-high field 1 H-detected fast MAS ssNMR spectroscopy, which exhibits increased sensitivity and spectral resolution, to further elucidate the atomic-level composition and structural arrangement of the cell wall of Schizophyllum commune, a mushroom-forming fungus from the Basidiomycota phylum. These advancements allowed us to reveal that Cu(II) ions and the antifungal peptide Cathelicidin-2 mainly bind to cell wall proteins at low concentrations while glucans are targeted at high metal ion concentrations. In addition, our data suggest the presence of polysaccharides containing N-acetyl galactosamine (GalNAc) and proteins, including the hydrophobin proteins SC3, shedding more light on the molecular make-up of cells wall as well as the positioning of the polypeptide layer. Obtaining such information may be of critical relevance for future research into fungi in material science and biomedical contexts.

The Transcription Factor Roc1 Is a Key Regulator of Cellulose Degradation in the Wood-Decaying Mushroom Schizophyllum commune.

Wood-decaying fungi of the class Agaricomycetes (phylum Basidiomycota) are saprotrophs that break down lignocellulose and play an important role in nutrient recycling. They secrete a wide range of extracellular plant cell wall degrading enzymes that break down cellulose, hemicellulose, and lignin, the main building blocks of plant biomass. Although the production of these enzymes is regulated mainly at the transcriptional level, no activating regulators have been identified in any wood-decaying fungus in the class Agaricomycetes. We studied the regulation of cellulase expression in the wood-decaying fungus Schizophyllum commune. Comparative genomics and transcriptomics on two wild isolates revealed a Zn2Cys6-type transcription factor gene (roc1) that was highly upregulated during growth on cellulose, compared to glucose. It is only conserved in the class Agaricomycetes. A roc1 knockout strain showed an inability to grow on medium with cellulose as sole carbon source, and growth on cellobiose and xylan (other components of wood) was inhibited. Growth on non-wood-related carbon sources was not inhibited. Cellulase gene expression and enzyme activity were reduced in the Δroc1 strain. ChIP-Seq identified 1474 binding sites of the Roc1 transcription factor. Promoters of genes involved in lignocellulose degradation were enriched with these binding sites, especially those of LPMO (lytic polysaccharide monooxygenase) CAZymes, indicating that Roc1 directly regulates these genes. A conserved motif was identified as the binding site of Roc1, which was confirmed by a functional promoter analysis. Together, Roc1 is a key regulator of cellulose degradation and the first identified in wood-decaying fungi in the phylum Basidiomycota. IMPORTANCE Wood-degrading fungi in the phylum Basidiomycota play a crucial role in nutrient recycling by breaking down all components of wood. Fungi have evolved transcriptional networks that regulate expression of wood-degrading enzymes, allowing them to prioritize one nutrient source over another. However, to date all these transcription factors have been identified in the phylum Ascomycota, which is only distantly related to the phylum Basidiomycota. Here, we identified the transcription factor Roc1 as a key regulator of cellulose degradation in the mushroom-forming and wood-degrading fungus Schizophyllum commune. Roc1 is highly conserved in the phylum Basidiomycota. Using comparative genomics, transcriptomics, ChIP-Seq and promoter analysis we have identified direct targets of Roc1, as well as other aspects of the transcriptional response to cellulose.

Fungal electronics.

Fungal electronics is a family of living electronic devices made of mycelium bound composites or pure mycelium. Fungal electronic devices are capable of changing their impedance and generating spikes of electrical potential in response to external control parameters. Fungal electronics can be embedded into fungal materials and wearables or used as stand alone sensing and computing devices.

On electrical gates on fungal colony.

Mycelium networks are promising substrates for designing unconventional computing devices providing rich topologies and geometries where signals propagate and interact. Fulfilling our long-term objectives of prototyping electrical analog computers from living mycelium networks, including networks hybridised with nanoparticles, we explore the possibility of implementing Boolean logical gates based on electrical properties of fungal colonies. We converted a 3D image-data stack of Aspergillus niger fungal colony to an Euclidean graph and modelled the colony as resistive and capacitive (RC) networks, where electrical parameters of edges were functions of the edges' lengths. We found that and, or and and-not gates are implementable in RC networks derived from the geometrical structure of the real fungal colony.

On Boolean gates in fungal colony.

A fungal colony maintains its integrity via flow of cytoplasm along mycelium network. This flow, together with possible coordination of mycelium tips propagation, is controlled by calcium waves and associated waves of electrical potential changes. We propose that these excitation waves can be employed to implement a computation in the mycelium networks. We use FitzHugh-Nagumo model to imitate propagation of excitation in a single colony of Aspergillus niger. Boolean values are encoded by spikes of extracellular potential. We represent binary inputs by electrical impulses on a pair of selected electrodes and we record responses of the colony from sixteen electrodes. We derive sets of two-inputs-on-output logical gates implementable the fungal colony and analyse distributions of the gates.

Spatial induction of genes encoding secreted proteins in micro-colonies of Aspergillus niger.

Aspergillus niger is used by the industry to produce enzymes and metabolites such as citric acid. In liquid cultures, it can grow as a dispersed mycelium or as micro-colonies with a width in the micrometer to millimeter range. Here, it was assessed whether expression of genes encoding secreted enzymes depends on mycelium morphology. To this end, expression of the reporter gene gfp from the promoters of the glucoamylase gene glaA, the feruloyl esterase gene faeA and the α-glucuronidase gene aguA was causally related to micro-colony size within a liquid shaken culture. Data could be fitted by hyperbolic functions, implying that the genes encoding these secreted proteins are expressed in a shell at the periphery of the micro-colony. The presence of such a shell was confirmed by confocal microscopy. Modelling predicted that the width of these zones was 13 to 156 µm depending on growth medium and micro-colony diameter. Together, data indicate that the highest productive micro-colonies are those colonies that have a radius ≤ the width of the peripheral expression zone.

Apical but not sub-apical hyphal compartments are self-sustaining in growth.

It was recently demonstrated that apical compartments of Aspergillus niger hyphae are self-sustaining in growth. This was shown by assessing the growth rate of individual hyphae before and after dissection of the second compartment. Using the same methodology, it is here demonstrated that single apical compartments of the septate fungi Penicillium chrysogenum and Schizophyllum commune as well as the 500-µm-apical region of the non-septate fungus Rhizopus stolonifer are also self-sustaining in growth. In contrast, single 2nd compartments (obtained by dissection of the first and third compartment) of the septate fungi or the region between 500 and 1000 µm from tips of R. stolonifer were severely impacted in their growth rate. In addition, it is shown that existing or newly formed branches originating from the 2nd compartments function as a backup system for hyphal growth when the apical part of the hypha of the three studied fungi is damaged. Together, it is concluded that the apical compartments/zones of the studied fungi are self-sustaining in growth. In contrast, the subapical region is not self-sustaining but functions as a backup once the apical zone is damaged. This back up system is relevant in nature because the apices of hyphae are the first to be exposed to (a)biotic stress conditions when entering an unexplored substrate.

Subpopulations of hyphae secrete proteins or resist heat stress in Aspergillus oryzae colonies.

Hyphae at the outer part of colonies of Aspergillus niger and Aspergillus oryzae are heterogeneous with respect to transcriptional and translational activity. This heterogeneity is maintained by Woronin body mediated closure of septal pores that block interhyphal mixing of cytoplasm. Indeed, heterogeneity between hyphae is abolished in ΔhexA strains that lack Woronin bodies. The subpopulation of hyphae with high transcriptional and translational activity secretes enzymes that degrade the substrate resulting in breakdown products that serve as nutrients. The role of hyphae with low transcriptional and translational activity was not yet known. Here, we show that this subpopulation is more resistant to environmental stress in A. oryzae, in particular to temperature stress, when compared to hyphae with high transcriptional and translational activity. Notably, all hyphae of the ΔhexA strain of A. oryzae were sensitive to heat stress explained by the reduced heterogeneity in this strain. Together, we show that different subpopulations of hypha secrete proteins and resist heat stress showing the complexity of a fungal mycelium.

Functional distinction of hyphal compartments.

Hyphae of higher fungi grow at their tips and are compartmentalized by porous septa that enable inter-compartmental cytoplasmic streaming. Woronin bodies discontinue cytoplasmic streaming by plugging the septal pores. Here, it was assessed whether apical compartments of Aspergillus niger sustain their own growth or whether their growth depends on subapical compartments. Hyphae of wildtype and the ΔhexA strain, lacking Woronin bodies, had a similar morphology and growth rate. A total of 58% and 17% of the hyphae continued growing, respectively, after dissecting the 2nd compartment. Extension rate of the apical compartments that continued growing was not affected, even when the carbon or nitrogen source was limiting. Thus, apical compartments are self-sustaining in growth. It was also shown that the first 8 subapical compartments of the wildtype, but not of the ΔhexA strain, function as a backup system for growth by forming new branches when their apical neighbouring compartment has been damaged. This backup system is pivotal in nature because of the life style of fungi to continuously explore their surrounding substrate that may prove hostile.

Maturation of conidia on conidiophores of Aspergillus niger.

Conidia of Aspergillus niger are produced on conidiophores. Here, maturation of conidia on these asexual reproductive structures was studied. Pigmented conidia that had developed on conidiophores for 2, 5, and 8days were similarly resistant to heat and were metabolically active as shown by CO2 release and conversion of the metabolic probe Tempone. A total number of 645-2421 genes showed a ⩾2-fold change in expression when 2-day-old conidia were compared to 5- and 8-day-old spores. Melanin was extracted more easily from the cell wall of 2-day-old conidia when compared to the older spores. In addition, mannitol content and germination rate of the 2-day-old conidia were higher. Dispersal efficiency by water was lower in the case of the 8-day-old conidia but no differences were observed in dispersal by wind and a hydrophobic moving object. These data and the fact that only a minor fraction of the conidia on a conidiophore were dispersed in the assays imply that a single colony of A. niger releases a heterogeneous population of conidia. This heterogeneity would provide a selective advantage in environments with rapidly changing conditions such as availability of water.