Cytoplasmic Continuity Revisited: Closure of Septa of the Filamentous Fungus Schizophyllum commune in Response to Environmental Conditions

Background: Mycelia of higher fungi consist of interconnected hyphae that are compartmentalized by septa. These septa contain large pores that allow streaming of cytoplasm and even organelles. The cytoplasm of such mycelia is therefore considered to be continuous. Methodology/Principal Findings: Here, we show by laser dissection that septa of Schizophyllum commune can be closed depending on the environmental conditions. The most apical septum of growing hyphae was open when this basidiomycete was grown in minimal medium with glucose as a carbon source. In contrast, the second and the third septum were closed in more than 50 % and 90 % of the cases, respectively. Interestingly, only 24 and 37 % of these septa were closed when hyphae were growing in the absence of glucose. Whether a septum was open or closed also depended on physical conditions of the environment or the presence of toxic agents. The first septum closed when hyphae were exposed to high temperature, to hypertonic conditions, or to the antibiotic nourseothricin. In the case of high temperature, septa opened again when the mycelium was placed back to the normal growth temperature. Conclusions/Significance: Taken together, it is concluded that the septal pores of S. commune are dynamic structures that open or close depending on the environmental conditions. Our findings imply that the cytoplasm in the mycelium of

Effect of fruiting-related genes on the formation of volatile organic compounds in the mushroom Schizophyllum commune

In fungi, little is known about connections between volatile organic compound (VOC) formation and developmental stages that are amongst others triggered by fruiting-related genes (FRGs). We analysed the volatilomes of Schizophyllum commune during different developmental stages in a variety of FRG-deletion strains and wild-type strains. The deletion strains Δtea1Δtea1, Δwc-2Δwc-2 and Δhom2Δhom2 were unable to develop fruiting bodies, and Δfst4Δfst4 formed only rudimentary fruiting body structures. Early developmental stages of these strains were dominated by esters, including methyl 2-methylbutanoate, ethyl 2-methylbutanoate, isobutyl 2-methylpropionate, and 2-methylbutyl acetate, of which the last three were not found in the headspace (HS) of the wild-type samples. Compared to the wild type, in the HS of hom2con samples, that are able to form fruiting bodies, methyl 2-methylbutanoate was the most abundant substance at early stages (68–81% of the total peak area). In contrast to fruiting body forming strains, Δtea1Δtea1, Δwc-2Δwc-2, Δhom2Δhom2 and Δfst4Δfst4 showed less sesquiterpenes in the HS. However, the sesquiterpenes found in the HS of FRG-deletion strains, namely, (E)-nerolidol, δ-cadinene, L-calamenene, α-bisabolol and β-bisabolene, were not present in hom2con or wild-type strains that mainly formed fruiting bodies and barely mycelium. Several sesquiterpenes, including α-guaiene, chamigrene and γ-gurjunene, were only found in presence of fruiting bodies. Our results show remarkable connections between FRGs, fruiting body development and VOC production in S. commune, especially counting for sesquiterpenes. Future studies are needed to reveal whether FRGs directly regulates VOC formation or indirectly by changing the phenotype

Inheritable CRISPR based epigenetic modification in a fungus

The CRISPRoff system was recently introduced as a programmable epigenetic memory writer that can be used to silence genes in human cells. The system makes use of a dead Cas9 protein (dCas9) that is fused with the ZNF10 KRAB, Dnmt3A, and Dnmt3L protein domains. The DNA methylation resulting from the CRISPRoff system can be removed by the CRISPRon system that consists of dCas9 fused to the catalytic domain of Tet1. Here, the CRISPRoff and CRISPRon systems were applied for the first time in a fungus. The CRISPRoff system resulted in an inactivation up to 100 % of the target genes flbA and GFP in Aspergillus niger. Phenotypes correlated with the degree of gene silencing in the transformants and were stable when going through a conidiation cycle, even when the CRISPRoff plasmid was removed from the flbA silenced strain. Introducing the CRISPRon system in a strain in which the CRISPRoff plasmid was removed fully reactivated flbA showing a phenotype similar to that of the wildtype. Together, the CRISPRoff and CRISPRon systems can be used to study gene function 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

Genome Sequence of the Model Mushroom Schizophyllum Commune

Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species\u27s unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals

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