Snowball: a novel gene family required for developmental patterning of fruiting bodies of mushroom-forming fungi (Agaricomycetes).

The morphogenesis of sexual fruiting bodies of fungi is a complex process determined by a genetically encoded program. Fruiting bodies reached the highest complexity levels in the Agaricomycetes; yet, the underlying genetics is currently poorly known. In this work, we functionally characterized a highly conserved gene termed snb1, whose expression level increases rapidly during fruiting body initiation. According to phylogenetic analyses, orthologs of snb1 are present in almost all agaricomycetes and may represent a novel conserved gene family that plays a substantial role in fruiting body development. We disrupted snb1 using CRISPR/Cas9 in the agaricomycete model organism Coprinopsis cinerea. snb1 deletion mutants formed unique, snowball-shaped, rudimentary fruiting bodies that could not differentiate caps, stipes, and lamellae. We took advantage of this phenotype to study fruiting body differentiation using RNA-Seq analyses. This revealed differentially regulated genes and gene families that, based on wild-type RNA-Seq data, were upregulated early during development and showed tissue-specific expression, suggesting a potential role in differentiation. Taken together, the novel gene family of snb1 and the differentially expressed genes in the snb1 mutants provide valuable insights into the complex mechanisms underlying developmental patterning in the Agaricomycetes. IMPORTANCE: Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are complex multicellular structures, with a spatially and temporally integrated developmental program that is, however, currently poorly known. In this study, we present a novel, conserved gene family, Snowball (snb), termed after the unique, differentiation-less fruiting body morphology of snb1 knockout strains in the model mushroom Coprinopsis cinerea. snb is a gene of unknown function that is highly conserved among agaricomycetes and encodes a protein of unknown function. A comparative transcriptomic analysis of the early developmental stages of differentiated wild-type and non-differentiated mutant fruiting bodies revealed conserved differentially expressed genes which may be related to tissue differentiation and developmental patterning fruiting body development.

Preassembled Cas9 Ribonucleoprotein-Mediated Gene Deletion Identifies the Carbon Catabolite Repressor and Its Target Genes in Coprinopsis cinerea.

Cre1 is an important transcription factor that regulates carbon catabolite repression (CCR) and is widely conserved across fungi. The cre1 gene has been extensively studied in several Ascomycota species, whereas its role in gene expression regulation in the Basidiomycota species remains poorly understood. Here, we identified and investigated the role of cre1 in Coprinopsis cinerea, a basidiomycete model mushroom that can efficiently degrade lignocellulosic plant wastes. We used a rapid and efficient gene deletion approach based on PCR-amplified split-marker DNA cassettes together with in vitro assembled Cas9-guide RNA ribonucleoproteins (Cas9 RNPs) to generate C. cinerea cre1 gene deletion strains. Gene expression profiling of two independent C. cinerea cre1 mutants showed significant deregulation of carbohydrate metabolism, plant cell wall degrading enzymes (PCWDEs), plasma membrane transporter-related and several transcription factor-encoding genes, among others. Our results support the notion that, like reports in the ascomycetes, Cre1 of C. cinerea orchestrates CCR through a combined regulation of diverse genes, including PCWDEs, transcription factors that positively regulate PCWDEs, and membrane transporters which could import simple sugars that can induce the expression of PWCDEs. Somewhat paradoxically, though in accordance with other Agaricomycetes, genes related to lignin degradation were mostly downregulated in cre1 mutants, indicating they fall under different regulation than other PCWDEs. The gene deletion approach and the data presented here will expand our knowledge of CCR in the Basidiomycota and provide functional hypotheses on genes related to plant biomass degradation. IMPORTANCE Mushroom-forming fungi include some of the most efficient lignocellulosic plant biomass degraders. They degrade dead plant materials by a battery of lignin-, cellulose-, hemicellulose-, and pectin-degrading enzymes, the encoding genes of which are under tight transcriptional control. One of the highest-level regulations of these metabolic enzymes is known as carbon catabolite repression, which is orchestrated by the transcription factor Cre1, and ensures that costly lignocellulose-degrading enzyme genes are expressed only when simple carbon sources (e.g., glucose) are not available. Here, we identified the Cre1 ortholog in a litter decomposer Agaricomycete, Coprinopsis cinerea, knocked it out, and characterized transcriptional changes in the mutants. We identified several dozen lignocellulolytic enzyme genes as well as membrane transporters and other transcription factors as putative target genes of C. cinerea cre1. These results extend knowledge on carbon catabolite repression to litter decomposer Basidiomycota.

Evolutionary Morphogenesis of Sexual Fruiting Bodies in Basidiomycota: Toward a New Evo-Devo Synthesis.

The development of sexual fruiting bodies is one of the most complex morphogenetic processes in fungi. Mycologists have long been fascinated by the morphological and developmental diversity of fruiting bodies; however, evolutionary developmental biology of fungi still lags significantly behind that of animals or plants. Here, we summarize the current state of knowledge on fruiting bodies of mushroom-forming Basidiomycota, focusing on phylogenetic and developmental biology. Phylogenetic approaches have revealed a complex history of morphological transformations and convergence in fruiting body morphologies. Frequent transformations and convergence is characteristic of fruiting bodies in contrast to animals or plants, where main body plans are highly conserved. At the same time, insights into the genetic bases of fruiting body development have been achieved using forward and reverse genetic approaches in selected model systems. Phylogenetic and developmental studies of fruiting bodies have each yielded major advances, but they have produced largely disjunct bodies of knowledge. An integrative approach, combining phylogenetic, developmental, and functional biology, is needed to achieve a true fungal evolutionary developmental biology (evo-devo) synthesis for fungal fruiting bodies.

CRISPR-Cas9-mediated disruption of the HMG-CoA reductase genes of Mucor circinelloides and subcellular localization of the encoded enzymes.

Terpenoid compounds, such as sterols, carotenoids or the prenyl groups of various proteins are synthesized via the mevalonate pathway. A rate-limiting step of this pathway is the conversion of 3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid catalyzed by the HMG-CoA reductase. Activity of this enzyme may affect several biological processes, from the synthesis of terpenoid metabolites to the adaptation to various environmental conditions. In this study, the three HMG-CoA reductase genes (i.e. hmgR1, hmgR2 and hmgR3) of the ß-carotene producing filamentous fungus, Mucor circinelloides were disrupted individually and simultaneously by a recently developed in vitro plasmid-free CRISPR-Cas9 method. Examination of the mutants revealed that the function of hmgR2 and hmgR3 are partially overlapping and involved in the general terpenoid biosynthesis. Moreover, hmgR2 seemed to have a special role in the ergosterol biosynthesis. Disruption of all three genes affected the germination ability of the spores and the sensitivity to hydrogen peroxide. Disruption of the hmgR1 gene had no effect on the ergosterol production and the sensitivity to statins but caused a reduced growth at lower temperatures. By confocal fluorescence microscopy using strains expressing GFP-tagged HmgR proteins, all three HMG-CoA reductases were localized in the endoplasmic reticulum.

South Indian Isolates of the Fusarium solani Species Complex From Clinical and Environmental Samples: Identification, Antifungal Susceptibilities, and Virulence.

Members of the Fusarium solani species complex (FSSC) are the most frequently isolated fusaria from soil. Moreover, this complex solely affects more than 100 plant genera, and is also one of the major opportunistic human pathogenic filamentous fungi, being responsible for approximately two-third of fusariosis cases. Mycotic keratitis due to Fusarium species is among the leading causes of visual impairment and blindness in South India, but its management is still challenging due to the poor susceptibility of the isolates to conventional antifungal drugs. Aims of the present study were to isolate South Indian clinical and environmental FSSC strains and identify them to species level, to determine the actual trends in their susceptibilities to antifungal therapeutic drugs and to compare the virulence of clinical and environmental FSSC members. Based on the partial sequences of the translation elongation factor 1α gene, the majority of the isolates-both from keratomycosis and environment-were confirmed as F. falciforme, followed by F. keratoplasticum and F. solani sensu stricto. In vitro antifungal susceptibilities to commonly used azole, allylamine and polyene antifungals were determined by the CLSI M38-A2 broth microdilution method. The first generation triazoles, fluconazole and itraconazole proved to be ineffective against all isolates tested. This phenomenon has already been described before, as fusaria are intrinsically resistant to them. However, our results indicated that despite the intensive agricultural use of azole compounds, fusaria have not developed resistance against the imidazole class of antifungals. In order to compare the virulence of different FSSC species from clinical and environmental sources, a Drosophila melanogaster model was used. MyD88 mutant flies having impaired immune responses were highly susceptible to all the examined fusaria. In wild-type flies, one F. falciforme and two F. keratoplasticum strains also reduced the survival significantly. Pathogenicity seemed to be independent from the origin of the isolates.

Development of a plasmid free CRISPR-Cas9 system for the genetic modification of Mucor circinelloides.

Mucor circinelloides and other members of Mucorales are filamentous fungi, widely used as model organisms in basic and applied studies. Although genetic manipulation methods have been described for some Mucoral fungi, construction of stable integrative transformants by homologous recombination has remained a great challenge in these organisms. In the present study, a plasmid free CRISPR-Cas9 system was firstly developed for the genetic modification of a Mucoral fungus. The described method offers a rapid but robust tool to obtain mitotically stable mutants of M. circinelloides via targeted integration of the desired DNA. It does not require plasmid construction and its expression in the recipient organism. Instead, it involves the direct introduction of the guide RNA and the Cas9 enzyme and, in case of homology directed repair (HDR), the template DNA into the recipient strain. Efficiency of the method for non-homologous end joining (NHEJ) and HDR was tested by disrupting two different genes, i.e. carB encoding phytoene dehydrogenase and hmgR2 encoding 3-hydroxy-3-methylglutaryl-CoA reductase, of M. circinelloides. Both NHEJ and HDR resulted in stable gene disruption mutants. While NHEJ caused extensive deletions upstream from the protospacer adjacent motif, HDR assured the integration of the deletion cassette at the targeted site.

Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates ß-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of ß-carotene (i.e. zeaxanthin and ß-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of ß-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than ß-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and ß-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the ß-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.

Transcription of the three HMG-CoA reductase genes of Mucor circinelloides.

BACKGROUND: Precursors of sterols, carotenoids, the prenyl groups of several proteins and other terpenoid compounds are synthesised via the acetate-mevalonate pathway. One of the key enzyme of this pathway is the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which catalyses the conversion of HMG-CoA to mevalonate. HMG-CoA reductase therefore affects many biological processes, such as morphogenesis, synthesis of different metabolites or adaptation to environmental changes. In this study, transcription of the three HMG-CoA reductase genes (designated as hmgR1, hmgR2 and hmgR3) of the ß-carotene producing Mucor circinelloides has been analysed under various culturing conditions; effect of the elevation of their copy number on the carotenoid and ergosterol content as well as on the sensitivity to statins has also been examined. RESULTS: Transcripts of each gene were detected and their relative levels varied under the tested conditions. Transcripts of hmgR1 were detected only in the mycelium and its relative transcript level seems to be strongly controlled by the temperature and the oxygen level of the environment. Transcripts of hmgR2 and hmgR3 are already present in the germinating spores and the latter is also strongly regulated by oxygen. Overexpression of hmgR2 and hmgR3 by elevating their copy numbers increased the carotenoid content of the fungus and decreased their sensitivity to statins. CONCLUSIONS: The three HMG-CoA reductase genes of M. circinelloides displayed different relative transcript levels under the tested conditions suggesting differences in their regulation. They seem to be especially involved in the adaptation to the changing oxygen tension and osmotic conditions of the environment as well as to statin treatment. Overexpression of hmgR2 and hmgR3 may be used to improve the carotenoid content.

Canthaxanthin production with modified Mucor circinelloides strains.

Canthaxanthin is a natural diketo derivative of ß-carotene primarily used by the food and feed industries. Mucor circinelloides is a ß-carotene-accumulating zygomycete fungus and one of the model organisms to study the carotenoid biosynthesis in fungi. In this study, the ß-carotene ketolase gene (crtW) of the marine bacterium Paracoccus sp. N81106 fused with fungal promoter and terminator regions was integrated into the M. circinelloides genome to construct stable canthaxanthin-producing strains. Different transformation methods including polyethylene glycol-mediated transformation with linear DNA fragments, restriction enzyme-mediated integration and Agrobacterium tumefaciens-mediated transformation were tested to integrate the crtW gene into the Mucor genome. Mitotic stability, site of integration and copy number of the transferred genes were analysed in the transformants, and several stable strains containing the crtW gene in high copy number were isolated. Carotenoid composition of selected transformants and effect of culturing conditions, such as temperature, carbon sources and application of certain additives in the culturing media, on their carotenoid content were analysed. Canthaxanthin-producing transformants were able to survive at higher growth temperature than the untransformed strain, maybe due to the effect of canthaxanthin on the membrane fluidity and integrity. With the application of glucose, trehalose, dihydroxyacetone and L-aspartic acid as sole carbon sources in minimal medium, the crtW-expressing M. circinelloides strain, MS12+pCA8lf/1, produced more than 200 µg/g (dry mass) of canthaxanthin.

Integration of a bacterial ß-carotene ketolase gene into the Mucor circinelloides genome by the Agrobacterium tumefaciens-mediated transformation method.

Plasmids introduced in Mucor circinelloides (and most transformable Mucorales) tend to replicate autonomously, and hardly ever integrate in the genome. This is critical if we want to express exogenous genes, because plasmids are easily lost during vegetative growth, and the ratio of plasmid molecules/nuclei is invariably low. Linearized molecules of DNA have been used to get their genomic integration but the transformation efficiency drops extremely. We have developed and highly optimized an efficient Agrobacterium-mediated transformation system for M. circinelloides to facilitate the integration of transforming DNA in the genome of the recipient strain that could also be used for other Mucorales.

Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the zygomycete Mucor circinelloides.

The zygomycete Mucor circinelloides accumulates ß-carotene as the main carotenoid compound. In this study, the applicability of some early genes of the general isoprenoid pathway to improve the carotenoid production in this fungus was examined. The isopentenyl pyrophosphate isomerase gene (ipi) was cloned and used together with the genes encoding farnesyl pyrophosphate synthase (isoA) and geranylgeranyl pyrophosphate synthase (carG) in overexpression studies. Transformation experiments showed that the first bottleneck in the pathway, from the aspect of carotenoid production, is the step controlled by the carG gene, but overexpression of the ipi and isoA genes also contributes to the availability of the precursors. Transformations with these isoprenoid genes in combination with a bacterial ß-carotene ketolase gene yielded Mucor strains producing canthaxanthin and echinenone.

Cloning of the Rhizomucor miehei 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene and its heterologous expression in Mucor circinelloides.

In this study, the gene hmgR encoding the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) was cloned and characterized in the zygomycete fungus Rhizomucor miehei. The hmgR gene comprises a total of 3,585 bp including the coding sequence of a 1,058 amino acids length putative protein and five introns (137, 83, 59, 60 and 69 bp in length) dispersed in the whole coding region. Southern hybridization analysis revealed that the gene is present only in one copy in the R. miehei genome. The isolated Rhizomucor gene was expressed in the related fungus, Mucor circinelloides. Transformants harbouring the Rhizomucor hmgR gene in an autoreplicative plasmid proved to be more tolerant to statins (e.g. lovastatin, simvastatin, and fluvastatin), the competitive inhibitors of the HMG-CoA reductase, than the original M. circinelloides strain. At the same time, heterologous expression of the Rhizomucor hmgR did not affect the carotenoid production of M. circinelloides.

Agrobacterium tumefaciens -mediated transformation of the zygomycete fungus Backusella lamprospora.

The Agrobacterium -mediated transformation was adapted to Backusella lamprospora, a zygomycete fungus closely related to Mucor. The transforming plasmid contained the hygromycin B resistance (hph) and the green fluorescent protein (gfp) genes under the control of the regulator regions of the Mucor circinelloides gpd1 gene. The presence of the hph and gfp genes in the transformants was detected by PCR. The introduced genes could also be amplified directly from the spores of the transformants. The transformation efficiency was investigated by fluorescence microscopy of the transformed spores. A gradual decrease in the hygromycin B resistance was observed during several cultivation cycles: the growth of the transformants on the selection medium became slower, and the detection of the introduced gene became more difficult.