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The Flba-Regulated Predicted Transcription Factor Fum21 of Aspergillus Niger Is Involved in Fumonisin Production

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The Flba-Regulated Predicted Transcription Factor Fum21 of Aspergillus Niger Is Involved in Fumonisin Production Downloaded from orbit.dtu.dk on: Oct 03, 2021 The FlbA-regulated predicted transcription factor Fum21 of Aspergillus niger is involved in fumonisin production Aerts, David; Hauer, Esther E.; Ohm, Robin A.; Arentshorst, Mark; Teertstra, Wieke R.; Phippen, Christopher; Ram, Arthur F. J.; Frisvad, Jens Christian; Wösten, Han A. B. Published in: Antonie Van Leeuwenhoek Link to article, DOI: 10.1007/s10482-017-0952-1 Publication date: 2018 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Aerts, D., Hauer, E. E., Ohm, R. A., Arentshorst, M., Teertstra, W. R., Phippen, C., Ram, A. F. J., Frisvad, J. C., & Wösten, H. A. B. (2018). The FlbA-regulated predicted transcription factor Fum21 of Aspergillus niger is involved in fumonisin production. Antonie Van Leeuwenhoek, 111(3), 311-322. https://doi.org/10.1007/s10482- 017-0952-1 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Antonie van Leeuwenhoek (2018) 111:311–322 https://doi.org/10.1007/s10482-017-0952-1 ORIGINAL PAPER The FlbA-regulated predicted transcription factor Fum21 of Aspergillus niger is involved in fumonisin production David Aerts . Esther E. Hauer . Robin A. Ohm . Mark Arentshorst . Wieke R. Teertstra . Christopher Phippen . Arthur F. J. Ram . Jens C. Frisvad . Han A. B. Wo¨sten Received: 1 June 2017 / Accepted: 25 September 2017 / Published online: 30 September 2017 Ó The Author(s) 2017. This article is an open access publication Abstract Aspergillus niger secretes proteins Whole genome expression analysis revealed that 63 throughout the colony except for the zone that forms and 11 genes were down- and up-regulated in Dfum21, asexual spores called conidia. Inactivation of flbA that respectively, when compared to the wild-type strain. encodes a regulator of G-protein signaling results in Notably, 24 genes predicted to be involved in colonies that are unable to reproduce asexually and secondary metabolism were down-regulated in that secrete proteins throughout the mycelium. In Dfum21, including 10 out of 12 genes of the fumonisin addition, the DflbA strain shows cell lysis and has cluster. This was accompanied by absence of fumon- thinner cell walls. Expression analysis showed that 38 isin production in the deletion strain and a 25% predicted transcription factor genes are differentially reduction in production of pyranonigrin A. Together, expressed in strain DflbA. Here, the most down- these results link FlbA-mediated sporulation-inhibited regulated predicted transcription factor gene, called secretion with mycotoxin production. fum21, was inactivated. Growth, conidiation, and protein secretion were not affected in strain Dfum21. Keywords Asexual development Á Aspergillus Á Fumonisin Á Fungus Á Mycotoxin Á Secondary metabolism Á Protein secretion Electronic supplementary material The online version of this article (doi:10.1007/s10482-017-0952-1) contains supple- mentary material, which is available to authorized users. D. Aerts Á E. E. Hauer Á R. A. Ohm Á M. Arentshorst Á A. F. J. Ram W. R. Teertstra Á H. A. B. Wo¨sten (&) Department of Molecular Microbiology and Microbiology, Department of Biology, Utrecht Biotechnology, Institute of Biology Leiden, Leiden University, Padualaan 8, 3584 CH Utrecht, The University, Sylviusweg 72, 2333 BE Leiden, The Netherlands Netherlands e-mail: [email protected] e-mail: [email protected] D. Aerts A. F. J. Ram e-mail: [email protected] e-mail: [email protected] E. E. Hauer C. Phippen Á J. C. Frisvad e-mail: [email protected] Department of Systems Biology, Technical University of R. A. Ohm Denmark, 2800 Lyngby, Denmark e-mail: [email protected] e-mail: [email protected] W. R. Teertstra J. C. Frisvad e-mail: [email protected] e-mail: [email protected] 123 312 Antonie van Leeuwenhoek (2018) 111:311–322 Introduction lysis. The DflbA strain therefore has a pleiotropic phenotype. Inactivation of flbA is accompanied by The genus Aspergillus consists of more than 300 differential expression of 38 transcription factor species (Samson et al. 2014) that are among the most genes, of which 18 are down-regulated and 20 are abundant fungi on the globe. Aspergilli degrade plant up-regulated (Krijgsheld and Wo¨sten 2013). These waste and as such play a role in carbon cycling in downstream regulatory genes may impact one or more nature. Moreover, the genus includes opportunistic of the processes affected by FlbA. Here, the role of the pathogens of plants, animals, and humans (Krijgsheld most down-regulated predicted transcription factor et al. 2013a). Enzymes secreted by Aspergillus play an gene, fum21, was studied. It is shown that fum21 important role in degradation of organic material and regulates production of the mycotoxins fumonisins. pathogenicity. The property to secrete high levels and Thus, sporulation-inhibited protein secretion is linked a diversity of enzymes make aspergilli such as to production of secondary metabolites via FlbA. Aspergillus niger important cell factories for the production of proteins and metabolites (Meyer et al. 2011;Wo¨sten et al. 2013). Materials and methods Aspergilli form a mycelium consisting of a network of hyphae that grow at their apex and that branch Strains and culture conditions subapically. The mycelium grows initially vegetative but at a certain moment asexual development is A. niger strain MA234.1 (transient ku70::amdS) (Park initiated (Krijgsheld et al. 2013a). The resulting et al. 2016) and its derivatives were grown at 30 °C conidia are the only spore type that are produced by using minimal medium (MM) (de Vries et al. 2004) A. niger. Conidiation starts with the formation of thick containing 25 mM xylose as a carbon source and aerial hyphae called stalks. When a stalk has reached a either (MM-XA) or not (MM-X) containing 1.5% certain height, its tip swells to form a vesicle. In many agar. Alternatively, strains were grown on Czapek aspergilli, this structure buds resulting in a layer of Yeast Auto-lysate (CYA) agar (Frisvad and Samson metulae. The metulae in turn form a layer of phialides, 2004), Yeast Extract Sucrose (YES) agar (Frisvad and from which chains of conidia develop. These spores Samson 2004), or transformation medium (TM; MM are heterogeneous in composition, water dispersal containing 0.5% yeast extract, 0.2% casaminoacids, efficiency, and germination rate (Teertstra et al. 2017) and 25 mM glucose, pH 6)(Kusters-van Someren et al. and give rise to new mycelia. 1991). The zone forming the asexual conidia within an A. Conidia were harvested from 3-day-old MM-XA niger colony does not secrete proteins, while non- cultures. Liquid cultures inoculated with 5 9 108 sporulating zones do release proteins into their envi- spores were pre-grown for 16 h at 200 rpm in 300 ml ronment (Krijgsheld et al. 2013b). Preventing conidi- TM in 500 ml Erlenmeyer flasks. After 16 h, 10 g wet ation of the colony by covering it with a polycarbonate weight mycelium was harvested, washed with 0.9% membrane does not impact the spatial secretion NaCl, and transferred to a 1 l Erlenmeyer flask pattern indicating that the capacity to sporulate but containing 150 ml MM-X. The culture was shaken at not the conidiation process itself inhibits secretion. 250 rpm and 30 °C for 4 h (RNA sequencing) or 24 h FlbA was shown to impact spatial secretion in the (SDS-PAGE). colony (Krijgsheld et al. 2013b). This protein regu- For growth on agar media, strains were inoculated lates the Ga subunit FadA by activating its GTPase directly on the medium or grown in a layer of 1.25% activity. By doing so, it promotes asexual develop- agarose in between two perforated polycarbonate ment and inhibits vegetative growth and autolysis membranes (pores of 0.1 lm, diameter 76 mm; Pro- (Wieser et al. 1994; Yu et al. 1996). Conidiation is filtra, Almere, The Netherlands) (Wo¨sten et al. 1991). abolished in the DflbA strain of A. niger and, as a These sandwiched cultures were inoculated in the consequence, protein secretion takes place throughout center of the agarose layer by placing a 2 ll drop the colony (Krijgsheld et al. 2013b). Moreover, the containing 104 conidia. The upper polycarbonate cell wall of DflbA is thinner and intracellular proteins membrane was placed on top of the agarose layer can be found in the culture medium, indicative of cell 24 h after inoculation to prevent spreading of conidia. 123 Antonie van Leeuwenhoek (2018) 111:311–322 313 Inactivation and complementation constructs contains the amdS gene and 30 regulatory sequences of of fum21 Aspergillus nidulans under control of the gpdA promoter. The amdS expression cassette (Meyer For the construction of the fum21 deletion strain, 50 et al. 2007) was PCR amplified with primer pair and 30 flanks of the gene were amplified from genomic 13/14 (Supplemental Table 1). Selection was done on DNA by PCR using primer pairs 1/2 and 3/4, MM containing 0.95 M sucrose, 15 mM CsCl, and respectively (Supplemental Table 1).
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