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Identification of a copper-transporting ATPase involved in biosynthesis of A. flavus conidial pigment

  • Applied genetics and molecular biotechnology
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Abstract

Conidia are asexual spores and play a crucial role in fungal dissemination. Conidial pigmentation is important for tolerance against UV radiation and contributes to survival of fungi. The molecular basis of conidial pigmentation has been studied in several fungal species. In spite of sharing the initial common step of polyketide formation, other steps for pigment biosynthesis appear to be species-dependent. In this study, we isolated an Aspergillus flavus spontaneous mutant that produced yellow conidia. The underlying genetic defect, a three-nucleotide in-frame deletion in the gene, AFLA_051390, that encodes a copper-transporting ATPase, was identified by a comparative genomics approach. This genetic association was confirmed by disruption of the wild-type gene. When yellow mutants were grown on medium supplemented with copper ions or chloride ions, green conidial color was partially and nearly completely restored, respectively. Further disruption of AFLA_045660, an orthologue of Aspergillus nidulans yA (yellow pigment) that encodes a multicopper oxidase, in wild type and a derived strain producing dark green conidia showed that it yielded mutants that produced gold conidia. The results placed formation of the gold pigment after that of the yellow pigment and before that of the dark green pigment. Using reported inhibitors of DHN-melanin (tricyclazole and phthalide) and DOPA-melanin (tropolone and kojic acid) pathways on a set of conidial color mutants, we investigated the involvement of melanin biosynthesis in A. flavus conidial pigment formation. Results imply that both pathways have no bearing on conidial pigment biosynthesis of A. flavus.

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References

  • Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bell AA, Wheeler MH (1986) Biosynthesis and functions of fungal melanins. Annu Rev Phytopathol 24:411–451

    Article  CAS  Google Scholar 

  • Bushnell B (2016) "BBMap short read aligner." University of California, Berkeley, California. URL http://sourceforge net/projects/bbmap

  • Cary JW, Harris-Coward PY, Ehrlich KC, Di Mavungu JD, Malysheva SV, De Saeger S, Dowd PF, Shantappa S, Martens SL, Calvo AM (2014) Functional characterization of a veA-dependent polyketide synthase gene in Aspergillus flavus necessary for the synthesis of asparasone, a sclerotium-specific pigment. Fungal Genet Biol 64:25–35

    Article  CAS  PubMed  Google Scholar 

  • Caten CE (1979) Genetic determination of conidial colour in Aspergillus heterocaryoticus and relationship of this species to Aspergillus amstelodami. Trans Br Mycol Soc 73(1):65–74

    Article  Google Scholar 

  • Chang P-K, Horn BW, Dorner JW (2005) Sequence breakpoints in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates. Fungal Genet Biol 42:914–923

    Article  CAS  PubMed  Google Scholar 

  • Chang P-K, Scharfenstein LL, Wei Q, Bhatnagar D (2010) Development and refinement of a high-efficiency gene-targeting system for Aspergillus flavus. J Microbiol Methods 81:240–246

    Article  CAS  PubMed  Google Scholar 

  • Chrysayi Tokousbalides M, Sisler HD (1979) Site of inhibition by tricyclazole in the melanin biosynthetic pathway of Verticillium dahliae. Pestic Biochem Physiol 11:64–73

    Article  Google Scholar 

  • Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6:80–92

    Article  CAS  Google Scholar 

  • Clutterbuck AJ (1972) Absence of laccase from yellow-spored mutants of Aspergillus nidulans. J Gen Microbiol 70:423–435

    Article  CAS  PubMed  Google Scholar 

  • Clutterbuck AJ (1990) The genetics of conidiophore pigmentation in Aspergillus nidulans. J Gen Microbiol 136:1731–1738

    Article  CAS  PubMed  Google Scholar 

  • Cordero RJB, Casadevall A (2017) Functions of fungal melanin beyond virulence. Fungal Biol Rev 31:99–112

    Article  PubMed  PubMed Central  Google Scholar 

  • Fujii I, Yasuoka Y, Tsai HF, Chang YC, Kwon-Chung KJ, Ebizuka Y (2004) Hydrolytic polyketide shortening by Ayg1p, a novel enzyme involved in fungal melanin biosynthesis. J Biol Chem 279:44613–44620

    Article  CAS  PubMed  Google Scholar 

  • Garrison E, Gabor M (2012) "Haplotype-based variant detection from short-read sequencing." arXiv preprint arXiv:1207.3907

  • Gaxiola RA, Yuan DS, Klausner RD, Fink GR (1998) The yeast CLC chloride channel functions in cation homeostasis. Proc Natl Acad Sci U S A 95:4046–4050

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Geib E, Brock M (2017) Comment on: “Melanisation of Aspergillus terreus-Is Butyrolactone I Involved in the Regulation of Both DOPA and DHN Types of Pigments in Submerged Culture? Microorganisms 2017, 5, 22”. Microorganisms 5:34

  • Geib E, Gressler M, Viediernikova I, Hillmann F, Jacobsen ID, Nietzsche S, Hertweck C, Brock M (2016) A non-canonical melanin biosynthesis pathway protects Aspergillus terreus conidia from environmental stress. Cell Chem Biol 23:587–597

    Article  CAS  PubMed  Google Scholar 

  • Griffith GW, Easton GL, Detheridge A, Roderick K, Edwards A, Worgan HJ, Nicholson J, Perkins WT (2007) Copper deficiency in potato dextrose agar causes reduced pigmentation in cultures of various fungi. FEMS Microbiol Lett 276:165–171

    Article  CAS  PubMed  Google Scholar 

  • Ha Huy K, Luckner M (1979) Structure and function of the conidiospore pigments of Penicillium cyclopium. Z Allg Mikrobiol 19:117–122

    Article  Google Scholar 

  • Horng JS, Chang PK, Pestka JJ, Linz JE (1990) Development of a homologous transformation system for Aspergillus parasiticus with the gene encoding nitrate reductase. Mol Gen Genet 224:294–296

    Article  CAS  PubMed  Google Scholar 

  • Hua SS, McAlpin CE, Chang P-K, Sarreal SB (2012) Characterization of aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates from pistachio. Mycotoxin Res 28:67–75

    Article  CAS  PubMed  Google Scholar 

  • Jentsch TJ, Friedrich T, Schriever A, Yamada H (1999) The CLC chloride channel family. Pflugers Arch 437:783–795

    Article  CAS  PubMed  Google Scholar 

  • Jorgensen TR, Park J, Arentshorst M, van Welzen AM, Lamers G, Vankuyk PA, Damveld RA, van den Hondel CA, Nielsen KF, Frisvad JC, Ram AF (2011) The molecular and genetic basis of conidial pigmentation in Aspergillus niger. Fungal Genet Biol 48:544–553

    Article  CAS  PubMed  Google Scholar 

  • Kubodera T, Yamashita N, Nishimura A (2000) Pyrithiamine resistance gene (ptrA) of Aspergillus oryzae: cloning, characterization and application as a dominant selectable marker for transformation. Biosci Biotechnol Biochem 64:1416–1421

    Article  CAS  PubMed  Google Scholar 

  • Kurtz MB, Champe SP (1981) Dominant spore color mutants of Aspergillus nidulans defective in germination and sexual development. J Bacteriol 148:629–638

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lawton TJ, Sayavedra-Soto LA, Arp DJ, Rosenzweig AC (2009) Crystal structure of a two-domain multicopper oxidase: implications for the evolution of multicopper blue proteins. J Biol Chem 284:10174–10180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li H (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv preprint arXiv 303.3997

  • Motoyama T, Yamaguchi I (2003) Fungicides, melanin biosynthesis inhibitors. In: Plimmer JR, Ragsdale NN, Gammon D (eds) Encyclopedia of agrochemicals, vol 3. Wiley, Hoboken

    Google Scholar 

  • Murakami H, Owaki K, Takase S (1966) An aflatoxin strain ATCC 15517. J Gen Appl Microbiol 12:195–206

    Article  CAS  Google Scholar 

  • Oddon DM, Diatloff E, Roberts SK (2007) A CLC chloride channel plays an essential role in copper homeostasis in Aspergillus nidulans at increased extracellular copper concentrations. Biochim Biophys Acta 1768:2466–2477

    Article  CAS  PubMed  Google Scholar 

  • O'Hara EB, Timberlake WE (1989) Molecular characterization of the Aspergillus nidulans yA locus. Genetics 121:249–254

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pal AK, Gajjar DU, Vasavada AR (2014) DOPA and DHN pathway orchestrate melanin synthesis in Aspergillus species. Med Mycol 52:10–18

    CAS  PubMed  Google Scholar 

  • Papa KE (1973) The parasexual cycle in Aspergillus flavus. Mycologia 65:1201–1205

    Article  CAS  PubMed  Google Scholar 

  • Raper KB, Thom CA (1968) A manual of the penicilla. Williams & Wilkins, Baltimore, USA

    Google Scholar 

  • Saitoh Y, Izumitsu K, Atsushi Morita A, Kiminori Shimizu K, Chihiro Tanaka C (2012) Cloning of sal1, a scytalone dehydratase gene involved in melanin biosynthesis in Cochliobolus heterostrophus. Mycoscience 53:330–334

    Article  CAS  Google Scholar 

  • Skory CD, Horng JS, Pestka JJ, Linz JE (1990) Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis. Appl Environ Microbiol 56:3315–3320

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sonnhammer EL, von Heijne G, Krogh A (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 6:175–182

    CAS  PubMed  Google Scholar 

  • Szewczyk E, Nayak T, Oakley CE, Edgerton H, Xiong Y, Taheri-Talesh N, Osmani SA, Oakley BR (2006) Fusion PCR and gene targeting in Aspergillus nidulans. Nat Protoc 1:3111–3120

    Article  CAS  PubMed  Google Scholar 

  • Takagi Y (1957) Studies on the conidial colour change in Aspergillus fungi. Part II. The role of copper and halogen ions in the formation of green conidia. J Gen Appl Microbiol 3:269–275

    Article  CAS  Google Scholar 

  • Takagi Y, Sakaguchi K (1957) Studies on the conidial colour change in Aspergillus fungi. Part I. Physiological modification of a genetic block between yellow and green color development. J Gen Appl Microbiol 3:125–136

    Article  CAS  Google Scholar 

  • Teertstra WR, Tegelaar M, Dijksterhuis J, Golovina EA, Ohm RA, Wosten HAB (2017) Maturation of conidia on conidiophores of Aspergillus niger. Fungal Genet Biol 98:61–70

    Article  PubMed  Google Scholar 

  • Tsai HF, Washburn RG, Chang YC, Kwon-Chung KJ (1997) Aspergillus fumigatus arp1 modulates conidial pigmentation and complement deposition. Mol Microbiol 26:175–183

    Article  CAS  PubMed  Google Scholar 

  • Tsai HF, Wheeler MH, Chang YC, Kwon-Chung KJ (1999) A developmentally regulated gene cluster involved in conidial pigment biosynthesis in Aspergillus fumigatus. J Bacteriol 181:6469–6477

    CAS  PubMed  PubMed Central  Google Scholar 

  • Varga J, Frisvad JC, Samson RA (2011) Two new aflatoxin producing species, and an overview of Aspergillus section Flavi. Stud Mycol 69:57–80

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Watanabe A, Fujii I, Tsai H, Chang YC, Kwon-Chung KJ, Ebizuka Y (2000) Aspergillus fumigatus alb1 encodes naphthopyrone synthase when expressed in Aspergillus oryzae. FEMS Microbiol Lett 192:39–44

    Article  CAS  PubMed  Google Scholar 

  • Wheeler MH, Klich MA (1995) The effects of tricyclazole, pyroquilon, phthalide, and related fungicides on the production of conidial wall pigments by Penicillium and Aspergillus species. Pestic Biochem Physiol 52:125–136

    Article  CAS  Google Scholar 

  • Yu X, Huo L, Liu H, Chen L, Wang Y, Zhu X (2015) Melanin is required for the formation of the multi-cellular conidia in the endophytic fungus Pestalotiopsis microspora. Microbiol Res 179:1–11

    Article  CAS  PubMed  Google Scholar 

  • Zhu C, Jiang N, Xiao D, Pan J, Zhu X (2010) Chloride channel-dependent copper acquisition of laccase in the basidiomycetous fungus Cryptococcus neoformans. Sci China Life Sci 53:125–130

    Article  CAS  PubMed  Google Scholar 

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  1. Southern Regional Research Center, Agricultural Research Service, U. S. Department of Agriculture, 1100 Robert E. Lee Boulevard, New Orleans, LA, 70124, USA

    Perng-Kuang Chang, Leslie L. Scharfenstein, Brian Mack, Qijian Wei, Matthew Gilbert, Matthew Lebar & Jeffrey W. Cary

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  1. Perng-Kuang Chang
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  2. Leslie L. Scharfenstein
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Correspondence to Perng-Kuang Chang.

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Chang, PK., Scharfenstein, L.L., Mack, B. et al. Identification of a copper-transporting ATPase involved in biosynthesis of A. flavus conidial pigment. Appl Microbiol Biotechnol 103, 4889–4897 (2019). https://doi.org/10.1007/s00253-019-09820-0

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