Fusarium graminearum is a plant pathogen infecting several important cereals, resulting in substantial yield losses and mycotoxin contamination of the grain. Triazole fungicides are used to control diseases caused by this fungus on a worldwide scale. Our previous microarray study indicated that 15 ABC transporter genes were transcriptionally upregulated in response to tebuconazole treatment. Here, we deleted four ABC transporter genes in two genetic backgrounds of F. graminearum representing the DON (deoxynivalenol) and the NIV (nivalenol) trichothecene chemotypes. Deletion of FgABC3 and FgABC4 belonging to group I of ABC-G and to group V of ABC-C subfamilies of ABC transporters, respectively, considerably increased the sensitivity to the class I sterol biosynthesis inhibitors triazoles and fenarimol. Such effects were specific since they did not occur with any other fungicide class tested. Assessing the contribution of the four ABC transporters to virulence of F. graminearum revealed that, irrespective of their chemotypes, deletion mutants of FgABC1 (ABC-C subfamily group V) and FgABC3 were impeded in virulence on wheat, barley and maize. Phylogenetic context and analyses of mycotoxin production suggests that FgABC3 may encode a transporter protecting the fungus from host-derived antifungal molecules. In contrast, FgABC1 may encode a transporter responsible for the secretion of fungal secondary metabolites alleviating defence of the host. Our results show that ABC transporters play important and diverse roles in both fungicide resistance and pathogenesis of F. graminearum.
References
[1]
Becher R, Miedaner T, Wirsel SGR (2013) Biology, diversity and management of FHB-causing Fusarium species in small-grain cereals. In: Kempken F, editor. The Mycota XI, Agricultural Applications. 2 ed. Berlin Heidelberg: Springer-Verlag. pp. in press, DOI 10.1007/1978–1003–1642–36821–36829_36828？.
[2]
Stepien L, Chelkowski J (2010) Fusarium head blight of wheat: pathogenic species and their mycotoxins. World Mycotoxin J 3: 107–119.
[3]
Willyerd KT, Li C, Madden LV, Bradley CA, Bergstrom GC, et al. (2012) Efficacy and stability of integrating fungicide and cultivar resistance to manage Fusarium head blight and deoxynivalenol in wheat. Plant Disease 96: 957–967.
[4]
Blandino M, Haidukowski M, Pascale M, Plizzari L, Scudellari D, et al. (2012) Integrated strategies for the control of Fusarium head blight and deoxynivalenol contamination in winter wheat. Field Crop Res 133: 139–149.
[5]
Paul PA, McMullen MP, Hershman DE, Madden LV (2010) Meta-analysis of the effects of triazole-based fungicides on wheat yield and test weight as influenced by Fusarium head blight intensity. Phytopathology 100: 160–171.
[6]
Klix MB, Verreet JA, Beyer M (2007) Comparison of the declining triazole sensitivity of Gibberella zeae and increased sensitivity achieved by advances in triazole fungicide development. Crop Prot 26: 683–690.
[7]
Yin Y, Liu X, Li B, Ma Z (2009) Characterization of sterol demethylation inhibitor-resistant isolates of Fusarium asiaticum and F. graminearum collected from wheat in China. Phytopathology 99: 487–497.
[8]
Becher R, Hettwer U, Karlovsky P, Deising HB, Wirsel SGR (2010) Adaptation of Fusarium graminearum to tebuconazole yielded descendants diverging for levels of fitness, fungicide resistance, virulence, and mycotoxin production. Phytopathology 100: 444–453.
[9]
Becher R, Weihmann F, Deising HB, Wirsel SGR (2011) Development of a novel multiplex DNA microarray for Fusarium graminearum and analysis of azole fungicide responses. BMC Genomics 12: 52.
[10]
Liu X, Yu F, Schnabel G, Wu J, Wang Z, et al. (2011) Paralogous Cyp51 genes in Fusarium graminearum mediate differential sensitivity to sterol demethylation inhibitors. Fungal Genet Biol 48: 113–123.
[11]
Fan J, Urban M, Parker JE, Brewer HC, Kelly SL, et al. (2013) Characterization of the sterol 14alpha-demethylases of Fusarium graminearum identifies a novel genus-specific CYP51 function. New Phytol 198: 821–835.
[12]
Cannon RD, Lamping E, Holmes AR, Niimi K, Baret PV, et al. (2009) Efflux-mediated antifungal drug resistance. Clin Microbiol Rev 22: 291–321.
[13]
Becher R, Wirsel SG (2012) Fungal cytochrome P450 sterol 14alpha-demethylase (CYP51) and azole resistance in plant and human pathogens. Appl Microbiol Biotechnol 95: 825–840.
[14]
De Waard MA, Andrade AC, Hayashi K, Schoonbeek HJ, Stergiopoulos I, et al. (2006) Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence. Pest Manag Sci 62: 195–207.
[15]
Bai GH, Shaner G (1996) Variation in Fusarium graminearum and cultivar resistance to wheat scab. Plant Dis 80: 975–979.
[16]
Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Dominguez Y, et al. (2004) Double-joint PCR: A PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41: 973–981.
[17]
Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CA (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56: 117–124.
[18]
Malonek S, Rojas MC, Hedden P, Gaskin P, Hopkins P, et al. (2004) The NADPH-cytochrome P450 reductase gene from Gibberella fujikuroi is essential for gibberellin biosynthesis. J Biol Chem 279: 25075–25084.
[19]
Namiki F, Matsunaga M, Okuda M, Inoue I, Nishi K, et al. (2001) Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxysporum f. sp. melonis. Mol Plant Microbe Interact 14: 580–584.
[20]
Nutz S, D？ll K, Karlovsky P (2011) Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: Application to qPCR assays for Fusarium verticillioides and F. proliferatum. Anal Bioanal Chem 401: 717–726.
[21]
Adejumo TO, Hettwer U, Karlovsky P (2007) Occurrence of Fusarium species and trichothecenes in Nigerian maize. Int J Food Microbiol 116: 350–357.
[22]
Adejumo TO, Hettwer U, Karlovsky P (2007) Survey of maize from south-western Nigeria for zearalenone, alpha- and beta-zearalenols, fumonisin B-1 and enniatins produced by Fusarium species. Food Addit Contam 24: 993–1000.
[23]
Kovalchuk A, Driessen AJ (2010) Phylogenetic analysis of fungal ABC transporters. BMC Genomics 11: 177.
[24]
Fincham JR (1989) Transformation in fungi. Microbiol Rev 53: 148–170.
[25]
Kang ZS, Huang LL, Krieg U, Mauler-Machnik A, Buchenauer H (2001) Effects of tebuconazole on morphology, structure, cell wall components and trichothecene production of Fusarium culmorum in vitro. Pest Manag Sci 57: 491–500.
[26]
Maier FJ, Miedaner T, Hadeler B, Felk A, Salomon S, et al. (2006) Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulence. Mol Plant Pathol 7: 449–461.
[27]
Prasad R, Goffeau A (2012) Yeast ATP-binding cassette transporters conferring multidrug resistance. Annu Rev Microbiol 66: 39–63.
[28]
Nakaune R, Adachi K, Nawata O, Tomiyama M, Akutsu K, et al. (1998) A novel ATP-binding cassette transporter involved in multidrug resistance in the phytopathogenic fungus Penicillium digitatum. Appl Environ Microbiol 64: 3983–3988.
[29]
Hayashi K, Schoonbeek HJ, De Waard MA (2002) Expression of the ABC transporter BcatrD from Botrytis cinerea reduces sensitivity to sterol demethylation inhibitor fungicides. Pestic Biochem Phys 73: 110–121.
[30]
Hulvey J, Popko JT, Sang H, Berg A, Jung G (2012) Overexpression of ShCYP51B and ShatrD in Sclerotinia homoeocarpa isolates exhibiting practical field resistance to a demethylation inhibitor fungicide. Appl Environ Microb 78: 6674–6682.
[31]
Zwiers LH, Stergiopoulos I, Gielkens MM, Goodall SD, De Waard MA (2003) ABC transporters of the wheat pathogen Mycosphaerella graminicola function as protectants against biotic and xenobiotic toxic compounds. Mol Genet Genomics 269: 499–507.
[32]
Lee S, Son H, Lee J, Lee YR, Lee YW (2011) A putative ABC transporter gene, ZRA1, is required for zearalenone production in Gibberella zeae. Curr Genet 57: 343–351.
[33]
Kim YT, Lee YR, Jin JM, Han KH, Kim H, et al. (2005) Two different polyketide synthase genes are required for synthesis of zearalenone in Gibberella zeae. Mol Microbiol 58: 1102–1113.
[34]
Gaffoor I, Brown DW, Plattner R, Proctor RH, Qi WH, et al. (2005) Functional analysis of the polyketide synthase genes in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum). Eukaryot Cell 4: 1926–1933.
[35]
Stergiopoulos I, Zwiers LH, De Waard MA (2003) The ABC transporter MgAtr4 is a virulence factor of Mycosphaerella graminicola that affects colonization of substomatal cavities in wheat leaves. Mol Plant Microbe Interact 16: 689–698.
[36]
Schoonbeek H, Del Sorbo G, De Waard MA (2001) The ABC transporter BcatrB affects the sensitivity of Botrytis cinerea to the phytoalexin resveratrol and the fungicide fenpiclonil. Mol Plant Microbe Interact 14: 562–571.
[37]
Stefanato FL, Abou-Mansour E, Buchala A, Kretschmer M, Mosbach A, et al. (2009) The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana. Plant J 58: 499–510.
[38]
Urban M, Bhargava T, Hamer JE (1999) An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease. EMBO J 18: 512–521.
[39]
Coleman JJ, White GJ, Rodriguez-Carres M, VanEtten HD (2011) An ABC transporter and a cytochrome P450 of Nectria haematococca MPVI are virulence factors on pea and are the major tolerance mechanisms to the phytoalexin pisatin. Mol Plant Microbe Interact 24: 368–376.
[40]
Skov J, Lemmens M, Giese H (2004) Role of a Fusarium culmorum ABC transporter (FcABC1) during infection of wheat and barley. Physiol Mol Plant Pathol 64: 245–254.
[41]
Fleissner A, Sopalla C, Weltring KM (2002) An ATP-binding cassette multidrug-resistance transporter is necessary for tolerance of Gibberella pulicaris to phytoalexins and virulence on potato tubers. Mol Plant Microbe Interact 15: 102–108.
[42]
Lysoe E, Seong KY, Kistler HC (2011) The transcriptome of Fusarium graminearum during the infection of wheat. Mol Plant Microbe Interact 24: 995–1000.
[43]
Kim Y, Park SY, Kim D, Choi J, Lee YH, et al. (2013) Genome-scale analysis of ABC transporter genes and characterization of the ABCC type transporter genes in Magnaporthe oryzae. Genomics 101: 354–361.
[44]
Zhang XW, Jia LJ, Zhang Y, Jiang G, Li X, et al. (2012) In planta stage-specific fungal gene profiling elucidates the molecular strategies of Fusarium graminearum growing inside wheat coleoptiles. Plant Cell 24: 5159–5176.
[45]
Jansen C, von Wettstein D, Sch？fer W, Kogel KH, Felk A, et al. (2005) Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum. Proc Natl Acad Sci USA 102: 16892–16897.
