Current Genetics

, Volume 45, Issue 4, pp 214–224 | Cite as

Analysis of a nonribosomal peptide synthetase gene from Alternaria brassicae and flanking genomic sequences

  • Thomas Guillemette
  • Adnane Sellam
  • Philippe Simoneau
Research Article


Very little information is currently available concerning the pathogenic determinants produced by Alternaria brassicae, the causal agent of the blackspot disease of crucifers. We screened a genomic library of this fungus and identified a nonribosomal peptide synthetase (NRPS) gene named AbrePsy1. The complete coding sequence is 22 kbp long and encodes a large protein (792 kDa) showing typical NRPS modular organization. Structural analysis of AbrePsy1 revealed four complete elongation modules, two of which have epimerization domains. In the vicinity of AbrePsy1, a second gene (named AbreAtr1), which encodes an ATP-binding cassette transporter was identified. Increased expression of AbrePsy1 and AbreAtr1 was observed during host-plant infection. However, while physically linked, these two genes are probably not functionally clustered, as their expression patterns differed.


Alternaria brassicae Seed-borne pathogen Pathogenic determinant ABC transporter Nonribosomal peptide synthetase Siderophore 


  1. Andrade AC, Del Sorbo G, Van Nistelrooy GM, De Waard MA (2000) The ABC transporter AtrB from Aspergillus nidulans mediates resistance to all major classes of fungicides and some natural toxic compounds. Microbiology 146:1987–1997PubMedGoogle Scholar
  2. Andrianapoulos A, Timberlake WE (1994) The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development. Mol Cell Biol 14:2503–2515PubMedGoogle Scholar
  3. Bailey AM, Kershaw MJ, Hunt BA, Paterson IC, Charnley AK, Reynolds SE, Clarkson JM (1996) Cloning and sequence analysis of an intron-containing domain from a peptide-synthetase-encoding gene of the entomopathogenic fungus Metarhizium anisopliae. Gene 173:195–197CrossRefPubMedGoogle Scholar
  4. Bains PS, Tewari JP (1987) Purification, chemical characterization and host-specificity of the toxin produced by Alternaria brassicae. Physiol Mol Plant Pathol 30:259–271Google Scholar
  5. Buchwaldt I, Green H (1992) Phytotoxicity of destruxin B and its possible role in the pathogenicity of Alternaria brassicae. Plant Pathol 41:55–63Google Scholar
  6. Challis GL, Ravel J (2000) Coelichelin, a new peptide siderophore encoded by the Streptomyces coelicolor genome: structure prediction from the sequence of its non-ribosomal peptide synthetase. FEMS Microbiol Lett 187:111–114CrossRefPubMedGoogle Scholar
  7. Challis GL, Ravel J, Townsend CA (2000) Predictive, structured-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. Chem Biol 7:211–224CrossRefPubMedGoogle Scholar
  8. Chiu HT, Hubbard BK, Shah AN, Eide J, Fredenburg RA, Walsh CT, Khosla C (2001) Molecular cloning and sequence analysis of the compelstatin biosynthetic gene cluster. Proc Natl Acad Sci USA 98:8548–8553CrossRefPubMedGoogle Scholar
  9. Conti E, Stachelhaus T, Mahariel MA, Brick P (1997) Structural basis for the activation of phenylalanine in the nonribosomal biosynthesis of gramicidin S. EMBO J 16:4174–4183PubMedGoogle Scholar
  10. Davies KA, De Lorono I, Foster SJ, Li D, Johnstone K, Ashby AM (2000) Evidence for a role of cutinase in pathogenicity of Pyrenopeziza brassicae on brassicas. Physiol Mol Plant Pathol 57:63–75CrossRefGoogle Scholar
  11. De Waard MA (1997) Significance of ABC transporters in fungicide sensitivity and resistance. Pest Sci 51:271–275CrossRefGoogle Scholar
  12. Dutton JR, John S, Miller BL (1997) StuAp is a sequence-specific transcription factor that regulates developmental complexity in Aspergillus nidulans. EMBO J 16:5710–5721PubMedGoogle Scholar
  13. Fu YH, Marzluf GA (1990) ni-t2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein. Proc Natl Acad Sci USA 87:5331–5335PubMedGoogle Scholar
  14. Glinski M, Urbanke C, Hornbogen T, Zocher R (2002) Enniatin synthetase is a monomer with extended structures: evidence for an intramolecular reaction mechanism. Arch Microbiol 178:267–273CrossRefPubMedGoogle Scholar
  15. Hayashi K, Schoonbeek HJ, De Waard MA (2002) Expression of the ABC-transporter BcatrD from Botrytis cinerea reduces sensitivity to sterol demethylation fungicides. Pest Biochem Physiol 73:110–121CrossRefGoogle Scholar
  16. Iacomi-Vasilescu B, Blanchard D, Guénard M, Molinero-Demilly V, Laurent E, Simoneau P (2002) Development of a PCR based diagnostic assay for detecting pathogenic Alternaria species in cruciferous seeds. Seed Sci Technol 30:87–95Google Scholar
  17. Iacomi-Vasilescu B, Avenot H, Bataillé-Simoneau N, Laurent E, Guénard M, Simoneau P (2003) In vitro fungicide sensitivity of Alternaria species pathogenic to crucifers and identification of Alternaria brassicicola field isolates highly resistant to both dicarboximides and phenylpyrroles. Crop Prot (in press)Google Scholar
  18. Keller NP, Hohn TM (1997) Metabolic pathway gene clusters in filamentous fungi. Fungal Genet Biol 21:17–29CrossRefPubMedGoogle Scholar
  19. Kershaw MJ, Moorhouse ER, Bateman R, Reynolds SE, Charnley AK (1999) The role of destruxins in the pathogenicity of Metarhizium anisopliae for three species of insect. J Invert Pathol 74:213–223CrossRefGoogle Scholar
  20. Konz D, Mahariel MA (1999) How do peptide synthetases generate structural diversity? Chem Biol 6:R39–R48CrossRefPubMedGoogle Scholar
  21. MacKinnon SL, Keifer P, Ayer WA (1999) Components from the phytotoxic extract of Alternaria brassicicola, a black spot pathogen of canola. Phytochemistry 51:215–221CrossRefGoogle Scholar
  22. Mahariel MA, Stachelhaus T, Mootz HD (1997) Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem Rev 97:2651–2673CrossRefPubMedGoogle Scholar
  23. Mai B, Breeden L (1997) Xbp1, a stress-induced transcriptional repressor of the Saccharomyces cerevisiae Swi4/Mbp1 family. Mol Cell Biol 17:6491–6501PubMedGoogle Scholar
  24. Möller EM, Bahnweg G, Sandermann H, Geiger HH (1992) A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucleic Acids Res 20:6115–6116PubMedGoogle Scholar
  25. Mootz HD, Schwarzer D, Mahariel MA (2002) Ways of assembling complex natural products on modular nonribosomal peptide synthetases. Chem Biochem 3:490–504Google Scholar
  26. Nikolskaya AN, Panaccione DG, Walton JD (1995) Identification of peptide synthetase-encoding genes from filamentous fungi producing host-selective phytotoxins or analogs. Gene 165:207–211CrossRefPubMedGoogle Scholar
  27. Otani H, Kohmoto K, Kodama M (1995) Alternaria toxins and their effects on host plants. Can J Bot 73:S453–S458Google Scholar
  28. Otani H, Kohnobe A, Kodama M, Kohmoto K (1998) Production of a host-specific toxin by germinating spores of Alternaria brassicicola. Physiol Mol Plant Pathol 52:285–295CrossRefGoogle Scholar
  29. Pedras MSC, Biesenthal CJ (2000) Vital staining of plant cell suspension cultures: evaluation of the phytotoxic activity of the phytotoxins phomalide and destruxin B. Plant Cell Rep 19:1135–1138CrossRefGoogle Scholar
  30. Pedras MSC, Zaharia IL, Gai Y, Zhou Y, Ward DE (2001) In planta sequential hydroxylation and glycosylation of a fungal phytotoxin: avoiding cell death and overcoming the fungal invader. Proc Natl Acad Sci USA 98:747–752CrossRefPubMedGoogle Scholar
  31. Pedras MSC, Zaharia IL, Ward DE (2002) The destruxins: synthesis, biosynthesis, biotransformation, and biological activity. Phytochemistry 59:579–596CrossRefPubMedGoogle Scholar
  32. Pitkin JW, Panaccione DG, Walton JD (1996) A putative cyclic peptide efflux pump encoded by the TOXA gene of the plant pathogenic fungus Cochliobolus carbonum. Microbiology 142:1557–1565PubMedGoogle Scholar
  33. Proctor RH, Brown DW, Plattner RD, Desjardins AE (2003) Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis. Fungal Genet Biol 38:237–249CrossRefPubMedGoogle Scholar
  34. Rea PA (1999) MRP subfamily ABC transporters from plants and yeast. J Exp Bot 50:895–913Google Scholar
  35. Renshaw JC, Robson GD, Trinci APJ, Wiebe MG, Livens FR, Collison D, Taylor RJ (2002) Fungal siderophores: structures, functions and applications. Mycol Res 106:1123–1142CrossRefGoogle Scholar
  36. Saharan GS (1993) Disease resistance. In: Labana KS, Banga SS, Banga SK (eds) Breeding oilseed brassicas. (Monographs on theoretical and applied genetics, vol 19) Springer, Berlin Heidelberg New York, pp 181–205Google Scholar
  37. Sambrook J, Fritsch EF, Maniatis TA (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  38. Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23:1087–1088PubMedGoogle Scholar
  39. Slaven JW, Anderson MJ, Sanglard D, Dixon GK, Bille J, Roberts IS, Denning DW (2002) Increased expression of a novel Aspergillus fumigatus ABC transporter gene, atrF, in the presence of itraconazole in an itraconazole resistant clinical isolate. Fungal Genet Biol 36:199–206CrossRefPubMedGoogle Scholar
  40. Sohn YS, Nam DH, Ryu DD (2001) Biosynthetic pathway of cephabacins in Lysobacter lactamgenus: molecular and biochemical characterization of the upstream region of the gene clusters for engineering of novel antibiotics. Metab Eng 3:380–392CrossRefPubMedGoogle Scholar
  41. Springer JP, Cole RJ, Dorner JW, Cox RH, Richard JL, Barnes CL, Helm D van der (1984) Structure and conformation of roseotoxin B. J Am Chem Soc 106:2388–2392Google Scholar
  42. Stachelhaus T, Mootz HD, Mahariel MA (1999) The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem Biol 6:493–505PubMedGoogle Scholar
  43. Stahl DJ, Theuerkauf A, Heitefuss R, Schafer W (1994) Cutinase of Nectria haematococca (Fusarium solani f.sp. pisi) is not required for fungal virulence or organ specificity on pea. Mol Plant-Microbe Interact 7:713–725Google Scholar
  44. Stergiopoulos I, Gielkens MMC, Goodall SD, Venema K, De Waard MA (2002) Molecular cloning and characterisation of three new ATP-binding cassette transporter genes from the wheat pathogen Mycosphaerella graminicola. Gene 289:141–149CrossRefPubMedGoogle Scholar
  45. Stevens R (1974) Mycology guidebook. University of Washington Press, SeattleGoogle Scholar
  46. Trail S, Xu JR, San Miguel P, Halgren RG, Kistler HC (2003) Analysis of expressed sequence tags from Giberella zeae (anamorph Fusarium graminearum). Fungal Genet Biol 38:187–197CrossRefPubMedGoogle Scholar
  47. Tudzynski P, Hölter K, Correia T, Arntz C, Grammel N, Keller U (1999) Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol Gen Genet 261:133–141CrossRefPubMedGoogle Scholar
  48. Unkles SE (1992) Gene organization in industrial filamentous fungi. In: Kinghorn JR, Turner G (eds) Applied molecular genetics of filamentous fungi. Blackie, Glasgow, pp 28–53Google Scholar
  49. Urban M, Bhargava T, Hamer JE (1999) An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease. EMBO J 18:512–521PubMedGoogle Scholar
  50. Van Kan JAL, Van’t Klooster JW, Wagemakers CAM, Dees DCT, Van der Vlugt-Bergmans CJB (1997) Cutinase A of Botrytis cinerea is expressed, but not essential, during penetration of gerbera and tomato. Mol Plant-Microbe Interact 10:30–38Google Scholar
  51. Venkatasubbaiah P, Tisserat NA, Chilton WS (1994) Metabolites of Ophiosphaerella herpotricha, a cause of spring dead spot of bermudagrass. Mycopathology 128:155–159Google Scholar
  52. Vermeulen T, Schoonbeek H, De Waard MA (2001) The ABC transporter BcatrB from Botrytis cinerea is a determinant of the activity of the phenylpyrrole fungicide fludioxonil. Pest Manag Sci 57:393–402PubMedGoogle Scholar
  53. Vogel HJ (1956) A convenient growth medium for Neurospora crassa (medium N). Microb Genet Bull 13:42–43Google Scholar
  54. Yao C, Kôller W (1994) Diversity of cutinases from plant pathogenic fungi: cloning and characterisation of a cutinase gene from Alternaria brassicicola. Physiol Mol Plant Pathol 44:81–92Google Scholar
  55. Yoder OC, Turgeon BG (2001) Fungal genomics and pathogenicity. Curr Opin Plant Biol 4:315–321PubMedGoogle Scholar
  56. Yuan WM, Gentil GD, Budde AD, Leong SA (2001) Characterization of the Ustilago maydis sid2 gene, encoding multidomain peptide synthetase in the ferrichrome biosynthetic gene cluster. J Bacteriol 183:4040–4051CrossRefPubMedGoogle Scholar
  57. Zhou LW, Haas H. Marzluf GA (1998) Isolation and characterization of a new gene, sre, which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa. Mol Gen Genet 259:532–540CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Thomas Guillemette
    • 1
  • Adnane Sellam
    • 1
  • Philippe Simoneau
    • 1
  1. 1.Faculté des SciencesUMR PAVE 77AngersFrance

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