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Molecular Genetics and Genomics

, Volume 272, Issue 2, pp 216–226 | Cite as

A reverse genetic approach for generating gene replacement mutants in Ustilago maydis

  • A. Brachmann
  • J. König
  • C. Julius
  • M. Feldbrügge
Original Paper

Abstract

We describe a versatile strategy for generating gene replacement mutants in the phytopathogenic fungus Ustilago maydis. The system includes the choice of 32 different insertion cassettes for genetic engineering purposes, such as gene disruption and more sophisticated insertions of reporter genes, heterologous promoters or combinations of the two. PCR-amplified flanking sequences needed for homologous recombination are ligated to the respective insertion cassettes via Sfi I sites. As proof of principle we generated two replacement mutants in which the endogenous promoter of the pheromone gene mfa1 drives expression of the Green Fluorescent Protein gene (gfp). Simultaneously, expression of the mfa1 ORF is controlled either by the carbon source-regulated crg1 promoter or the nitrogen source-regulated nar1 promoter. In both cases gfp expression was pheromone-inducible and pheromone expression was only detected when the heterologous promoters were active.

Keywords

Ustilago maydis Gene replacement Knock out/knock in Mating factor Regulated gene expression 

Notes

Acknowledgements

We acknowledge Dr. R. Kahmann for critically reading the manuscript, and thank J. Hohenner, S. Hester and P. Roth for excellent technical assistance. This work was supported by the DFG through Sonderforschungsbereich 369 and by funding from Bayer CropScience

References

  1. Banks GR, Shelton PA, Kanuga N, Holden DW, Spanos A (1993) The Ustilago maydis nar1 gene encoding nitrate reductase activity: sequence and transcriptional regulation. Gene 131:69–78CrossRefPubMedGoogle Scholar
  2. Banuett F (1992) Ustilago maydis, the delightful blight. Trends Genet 8:174–180PubMedGoogle Scholar
  3. Banuett F (1995) Genetics of Ustilago maydis, a fungal pathogen that induces tumors in maize. Annu Rev Genet 29:179–208CrossRefPubMedGoogle Scholar
  4. Banuett F, Herskowitz I (1989) Different a alleles are necessary for maintenance of filamentous growth but not for meiosis. Proc Natl Acad Sci USA 86:5878–5882Google Scholar
  5. Barrett KJ, Gold SE, Kronstad JW (1993) Identification and complementation of a mutation to constitutive filamentous growth in Ustilago maydis. Mol Plant Microbe Interact 6:274–283PubMedGoogle Scholar
  6. Basse CW, Kolb S, Kahmann R (2002) A maize-specifically expressed gene cluster in Ustilago maydis. Mol Microbiol 43:75–93CrossRefPubMedGoogle Scholar
  7. Baudin A, Ozier-Kalogeropoulos O, Denouel A, Lacroute F, Cullin C (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21:3329–3330PubMedGoogle Scholar
  8. Bevan M, Barnes WM, Chilton MD (1983) Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res 11:369–385PubMedGoogle Scholar
  9. Bölker M (2001) Ustilago maydis - a valuable model system for the study of fungal dimorphism and virulence. Microbiology 147:1395–1401PubMedGoogle Scholar
  10. Bölker M, Urban M, Kahmann R (1992) The a mating type locus of U. maydis specifies cell signaling components. Cell 68:441–450CrossRefPubMedGoogle Scholar
  11. Bottin A, Kämper J, Kahmann R (1996) Isolation of a carbon source-regulated gene from Ustilago maydis. Mol Gen Genet 253:342–352CrossRefPubMedGoogle Scholar
  12. Brachmann A, Weinzierl G, Kämper J, Kahmann R (2001) Identification of genes in the bW/bE regulatory cascade in Ustilago maydis. Mol Microbiol 42:1047–1063CrossRefPubMedGoogle Scholar
  13. Brachmann A, Schirawski J, Müller P, Kahmann R (2003) An unusual MAP kinase is required for efficient penetration of the plant surface by Ustilago maydis. EMBO J 22:2199–2210CrossRefPubMedGoogle Scholar
  14. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99:7877–7882Google Scholar
  15. Davidson RC, Blankenship JR, Kraus PR, de Jesus Berrios M, Hull CM, D’Souza C, Wang P, Heitman J (2002) A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148:2607–2615PubMedGoogle Scholar
  16. Gage MJ, Bruenn J, Fischer M, Sanders D, Smith TJ (2001) KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake. Mol Microbiol 41:775–785CrossRefPubMedGoogle Scholar
  17. Garrido E, Pérez-Martín J (2003) The crk1 gene encodes an Ime2-related protein that is required for morphogenesis in the plant pathogen Ustilago maydis. Mol Microbiol 47:729–743CrossRefPubMedGoogle Scholar
  18. Giaever G, et al (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387–391CrossRefPubMedGoogle Scholar
  19. Gillissen B, Bergemann J, Sandmann C, Schroeer B, Bölker M, Kahmann R (1992) A two-component regulatory system for self/non-self recognition in Ustilago maydis. Cell 68:647–657CrossRefPubMedGoogle Scholar
  20. Goffeau A (1994) Yeast genes in search of functions. Nature 369:101–102CrossRefPubMedGoogle Scholar
  21. Gold S, Duncan G, Barrett K, Kronstad J (1994a) cAMP regulates morphogenesis in the fungal pathogen Ustilago maydis. Genes Dev 8:2805–2816PubMedGoogle Scholar
  22. Gold SE, Bakkeren G, Davies JE, Kronstad JW (1994b) Three selectable markers for transformation of Ustilago maydis. Gene 142:225–230CrossRefPubMedGoogle Scholar
  23. Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551Google Scholar
  24. Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272CrossRefPubMedGoogle Scholar
  25. Holliday R (1974) Ustilago maydis. In: King RC (ed) Handbook of genetics, vol 1. Plenum Press, New York, pp 575–595Google Scholar
  26. Huber SM, Lottspeich F, Kämper J (2002) A gene that encodes a product with similarity to dioxygenases is highly expressed in teliospores of Ustilago maydis. Mol Genet Genomics 267:757–771CrossRefPubMedGoogle Scholar
  27. Hull CM, Heitman J (2002) Genetics of Cryptococcus neoformans. Annu Rev Genet 36:557–615CrossRefPubMedGoogle Scholar
  28. Kaffarnik F, Müller P, Leibundgut M, Kahmann R, Feldbrügge M (2003) PKA and MAPK phosphorylation of Prf1 allows promoter discrimination in Ustilago maydis. EMBO J 22:5817–5826CrossRefPubMedGoogle Scholar
  29. Kahmann R, Steinberg G, Basse C, Feldbrügge M, Kämper J (2000) Ustilago maydis, the causative agent of corn smut disease. In: Kronstad JW (ed) Fungal pathology. Kluwer Academic Publishers, Dordrecht, pp 347–371Google Scholar
  30. Kämper J (2004) A PCR-based system for highly efficient generation of gene replacement mutants in Ustilago maydis. Mol Genet Genomics 271:103–110CrossRefPubMedGoogle Scholar
  31. Kämper J, Reichmann M, Romeis T, Bölker M, Kahmann R (1995) Multiallelic recognition: nonself-dependent dimerization of the bE and bW homeodomain proteins in Ustilago maydis. Cell 81:73–83CrossRefPubMedGoogle Scholar
  32. Keon JP, White GA, Hargreaves JA (1991) Isolation, characterization and sequence of a gene conferring resistance to the systemic fungicide carboxin from the maize smut pathogen, Ustilago maydis. Curr Genet 19:475–481PubMedGoogle Scholar
  33. Kojic M, Holloman WK (2000) Shuttle vectors for genetic manipulations in Ustilago maydis. Can J Microbiol 46:333–338CrossRefPubMedGoogle Scholar
  34. Kojic M, Kostrub CF, Buchman AR, Holloman WK (2002) BRCA2 homolog required for proficiency in DNA repair, recombination, and genome stability in Ustilago maydis. Mol Cell 10:683–691CrossRefPubMedGoogle Scholar
  35. Kronstad JW (2003) Castles and cuitlacoche: the first international Ustilago conference. Fungal Genet Biol 38:265–271CrossRefPubMedGoogle Scholar
  36. Kronstad JW, Leong SA (1990) The b mating-type locus of Ustilago maydis contains variable and constant regions. Genes Dev 4:1384–1395PubMedGoogle Scholar
  37. Krügel H, Fiedler G, Smith C, Baumberg S (1993) Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei. Gene 127:127–131CrossRefPubMedGoogle Scholar
  38. Kuwayama H, Obara S, Morio T, Katoh M, Urushihara H, Tanaka Y (2002) PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors. Nucleic Acids Res 30:e2CrossRefPubMedGoogle Scholar
  39. Lorenz MC, Muir RS, Lim E, McElver J, Weber SC, Heitman J (1995) Gene disruption with PCR products in Saccharomyces cerevisiae. Gene 158:113–117CrossRefPubMedGoogle Scholar
  40. Loubradou G, Brachmann A, Feldbrügge M, Kahmann R (2001) A homolog of the transcriptional repressor Ssn6p antagonizes cAMP signalling in Ustilago maydis. Mol Microbiol 40:719–730CrossRefPubMedGoogle Scholar
  41. Sambrook J, Frisch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  42. Sánchez-Martínez C, Pérez-Martín J (2001) Dimorphism in fungal pathogens: Candida albicans and Ustilago maydis - similar inputs, different outputs. Curr Opin Microbiol 4:214–221CrossRefPubMedGoogle Scholar
  43. Schulz B, Banuett F, Dahl M, Schlesinger R, Schafer W, Martin T, Herskowitz I, Kahmann R (1990) The b alleles of U. maydis, whose combinations program pathogenic development, code for polypeptides containing a homeodomain-related motif. Cell 60:295–306PubMedGoogle Scholar
  44. Sheen J, Hwang S, Niwa Y, Kobayashi H, Galbraith DW (1995) Green-fluorescent protein as a new vital marker in plant cells. Plant J 8:777–784CrossRefPubMedGoogle Scholar
  45. Spellig T, Bölker M, Lottspeich F, Frank RW, Kahmann R (1994) Pheromones trigger filamentous growth in Ustilago maydis. EMBO J 13:1620–1627PubMedGoogle Scholar
  46. Spellig T, Bottin A, Kahmann R (1996) Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis. Mol Gen Genet 252:503–509CrossRefPubMedGoogle Scholar
  47. Straube A, Enard W, Berner A, Wedlich-Söldner R, Kahmann R, Steinberg G (2001) A split motor domain in a cytoplasmic dynein. EMBO J 20:5091–5100CrossRefPubMedGoogle Scholar
  48. Szabó Z, Tönnis M, Kessler H, Feldbrügge M (2002) Structure-function analysis of lipopeptide pheromones from the plant pathogen Ustilago maydis. Mol Genet Genomics 268:362–370CrossRefPubMedGoogle Scholar
  49. Timberlake WE, Marshall MA (1989) Genetic engineering of filamentous fungi. Science 244:1313–1317PubMedGoogle Scholar
  50. Tsukuda T, Carleton S, Fotheringham S, Holloman WK (1988) Isolation and characterization of an autonomously replicating sequence from Ustilago maydis. Mol Cell Biol 8:3703–3709PubMedGoogle Scholar
  51. Wedlich-Söldner R, Straube A, Friedrich MW, Steinberg G (2002) A balance of KIF1A-like kinesin and dynein organizes early endosomes in the fungus Ustilago maydis. EMBO J 21:2946–2957CrossRefPubMedGoogle Scholar
  52. Wendland J (2003) PCR-based methods facilitate targeted gene manipulations and cloning procedures. Curr Genet 44:115–123CrossRefPubMedGoogle Scholar
  53. Williams SA, Halford SE (2001) Sfi I endonuclease activity is strongly influenced by the non-specific sequence in the middle of its recognition site. Nucleic Acids Res 29:1476–1483CrossRefPubMedGoogle Scholar
  54. Winzeler EA, et al (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906CrossRefPubMedGoogle Scholar
  55. Yuan WM, Gentil GD, Budde AD, Leong SA (2001) Characterization of the Ustilago maydis sid2 gene, encoding a multidomain peptide synthetase in the ferrichrome biosynthetic gene cluster. J Bacteriol 183:4040–4051CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • A. Brachmann
    • 1
    • 2
  • J. König
    • 1
  • C. Julius
    • 1
  • M. Feldbrügge
    • 1
  1. 1.Department of Organismic InteractionsMax Planck Institute for Terrestrial MicrobiologyMarburgGermany
  2. 2.National Institute for Diabetes and Digestive and Kidney DiseasesNIHBethesdaUSA

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