Abstract
Ustilago maydis, the causative agent of corn smut disease, is one of the most versatile model systems for the study of plant pathogenic fungi. With the availability of the complete genomic sequence there is an increasing need to improve techniques for the generation of deletion mutants in order to elucidate the functions of unknown genes. Here a method is presented which allows one to generate constructs for gene replacement without the need for cloning. The 5′ and 3′-regions of the target gene are first amplified by PCR, and subsequently ligated directionally to a marker cassette via two distinct Sfi I sites, providing the flanking homologies needed for homologous recombination in U. maydis. Then the ligation product is used as a template for the amplification of the deletion construct, which can be used directly for transformation of U. maydis. The use of the fragments generated by PCR drastically increases the frequency of homologous recombination when compared to the linearized plasmids routinely used for gene replacement in U. maydis.
Similar content being viewed by others
References
Banks GR, Taylor SY (1988) Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation. Mol Cell Biol 8:5417–5424
Banuett F (1995) Genetics of Ustilago maydis, a fungal pathogen that induces tumors in maize. Annu Rev Genet 29:179–208
Banuett F, Herskowitz I (1989) Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis. Proc Natl Acad Sci USA 86:5878–5882
Bölker M (2001) Ustilago maydis —a valuable model system for the study of fungal dimorphism and virulence. Microbiology 147:1395–1401
Bölker M, Urban M, Kahmann R (1992) The a mating type locus of Ustilago maydis specifies cell signaling components. Cell 68:441–450
Bölker M, Genin S, Lehmler C, Kahmann R (1995) Genetic regulation of mating, and dimorphism in Ustilago maydis. Can J Bot 73:320–325
Brachmann A, Weinzierl G, Kämper J, Kahmann R (2001) Identification of genes in the bW/bE regulatory cascade in Ustilago maydis. Mol Microbiol 42:1047–1063
Broomfield PL, Hargreaves JA (1992) A single amino-acid change in the iron-sulphur protein subunit of succinate dehydrogenase confers resistance to carboxin in Ustilago maydis. Curr Genet 22:117–121
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 148:2607–2615
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 68:647–657
Gold SE, Bakkeren G, Davies JE, Kronstad JW (1994) Three selectable markers for transformation of Ustilago maydis. Gene 142:225–230
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59
Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of E. coli. Gene 57:267–272
Holliday R (1974) Ustilago maydis. In: King RC (ed) Handbook of genetics, vol. 1. Plenum Press, New York, pp 575–595
Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68
Kahmann R, Steinberg G, Basse C, Kämper J (2000) Ustilago maydis, the causative agent of corn smut disease. In: Kronstad JW (ed) Fungal pathology. Kluwer Academic Publishers, Dordrecht, pp 347–371
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 19:475–481
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:E2
Martinez-Espinoza AD, Garcia-Pedrajas MD, Gold SE (2002) The Ustilaginales as plant pests and model systems. Fungal Genet Biol 35:1–20.
Orr-Weaver TL, Szostak JW, Rothstein RJ (1983) Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol 101:228–245.
Sambrook J, Frisch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Schulz B, Banuett F, Dahl M, Schlesinger R, Schäfer 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 60:295–306
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 252:503–509.
Tsukuda T, Carleton S, Fotheringham S, Holloman WK (1988) Isolation and characterization of an autonomously replicating sequence from Ustilago maydis. Mol Cell Biol 8:3703–3709
Urban M, Kahmann R, Bölker M (1996) Identification of the pheromone response element in Ustilago maydis. Mol Gen Genet 251:31–37
Wach A (1996) PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast 12:259–265
Wach A, Brachat A, Pohlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808
Wang J, Holden DW, Leong SA (1988) Gene transfer system for the phytopathogenic fungus Ustilago maydis. Proc Natl Acad Sci USA 85:865–869
Wendland J (2003) PCR-based methods facilitate targeted gene manipulations and cloning procedures. Curr Genet 44:115–123
Winzeler EA, et al (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906
Acknowledgments
I would like to thank R. Kahmann and U. Kämper for critically reading the manuscript and for helpful suggestions, and A. Waldschmidt and V. Vincon for excellent technical assistance. This work was supported by funding from Bayer CropScience AG
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by G. Jürgens
Rights and permissions
About this article
Cite this article
Kämper, J. A PCR-based system for highly efficient generation of gene replacement mutants in Ustilago maydis . Mol Genet Genomics 271, 103–110 (2004). https://doi.org/10.1007/s00438-003-0962-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-003-0962-8