Molecular and General Genetics MGG

, Volume 248, Issue 2, pp 142–150 | Cite as

Efficient gene targeting in the filamentous fungusAlternaria alternata

  • Hiroshi Shiotani
  • Takashi Tsuge
Original Paper


To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1) transformants accounted for 30 to 80% of hygromycin B-resistant (HyR) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1 transformants accounted for only 5% of HyR transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.

Key words

Alternaria alternata Gene targeting Melanin biosynthesis gene 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Adachi Y, Watanabe Y, Tanabe K, Doke N, Nishimura S, Tsuge T (1993) Nuclear ribosomal DNA as a probe for genetic variability in the Japanese pear pathotype ofAlternaria alternata. Appl Environ Microbiol 59:3197–3205Google Scholar
  2. Bell AA, Wheeler MH (1986) Biosynthesis and function of fungal melanins. Annu Rev Phytopathol 24:411–451Google Scholar
  3. Bell AA, Puhalla WJ, Tolmsoff WJ, Stipanovic RD (1976) Use of mutants to establish (+)-scytalone as an intermediate in melanin biosynthesis byVerticillium dahliae. Can J Microbiol 22:787–799Google Scholar
  4. Capecchi MR (1989) Altering the genome by homologous recombination. Science 244:1288–1292Google Scholar
  5. Cullen D, Leong SA, Wilson J, Henner DJ (1987) Transformation ofAspergillus nidulans with the hygromycin-resistance gene,hph. Gene 57:21–26Google Scholar
  6. Desjardins AE, Hohn TM, McCormick SP (1992) Effect of gene disruption of trichodiene synthase on the virulence ofGibberella pulicaris. Mol Plant-Microbe Interact 5:214–222Google Scholar
  7. Feinberg AP, Vogelstein B (1983) A technique for radio-labeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13Google Scholar
  8. Fincham IRS (1989) Transformation in fungi. Microbiol Rev 53:148–170Google Scholar
  9. Fotheringham S, Holloman WK (1989) Cloning and disruption ofUstilago maydis genes. Mol Cell Biol 9:4052–4055Google Scholar
  10. Gritz L, Davies J (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression inEscherichia coli andSaccharomyces cerevisiae. Gene 25:179–188Google Scholar
  11. Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933Google Scholar
  12. Hynes MJ (1986) Transformation of filamentous fungi. Exp Mycol 10:1–8Google Scholar
  13. Kimura N, Tsuge T (1993) Gene cluster involved in melanin biosynthesis of the filamentous fungusAlternaria alternata. J Bacteriol 175:4427–4435Google Scholar
  14. Kingsman A, Gimlich R, Clarke L, Chinault A, Carbon J (1981) Sequence variation in dispersed repetitive sequences inSaccharomyces cerevisiae. J Mol Biol 145:619–632Google Scholar
  15. Kronstad JW, Wang J, Covert SF, Holden DW, McKnight GL, Leong SA (1989) Isolation of metabolic genes and demonstration of gene disruption in the phytopathogenic fungusUstilago maydis. Gene 79:97–106Google Scholar
  16. Kusaba M, Tsuge T (1994) Nuclear ribosomal DNA variation and pathogenic specialization inAlternaria fungi known to produce host-specific toxins. Appl Environ Microbiol 60:3055–3062Google Scholar
  17. Lopes TS, Hakkaart GJAJ, Koerts BL, Raué HA, Planta RJ (1991) Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA ofSaccharomyces cerevisiae. Gene 105:83–90Google Scholar
  18. Marmeisse R, van den Ackerveken GFJM, Goosen T, de Wit PJGM, van den Broek HWJ (1993) Disruption of the avirulence geneavr9 in two races of the tomato pathogenCladosporium fulvum causes virulence on tomato genotypes with the complementary resistance geneCf9. Mol Plant-Microbe Interact 6:412–417Google Scholar
  19. Miller BL, Miller KY, Timberlake WE (1985) Direct and indirect gene replacement inAspergillus nidulans. Mol Cell Biol 5:1714–1721Google Scholar
  20. Moerschell RP, Tsunasawa S, Sherman F (1988) Transformation of yeast with synthetic oligonucleotides. Proc Natl Acad Sci USA 85:524–528Google Scholar
  21. Mullaney EJ, Hamer JE, Roberti KA, Yelton MM, Timberlake WE (1985) Primary structure of thetrpC gene fromAspergillus nidulans. Mol Gen Genet 199:37–45Google Scholar
  22. Nakashima T, Ueno T, Fukami H, Taga T, Masuda H, Osaki K, Otani H, Kohmoto K, Nishimura S (1985) Isolation and structures of AK-toxin I and II, host-specific phytotoxic metabolites produced byAlternaria alternata Japanese pear pathotype. Agric Biol Chem 49:807–815Google Scholar
  23. Nishimura S, Kohmoto K (1983) Host-specific toxins and chemical structures fromAlternaria species. Annu Rev Phytopathol 21:87–116Google Scholar
  24. Orr-Weaver TL, Szostak JW (1983) Yeast recombination: the association between double-strand gap repair and crossing-over. Proc Natl Acad Sci USA 80:4417–4421Google Scholar
  25. Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358Google Scholar
  26. Orr-Weaver TL, Szostak JW, Rothstein RJ (1983) Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol 101:228–245Google Scholar
  27. Otani H, Kohmoto K, Nishimura S, Nakashima T, Ueno T, Fukami H (1985) Biological activities of AK-toxins I and II, host-specific toxins fromAlternaria alternata Japanese pear pathotype. Ann Phytopath Soc Jpn 51:285–293Google Scholar
  28. Paietta JV, Marzluf GA (1985) Gene disruption by transformation inNeurospora crassa. Mol Cell Biol 5:1554–1559Google Scholar
  29. Panaccione DG, Scott-Craig JS, Pocard JA, Walton JD (1992) A cyclic peptide synthetase gene required for pathogenicity of the fungusCochliobolus carbonum on maize. Proc Natl Acad Sci USA 89:6590–6594Google Scholar
  30. Reed KC, Mann DA (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13:7207–7221Google Scholar
  31. Rogers LM, Flaishman MA, Kolattukudy PE (1994) Cutinase gene disruption inFusarium solani f. sp.pici decreases its virulence on pea. Plant Cell 6:935–945Google Scholar
  32. Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol 194:281–301Google Scholar
  33. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  34. Scott-Craig JS, Panaccione DG, Cervone F, Walton JD (1990) Endopolygalacturonase is not required for pathogenicity ofCochliobolus carbonum on maize. Plant Cell 2:1191–1200Google Scholar
  35. Schiestl RH, Petes TD (1991) Integration of DNA fragments by illegitimate recombination inSaccharomyces cerevisiae. Proc Natl Acad Sci USA 88:7585–7589Google Scholar
  36. Shortle D, Haber JE, Botstein D (1982) Lethal disruption of the yeast actin gene by integrative DNA transformation. Science 217:371–373Google Scholar
  37. Stahl DJ, Schäfer W (1992) Cutinase is not required for fungal pathogenicity on pea. Plant Cell 4:621–629Google Scholar
  38. Sweigard JA, Chumley FG, Valent B (1992) Disruption of a Magnaporthe grisea cutinase gene. Mol Gen Genet 232:183–190Google Scholar
  39. Szostak JW, Wu R (1980) Unequal crossing over in the ribosomal DNA ofSaccharomyces cerevisiae. Nature 284:426–430Google Scholar
  40. Szostak JW, Orr-Weaver TL, Rothstein RJ, Stahl FW (1983) The double-strand-break repair model for recombination. Cell 33:25–35Google Scholar
  41. Talbot NJ, Ebbole DJ, Hamer JE (1993) Identification and characterization ofMPG1, a gene involved in pathogenicity from the rice blast fungusMagnaporthe grisea. Plant Cell 5:1575–1590Google Scholar
  42. Tanabe K, Nishimura S, Kohmoto K (1988) Pathogenicity of melanin-deficient mutants ofAlternaria alternata Japanese pear pathotype. Ann Phytopath Soc Jpn 54:54Google Scholar
  43. Tanaka S (1933) Studies on black spot disease of Japanese pear (Pyrus serotina Rehd.). Mem Coll Agric Kyoto Imp Univ 28:1–31Google Scholar
  44. Tsuge T, Kobayashi H, Nishimura S (1989) Organization of ribosomal RNA genes inAlternaria alternata Japanese pear pathotype, a host-selective AK-toxin-producing fungus. Curr Genet 16:267–272Google Scholar
  45. Tsuge T, Nishimura S, Kobayashi H (1990) Efficient integrative transformation of the phytopathogenic fungusAlternaria alternata mediated by the repetitiverDNA sequences. Gene 90:207–214Google Scholar
  46. Turgeon BG, Garber RC, Yoder OC (1987) Development of a fungal transformation system based on selection of sequences with promoter activity. Mol Cell Biol 7:3297–3305Google Scholar
  47. Wilson JH, Leung WY, Bosco G, Dieu D, Haber JE (1994) The frequency of gene targeting in yeast depends on the number of target copies. Proc Natl Acad Sci USA 91:177–181Google Scholar
  48. Winston F, Chumley F, Fink GR (1983) Eviction and transplacement of mutant genes in yeast. Methods Enzymol 101:211–228Google Scholar
  49. Zheng H, Wilson JH (1990) Gene targeting in normal and amplified cell lines. Nature 344:170–173Google Scholar
  50. Zimmer A (1992) Manipulating the genome by homologous recombination in embryonic stem cells. Annu Rev Neurosci 15:115–137Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Hiroshi Shiotani
    • 1
  • Takashi Tsuge
    • 1
  1. 1.School of Agricultural SciencesNagoya UniversityChikusa, NagoyaJapan

Personalised recommendations