Current Genetics

, Volume 25, Issue 1, pp 41–46 | Cite as

Restoration of pathogenicity of a penetration-deficient mutant ofCollectotrichum lagenarium by DNA complementation

  • Noel S. Perpetua
  • Yasuyuki Kubo
  • Tetsuro Okuno
  • Iwao Furusawa
Original Articles


Infection byColletotrichum lagenarium requires formation of an appressorium and of a penetration peg. A mutant, 83 348, defective in morphogenesis of the penetration peg was unable to penetrate into cellulose membranes or infect cucumber leaves. DNA transformation using a wild-type genomic library constructed in pKVB resulted in two transformants, Ppr1 and Ppr2, with restored penetration peg formation, from 2,000 benomylresistant transformants. However, penetration into cellulose membranes by these transformats ranged from 30 to 40% compared to greater than 90% by wild-type. Southern-blot hybridization showed that a single copy of a cosmid clone had integrated into the genome of the transformants. A 12.0-kbp fragment of the cosmid vector with the flanking region of wild-type genomic DNA was recovered by plasmid rescue from Ppr1. Using the flanking DNA sequences as a probe for colony blot hybridization, a genomic clone was identified and designated pRP46. Transformants obtained following transformation with pRP46 were able to penetrate cellulose membranes. The penetration frequency of pRP46 transformants ranged from 25 to 65%. Transformants were also pathogenic on cucumber.

Key words

Fungal transformation Colletotrichum lagenarium Penetration peg formation DNA complementation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Chumley F, Valent B (1990) Mol Plant Microbe 3:135–143Google Scholar
  2. Dickman MB (1988) Curr Genet 14:241–246Google Scholar
  3. Fincham JRS (1989) Microbiol Rev 53:148–170Google Scholar
  4. Hinnen A, Hicks JB, Finks GR (1978) Proc Natl Acad Si USA 75:1929–1933Google Scholar
  5. Hoch HC, Staples RC (1991) Signaling for infection structure formation in fungi. In: Cole GT, Hoch HC (eds) The fungal spore and disease initiation in plants and animals. Plenum Publishing Corporation, New York, pp 25–46Google Scholar
  6. Holm C, Meeks-Wagner DW, Fangman WL, Botstein D (1986) Gene 42:169–173Google Scholar
  7. Johnstone IL, Hughes SG, Clutterbuck AJ (1985) EMBO J 4:1307–1311Google Scholar
  8. Katoh M, Hirose I, Kubo Y, Hikichi Y, Kunoh H, Furusawa I, Shishiyama (1988) Phys Mol Plant Pathol 32:177–184Google Scholar
  9. Kistler HC, Benny VK (1988) Curr Gen 13:145–149Google Scholar
  10. Koller W (1991) The Plant Cuticle, a barrier to be overcome by fungal plant pathogens. In: Cole GT, Hoch HC (eds) The fungal spore disease initiation in plants and animals. Plenum Publishing Corporation, New York, pp 219–246Google Scholar
  11. Koller W, Allan CR, Kolattukudy PE (1988) Physiol Plant Pathol 20:47–60Google Scholar
  12. Kronstad JW, Leong SA (1989) Proc Natl Acad Sci USA 86:978–982Google Scholar
  13. Kubo Y, Furusawa I (1991) Prerequisite for successful invasion of the plant host by appressoria ofColletotrichum andPyricularia. In: Cole GT, Hoch HC (eds) The fungal spore and disease initiation in plants and animals. Plenum Publishing Corporation, New York, pp 205–218Google Scholar
  14. Kubo Y, Susuki K, Furusawa I, Ishida I, Yamamoto M (1982) phytopathology 72:489–501Google Scholar
  15. Kubo Y, Nakamura H, Kobayashi K, Okuno T, Furusawa I (1991) Mol Plant-Microbe Int 5:440–445Google Scholar
  16. Leong SA, Holden DW (1989) Annu Rev Phytopathol 27:403–481Google Scholar
  17. Lim SM, Hooker AL (1971) Genetics 69:115–117Google Scholar
  18. Mishra NC (1979) J Gen Microbiol 113:255–259Google Scholar
  19. Parsons KA, Chumley FG, Valent B (1987) Proc Natl Acad Sci USA 84:4161–4165Google Scholar
  20. Rodriguez RJ, Yoder OC (1987) Gene 54:73–81Google Scholar
  21. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  22. Southern EM (1975) J Mol Biol 98:503–517Google Scholar
  23. Stohl LL, Lamboritz AM (1983) Nucleic Acids Res 12:6169–6178Google Scholar
  24. Turgeon BG, Garber RC, Yoder OC (1985) Mol Gen Genet 201:450–453Google Scholar
  25. Van Kran JAL, Van den Ackerveken GFJM, de Wit PJGM (1991) Mol Plant-Microbe Interact 4:52–59Google Scholar
  26. Vollmer SJ, Yanofsky C (1986) Proc Natl Acad Sci USA 83:4809–4873Google Scholar
  27. Walton JO (1987) Proc Natl Acad Sci USA 84:8444–8447Google Scholar
  28. Wang J, Holden DW, Leong SA (1988) Proc Natl Acad Sci USA 85:865–869Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Noel S. Perpetua
    • 1
  • Yasuyuki Kubo
    • 1
  • Tetsuro Okuno
    • 1
  • Iwao Furusawa
    • 1
  1. 1.Laboratory of Plant Pathology, Faculty of AgricultureKyoto UniversityKyotoJapan

Personalised recommendations