Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola

M. R. Thon, E. M. Nuckles, L. J. Vaillancourt

Research output: Contribution to journalArticlepeer-review

74 Scopus citations


We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations with linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme-recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.

Original languageEnglish
Pages (from-to)1356-1365
Number of pages10
JournalMolecular Plant-Microbe Interactions
Issue number12
StatePublished - 2000


  • Anthracnose leaf blight
  • Anthracnose stalk rot
  • Corn
  • Glomerella graminicola

ASJC Scopus subject areas

  • Physiology
  • Agronomy and Crop Science


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