December
2000
, Volume
13
, Number
12
Pages
1,356
-
1,365
Authors
M. R.
Thon
,
E. M.
Nuckles
,
and
L. J.
Vaillancourt
Affiliations
Department of Plant Pathology, S-305 Agricultural Sciences Center-North, University of Kentucky, Lexington 40546-0091, U.S.A.
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RelatedArticle
Accepted 29 August 2000.
Abstract
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.
JnArticleKeywords
Additional keywords:
anthracnose leaf blight,
anthracnose stalk rot,
corn,
Glomerella graminicola.
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ArticleCopyright
© 2000 The American Phytopathological Society