TY - JOUR
T1 - Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola
AU - Thon, M. R.
AU - Nuckles, E. M.
AU - Vaillancourt, L. J.
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2000
Y1 - 2000
N2 - 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.
AB - 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.
KW - Anthracnose leaf blight
KW - Anthracnose stalk rot
KW - Corn
KW - Glomerella graminicola
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U2 - 10.1094/MPMI.2000.13.12.1356
DO - 10.1094/MPMI.2000.13.12.1356
M3 - Article
C2 - 11106028
AN - SCOPUS:0033670641
SN - 0894-0282
VL - 13
SP - 1356
EP - 1365
JO - Molecular Plant-Microbe Interactions
JF - Molecular Plant-Microbe Interactions
IS - 12
ER -