Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola.
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.
Thon
M R
MR
Department of Plant Pathology, University of Kentucky, Lexington 40546-0091, USA.
Nuckles
E M
EM
Vaillancourt
L J
LJ
D015139:Blotting, Southern; D020231:Colletotrichum / Q000235:genetics* / Q000472:pathogenicity*; D005816:Genetic Complementation Test; D016254:Mutagenesis, Insertional; D010935:Plant Diseases; D018515:Plant Leaves / Q000382:microbiology; D010957:Plasmids; D011523:Protoplasts / Q000502:physiology; D015183:Restriction Mapping; D014169:Transformation, Bacterial; D003313:Zea mays / Q000382:microbiology*