title: A critical assessment of Agrobacterium tumefaciens-mediated transformation as a tool for pathogenicity gene discovery in the phytopathogenic fungus Leptosphaeria maculans.
abstract: We evaluated the usefulness and robustness of Agrobacterium tumefaciens-mediated transformation (ATMT) as a high-throughput transformation tool for pathogenicity gene discovery in the filamentous phytopathogen Leptosphaeria maculans. Thermal asymmetric interlaced polymerase chain reaction allowed us to amplify the left border (LB) flanking sequence in 135 of 400 transformants analysed, and indicated a high level of preservation of the T-DNA LB. In addition, T-DNA preferentially integrated as a single copy in gene-rich regions of the fungal genome, with a probable bias towards intergenic and/or regulatory regions. A total of 53 transformants out of 1388 (3.8%) showed reproducible pathogenicity defects when inoculated on cotyledons of Brassica napus, with diverse altered phenotypes. Co-segregation of the altered phenotype with the T-DNA integration was observed for 6 of 12 transformants crossed. If extrapolated to the whole collection, this indicates that 1.9% of the collection actually corresponds to tagged pathogenicity mutants. The preferential insertion into gene-rich regions along with the high ratio of tagged mutants renders ATMT a tool of choice for large-scale gene discovery in L. maculans.
authors.affiliation: Institut National de la Recherche Agronomique, Phytopathologie et Méthodologies de la Détection, Route de St-Cyr, F-78026 Versailles Cedex, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Blaise
authors.forename: Estelle
authors.initials: E
authors.lastname: Rémy
authors.forename: Michel
authors.initials: M
authors.lastname: Meyer
authors.forename: Ligang
authors.initials: L
authors.lastname: Zhou
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Narcy
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Roux
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Balesdent
authors.forename: Thierry
authors.initials: T
authors.lastname: Rouxel
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001203:Ascomycota / Q000235:genetics* / Q000472:pathogenicity*; D001483:Base Sequence; D008969:Molecular Sequence Data; D010641:Phenotype; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D014169:Transformation, Bacterial / Q000235:genetics*