A critical assessment of Agrobacterium tumefaciens-mediated transformation as a tool for pathogenicity gene discovery in the phytopathogenic fungus Leptosphaeria maculans

• Published in: Fungal genetics and biology Vol. 44; no. 2; pp. 123 - 138
• Main Authors: Blaise, Françoise, Rémy, Estelle, Meyer, Michel, Zhou, Ligang, Narcy, Jean-Paul, Roux, Jacqueline, Balesdent, Marie-Hélène, Rouxel, Thierry
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2007; Elsevier
• Subjects: Agrobacterium; Agrobacterium tumefaciens; Agrobacterium tumefaciens - genetics; Agrotransformation; Ascomycota - genetics; Ascomycota - pathogenicity; Base Sequence; Brassica napus; Formal genetics; fungal diseases of plants; Gene tagging; genes; genetic recombination; genetic transformation; Genetics; Leptosphaeria maculans; Life Sciences; Molecular Sequence Data; mutants; pathogenicity; Pathogenicity gene; Phenotype; Plant Diseases - microbiology; plant pathogenic fungi; Plant–fungus interaction; Polymerase Chain Reaction - methods; SON-PCR; T-DNA left border flanking sequence; TAIL-PCR; transfer DNA; Transformation, Bacterial - genetics; SON-PCR; Plant–fungus interaction; Gene tagging; Agrotransformation; Leptosphaeria maculans; Pathogenicity gene; Formal genetics; TAIL-PCR; LEPTOSPHAERIA MACULANS; PLANT-FUNGUS INTERACTION; AGROTRANSFORMATION; PATHOGENICITY GENE; GENE TAGGINS; FORMAL GENETICS
• ISSN: 1087-1845; 1096-0937
• DOI: 10.1016/j.fgb.2006.07.006

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.