Agrobacterium T-DNA-mediated integration and gene replacement in the brown rot pathogen Monilinia fructicola.
A transformation system utilizing Agrobacterium tumefaciens was developed for targeted gene disruption in Monilinia fructicola, a fungal pathogen that causes brown rot disease of stone fruits. Transformation with a vector containing the neomycin phosphotransferase II (nptII) cassette flanked with 4 kb cutinase gene (Mfcut1) flanking sequences resulted in an average of 13 transformants per 10(5) spores. When assayed by PCR and DNA blot analyses, more than 50% of the transformants recovered had integrated in the targeted Mfcut1 locus. Both target-gene-specific and non-specific integrations carried direct (head-to-tail) repeat T-DNA integrations. Sequence analysis of these T-DNA integrations revealed that 26 bp of the T-DNA right border were missing at the junctions between direct repeats in all cases. The recombination event during non-specific T-DNA integration in this fungus was unlike that reported in Agrobacterium-mediated transformation in plants.
Lee
Miin-Huey
MH
Department of Plant Pathology, University of California, One Shields Ave, Davis, CA 95616, USA.
Bostock
Richard M
RM
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001203:Ascomycota / Q000187:drug effects / Q000235:genetics* / Q000648:ultrastructure; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics; D002467:Cell Nucleus / Q000648:ultrastructure; D004269:DNA, Bacterial / Q000235:genetics*; D025141:Drug Resistance, Fungal / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D016681:Genome, Fungal / Q000235:genetics; D019868:Kanamycin Kinase / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D009355:Neomycin / Q000494:pharmacology; D010935:Plant Diseases / Q000382:microbiology*; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D014170:Transformation, Genetic
D004269:DNA, Bacterial; C036483:T-DNA; D019868:Kanamycin Kinase; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D009355:Neomycin