Involvement of KU80 in T-DNA integration in plant cells.
In Agrobacterium-mediated genetic transformation of plant cells, the bacterium exports a well defined transferred DNA (T-DNA) fragment and a series of virulence proteins into the host cell. Following its nuclear import, the single-stranded T-DNA is stripped of its escorting proteins, most likely converts to a double-stranded (ds) form, and integrates into the host genome. Little is known about the precise mechanism of T-DNA integration in plants, and no plant proteins specifically associated to T-DNA have been identified. Here we report the direct involvement of KU80, a protein that binds dsT-DNA intermediates. We show that ku80-mutant Arabidopsis plants are defective in T-DNA integration in somatic cells, whereas KU80-overexpressing plants exhibit increased susceptibility to Agrobacterium infection and increased resistance to DNA-damaging agents. The direct interaction between dsT-DNA molecules and KU80 in planta was confirmed by immunoprecipitation of KU80 dsT-DNA complexes from Agrobacterium-infected plants. Transformation of KU80-overexpressing plants with two separate T-DNA molecules resulted in an increased rate of extrachromosomal T-DNA to T-DNA recombination, indicating that KU80 bridges between dsT-DNAs and double-strand breaks. This last result further supports the notion that integration of T-DNA molecules occurs through ds intermediates and requires active participation of the host's nonhomologous end-joining repair machinery.
Li
Jianxiong
J
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA.
Vaidya
Manjusha
M
White
Charles
C
Vainstein
Alexander
A
Citovsky
Vitaly
V
Tzfira
Tzvi
T
D034961:Antigens, Nuclear / Q000096:biosynthesis* / Q000737:chemistry*; D017360:Arabidopsis / Q000235:genetics* / Q000382:microbiology*; D029681:Arabidopsis Proteins / Q000737:chemistry; D004247:DNA / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D018744:DNA, Plant; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D017343:Genes, Plant / Q000235:genetics*; D047468:Immunoprecipitation; D000072200:Ku Autoantigen; D008957:Models, Genetic; D010641:Phenotype; D010944:Plants / Q000378:metabolism; D011485:Protein Binding; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012231:Rhizobium / Q000378:metabolism*; D014170:Transformation, Genetic; D019076:Transgenes
D034961:Antigens, Nuclear; D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D018744:DNA, Plant; D004268:DNA-Binding Proteins; C036483:T-DNA; D004247:DNA; D000072200:Ku Autoantigen