Formation of complex extrachromosomal T-DNA structures in Agrobacterium tumefaciens-infected plants.
Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The integration of transferred DNA (T-DNA) and the formation of complex insertions in the genome of transgenic plants during A. tumefaciens-mediated transformation are still poorly understood. Here, we show that complex extrachromosomal T-DNA structures form in A. tumefaciens-infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules can circularize in planta. We recovered circular T-DNA molecules (T-circles) using a novel plasmid-rescue method. Sequencing analysis of the T-circles revealed patterns similar to the insertion patterns commonly found in transgenic plants. The patterns include illegitimate DNA end joining, T-DNA truncations, T-DNA repeats, binary vector sequences, and other unknown "filler" sequences. Our data suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a double-stranded form. We propose that termini of linear double-stranded T-DNAs are recognized and repaired by the plant's DNA double-strand break-repair machinery. This can lead to circularization, integration, or the formation of extrachromosomal complex T-DNA structures that subsequently may integrate.
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, USA. kamys@umich.edu
Kamy
K
Singer
Yoel M
YM
Shiboleth
Jianming
J
Li
Tzvi
T
Tzfira
D016960:Agrobacterium tumefaciens / Q000472:pathogenicity*; D000667:Ampicillin / Q000494:pharmacology; D003001:Cloning, Molecular; D059766:DNA End-Joining Repair; D004269:DNA, Bacterial / Q000235:genetics*; D004270:DNA, Circular / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D010935:Plant Diseases / Q000382:microbiology*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism / Q000382:microbiology*; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D004270:DNA, Circular; D004277:DNA, Single-Stranded; C036483:T-DNA; D000667:Ampicillin