Abstract
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.
Highlights
Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants
To recover complex circular transferred DNA (T-DNA) structures potentially forming in A. tumefaciens-infected plants, we developed a direct plasmid-rescue method in which circular T-DNA molecules (T-circles) are captured as plasmids in Escherichia coli
In order to distinguish between E. coli colonies that acquired binary plasmids from the A. tumefaciens in the infected plant tissue and E. coli colonies that potentially acquired T-circles from plant cells, each colony was tested for spectinomycin/streptomycin resistance (Fig. 1B, 6)
Summary
Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The transformation process is initiated by the transfer of a single-stranded (ss) DNA segment derived from the A. tumefaciens tumor-inducing (Ti) plasmid into the plant cell nucleus. This transferred DNA (T-DNA) is delimited by two 25-bp direct imperfect repeats known as the right border (RB) and left border (LB), which are required for its transfer. To study the last step in the transformation process, T-DNA integration into the plant genome, T-DNA insertion events have been extensively characterized by sequencing of the T-DNA/chromosome junctions. De Neve et al (1997) proposed that complex T-DNA structures, such as those that involve T-DNA repeats, form when two or more double-stranded (ds) T-DNA intermediates ligate in the plant nucleus prior to integration. The experimental setup of that indirect virus-based system, which may have resulted from recombination by viral components within the T-DNA molecules, did not allow the detection of potential complex T-DNA structures
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