Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants

• Published in: Plant physiology (Bethesda) Vol. 160; no. 1; pp. 511 - 522
• Main Authors: Singer, Kamy, Shiboleth, Yoel M., Li, Jianming, Tzfira, Tzvi
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Biologists 01.09.2012
• Subjects: Agrobacterium; Agrobacterium tumefaciens; Agrobacterium tumefaciens - pathogenicity; Ampicillin - pharmacology; Biological and medical sciences; Cloning, Molecular; DNA; DNA End-Joining Repair; DNA, Bacterial - genetics; DNA, Circular - genetics; DNA, Single-Stranded - genetics; Drug Resistance, Bacterial; Escherichia coli - drug effects; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genetic Vectors - genetics; Genomes; Molecules; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana - microbiology; Plant architecture; Plant cells; Plant Diseases - microbiology; Plant physiology and development; Plants; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Plants, Genetically Modified - microbiology; Plasmids; Plasmids - genetics; Sequence Analysis, DNA - methods; Sequencing; SYSTEMS BIOLOGY, MOLECULAR BIOLOGY, AND GENE REGULATION; Transformation, Genetic; Infection; Agrobacterium tumefaciens; Bacteria; Rhizobiaceae; T-DNA; Plant physiology
• ISSN: 0032-0889; 1532-2548; 1532-2548
• DOI: 10.1104/pp.112.200212

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. tumefaciensmediated 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.