T-DNA Integration during Agrobacterium-Mediated Transformation of Plants.

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 Agrobacterium-mediated transformation are still poorly understood. Here, I show that complex extrachromosomal T-DNA structures form in Agrobacterium-infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules can circularize in-planta. I recovered circular T-DNA molecules (T-circles) using a novel plasmid-rescue method that I have developed. 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. Data obtained in this study suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a double stranded form. It is proposed 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. Interestingly, extrachromosomal end-joining between T-DNAs occurred predominantly in a “head-head”/”tail-tail” configuration, which may suggest that the two ends of a T-DNA have different properties. It has also been revealed that a mutant strain of Agrobacterium, which was previously reported to be deficient in the ability to integrate T-DNA into plants, is deficient in the ability to produce T-circles in plants. Because this mutant strain is deleted in VirD2, a protein secreted from Agrobacterium to plants during transformation, results presented in this thesis may provide evidence for a mechanistic role of VirD2 in T-DNA integration in plants. Based on these results, a revised model for T-DNA integration is proposed. According to the proposed model, a double-stranded T-DNA is arranged in a circular - but not fully closed - mode during integration. In addition to revealing more details of an important aspect of natural horizontal gene transfer between prokaryotes and eukaryotes, the data obtained in this project may be important for future research involving applications of Agrobacterium-mediated transformation.