Targeted Integration of T-DNA into the Tobacco Genome at Double-Stranded Breaks: New Insights on the Mechanism of T-DNA Integration

Abstract

Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome. We have investigated targeting of T-DNA by nonhomologous end joining process to a specific double-stranded break created in the plant genome by I-CeuI endonuclease. Sequencing of genomic DNA/T-DNA junctions in targeted events revealed that genomic DNA at the cleavage sites was usually intact or nearly so, whereas donor T-DNA ends were often resected, sometimes extensively, as is found in random T-DNA inserts. Short filler DNAs were also present in several junctions. When an I-CeuI site was placed in the donor T-DNA, it was often cleaved by I-CeuI endonuclease, leading to precisely truncated targeted T-DNA inserts. Their structure requires that T-DNA cutting occurred before or during integration, indicating that T-DNA is at least partially double stranded before integration is complete. This method of targeting full-length T-DNA with considerable fidelity to a chosen break point in the plant genome may have experimental and practical applications. Our findings suggest that insertion at break points by nonhomologous end joining is one normal mode of entry for T-DNA into the plant genome

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

“…More importantly, T-DNA molecules can be digested by rarecutting restriction enzymes prior to their final integration into the plant genome (Chilton and Que, 2003;Tzfira et al, 2003). These observations indicate that it is the dsT-DNA intermediates that function as substrates for the NHEJ integration machinery (Chilton and Que, 2003;Tzfira et al, 2003). Furthermore, sequencing analysis indicates that the digested dsT-DNA molecules may be integrated into the rare-cutter-induced genomic DSBs by a simple NHEJ ligation-like mechanism (Chilton and Que, 2003;Tzfira et al, 2003).…”

“…These observations indicate that it is the dsT-DNA intermediates that function as substrates for the NHEJ integration machinery (Chilton and Que, 2003;Tzfira et al, 2003). Furthermore, sequencing analysis indicates that the digested dsT-DNA molecules may be integrated into the rare-cutter-induced genomic DSBs by a simple NHEJ ligation-like mechanism (Chilton and Que, 2003;Tzfira et al, 2003). These observations led us to suggest that NHEJ-mediated gene replacement might be achieved by coupling the release of a target DNA portion (by the expression of ZFN enzymes) with the delivery of donor T-DNA molecules.…”

“…Induction of DSBs by the transient expression of naturally occurring rare-cutting restriction enzymes results in the incorporation of the T-DNA molecules into a predetermined integration site in the plant cell (Salomon and Puchta, 1998;Chilton and Que, 2003;Tzfira et al, 2003). More importantly, T-DNA molecules can be digested by rarecutting restriction enzymes prior to their final integration into the plant genome (Chilton and Que, 2003;Tzfira et al, 2003).…”

“…More recently, ZFNs have been used for NHEJmediated targeted chromosomal deletions (Lee et al, 2010;Söllü et al, 2010), transgene removal (Petolino et al, 2010), and even NHEJ-mediated targeted DNA integration in mammalian genomes (Orlando et al, 2010). Since genomic DSBs can function as traps for the integration of A. tumefaciens transferred DNA (T-DNA) molecules via NHEJ (Salomon and Puchta, 1998;Chilton and Que, 2003;Tzfira et al, 2003), we decided to explore the possible use of the NHEJ DNA-repair pathway not only for site-specific mutagenesis and targeted gene insertion but also for gene replacement.…”

Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones

“…However, evidence for such a reaction has not been obtained. It has been shown that T-DNA preferentially integrates at double-strand breaks in plant cells (Salomon & Puchta, 1998;Chilton & Que, 2003) and in yeast (van Attikum & Hooykaas, 2003). A role of VirD2 in integration at double-strand breaks has not yet been shown.…”