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.

Figure 1.

Schematic drawing of vector T-DNA regions. LB, T-DNA left border repeat; RB, T-DNA right border repeat; BAR, Basta resistance gene; N, the 5′-region of NPT (neomycin phosphotransferase) gene conferring kanamycin resistance; PT, the 3′-end of NPT gene. The first intron of Arabidopsis CHC1 gene (http://www.chromdb.org/) was used to separate the 5′ and 3′ region of the NPT gene. HPT, Hygromycin phosphotransferase; I-CeuI, I-CeuI endonuclease cleavage sequence; Phsp80, Brassica oleracea HSP80 promoter; Pubq3, Arabidopsis ubiquitin 3 promoter; Psmas, super-MAS promoter; Tnos, nopaline synthase 3′-untranslated region (UTR); Tpal, Arabidopsis phenylalanine aminolyase-1 3′-UTR; Tubq3, Arabidopsis ubiquitin-3 3′-UTR; I-CeuIEN, synthetic I-CeuI endonuclease gene. The following vectors have VS1 origin of replication: pNOV034, pNOV039, pNOV2729, pNOV5028, and pNOV5040. These vectors have RK2 origin of replication: pNOV035, pNOV5025, and pNOV5041.

Figure 2.

Schematic representation of targeted T-DNA insertion and primers used for PCR analysis. A, Whole-donor T-DNA is integrated into the cleaved I-CeuI site in the target locus if the I-CeuI site in the donor T-DNA is not cleaved. B, I-CeuI site in the donor T-DNA is cleaved, and the truncated T-DNA is integrated at the cleaved I-CeuI site of the target locus. PCR primers used for amplification: 1, PSMASFW2 (5′-CCG GTG AGT AAT ATT GTA CGG CTA AGA-3′); 2, NPTR6 (5′-AGA TCC TCA GAA GAA CTC GTC AAG AAG-3′); 3, NPTFA (5′-GAT CTC TAG AAT GAT TGA ACA AGA TGG ATT–3′); 4, INTBAFRV (5′-GCC GCG CTG CCT CGT CCT GAA AAA TTC AGA AA-3′) or NPTR2 (5′-GAA TAG TAC TAA TAC CTG GCA CTT CGC CCA ATA G-3′); 5, BAR78FW (5′-CAC TAC ATC GAG ACA AGC ACG GTC AAC T-3′); 6, HYG707RV (5′-CGG CCT CCA GAA GAA GAT GT–3′); 7, KAN97RV (5′-CAG AGC AGC CGA TTG TCT GT-3′); and 8, UBQPR182rv (5′-CTC CTC ACT CTT CTG CTA CAG ACT CGG AAC TC-3′).

Figure 3.

DNA sequence at right junctions of targeted insertions with pNOV035 T-DNA. Donor T-strand and host plant I-CeuI cleavage site are illustrated at the top of the figure, together with a theoretically “perfect” junction of T-strand LB to the long arm of the I-CeuI site. In the donor T-DNA, the LB repeat sequence is underlined, and its VirD2-nick site is indicated by a slash. In the target DNA, the I-CeuI recognition sequence is highlighted in red, and the 4-bp overhang sequence is highlighted in blue. Short filler sequences of unknown origin found in these events are shown in lowercase letters and highlighted in green. In several cases where 1 or 2 bp could be attributed either to the insert or to the host DNA (termed microhomology regions by other authors), we have assigned them to the host. This was noted in a minority of events and may be mechanistically significant but clearly is not a requirement for integration. Event 12CG.1 has an 80-bp of filler DNA inserted at “**” that is a direct repeat of part of the intron just beyond the I-CeuI target site.