Site-specific integration of Agrobacterium tumefaciens T-DNA via double-stranded intermediates

Abstract

Agrobacterium tumefaciens-mediated genetic transformation involves transfer of a single-stranded T-DNA molecule (T strand) into the host cell, followed by its integration into the plant genome. The molecular mechanism of T-DNA integration, the culmination point of the entire transformation process, remains largely obscure. Here, we studied the roles of double-stranded breaks (DSBs) and double-stranded T-DNA intermediates in the integration process. We produced transgenic tobacco (Nicotiana tabacum) plants carrying an I-SceI endonuclease recognition site that, upon cleavage with I-SceI, generates DSB. Then, we retransformed these plants with two A. tumefaciens strains: one that allows transient expression of I-SceI to induce DSB and the other that carries a T-DNA with the I-SceI site and an integration selection marker. Integration of this latter T-DNA as full-length and I-SceI-digested molecules into the DSB site was analyzed in the resulting plants. Of 620 transgenic plants, 16 plants integrated T-DNA into DSB at their I-SceI sites; because DSB induces DNA repair, these results suggest that the invading T-DNA molecules target to the DNA repair sites for integration. Furthermore, of these 16 plants, seven plants incorporated T-DNA digested with I-SceI, which cleaves only double-stranded DNA. Thus, T-strand molecules can be converted into double-stranded intermediates before their integration into the DSB sites within the host cell genome.

Figure 1.

Maps of the T-DNA regions. A, pBNE3I. B, pBIG-HYG-terGUS/I-SceI. C, Nucleotide sequence of the I-SceI site between the T-DNA left border and the uidA gene in pBIG-HYG-terGUS/I-SceI. LB, T-DNA left border; RB, T-DNA right border; 35S, cauliflower mosaic virus 35S RNA promoter; nptII, kanamycin resistance gene; codA, cytosine deaminase gene, uidA; β-glucoronidase (GUS) gene; hpt, hygromycin resistance gene;NOS5′, nopaline synthase promoter;NOS3′, nopaline synthase terminator; pAg7, agropine synthase terminator.

Figure 2.

Orientation of the integrated T-DNA within DSB sites. A and B, Two possible orientations for integration of the I-SceI-uidA-hpt T-DNA. C and D, Two possible orientations for integration of the I-SceI-digested I-SceI-uidA-hpt T-DNA. I-SceI sites are shown within the integrating T-DNA and in the DSB site. Arrowheads indicate the left-to-right border direction of T-DNA. Designations of each T-DNA border/DSB site integration junction are indicated.

Figure 3.

Nucleotide sequence of integration junctions between the I-SceIORF T-DNA and DSB. Upper strands of the T-DNA and the DSB site are shown. The I-SceI site is indicated in capital letters, and homologous base pairs are highlighted in gray. Numbers within the nucleotide sequences indicate the size of large deletions (see also Table III).

Figure 4.

Nucleotide sequence of integration junctions between the I-SceI-uidA-hpt T-DNA and DSB. Upper strands of the T-DNA and the DSB site are shown. The I-SceI site is indicated in capital letters, and homologous base pairs are highlighted in gray.

Figure 5.

Nucleotide sequence of integration junctions between the I-SceI-digested I-SceI-uidA-hpt T-DNA and DSB. Upper strands of the T-DNA and the DSB site are shown. The I-SceI site is indicated in capital letters, and homologous base pairs are highlighted in gray.