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Site-Specific Integration of Agrobacterium tumefaciens T-DNA via Double-Stranded Intermediates¹

Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794 (T.T., M.V., V.C.); and Westhampton Beach High School, 340 Mill Road, Westhampton Beach, New York 11978 (L.R.F.)

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.

¹ This work was supported by the U.S.-Israel Binational Research and Development Fund (grant to T.T.), by the National Institutes of Health (grant to V.C.), by the National Science Foundation (grant to V.C.), by the U.S. Department of Agriculture (grant to V.C.), and by the U.S.-Israel Binational Science Foundation (grant to V.C.).

^* Corresponding author; e-mail ttzfira{at}notes.cc.sunysb.edu; fax 631–632–8575.

Received August 21, 2003; returned for revision August 28, 2003; accepted August 28, 2003.

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