Plant Physiology Preview
Published on October 9, 2003; 10.1104/pp.103.032128
Received August 21, 2003
Returned for revision August 28, 2003
Accepted August 28, 2003
Site-Specific Integration of Agrobacterium tumefaciensT-DNA via Double-Stranded Intermediates
Tzvi Tzfira *, Leah Renée Frankman , Manjusha Vaidya , and Vitaly Citovsky
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York11794-5215 (T.T., M.V., V.C.); and Westhampton Beach High School, 340 Mill Road, Westhampton Beach,New York 11978 (L.R.F.)
* Corresponding author; email: ttzfira{at}notes.cc.sunysb.edu.
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.
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