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

Tzvi Tzfira, Leah Renée Frankman, Manjusha Vaidya, Vitaly Citovsky

Research output: Contribution to journalArticlepeer-review

126 Scopus citations


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.

Original languageEnglish
Pages (from-to)1011-1023
Number of pages13
JournalPlant Physiology
Issue number3
StatePublished - 1 Jan 2003
Externally publishedYes

ASJC Scopus subject areas

  • Physiology
  • Genetics
  • Plant Science


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