Site-specific integration of Agrobacterium tumefaciens T-DNA via double-stranded intermediates.
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.
Tzfira
Tzvi
T
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794, USA. ttzfira@notes.cc.sunysb.edu
Frankman
Leah Renée
LR
Vaidya
Manjusha
M
Citovsky
Vitaly
V
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D029701:Saccharomyces cerevisiae Proteins; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics
D004269:DNA, Bacterial; D029701:Saccharomyces cerevisiae Proteins; C036483:T-DNA; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific