Dobule-strand break-induced recombination between ectopic homologous sequences in somatic plant cells

Homologous recombination between ectopic sites is rare in higher eukaryotes . To test whether double-strand breaks (DSBs) can induce ectopic recombinat ion, transgenic tobacco plants harboring two unlinked, nonfunctional homolo gous parts of a kanamycin resistance gene were produced. To induce homologo us recombination between the recipient locus (containing an I-SceI site wit hin homologous sequences) and the donor locus, the rare cutting restriction enzyme I-SceI was transiently expressed via Agrobacterium in these plants. Whereas without I-SceI expression no recombination events were detectable, four independent recombinants could be isolated after transient I-SceI exp ression, corresponding to approximately one event in 10(5) transformations. After regeneration, the F-1 generation of all recombinants showed Mendelia n segregation of kanamycin resistance, Molecular analysis of the recombinan ts revealed that the resistance gene was indeed restored via homologous rec ombination. Three different kinds of reaction products could be identified. In one recombinant a classical gene conversion without exchange of flankin g markers occurred. In the three other cases homologous sequences were tran sferred only to one end of thr break. Whereas in three cases the ectopic do nor sequence remained unchanged, in one case rearrangements were found in r ecipient and donor loci. Thus, ectopic homologous recombination, which seem s to be a minor repair pathway for DSBs in plants, is described best by rec ombination models that postulate independent roles for the break ends durin g the repair process.