Targeted recombination in plants using Agrobacterium coincides with additional rearrangements at the target locus

Abstract

The use of Agrobacterium for gene targeting in plants has been investigated. Leaf protoplasts of five transgenic tobacco lines, containing a T-DNA insertion with a defective npt-II gene at different positions in the plant genome, were transformed via Agrobacterium with a T-DNA containing a npt-II repair gene. After selection for kanamycin resistance and PCR analysis six recombinants were derived from four of the target lines. The recombination frequencies were similar for the different target lines with one recombinant from approximately 3 x 10(5) transformants. Apparently gene targeting is more or less independent of the location of the target construct in the plant genome. Molecular analysis revealed that gene targeting had occurred in five of the six recombinant lines. However precise recombination had occurred in only one line, while in the other four lines restoration of the npt-II gene was accompanied by a deletion of part of the target locus. The sixth recombinant line showed restoration of the npt-II gene of the incoming T-DNA construct which was inserted in the plant genome at a position closely linked to the target locus. The different recombination products favour a model in which recombination is via gene conversion followed by reintegration of the synthesized DNA via homologous or illegitimate recombination rather than a reciprocal exchange of DNA between two cross-overs.

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium

“…This process seemed to be restricted to the target locus itself in one case, whereas in the other two cases also sequences flanking the target locus were included. Similar events have been described before in tobacco (27,28).…”

“…Therefore, the modified nptII gene had to reside in an additional T-DNA copy in which the incoming T-DNA was converted by the target locus and then integrated elsewhere. This phenomenon was described before for plants (27,28) and mammals (29) as ectopic targeting. Further PCR analyses showed that two of these modified target locus copies also contained the flanking tobacco DNA sequences of the original B18͞4 locus.…”

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

“…In most of these studies, DNA was introduced into cultured cells by direct gene transfer (10,11) or Agrobacterium-mediated transformation (12)(13)(14)(15). In general, GT efficiencies were low, on both a per-cell basis (Ͻ10 Ϫ7 GT events per cell) and per-integration basis (10 Ϫ6 to 10 Ϫ4 GT events per integration), and negative selection to select against random integrations was not successful (16)(17)(18)(19).…”

High-frequency gene targeting in Arabidopsis plants expressing the yeast RAD54 gene

“…The number of gene-targeting events detected in early studies in plants was too small to permit a statistically reliable estimate of gene-targeting efficiency (3)(4)(5)(6)(7)(8). The recent report of several additional gene-targeting events in two studies has made it possible to estimate the gene-targeting frequency with greater accuracy.…”