Proc. Natl. Acad. Sci. USA, 96:8321-8323, June 1999

Stimulation of homologous recombination in plants by expression of the bacterial resolvase RuvC

Gil Shalev, Yaron Sitrit, Naomi Avivi-Ragolski, Conrad Lichtenstein, and Avraham A. Levy

*Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel; and School of Biological Sciences, Queen Mary and Westfield College, University of London, London E1 4NS, United Kingdom

Targeted gene disruption exploits homolo-gous recombination (HR) as a powerful reverse genetic tool, for example, in bacteria, yeast, and transgenic knockout mice, but it has not been applied to plants, owing to the low frequency of HR and the lack of recombinogenic mutants. To increase the frequency of HR in plants, we constructed transgenic tobacco lines carrying the Escherichia coli RuvC gene fused to a plant viral nuclear localization signal. We show that RuvC, encoding an endonuclease that binds to and resolves recombination intermediates (Holliday junctions) is properly transcribed in these lines and stimulates HR. We observed a 12-fold stimulation of somatic crossover between genomic sequences, a 11-fold stimulation of intrachromo-somal recombination, and a 56-fold increase for the frequency of extrachromosomal recombination between plasmids co-transformed into young leaves via particle bombardment. This stimulating effect may be transferred to any plant species to obtain recombinogenic plants and thus constitutes an impor-tant step toward gene targeting.

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