A transient assay in plant cells reveals a positive correlation between extrachromosomal recombination rates and length of homologous overlap

Abstract

An assay to monitor homologous recombination in plant cells has been established by cotransfecting Nicotiana plumbaginifolia protoplasts with different topological forms of plasmids of various deletion mutants of a non-selectable marker gene, the beta-glucuronidase (GUS) gene. Transient GUS enzyme activities were measured by a sensitive assay. In the nuclear DNA of the cotransfected protoplasts the recombined complete GUS gene could be detected by a specially modified PCR analysis. In comparison to the standard assay, which monitors homologous recombination by integration of a selectable marker, the described assay avoids position effects of gene expression, is fast, easy to handle and large numbers of samples can be processed simultaneously. We were able to demonstrate a positive correlation between the length of overlapping homology (up to 1200 base pairs) of the transfected supercoiled circular or linearized plasmids and the respective GUS activities. We found a significant drop in the recombination rates when the overlap of both substrates was reduced to 456 basepairs or less. The requirement for such a long stretch of homology for efficient recombination might ensure the stability of the rather repetitive plant genome.