A new transgene assay to study microsatellite instability in wild-type and mismatch-repair defective plant progenies

DNA mismatch repair (MMR) genes are essential for maintaining the fidelity of DNA replication in prokaryotes and eukaryotes. In most species, from Escherichia coli to human, alterations in this system result in elevated frequencies of microsatellite length alterations. In the present report, we designed an in planta assay system to measure the frequencies of microsatellite instability in Arabidopsis thaliana. A transgenic reporter line was selected in which a (CT)(23) dinucleotide disrupts the reading frame of a downstream protein (phosphinotricin acetyltransferase). Microsatellite variations that restore the reading frame can be scored as glufosinate-resistant seedlings in the progenies. The spontaneous frequency of reversion at the transgene in the transgenic line is about 4 x 10(-4) and is stable over at least four generations. We used this assay system to evaluate the effect of an alteration of the function of ATMSH2, the Arabidopsis homologue of MSH2, on microsatellite stability. Homozygous plants of the ATMSH2 insertion mutant line Salk_002708 show a 60-fold increase of the reversion frequency in their progenies. This effect was confirmed by RNAi inactivation of ATMSH2. The reporter line can thus be used either to perform functional analyses of genes putatively involved in MMR control or to evaluate the effect of biotic and abiotic stress on microsatellite stability in plant progenies. (c) 2004 Elsevier Ireland Ltd. All rights reserved.