Positive–Negative selection for homologous recombination in Arabidopsis

Abstract

In plants gene knock-outs and targeted mutational analyses are hampered by the inefficiency of homologous recombination. We have developed a strategy to enrich for rare events of homologous recombination in Arabidopsis using combined positive and negative selection. The T-DNA targeting construct contained two flanking regions of the target alcohol dehydrogenase gene as homologous sequences, and neomycin phosphotransferase and cytosine deaminase as positive and negative markers, respectively. A root explant transformation procedure was used to obtain transgenic calli. Among 6250 transformants isolated by positive selection, 39 were found to be resistant to negative selection as well. Of these 39, at least one had undergone homologous recombination correlated with a unidirectional transfer of information. Although the ADH locus was not changed, our data demonstrate that a homologous recombination event can be selected by positive negative selection in plants.

Introduction

Several studies on homologous recombination in plants have been reported (Mengiste and Paszkowski, 1999), most of them conducted in protoplast systems either by direct DNA transfer (Halfter et al., 1992, Offringa et al., 1993, Paszkowski et al., 1988) or by Agrobacterium-mediated transformation (Offringa et al., 1990, Risseeuw et al., 1995, Risseeuw et al., 1997). Targeted recombination among random insertion events has been found, in most of the cases, at frequencies of the order of 10⁻⁵ (Lee et al., 1990, Miao and Lam, 1995), while in a single report a knock-out event was achieved among 750 transformants (Kempin et al., 1997).

Combined positive and negative selection (PNS) was first developed to enrich for rare homologous recombinants in mouse knockout studies (Mansour et al., 1988). The donor construct includes the targeting sequence, as well as both a positive and a negative selective marker. The positive marker ensures that the recipient cell has integrated the targeting sequence. The negative marker flanks the targeting sequence, so that transformants integrating randomly the entire donor construct are sensitive to negative selection, whereas transformants integrating only the targeting sequence by homologous recombination necessarily do not integrate the negative selective marker as well, and therefore are resistant to negative selection (Fig. 1).

For plants, appropriate markers are available. The widely used positive selective marker NPT (neomycin phosphotransferase) encodes resistance to kanamycin. The negative selective marker codA (cytosine deaminase) catalyses the conversion of non-toxic 5-fluorocytosine (5-FC) to toxic 5′ fluorouracyl (5′-FU) and has been developed for Arabidopsis thaliana (Perera et al., 1993), tobacco and Lotus japonicus (Stougaard, 1993). Recently PNS has been applied to A. thaliana and L. japonicus where a significant enrichment due to the use of codA as negative marker has been described (Gallego et al., 1999, Thykjar et al., 1997). Nevertheless, neither gene replacement nor other types of homologous recombination events were selected despite the use of a long donor targeting sequence (23 kb) (Thykjar et al., 1997), and the analysis of two different loci in both plants.

Here we describe a different approach for PNS in Arabidopsis. In our approach the PNS was applied to the agrobacterial transformation of root explants cultures that have been reported to achieve a higher frequency of single T-DNA insertion (Grevelding et al., 1995), a key factor in the success of a PNS strategy. Embryogenic calli were obtained by the root explants and transformed with the Agrobacterium strain carrying the targeting construct. The target locus in our experimental system is the alcohol deydrogenase gene (ADH) whose activity can be easily evaluated (Jacobs et al., 1988), allowing the analysis of the target functional destruction on the doubly-selected transformants. We were able to select and characterize a single non-reciprocal homologous recombination event with a large transfer of information from one flanking region of the target gene to the incoming T-DNA. As a consequence, an ectopically repaired copy of the ADH gene was integrated elsewhere into the genome. The reported event is the first example of a homologous recombination event selected by PNS in plants and represents a step forward in the development of such a system in plants.