T‐DNA recombination and replication in maize cells

Abstract

SummaryT-DNA recombination and replication was analyzed in 'black mexican sweet' (BMS) cells transformed with T-DNAs containing the replication system from wheat dwarf virus (WDV). Upon recombination between the T-DNA ends, a promoterless marker gene (gusA) was activated. Activation of the recombination marker gene was delayed and increased exponentially over time, suggesting that recombination and amplification of the T-DNA occurred in maize cells. Mutant versions of the viral initiator gene (rep), known to be defective in the replication function, failed to generate recoverable recombinant T-DNA molecules. Circularization of T-DNA by the FLP/FRT site-specific recombination system and/or homologous recombination was not necessary to recover circular T-DNAs. However, replicating T-DNAs appeared to be suitable substrates for site-specific and homologous recombination. Among 33 T-DNA border junctions sequenced, only one pair of identical junction sites was found implying that the population of circular T-DNAs was highly heterogenous. Since no circular T-DNA molecules were detected in treatments without rep, it suggested that T-DNA recombination was linked to replication and might have been stimulated by this process. The border junctions observed in recombinant T-DNA molecules were indicative of illegitimate recombination and were similar to left-border recombination of T-DNA into the genome after Agro-mediated plant transformation. However, recombination between T-DNA molecules differed from T-DNA/genomic DNA junction sites in that few intact right borders were observed. The replicating T-DNA molecules did not enhance genomic random integration of T-DNA in the experimental configuration used for this study.

Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants

“…Previous studies have shown that linearized plasmids recircularize in protoplasts (Gorbunova and Levy, 1997;Gallego et al, 2003). However, circularization of ds T-DNAs in plants was considered a relatively rare event until now, because evidence for circularization was indirect and required a viral amplification system (Bakkeren et al, 1989;Zhao et al, 2003). Our ability to detect T-circles directly from infected plants using a plasmid-rescue approach suggests that circularization of ds T-DNAs may not be as rare as previously thought.…”

“…Sequencing of end-joining junctions in our T-circles revealed that their ends had none or only a few base pairs of overlapping microhomology (Supplemental Fig. S3), thus indicating that our T-circles did not form through homologous recombination, as occurs in A. tumefaciens, but through illegitimate end joining, which is typical of the circularization of T-DNAs in infected plants (Bakkeren et al, 1989;Zhao et al, 2003). Moreover, T-circles containing T-DNAs from different A. tumefaciens strains were generated following coagroinfiltration (Figs.…”

Stable Recombinase-Mediated Cassette Exchange in Arabidopsis UsingAgrobacterium tumefaciens

“…1D). In the case of T-DNA with the simple RMCE cassette, T-DNA border ligation may result in a similar circular substrate, but probably this does not occur frequently (Bundock et al, 1995;Vergunst and Hooykaas, 1998;Zhao et al, 2003). Here, we used a cotransformation approach to introduce Cre recombinase in the same cell as the exchange T-DNAs by means of an Agrobacterium strain harboring a T-DNA vector containing a strong expression unit for the cre gene.…”

“…Based on several studies, it is known that T-DNA becomes double stranded prior to integration (Offringa et al, 1990;Mozo and Hooykaas, 1992;Narasimhulu et al, 1996;Chilton and Que, 2003;Tzvira et al, 2003). Although circle formation of T-DNA by ligation of the two T-DNA borders has been reported (Bakkeren et al, 1989;Bundock et al, 1995;Zhao et al, 2003), the frequency of occurrence remains unclear. Pansegrau and coworkers (1993) have shown that VirD2 can mediate ligation of the two T-DNA borders in vitro.…”

“…In vivo, circular T-DNA was only recovered when it was adapted with a replication origin. This was the case when a viral replicon was placed on the T-DNA in maize (Zea mays) cells (Zhao et al, 2003) and turnip (Brassica napus; Bakkeren et al, 1989), or in the presence of the replicator from the yeast (Saccharomyces cerevisiae) 2m plasmid (Bundock et al, 1995). These data suggest that border ligation may occur, but is not efficient.…”