Characterization of T-DNA integration in creeping bentgrass

Abstract

Agrobacterium-mediated transformation provides a powerful tool for plant biotechnology. Great efforts have been made to unravel the underlying mechanisms of T-DNA integration. Creeping bentgrass ( Agrostis palustris Huds.), an economically important turfgrass species, has been widely studied for genetic transformation. In order to characterize T-DNA integration in this species, we analyzed T-DNA junction sequences in transgenic bentgrass obtained by transformation with A. tumefaciens strain EHA101 carrying a pPZP201-derived plasmid. Using T-DNA specific primers, inverse polymerase chain reaction (IPCR) and standard PCR were performed to identify T-DNA/plant junctions and T-DNA/T-DNA junctions, respectively. Sequence comparisons revealed that T-DNA right border (RB) termini were more intact, represented by a smaller range of border deletions and no backbone integration, compared to T-DNA left border (LB) termini. Multiple T-DNA arrays inserted at a single chromosome locus were detected in various configurations. It was shown that the formation of the multiple T-DNA arrays was governed by illegitimate recombination (IR), with sequence microsimilarities over and/or close to the T-DNA breaking points. It was also demonstrated that the fusion of two T-DNA copies was frequently associated with the formation of filler DNA and significant T-DNA truncation. Compositional analysis over the bentgrass sequences revealed a similar A/ T ( A + T) content but a polarized distribution of A and T associated with LB flanking sequence tags (FSTs) and RB FSTs, which was further documented in Arabidopsis T-DNA FSTs. DNA folding analysis on T-DNA FSTs suggested that the T-DNA target region had a potential of forming secondary structures, which may designate an active region for T-DNA integration. The results presented in this study shed new light on microsimilarity-based illegitimate recombination and advance our understanding of T-DNA integration.