Agrobacterium tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism

Abstract

During crown gall tumorigenesis, part of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid, the T-DNA, integrates into plant DNA. Direct repeats define the left and right ends of the T-DNA, but tumorigenesis requires only the right-hand repeat. Virulence (vir) genes act in trans to mobilize the T-DNA into plant cells. Transfer of T-DNA begins when the VirD endonuclease cleaves within the right-hand border repeat. Although the T-DNA right-border repeat promotes T-DNA transmission best in its normal orientation, an inverted right border exhibits reduced but significant activity. Two models may account for this diminished tumorigenesis. The right border may function bidirectionally, with strong activity only in its wild-type orientation, or it may promote T-DNA transfer in a unidirectional manner such that, with an inverted right border, transfer proceeds around the entire Ti plasmid before reaching the T-DNA. To determine whether a substantial portion of the Ti plasmid is transferred to plant cells, as predicted by the unidirectional-transfer hypothesis, we examined T-DNAs in tumors induced by strains containing a Ti plasmid with a right border inverted with respect to the T-DNA oncogenes. These tumors contained extremely long T-DNAs corresponding to most or all of the Ti plasmid. To test whether the right border can function bidirectionally, we inserted T-DNAs with either a properly oriented or an inverted right border into a specific site in the A. tumefaciens chromosome. A border situated to transfer the oncogenes first directed T-DNA transfer even from the bacterial chromosome, whereas a border in the opposite (inverted) orientation did not transfer the oncogenes to plant cells. Our results indicate that the right-border repeat functions in a unidirectional manner.

Integration of T‐DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration

“…Because T-DNA transfer is apparently a polar process, initiating at the right border and continuing to (and perhaps beyond) the left border (Caplan et al, 1985;Joos et al, 1983;Miranda et al, 1992;Peralta et al, 1986), we expected that plants generated using pBSG-t w o u l d have a higher probability of receiving and integrating the mas2'-gusA gene than would plants generated using pBSG-2. Our data, however, indicated that there was no major difference in the transfer of the gusA gene whether this gene were located close to the left or to the right T-DNA border.…”

Intragenic vectors for gene transfer without foreign DNA

“…Agrobacterium-mediated transformation is the preferred method for gene transfer because it is driven by biological processes and results in a high frequency of single locus insertion events without rearrangements of the transferred DNA (Gheysen et al 1998;Gelvin 2003). Furthermore, Agrobacterium-mediated transformation can effect the transfer of very large fragments (150-200 kb) into plant genomes (Miranda et al 1992;Hamilton et al 1996). …”

T-DNA–mediated transfer of Agrobacterium tumefaciens chromosomal DNA into plants

“…Additionally, T-DNA backbone sequences highly similar to AchrDNA (such as IS and their remnants) could be involved in homologous recombination of the entire T-DNA vector into AchrDNA. T-DNA with intact border sequences, which was deliberately inserted into bacterial chromosomes by homologous recombination, can be transferred to plants 13 .…”