Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation (original) (raw)
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The EMBO Journal
Communicated by C.J.Leaver We determined whether T-DNA molecules introduced into plant cells using Agrobacterium are suitable substrates for homologous recombination. For the detection of such recombination events different mutant versions of a NPII construct were used. In a first set of experiments protoplasts of Nicotiana tabacum SR1 were cocultivated with two Agrobacterium tumefaciens strains. Each strain contained a different T-DNA, one carrying a 5' deleted NPTII gene and the other a NPMII gene with a 3' deletion. A restored NPMII gene was found in 1-4% of the protoplasts that had been cotransformed with both T-DNAs. Restoration of the NPMI gene could only be the consequence of homologous recombination between the two different T-DNAs in the plant cell, since the possibility of recombination in Agrobacterium was excluded in control experiments. In subsequent experiments we investigated the potential use of Agrobacterium for gene targeting in plants. A transgenic tobacco line with a T-DNA insertion carrying a defective NPTII gene with a 3' deletion was transformed via Agrobacterium with a T-DNA containing a defective NP1'l repair gene. Several kanamycin resistant plant lines were obtained with an intact NPTIM gene integrated in their genome. In one of these lines the defective NPTII gene at the target locus had been properly restored. Our results show that in plants recombination can occur between a chromosomal locus and a homologous T-DNA introduced via A.tumefaciens. This opens the possibility of using the Agrobacterium transformation system for site directed mutagenesis of the plant genome.
Delivery of T-DNA from the Agrobacterium tumefaciens chromosome into plant cells
The EMBO journal, 1984
The intact T-region of the B6Ti plasmid of Agrobacterium tumefaciens was stepwise cloned into a site in transposon Tn3. In this way a suitable vehicle (Tn1882) was obtained for translocating the T-region to different replicons, i.e., to other plasmids or the chromosome. The IncP plasmid R772::Tn1882 conferred tumorigenicity on Agrobacterium if the virulence genes were provided in trans in the same cell. This result showed that the T-region present on Tn1882 was transferred efficiently to plant cells. Normal tumor development also occurred if the T-region was placed in the chromosome of A. tumefaciens and an R' plasmid was present carrying virA-E or virA-F. We conclude that the plasmid location of the T-region is not a prerequisite for transfer to the plant cell. The apparently normal delivery of the T-DNA from a bacterial chromosomal location supports a model involving a processing step within Agrobacterium effecting transfer of the T-region as a separate entity.
T-DNA integration: a mode of illegitimate recombination in plants. EMBO J
The EMBO Journal
Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp. In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides. Formation of precise junctions at the right T-DNA border, and DNA sequence homology between the left termini of T-DNA segments and break-points of target deletions were observed in those cases where full-length canonical T-DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T-DNA segments showed no homology to break-points of target sequence deletions. Homology between short segments within target sites and T-DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T-DNA integration results from illegitimate recombination. The data suggest that while the left T-DNA terminus and both target termini participate in partial pairing and DNA repair, the right T-DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.
Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase
Nucleic Acids Research, 1998
In this study Agrobacterium tumefaciens transferred DNA (T-DNA) was targeted to a chromosomally introduced lox site in Arabidopsis thaliana by employing the Cre recombinase system. To this end, Arabidopsis target lines were constructed which harboured an active chimeric promoter-lox-cre gene stably integrated in the plant genome. A T-DNA vector with a promoterless lox-neomycin phosphotransferase (nptII) fusion was targeted to this genomic lox site with an efficiency of 1.2-2.3% of the number of random events. Cre-catalyzed site-specific recombination resulted in restoration of nptII expression by translational fusion of the lox-nptII sequence in the integration vector with the transcription and translation initiation sequences present at the target site, allowing selective enrichment on medium containing kanamycin. Simultaneously, the coding sequence of the Cre recombinase was disconnected from these same transcription and translation initiation signals by displacement, aimed at preventing the efficient reversible excision reaction. Of the site-specific recombinants, 89% were the result of precise integration. Furthermore, ∼50% of these integrants were single copy transformants, based on PCR analysis. Agrobacterium T-DNA, which is transferred to plant cells as a singlestranded linear DNA structure, is in principle incompatible with Cre-mediated integration. Nevertheless, the results presented here clearly demonstrate the feasibility of the Agrobacterium-mediated transformation system, which is generally used for transformation of plants, to obtain site-specific integration.
Horizontal gene transfer from Agrobacterium to plants
Frontiers in Plant Science, 2014
Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named "cellular T-DNA" (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role.
Illegitimate recombination in plants: a model for T-DNA integration
Genes & Development, 1991
Agrobacterium tumefaciens is a soil bacterium capable of transferring DNA (the T-DNA) to the genome of higher plants, where it is then stably integrated. Six T-DNA inserts and their corresponding preinsertion sites were cloned from Arabidopsis thaliana and analyzed. Two T-DNA integration events from Nicotiana tabacum were included in the analysis. Nucleotide sequence comparison of plant target sites before and after T-DNA integration showed that the T-DNA usually causes only a small (13-28 bp) deletion in the plant DNA, but larger target rearrangements can occur. Short homologies between the T-DNA ends and the target sites, as well as the presence of filler sequences at the junctions, indicate that T-DNA integration is mediated by illegitimate recombination and that these processes in plants are very analogous to events in mammalian cells. We propose a model for T-DNA integration on the basis of limited base-pairing for initial synapsis, followed by DNA repair at the junctions. Variations of the model can explain the formation of filler DNA at the junctions by polymerase slipping and template switching during DNA repair synthesis and the presence of larger plant target DNA rearrangements.