T-DNA Border Sequences Research Articles

Role of the overdrive sequence in T-DNA border cleavage in Agrobacterium.

The T-DNA of the Ti plasmid of Agrobacterium is flanked by 25-base-pair imperfect direct repeats that are required in cis for transfer to the genome of the plant host. Another sequence, designated overdrive, is located adjacent to the right-border repeats and functions in cis to enhance tumor formation. We have examined the effect of the overdrive sequence on the early steps in T-DNA processing. We report here that overdrive greatly enhances cleavage by the site-specific endonuclease in Agrobacterium, perhaps by directing the endonuclease to the adjacent border sequences. We also show by a gel mobility-shift assay that overdrive affinity-purified proteins from acetosyringone-induced Agrobacterium cells interact with T-DNA border and overdrive sequences. Further, we show that in vivo the virC operon enhances cleavage at the T-DNA borders, most likely by interaction between the VirC1 protein and the overdrive sequence.

Vir-induced recombination in Agrobacterium: Physical characterization of precise and imprecise T-circle formation

Induction of Ti plasmid virulence ( vir) gene expression during the early stages of plant cell transformation by Agrobacterium tumefaciens initiates the generation of several T-DNA-associated molecular events: (1) site-specific nicks at T-DNA border sequences (border nicks); (2) free, unipolar, linear, single-stranded T-DNA copies (T-strands); and (3) double-stranded, circular T-DNA molecules (T-circles). The first two T-DNA products have been detected in A. tumefaciens, while T-circles have only been detected following Escherichia coli transformation or transduction. The relationship between the three events has not been evaluated since the genesis of T-circles in A. tumefaciens has not been clarified. Evidence is presented here that T-circles are not an artefact of E. coli transformation, but are present as free, double-stranded molecules in A. tumefaciens resulting from site-specific reciprocal recombination between the left and right 25-base-pair border sequences that flank the T-DNA. Furthermore, the frequency of T-circle formation correlates with the frequency of formation of its reciprocal product, the Ti plasmid deleted in the T-DNA region. Several types of recombinant T-DNA circles arise after activation of vir gene expression, a major class representing precise site-specific recombination between both T-DNA borders, and a minor class representing recombination events either utilizing only one T-DNA border sequence and other Ti plasmid sequences, or utilizing only Ti plasmid sequences (i.e. no T-DNA borders). Nucleotide sequence analyses show that when one (nicked) border recombines with other Ti plasmid sequences, a small stretch (16 to 17 base-pairs) of local homology suffices to allow crossing over.

Agroinfection of Nicotiana spp. with Cloned DNA of Tomato Golden Mosaic Virus

Summary Head-to-tail dimers of cloned DNA A and DNA B of tomato golden mosaic virus (TGMV) were integrated between the T-DNA border sequences of a broad host range binary vector and transferred into cells of Nicotiana benthamiana seedlings using an Agrobacterium tumefaciens-mediated delivery system. Most of the inoculated plants developed golden-yellow mosaic and leaf curling symptoms typical of TGMV infection. Extracts of infected leaves were shown to contain both double-stranded and single-stranded TGMV DNA forms of genome length and the virus capsid polypeptide. Infection was also achieved by inoculating plants with mixtures of Agrobacterium strains containing dimers of DNA A or DNA B, with a strain containing a partial dimer of DNA A and a dimer of DNA B and with a strain containing a dimer of DNA B and a partial dimer of DNA A with a 603 bp deletion in the coat protein gene. In the latter case, mosaic symptoms were mild and leaves did not curl. Transgenic N. tabacum cv. Samsun plants containing head-to-tail dimers of DNA A (A2 plants) or DNA B (B2 plants) were produced by transformation with Ti plasmid vectors. A2 plants and B2 plants were agroinfected with dimeric DNA B and dimeric DNA A, respectively. In both cases, symptoms typical of TGMV infection were induced and viral single-stranded DNA of both components was detected in the systemically infected tissue.

The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA.

Agrobacterium tumefaciens transfers T-DNA into the plant genome by a process mediated by Ti plasmid-encoded vir genes. Cleavage at T-DNA border sequences by the VirD endonuclease generates linear, single-stranded T-DNA molecules. In the work described in this report, we used electrophoretic mobility shift assays to show that the purified virE2 gene product binds to single-stranded DNA. VirE2 protein associates with T-DNA as shown by immunoprecipitation studies with VirE2-specific antiserum. The VirE2 protein was detected primarily in the cytoplasm, but also in the inner and outer membrane and periplasmic fractions. Virulence of a virE2 mutant was restored by mixed infection with strains carrying an intact vir region, but not with virA, virB, virD, virE, or virG mutants or chvA, chvB, or exoC mutants. We propose that the VirE2 protein is involved in the processing of T-DNA and in T-strand protection during transfer to the plant cell.

Agroinfection of Triticum aestivum with Cloned DNA of Wheat Dwarf Virus

Summary A head-to-tail dimer of cloned DNA from a Swedish isolate of wheat dwarf virus (WDV) was integrated between the T-DNA border sequences of a broad host range binary vector and transferred into cells of wheat seedlings using an Agrobacterium-mediated delivery system. Two-thirds of the inoculated plants developed a systemic infection. Extracts of infected leaves were shown to contain the virus double-stranded (supercoiled, open circular and linear) and single-stranded DNA forms of unit genome length and the virus capsid polypeptide. The results demonstrate the infectivity of a previously sequenced clone of WDV DNA.

Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5' virD gene products.

Agrobacterium tumefaciens transfers its Ti-plasmid T-DNA to plant cells. This process is initiated by plant-induced activation of the Ti-plasmid virulence loci, resulting in the generation of single stranded (ss) cleavages of the Ti-plasmid T-DNA border sequences (border nicks) and ss linear unipolar T-DNA molecules (T-strands). A single T-strand is produced from the two-border T-region of the pGV3850 nopaline plasmid. In this paper the induced molecular events for the complex T-region of the pTiA6 octopine plasmid are analyzed. This T-region carries four T-DNA borders delimiting three T-DNA elements (TR, TC and TL). Induction of pTiA6 generates cleavages independently at its border repeats, and six distinct T-strand species corresponding to TR, TR/TC, TR/TC/TL, TC, TC/TL and TL. These T-strand molecules are linear and correspond to the bottom strand of the pTiA6 T-region. Thus, borders can function for both initiation and termination of T-strand synthesis. We propose that the different pTiA6 T-strands are independently generated, and that the distribution of border nicks within the parental T-region determines which T-strand is produced. To identify genes involved in T-strand production, pTiA6 virulence (vir) and chromosomal virulence (chv) mutant strains were analyzed. VirA and VirG, the vir regulatory loci are required. Furthermore, the two 5' cistrons of virD are required for both border nicks and T-strands, suggesting that these genes encode the border endonuclease, and that T-strand production is dependent on border nicks. That no mutants are defective for T-strands alone suggests that functions encoded outside of vir and chv might mediate some of the later reactions of T-strand synthesis.

Erratum: Site-Specific Nick in the T-DNA Border Sequence as a Result of Agrobacterium vir Gene Expression

In the report "Site-specific nick in the T-DNA border sequence as a result of Agrobacterium vir gene expression" by K. Wang et al. (30 Jan., p. 587), figure 2A on page 588 should have shown a 3.2-kilobase band in lane b that was not visible. Also, 1.9-kilobase bands in lanes e and g were not visible. A print that shows the bands in figure 2, A and B, appears below. In addition, reference 24 should have been to J. L. Slightom, L. Jouanin, F. Leach, R. F. Drong, D. Tepfer, EMBO J. 4, 3069 (1985). [See table in the PDF file]

Erratum

In the report "Site-specific nick in the T-DNA border sequence as a result of Agrobacterium vir gene expression" by K. Wang et al. (30 Jan., p. 587), figure 2A on page 588 should have shown a 3.2-kilobase band in lane b that was not visible. Also, 1.9-kilobase bands in lane c and g were not visible. A Print that shows the bands in figure 2, A and B, appears below. In addition, reference 24 should have been to J. L. Slightom, L. Jouanin, F. Leach, R. F. Drong, D. Tepfer, EMBO J. 4, 3069 (1985). [See Table A and B in Source PDF].

Site-Specific Nick in the T-DNA Border Sequence as a Result of Agrobacterium vir Gene Expression

The T-DNA transfer process of Agrobacterium tumefaciens is activated by the induction of the expression of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. The vir gene products act in trans to mobilize the T-DNA element from the bacterial Ti plasmid. The T-DNA is bounded by 25-base pair direct repeat sequences, which are the only sequences on the element essential for transfer. Thus, specific reactions must occur at the border sites to generate a transferable T-DNA copy. The T-DNA border sequences were shown in this study to be specifically nicked after vir gene activation. Border nicks were detected on the bottom strand just after the third or fourth base (+/- one or two nucleotides) of the 25-base pair transferpromoting sequence. Naturally occurring and base-substituted derivatives of the 25-base pair sequences are effective substrates for acetosyringone-induced border cleavage, whereas derivatives carrying only the first 15 or last 19 base pairs of the 25-base pair sequence are not. Site-specific border cleavages occur within 12 hours after acetosyringone induction and probably represent an early step in the T-DNA transfer process.

Locations and stability of Agrobacterium-mediated T-DNA insertions in the Lycopersicon genome

The genomic distribution and genetic behavior of DNA sequences introduced into the tomato genome by Agrobacterium tumefaciens were investigated in the backcross progeny of 10 transformed Lycopersicon esculentum x L. pennellii hybrids. All transformants were found to represent single locus insertions based on the co-segregation of restriction fragments corresponding to the T-DNA left and right border sequences in the backcross progeny. Isozyme and restriction fragment length polymorphism (RFLP) markers were used to test linkage relationships of the insertion in each backcross family. The T-DNA inserts in 9 of the 10 transformants were mapped in relation to one or more of these markers, and each mapped to a different chromosomal location. Because only one insertion did not show linkage with the markers employed, it must be located somewhere other than the genomic regions covered by the markers assayed. We conclude that Agrobacterium-mediated insertion in the Lycopersicon genome appears to be random at the chromosomal level. No discrepancies were found between the T-DNA genotype and the nopaline phenotype in the 322 backcross progeny of the nopaline positive transformants. Backcross progeny of two nopaline negative transformants showed incomplete correspondence between the T-DNA genotype and the kanamycin resistance phenotype. No alteration of T-DNA was observed in progeny showing a discrepancy between T-DNA and kanamycin resistance. However, two kanamycin resistant progeny plants of one of these two transformants possessed altered T-DNA restriction patterns, indicating genetic instability of the T-DNA in this transformant.

Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens : Role of T-DNA borders in the transfer process

We have developed a sensitive leaf disc transformation procedure for studying early and/or transient T-DNA expression during Agrobacterium tumefaciens-mediated transformation of plant cells. Using this system, we have examined the function of T-DNA border sequences on the early expression of T-DNA genes and on the stable integration of those genes in infected cells. Deletion of the right border from the T-DNA appears to permit transfer of T-DNA genes from the tumor-inducing (Ti) plasmid but greatly reduces the frequency of their stable integration. A binary vector has been constructed to permit examination of T-DNA border function in trans to the Ti plasmid. In this situation, a single T-DNA border is necessary for early expression of T-DNA genes and is sufficient for stable integration in any orientation.

Activity of T-DNA borders in plant cell transformation by mini-T plasmids.

By using a binary vector system, we examined the requirements for border sequences in T-DNA transformation of plant genomes. Mini-T plasmids consisting of small replicons with different extents of pTiT37 T-DNA were tested for plant tumor-inducing ability in Agrobacterium tumefaciens strain LBA4404 containing helper plasmid pAL4404 (which encodes virulence genes needed for T-DNA transfer). Assays of these bacteria on carrot disks, Kalanchoë leaves, and SR1 Nicotiana tabacum plantlets showed that mini-T plasmid containing full length T-DNA including left and right borders was highly virulent, as were mini-T plasmids containing all onc (oncogenicity) genes and only the right border. In contrast, mini-T plasmids containing all onc genes and only the left border induced tumors only rarely, and a mini-T plasmid containing all onc genes but no T-DNA borders was completely avirulent. Southern hybridization analyses of tumor DNA showed that T-DNA border sequences delimited the extent of the two-border mini-T plasmid transferred and integrated into the plant genome. When only one T-DNA border was present, it formed one end of the transferred DNA, and the other end mapped in the vector sequences. The implications of these results for the mechanism of T-DNA transfer and integration are discussed.

Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts

DNA from a bacterial plasmid containing the T-DNA border sequences of Agrobacterium tumefaciens was transferred into the nucleus or the cytoplasm of tobacco mesophyll protoplasts by microinjection. Following culture in hanging drops, some of these protoplasts produced calli containing the foreign DNA sequences. Evidence for the presence of the injected plasmid DNA in these calli was provided by Southern hybridization analysis. The results demonstrated that random portions of the bacterial plasmid were linked to plant DNA and that integration did not occur at the T-DNA borders present on the injected plasmid. The average number of integrated copies ranged from less than one to 1–2 per tobacco genome. The frequency of integration averaged 14% with intranuclear injections compared to 6% with cytoplasmic injections. With further refinement, the use of microinjection may allow the introduction of many different types of genetic elements into plants.

Vectors for Transformation of Higher Plants

We have constructed several vectors for the cloning of foreign DNA into plant genomes. These vectors are based on the transformation system of Agrobacterium tumefaciens and incorporate a number of useful features for plant cell transformation. The prototype vector, pEND4K, contains the left and right T-DNA border sequences and a chimeric gene which confers kanamycin resistance on transformed plant cells. The vector also includes the following features that facilitate the bacterial manipulations: the bacteriophage lambda cos site for in vitro packaging of large inserts, alpha complementation for direct screening of cloned inserts, and the wide host range RK2 replication and transfer origins. The vectors have been demonstrated to efficiently transform plant cells by several criteria.

The SEV System: A New Disarmed Ti Plasmid Vector System for Plant Transformation

A derivative of the octopine-type Ti plasmid, pTiB6S3, was constructed in which the TL-DNA oncogenic functions, the TL-DNA right border sequence and all of TR-DNA were deleted and replaced with the kanamycin antibiotic resistance marker from Tn903 (601). The resulting avirulent plasmid, pTiB6S3–SE contains only the TL-DNA left border sequence and a region (1.6 kb) of homologous DNA (LIH) to allow recombination with intermediate vectors such as pMON120 or pMON200. Cointegrate formation between pTiB6S3–SE and pMON200, which contains a chimeric plant kanamycin resistance gene (Kmr), an intact nopaline synthase gene (NOS), a poly linker region to facilitate the insertion of foreign genes into the vector and a functional T-DNA right border sequence, results in the formation of a selectable, avirulent T-DNA. This vector system is referred to as the SEV system (Split End Vector) since the T-DNA border sequences are present on separate plasmids prior to recombination. Plant cells (tobacco, petunia and tomato) transformed with SEV T-DNA are easily identified by their resistance to kanamycin and their ability to synthesize nopaline. A major advantage of the new disarmed system is that all transformed colonies can potentially be regenerated into intact plants that normally express and segregate the inserted foreign DNA sequences.

Binary Agrobacterium vectors for plant transformation.

A vector molecule for the efficient transformation of higher plants has been constructed with several features that make it efficient to use. It utilizes the trans acting functions of the vir region of a co-resident Ti plasmid in Agrobacterium tumefaciens to transfer sequences bordered by left and right T-DNA border sequences into the nuclear genome of plants. The T-region contains a dominant selectable marker gene that confers high levels of resistance to kanamycin, and a lac alpha-complementing region from M13mp19 that contains several unique restriction sites for the positive selection of inserted DNA.