Molecular characterization of T-DNA integration sites in transgenic birch

The integration and structure of a transgene locus can have profound effects on the level and stability of transgene expression. We screened 28 transgenic birch (Betula platyphylla Suk.) lines transformed with an insect-resistance gene (bgt) using Agrobacterium tumefaciens. Among the transgenic plants, the copy number of transgene varied from one to four. A rearrangement or partial deletion had occurred in the process of T-DNA integration. T-DNA repeat formation, detected by reverse primer PCR, was found among randomly screened transgenic lines. Sequencing of the junctions between the T-DNA inserts revealed deletions of 19–589 bp and an additional 45 bp filler DNA sequence was inserted between the T-DNA repeats at one junction. Micro-homologous sequences (1–6 bp) were observed in the junctions between the T-DNA inserts. Using SiteFinding-PCR, a relatively high percentage of AT value was found for the flanking regions. Deletion of the right border repeat was observed in 12/18 of the T-DNA/plant junctions analyzed. The number of nucleotides deleted varied from 3 to 712. Deletions of 17–89 bp were observed in all left T-DNA/plant junctions analyzed. A vector backbone DNA sequence in the transgene loci was also detected using primer pairs outside the left and right T-DNA borders. Approximately 89.3% of the lines contained some vector backbone DNA. These observations revealed that it is important to check the specificity of the integration. A mechanism of T-DNA transport and integration is proposed for this long-lived tree species.     

Microhomologies between T-DNA ends and target sites often occur in inverted orientation and may be responsible for the high frequency of T-DNA-associated inversions

Sequence analysis of left and right border integration sites of independent, single-copy T-DNA inserts in Arabidopsis thaliana revealed three previously unrecognized concomitants of T-DNA integration. First, genomic pre-insertion sites shared sequence similarity not only with the T-DNA left and right border regions, as was previously reported, but also at high frequency with the inverted complement of the T-DNA right border region. Second, palindromic sequences were frequently found to overlap or lie adjacent to genomic target sites, suggesting a high recombinogenic potential for palindromic elements during T-DNA integration and a possible role during the primary contact between the T-DNA and the target DNA. Third, “filler” DNA sequences between genomic pre-insertion site DNA and T-DNA often derive from sequences in the T-DNA left and right border regions that are clustered around palindromic sequences in these T-DNA regions, suggesting that these palindromic elements are “hot spots” for filler DNA formation. The discovery of inverted sequence similarities at the right border suggests a previously unrecognized mode of T-DNA integration that involves heteroduplex formation at both T-DNA borders and with opposite strands of the target DNA. Scanning for sequence similarities in both direct and inverted orientation may increase the probability and/or effectiveness of anchoring the T-DNA to the target DNA. Variations on this scheme may also account for inversion events at the target site of T-DNA integration and inverted T-DNA repeat formation, common sequence organization patterns associated with T-DNA integration. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions

Plant transformation via Agrobacterium frequently results in formation of multiple copy T-DNA arrays at one target site of the chromosome. The T-DNA copies are arranged in repeats, direct or inverted around one of the T-DNA borders. A Ti plasmid-derived transformation vector has been constructed enabling direct selection of transformants carrying at least two linked copies of T-DNA in the same orientation. The selection is based on expression of a promoterless neomycin phosphotransferase gene on one T-DNA copy from a promoter located on the other T-DNA copy. After co-cultivation of tobacco protoplasts with Agrobacterium, as many as 30% of regenerated transformed plants carried directly repeated T-DNA copies. The junction regions between two T-DNAs were amplified and 13 amplified fragments were cloned and sequenced. The involvement of T-DNA left and right border sequences in direct repeat junctions was determined. In some junctions, additional filler DNA was detected. The length of filler DNA varied from a few up to almost 300 bp. The longer filler DNAs from two clones were found to be T-DNA fragments in direct or reverse orientation. We discuss the recently suggested models for T-DNA integration and propose that the formation of direct repeats in genomes does not necessarily result from ligation of intermediates (i.e. T-strands), but more likely from the co-integration of several intermediates into one target site.     

T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation

Transgenic Arabidopsis and tobacco plants (125) derived from seven Agrobacterium-mediated transformation experiments were screened by polymerase chain reaction and DNA gel blot analysis for the presence of vector `backbone' sequences. The percentage of plants with vector DNA not belonging to the T-DNA varied between 20% and 50%. Neither the plant species, the explant type used for transformation, the replicon type nor the selection seem to have a major influence on the frequency of vector transfer. Only the border repeat sequence context could have an effect because T-DNA vector junctions were found in more than 50% of the plants of three different transformation series in which T-DNAs with octopine borders without inner border regions were used. Strikingly, many transgenic plants contain vector backbone sequences linked to the left T-DNA border as well as vector junctions with the right T-DNA border. DNA gel blots indicate that in most of these plants the complete vector sequence is integrated. We assume that integration into the plant genome of complete vector backbone sequences could be the result of a conjugative transfer initiated at the right border and subsequent continued copying at the left and right borders, called read-through. This model would imply that the left border is not frequently recognized as an initiation site for DNA transfer and that the right border is not efficiently recognized as a termination site for DNA transfer.     

Transgene repeats in aspen: molecular characterisation suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome

Rearrangements of T-DNAs during genetic transformation of plants can result in the insertion of transgenes in the form of repeats into the host genome and frequently lead to loss of transgene expression. To obtain insight into the mechanism of repeat formation we screened 45 transgenic lines of aspen and hybrid aspen transformed with six different gene constructs. The frequency of T-DNA repeat formation among randomly screened transgenic lines was found to be about 21%. In ten transgenic lines direct repeats were detected. An inverted repeat was found in one other transgenic line. Sequencing of the junctions between the T-DNA inserts revealed identical residual right-border repeat sequences at the repeat junctions in all ten transgenic lines that had direct repeats. Formation of "precise" junctions based on short regions of sequence similarity between recombining strands was observed in three transgenic lines transformed with the same plasmid. Additional DNA sequences termed filler DNAs were found to be inserted between the T-DNA repeats at eight junctions where there was no similarity between recombining ends. The length of the filler DNAs varied from 4 to almost 300 bp. Small filler DNAs--a few base pairs long--were in most cases copied from T-DNA near the break points. The large filler sequences of about 300 bp in two transgenic lines were found to be of host plant origin, suggesting that transgene repeat formation occurred as a result of the simultaneous invasion of a receptive site in the host genome by two independent T-DNA strands. On the basis of the results obtained, and in the light of previous reports on T-DNA/plant DNA junctions in aspen and other crop plants, a mechanistic model for transgene rearrangement and filler formation is suggested.     

Analysis of TR-DNA/plant junctions in the genome of a Convolvulus arvensis clone transformed by Agrobacterium rhizogenes strain A4

A Charon 4A phage library, containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant genetically transformed by Ri T-DNA from Agrobacterium rhizogenes strain A4, was used to isolate a lambda clone that contains part of the Ri TL-DNA and the complete TR-DNA. The two Ri T-DNAs were recovered adjacent to each other in a tail-to-tail configuration (i.e. with the TR-DNA inverted with respect to the TL-DNA). Comparison of nucleotide sequences from this lambda clone with the corresponding sequences from the Ri plasmid allowed us to determine the location of the T-DNA/plant junction for the right end of the TL-DNA and the left and right ends of the TR-DNA. We located, near each of these borders, a 24 bp sequence that is similar to the 24 bp consensus sequence found near the pTi T-DNA extremities. In addition, sequences similar to the core overdrive sequence from pTi are located near each right border. Hybridization and nucleotide sequence analysis of the DNA adjacent to the TL/TR junction shows that no plant DNA is located between the TL and TR-DNAs and suggests that the plant DNA adjacent to the end of the TR-DNA may have been rearranged during the integration into the plant genome.     

Characterization of T-DNA insertions in transgenic grapevines obtained by Agrobacterium-mediated transformation

T-DNA integration patterns in 49 transgenic grapevines produced via Agrobacterium-mediated transformation were analyzed. Inverse PCR (iPCR) was performed to identify T-DNA/plant junctions. Sequence comparison revealed several deletions in the T-DNA right border (RB) and left border (LB), and filler DNA and duplications or deletions of grapevine DNA at the T-DNA insertion loci. In 20 T-DNA/grapevine genome junctions microsimilarities were found associated with the joining points and in all grapevine lines microsimilarities were present near the breaking points along the 30 bases of T-DNA adjacent to the two borders. Analysis of target site preferences of T-DNA insertions indicated a non-random distribution of the T-DNA, with a bias toward the intron regions of the grapevine genes. Compositional analysis of grapevine DNA around the T-DNA insertion sites revealed an inverse relationship between the CG and AT-skews and AT rich sequences present at 300–500 bp upstream the insertion points, near the RB of the T-DNA. PCR assays showed that vector backbone sequences were integrated in 28.6% of the transgenic plants analyzed and multiple T-DNAs frequently integrated at the same position in the plant genome, resulting in the formation of tandem and inverted repeats.     

The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration

After Agrobacterium-mediated plant transformation, multiple T-DNAs frequently integrate at the same position in the plant genome, resulting in the formation of inverted and direct repeats. Because these inverted repeats cannot be amplified and analyzed by PCR, Arabidopsis root cells were co-transformed with two different T-DNAs with distinct sequences adjacent to the T-DNA borders. Nine direct or inverted T-DNA border junctions were analyzed at the sequence level. Precise end-to-end fusions were found between two right border ends, whereas imprecise fusions and filler DNA were present in T-DNA linkages containing a left border end. The results suggest that end-to-end ligation of double-stranded T-DNAs occurs especially between right T-DNA ends and that illegitimate recombination on the basis of microhomology, deletions, repair activities and insertions of filler DNA is involved in the formation of left border T-DNA junctions. Therefore, a similar illegitimate recombination mechanism is proposed that is involved in the formation of complex T-DNA inserts as well as in the integration of the T-DNA in the plant genome.     

Deviating T-DNA transfer fromAgrobacterium tumefaciens to plants

We analyzed 29 T-DNA inserts in transgenicArabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA. DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data. Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences. Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T-region. On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat.     

Sequence context of the T-DNA border repeat element determines its relative activity during T-DNA transfer to plant cells

Summary We present a detailed analysis of the function of the right and left T-DNA border regions of the nopaline Ti plasmid of Agrobacterium tumefaciens. An avirulent deletion of the right border of the nopaline Ti plasmid (pGV3852) was used as an acceptor for 14 different T-DNA border constructs. The functional activities of these constructs were assayed by their ability to restore virulence, i.e. transformation on inoculated plants. Tumorigenicities were measured in several independent experiments over a 2 year period and the statistical significance of their relative levels was evaluated. The data indicate: (i) the entire sequence of the 25 bp direct repeat of the T-DNA is required to provide an efficient substrate for mediating T-DNA transfer events; deletion derivatives of either the conserved or the vaiable domain of the repeat are defective in T-DNA transfer; (ii) while the 25 bp direct repeat alone can promote the T-DNA transfer, the flanking sequences of the repeats enhance (on the right) or attenuate (on the left) their activity; and (iii) tumorigenicity measurements vary depending on the plant assay system: potato discs are more sensitive than wounded tobacco leaves in detecting differences in T-DNA border activity.     

Transgene integration in aspen: structures of integration sites and mechanism of T-DNA integration

To obtain insight into the mechanism of transferred DNA (T-DNA) integration in a long-lived tree system, we analysed 30 transgenic aspen lines. In total, 27 right T-DNA/plant junctions, 20 left T-DNA/plant junctions, and 10 target insertions from control plants were obtained. At the right end, the T-DNA was conserved up to the cleavage site in 18 transgenic lines (67%), and the right border repeat was deleted in nine junctions. Nucleotides from the left border repeat were present in 19 transgenic lines out of 20 cases analysed. However, only four (20%) of the left border ends were conserved to the processing end, indicating that the T-DNA left and right ends are treated mechanistically differently during the T-DNA integration process. Comparison of the genomic target sites prior to integration to the T-DNA revealed that the T-DNA inserted into the plant genome without any notable deletion of genomic sequence in three out of 10 transgenic lines analysed. However, deletions of DNA ranging in length from a few nucleotides to more than 500 bp were observed in other transgenic lines. Filler DNAs of up to 235 bp were observed on left and/or right junctions of six transgenic lines, which in most cases originated from the nearby host genomic sequence or from the T-DNA. Short sequence similarities between recombining strands near break points, in particular for the left T-DNA end, were observed in most of the lines analysed. These results confirm the well-accepted T-DNA integration model based on single-stranded annealing followed by ligation of the right border which is preserved by the VirD2 protein. However, a second category of T-DNA integration was also identified in nine transgenic lines, in which the right border of the T-DNA was partly truncated. Such integration events are described via a model for the repair of genomic double-strand breaks in somatic plant cells based on synthesis-dependent strand-annealing. This report in a long-lived tree system provides major insight into the mechanism of transgene integration.     

DNA from the A6S/2 crown gall tumor contains scrambled Ti-plasmid sequences near its junctions with plant DNA

The A6S/2 tumor incited on tobacco by Agrobacterium tumefaciens harboring the octopine-type A6 Ti plasmid contains one insert of Ti-plasmid sequences (the T DNA). This 13 kb insert is derived from a colinear sequence in the Ti plasmid (the T region) and becomes attached to plant DNA in the nucleus of the host cell. We have determined the DNA sequence encompassing the left end of the T region of the A6 Ti plasmid and the corresponding portion of the A6S/2 T DNA. The two sequences are identical for at least 806 bp. To the left of the divergence point, the tumor contains five partially overlapping sequences that are direct or inverted repeats of sequences to the right of the divergence point. The Ti plasmid contains only the right member of each of these repeats. We have also performed heteroduplex studies that indicate that this T DNA has a 520 bp inverted repeat of an internal sequence at the right end near its junction with plant DNA. The repeated sequences near the ends of the T DNA resemble the repeats of adenovirus type 12 sequences found near its junction with host DNA. We discuss data suggesting that the 23 bp to the immediate right of the divergence point of the A6 left junction form a site important in some step in the transfer of T-region DNA from the bacteria to the plant.     

Transgene structures in T-DNA-inserted rice plants

T-DNA is commonly used for delivery of foreign genes and as an insertional mutagen. Although ample information exists regarding T-DNA organization in dicotyledonous plants, little is known about the monocot rice. Here, we investigated the structure of T-DNA in a large number of transgenic rice plants. Analysis of the T-DNA borders revealed that more than half of the right ends were at the cleavage site, whereas the left ends were not conserved and were deleted up to 180 bp from the left border (LB) cleavage site. Three types of junctions were found between T-DNA and genomic DNA. In the first, up to seven nucleotide overlaps were present. The frequency of this type was much higher in the LB region than at the right border (RB). In the second type, which was more frequent in RB, the link was direct, without any overlaps or filler DNA. Finally, the third type showed filler DNA between T-DNA and the plant sequences. Out of 171 samples examined, 77 carried the vector backbone sequence, with the majority caused by the failure of T-strand termination at LB. However, a significant portion also resulted from co-integration of T-DNA and the vector backbone to a single locus. Most linkages between T-DNA and the vector backbone were formed between two 3 ends or two 5 ends of the transferred DNAs. The 3 ends were mostly linked through 3–6 bp of the complementing sequence, whereas the 5 ends were linked through either precise junctions or imprecise junctions with filler DNA.     

Insights into recognition of the T-DNA border repeats as termination sites for T-strand synthesis by Agrobacterium tumefaciens

The recognition of the T-DNA left border (LB) repeat is affected by its surrounding sequences. Here, the LB regions were further characterized by molecular analysis of transgenic plants, obtained after Agrobacterium tumefaciens-mediated transformation with T-DNA vectors that had been modified in this LB region. At least the 24-bp LB repeat by itself was insufficient to terminate the T-strand synthesis. Addition of the natural inner and/or outer border regions to at least the LB repeat, even when present at a distance, enhanced the correct recognition of the LB repeat, reducing the number of plants containing vector backbone sequences. In tandem occurrence of both the octopine and nopaline LB regions with their repeats terminated the T-strand synthesis most efficiently at the LB, yielding a reproducibly high number of plants containing only the T-DNA. Furthermore, T-strand synthesis did not terminate efficiently at the right border (RB) repeat, which might indicate that signals in the outer RB region inhibit the termination of T-strand synthesis at the RB repeat.     

Molecular analysis of <Emphasis Type="Italic">Agrobacterium</Emphasis> T-DNA integration in tomato reveals a role for left border sequence homology in most integration events

Studies in several plants have shown that Agrobacterium tumefaciens T-DNA can integrate into plant chromosomal DNA by different mechanisms involving single-stranded (ss) or double-stranded (ds) forms. One mechanism requires sequence homology between plant target and ssT-DNA border sequences and another double-strand-break repair in which preexisting chromosomal DSBs “capture” dsT-DNAs. To learn more about T-DNA integration in Solanum lycopersicum we characterised 98 T-DNA/plant DNA junction sequences and show that T-DNA left border (LB) and right border transfer is much more variable than previously reported in Arabidopsis thaliana and Populus tremula. The analysis of seven plant target sequences showed that regions of homology between the T-DNA LB and plant chromosomal DNA plays an important role in T-DNA integration. One T-DNA insertion generated a target sequence duplication that resulted from nucleolytic processing of a LB/plant DNA heteroduplex that generated a DSB in plant chromosomal DNA. One broken end contained a captured T-DNA that served as a template for DNA repair synthesis. We propose that most T-DNA integrations in tomato require sequence homology between the ssT-DNA LB and plant target DNA which results in the generation of DSBs in plant chromosomal DNA.     

A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA

Approximately 6000 specific DNA deletion events occur during development of the somatic macronucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis-acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetrahymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis-acting sequences outside the deleted DNA.     

Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression

Analysis of Agrobacterium-transferred DNA (T-DNA) revealed strong correlations between transgene structures and floral pigmentation patterns from chalcone synthase (chs) co-suppression among 47 Petunia transformants. Presented here are the full details of T-DNA structural organization in that population. Sixteen transformants (34%) carried one T-DNA copy while 31 (66%) carried 106 complete and partial T-DNA elements in 54 linkage groups. Thirty linkage groups contained multiple T-DNA copies; 15 of these contained only contiguously repeated copies, 8 contained only dispersed copies and 7 contained both. Right-border inverted repeats were three times more frequent than left-border inverted or direct repeats. Large fragments of binary-vector sequences were linked to the T-DNA in seven plants.     

The structure of an integrated copy of the giant linear plasmid SCP1 in the chromosome of Streptomyces coelicolor 2612.

Summary In NF strain 2612 of Streptomyces coelicolor, a giant linear plasmid SCP1 is integrated into the chromosome at the 9 o'clock position. To characterize the integrated structure of SCP1, cloning and sequence analysis of the two junctions between the SCP1 DNA and the chromosomal DNA was carried out. The left junction was revealed to retain an almost intact left terminus of SCP1. On the other hand, the right junction was composed of IS466, deleting completely the right terminal inverted repeat of SCP1. This junction might have been formed by recombination of two IS466 elements, one present at the end of the right terminal inverted repeat of SCP1 and one on the chromosome. Based on these results, we have proposed a model for the integration of SCP1 into the chromosome. The unique conjugal transfer of NF strains and the origin of the chromosomal antibiotic biosynthetic genes in Streptomyces species are also discussed in relation to this model.     

Agrobacterium-mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA∝barley genomic DNA junctions

Agrobacterium-mediated barley transformation promises many advantages compared to alternative gene transfer methods, but has so far been established in only a few laboratories. We describe a protocol that facilitates rapid establishment and optimisation of Agrobacterium-mediated transformation for barley by instant monitoring of the transformation success. The synthetic green fluorescent protein (sgfpS65T) reporter gene was introduced in combination with thehpt selectable marker gene into immature embryos of barley (Hordeum vulgare L.) by cocultivation with Agrobacterium tumefaciens strain AGLO harboring binary vector pYF133. Using green fluorescent protein (GFP) as a non-destructive visual marker allowed us to identify single-cell recipients of T-DNA at an early stage, track their fate and evaluate factors that affect T-DNA delivery. GFP screening was combined with a low level hygromycin selection. Consequently, transgenic plantlets ready to transfer to soil were obtained within 50 days of explant culture. Southern blot- and progeny segregation analyses revealed a single copy T-DNA insert in more than half of the transgenic barley plants. T-DNA/barley genomic DNA junctions were amplified and sequenced. The right T-DNA ends were highly conserved and clustered around the first 4 nucleotides of the right 25 bp border repeat, while the left T-DNA ends were more variable, located either in the left 25 bp border repeat or within 13 bp from the left repeat. T-DNAs were transferred from Agrobacterium to barley with exclusion of vector sequence suggesting a similar molecular T-DNA transfer mechanism as in dicotyledonous plants.     

Single-copy T-DNA insertions in Arabidopsis are the predominant form of integration in root-derived transgenics,whereas multiple insertions are found in leaf discs

Different patterns of T-DNA integration in Arabidopsis were obtained that depended on whether a root or a leaf-disc transformation method was used. An examination of 82 individual transgenic Arabidopsis plants, derived from 15 independent Agrobacterium-mediated transformations in which different cointegrate and binary constructs were used, indicated that the transformation method had a significant influence on the type and copy number of T-DNA integration events. Southern hybridizations showed that most of the transgenic plants produced by a leaf-disc method contained multiple T-DNA insertions (89%), the majority of which were organized as right-border inverted repeat structures (58%). In contrast, a root transformation method mostly resulted in single T-DNA insertions (64%), with fewer right-border inverted repeats (38%). The transformation vectors, including cointegrate and binary types, and the plant selectable markers, hygromycin phosphotransferase and dihydrofolate reductase, did not appear to influence the T-DNA integration patterns.