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.     

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.     

Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation.

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 NPTII 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 NPTII gene with a 3' deletion. A restored NPTII gene was found in 1-4% of the protoplasts that had been cotransformed with both T-DNAs. Restoration of the NPTII 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 was 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 NPTII repair gene. Several kanamycin resistant plant lines were obtained with an intact NPTII 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.     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome.

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.     

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.     

Gene conversion in transgenic maize plants expressing FLP/FRT and Cre/loxP site-specific recombination systems

DNA recombination reactions (site-specific and homologous) were monitored in the progeny of transgenic maize plants by bringing together two recombination substrates (docking sites and shuttle vectors) in the zygotes. In one combination of transgenic events, the recombination marker gene (yellow fluorescent protein gene, YFP) was activated in 1%-2% of the zygotes receiving both substrates. In other crosses, chimeric embryos and plants were identified, indicative of late recombination events taking place after the first mitotic division of the zygotes. The docking site structure remained unchanged; therefore, all recovered recombination events were classified as gene conversions. The recombinant YFP-r gene segregated as a single locus in subsequent generations. The recombination products showed evidence of homologous recombination at the 5' end of the YFP marker gene and recombinational rearrangements at the other end, consistent with the conclusion that DNA replication was involved in generation of the recombination products. Here, we demonstrate that maize zygotes are efficient at generating homologous recombination products and that the homologous recombination pathways may successfully compete with other possible DNA repair/recombination mechanisms such as site-specific recombination. These results indicate that maize zygotes provide a permissive environment for homologous recombination, offering a new strategy for gene targeting in maize.     

Localization of T-DNA Insertions in Petunia by Fluorescence in Situ Hybridization: Physical Evidence for Suppression of Recombination

Using fluorescence in situ hybridization (FISH) with metaphase preparations, we localized a 4-kb single-copy T-DNA sequence in a group of petunia transformants. The selected T-DNAs previously had been shown to be linked to the phenotypic marker FI on chromosome II. Linkage analysis had revealed that recombination around the FI locus is suppressed in a wide cross relative to an inbred recombination assay. The localization of six FI-linked T-DNAs and the FI locus itself, using FISH, revealed a number of aspects of recombination in petunia: (1) the central region of chromosome II showed at least a 10-fold suppression of recombination in wide crosses relative to the distal region; (2) recombination in wide hybrids over two-thirds of the chromosome was extremely low; and (3) recombination between completely homologous chromosomes in an inbred cross also was suppressed in the central region. In addition, the T-DNAs were not evenly distributed along the chromosome, suggesting a possible preference for a distal position for T-DNA integration. Implications for such a preference are discussed.     

Efficient transfer of base changes from a vector to the rice genome by homologous recombination: involvement of heteroduplex formation and mismatch correction

Gene targeting refers to the alteration of a specific DNA sequence in an endogenous gene at its original locus in the genome by homologous recombination. Through a gene-targeting procedure with positive–negative selection, we previously reported the generation of fertile transgenic rice plants with a positive marker inserted into the Adh2 gene by using an Agrobacterium-mediated transformation vector containing the positive marker flanked by two 6-kb homologous segments for recombination. We describe here that base changes within the homologous segments in the vector could be efficiently transferred into the corresponding genomic sequences of rice recombinants. Interestingly, a few sequences from the host genome were flanked by the changed sequences derived from the vector in most of the recombinants. Because a single-stranded T-DNA molecule in Agrobacterium-mediated transformation is imported into the plant nucleus and becomes double-stranded, both single-stranded and double-stranded T-DNA intermediates can serve in gene-targeting processes. Several alternative models, including the occurrence of the mismatch correction of heteroduplex molecules formed between the genomic DNA and either a single-stranded or double-stranded T-DNA intermediate, are compared to explain the observation, and implications for the modification of endogenous genes for functional genomic analysis by gene targeting are discussed.     

Plasmid construction by homologous recombination in yeast

We describe a convenient method for constructing new plasmids that relies on interchanging parts of plasmids by homologous recombination in Saccharomyces cerevisiae. A circular recombinant plasmid of a desired structure is regenerated after transformation of yeast with a linearized plasmid and a DNA restriction fragment containing appropriate homology to serve as a substrate for recombinational repair. The free ends of the input DNA molecules need not be homologous in order for efficient recombination between internal homologous regions to occur. The method is particularly useful for incorporating into or removing from plasmids selectable markers, centromere or replication elements, or particular alleles of a gene of interest. Plasmids constructed in yeast can subsequently be recovered in an Escherichia coli host. Using this method, we have constructed an extended series of new yeast centromere, episomal and replicating (YCp, YEp, and YRp) plasmids containing, in various combinations, the selectable yeast markers LEU2, HIS3, LYS2, URA3 and TRP1.     

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.     

Transformed cell clones as a tool to study T-DNA integration mediated by Agrobacterium tumefaciens

A large number of tobacco SR1 cell clones transformed by the wild-type Agrobacterium C58 have been analysed for the presence of screenable markers such as tumour morphology, opine synthesis and hormone dependence. Distinct phenotypic classes were observed depending upon whether the cell clones were isolated from primary tumours or were obtained via cocultivation of protoplasts. These classes of tobacco SR1-C58 transformants appear to arise from errors in the Ti plasmid (T-DNA) transfer and integration mechanism itself rather than from subsequent T-DNA rearrangements, since 900 subclones, obtained by recloning a wild-type SR1-C58-transformed cell clone, yielded no variation in the phenotypes. A detailed genomic T-DNA analysis showed the presence of characteristic, abnormally short T-DNAs in the teratoma-forming, Acs- class and also in the Nos- class. The abnormal right border in two Nos- clones ends close to a sequence that resembles the normal T-DNA terminus and lies adjacent to the nos promoter, suggesting that this sequence could have functioned as a recognition site directing these particular T-DNA transfers. On the basis of the phenotypic and genomic blotting data it is clear that the short T-DNAs are characteristic of the cocultivation method. Other phenomena causing phenotypic variation, such as the loss of the T-DNA, and the gradual repression of T-DNA gene expression by methylation, are the main causes of aberrations in primary tumours. Moreover, the physical data suggest that early in the transformation cycle of Agrobacterium a replication step of a preselected T-DNA occurs before integration into the plant genome.     

Illegitimate recombination in the histone multigenic family generates circular DNAs in Drosophila embryos.

From extrachromosomal covalently closed circular DNA molecules purified from Drosophila melanogaster embryos, we have isolated 24 clones homologous to the histone tandemly repeated gene family. Some of the clones harbor one of the two main types of genomic repeated units of 4.8 and 5.0 kb. and probably result from homologous recombination. The remaining clones have a size ranging from 0.2 to 2.5 kb. and most of them carry a single fragment of the repeated unit. Nucleotide sequences of the junction region of six of these clones indicate they are generated by illegitimate recombination between short (8-15 bp.) imperfect direct repeats. The data suggest that most of the histone homologous circular DNA molecules are deleted histone units.     

Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants

A major limitation of crop biotechnology and breeding is the lack of efficient molecular technologies for precise engineering of target genomic loci. While transformation procedures have become routine for a growing number of plant species, the random introduction of complex transgenenic DNA into the plant genome by current methods generates unpredictable effects on both transgene and homologous native gene expression. The risk of transgene transfer into related plant species and consumers is another concern associated with the conventional transformation technologies. Various approaches to avoid or eliminate undesirable transgenes, most notably selectable marker genes used in plant transformation, have recently been developed. These approaches include cotransformation with two independent T-DNAs or plasmid DNAs followed by their subsequent segregation, transposon-mediated DNA elimination, and most recently, attempts to replace bacterial T-DNA borders and selectable marker genes with functional equivalents of plant origin. The use of site-specific recombination to remove undesired DNA from the plant genome and concomitantly, via excision-mediated DNA rearrangement, switch-activate by choice transgenes of agronomical, food or feed quality traits provides a versatile “transgene maintenance and control” strategy that can significantly contribute to the transfer of transgenic laboratory developments into farming practice. This review focuses on recent reports demonstrating the elimination of undesirable transgenes (essentially selectable marker and recombinase genes) from the plant genome and concomitant activation of a silent transgene (e.g., a reporter gene) mediated by different site-specific recombinases driven by constitutive or chemically, environmentally or developmentally regulated promoters. These reports indicate major progress in excision strategies which extends application of the technology from annual, sexually propagated plants towards perennial, woody and vegetatively propagated plants. Current trends and future prospects for optimization of excision-activation machinery and its practical implementation for the generation of transgenic plants and plant products free of undesired genes are discussed.     

Generation of single-copy T-DNA transformants in Arabidopsis by the CRE/loxP recombination-mediated resolution system

We investigated whether complex T-DNA loci, often resulting in low transgene expression, can be resolved efficiently into single copies by CRE/loxP-mediated recombination. An SB-loxP T-DNA, containing two invertedly oriented loxP sequences located inside and immediately adjacent to the T-DNA border ends, was constructed. Regardless of the orientation and number of SB-loxP-derived T-DNAs integrated at one locus, recombination between the outermost loxP sequences in direct orientation should resolve multiple copies into a single T-DNA copy. Seven transformants with a complex SB-loxP locus were crossed with a CRE-expressing plant. In three hybrids, the complex T-DNA locus was reduced efficiently to a single-copy locus. Upon segregation of the CRE recombinase gene, only the simplified T-DNA locus was found in the progeny, demonstrating DNA had been excised efficiently in the progenitor cells of the gametes. In the two transformants with an inverted T-DNA repeat, the T-DNA resolution was accompanied by at least a 10-fold enhanced transgene expression. Therefore, the resolution of complex loci to a single-copy T-DNA insert by the CRE/loxP recombination system can become a valuable method for the production of elite transgenic Arabidopsis thaliana plants that are less prone to gene silencing.     

Unusual Telomeric DNAs in Human Telomerase-Negative Immortalized Cells

A significant fraction of human cancer cells and immortalized cells maintain telomeres in a telomerase-independent manner called alternative lengthening of telomeres (ALT). It has been suggested that ALT involves homologous recombination that is expected to generate unique intermediate DNAs. However, the precise molecular mechanism of ALT is not known. To gain insight into how telomeric DNAs (T-DNAs) are maintained in ALT, we examined the physical structures of T-DNAs in ALT cells. We found abundant single-stranded regions in both G and C strands of T-DNAs. Moreover, two-dimensional gel electrophoreses and native in-gel hybridization analyses revealed novel ALT-specific single-stranded T-DNAs, in addition to previously reported t-circles. These newly identified ALT-specific T-DNAs include (i) the t-complex, which consists of highly branched T-DNAs with large numbers of internal single-stranded portions; (ii) ss-G, which consists of mostly linear single-G-strand T-DNAs; and (iii) ss-C, which consists of most likely circular single-C-strand T-DNAs. Cellular-DNA fractionation by the Hirt protocol revealed that t-circles and ss-G exist in ALT cells as extrachromosomal and chromatin-associated DNAs. We propose that such ALT-specific T-DNAs are produced by telomere metabolism specific to ALT, namely, homologous recombination and the rolling-circle replication mechanism.     

Transfer of the symbiotic plasmid of Rhizobium etli CFN42 requires cointegration with p42a, which may be mediated by site-specific recombination

Plasmid p42a from Rhizobium etli CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym). Most pSym transconjugants also inherit p42a. pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms*. RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions. These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, and self-transmissible. The symbiotic features of derivatives simultaneously containing a wild-type pSym and an RpSym* were analyzed. Structural analysis of 10 RpSyms* showed that 7 shared one of the two pSym-p42a junctions. Sequencing of this common junction revealed a 53-bp region that was 90% identical in pSym and p42a, including a 5-bp central region flanked by 9- to 11-bp inverted repeats reminiscent of bacterial and phage attachment sites. A gene encoding an integrase-like protein (intA) was localized downstream of the attachment site on p42a. Mutation or the absence of intA abolished pSym transfer from a recA mutant donor. Complementation with the wild-type intA gene restored transfer of pSym. We propose that pSym-p42a cointegration is required for pSym transfer; cointegration may be achieved either through homologous recombination among large reiterated sequences or through IntA-mediated site-specific recombination between the attachment sites. Cointegrates formed through the site-specific system but resolved through RecA-dependent recombination or vice versa generate RpSyms*. A site-specific recombination system for plasmid cointegration is a novel feature of these large plasmids and implies that there is unique regulation which affects the distribution of pSym in nature due to the role of the cointegrate in conjugative transfer.     

T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs

Nicotiana protoplasts and Arabidopsis leaf discs or roots were co-cultivated with two Agrobacterium strains each carrying a different T-DNA. Co-transformed plants were selected and the integration of the different T-DNAs was analysed at the genetic and genomic level. Genetic analysis showed that the T-DNAs derived from different bacteria were frequently integrated at the same locus, independent of the plant species or transformation method used. Southern analysis revealed that 12 out of 27 Arabidopsis transformants contained the co-transferred T-DNAs linked to each other in all possible configurations but with a preference for those with at least one right border involved in linkage. Overall, our data support the hypothesis that ligation of separate T-DNAs is a dominant mechanism in formation of the frequently observed repeats of identical T-DNAs. We propose a scheme which could explain the formation of T-DNA repeats and the preferential involvement of right borders in T-DNA linkages.     

Gene targeting and instability of Agrobacterium T-DNA loci in the plant genome

To develop a model system for studies of homologous recombination in plants, transgenic Nicotiana tabacum and Nicotiana plumbaginifolia lines were generated harbouring a single target T-DNA containing the negative selective codA gene encoding cytosine deaminase (CD) and the β-glucuronidase (GUS) gene. Subsequently, the target lines were transformed with a replacement-type T-DNA vector in which the CD gene and the GUS promoter had been replaced with a kanamycin-resistance gene. For both Nicotiana species kanamycin-resistant lines were selected which had lost the CD gene and the GUS activity. One tobacco line was the result of a precise gene targeting event. However, most other lines were selected due to a chromosomal deletion of the target locus. The deletion frequency of the target locus varied between target lines, and could be present in up to 20% of the calli which were grown from leaf protoplasts. T-DNA transfer was not required for induction of the deletions, indicating that the target loci were unstable. A few lines were obtained in which the target locus had been deleted partially. Sequence analysis of the junctions revealed deletion of DNA sequences between microhomologies. We conclude that T-DNAs, which are stable during plant development as well as in transmission to the offspring, may become unstable during propagation in callus tissue. The relationships between callus culture, genetic instability and the process of T-DNA integration and deletion in the plant genome are discussed.     

RuvABC-dependent double-strand breaks in dnaBts mutants require recA

Replication fork arrest can cause DNA double-strand breaks (DSBs). These DSBs are caused by the action of the Holliday junction resolvase RuvABC, indicating that they are made by resolution of Holliday junctions formed at blocked forks. In this work, we study the homologous recombination functions required for RuvABC-mediated breakage in cells deficient for the accessory replicative helicase Rep or deficient for the main Escherichia coli replicative helicase DnaB. We show that, in the rep mutant, RuvABC-mediated breakage occurs in the absence of the homologous recombination protein RecA. In contrast, in dnaBts mutants, most of the RuvABC-mediated breakage depends on the presence of RecA, which suggests that RecA participates in the formation of Holliday junctions at forks blocked by the inactivation of DnaB. This action of RecA does not involve the induction of the SOS response and does not require any of the recombination proteins essential for the presynaptic step of homologous recombination, RecBCD, RecF or RecO. Consequently, our observations suggest a new function for RecA at blocked replication forks, and we propose that RecA acts by promoting homologous recombination without the assistance of known presynaptic proteins.