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First published online October 9, 2003; 10.1104/pp.103.032128 Plant Physiology 133:1011-1023 (2003) © 2003 American Society of Plant Biologists Site-Specific Integration of Agrobacterium tumefaciens T-DNA via Double-Stranded Intermediates1Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794 (T.T., M.V., V.C.); and Westhampton Beach High School, 340 Mill Road, Westhampton Beach, New York 11978 (L.R.F.)
Agrobacterium tumefaciens-mediated genetic transformation involves transfer of a single-stranded T-DNA molecule (T strand) into the host cell, followed by its integration into the plant genome. The molecular mechanism of T-DNA integration, the culmination point of the entire transformation process, remains largely obscure. Here, we studied the roles of double-stranded breaks (DSBs) and double-stranded T-DNA intermediates in the integration process. We produced transgenic tobacco (Nicotiana tabacum) plants carrying an I-SceI endonuclease recognition site that, upon cleavage with I-SceI, generates DSB. Then, we retransformed these plants with two A. tumefaciens strains: one that allows transient expression of I-SceI to induce DSB and the other that carries a T-DNA with the I-SceI site and an integration selection marker. Integration of this latter T-DNA as full-length and I-SceI-digested molecules into the DSB site was analyzed in the resulting plants. Of 620 transgenic plants, 16 plants integrated T-DNA into DSB at their I-SceI sites; because DSB induces DNA repair, these results suggest that the invading T-DNA molecules target to the DNA repair sites for integration. Furthermore, of these 16 plants, seven plants incorporated T-DNA digested with I-SceI, which cleaves only double-stranded DNA. Thus, T-strand molecules can be converted into double-stranded intermediates before their integration into the DSB sites within the host cell genome.
Agrobacterium tumefaciens is the only known organism capable of inter-kingdom DNA transfer (Stachel and Zambryski, 1989  ). In nature, this bacterium causes crown gall disease in many dicotyledonous plant species by transferring a specific DNA fragment, the transferred DNA (T-DNA), from its tumor-inducing (Ti) plasmid into the host cell (for review, see Gelvin, 2000; Tzfira et al., 2000; Zupan et al., 2000; Gelvin, 2003). The wild-type T-DNA carries two types of genes: oncogenes and genes for biosynthesis of amino acid derivatives, opines. Expression of the integrated T-DNA, therefore, results in uncontrolled cell division and formation of tumors (Gaudin et al., 1994; Das, 1998) that produce and secrete opines that are utilized by A. tumefaciens and several other microorganisms as the source of carbon and nitrogen (for review, see Savka et al., 2002). The T-DNA itself does not encode genes required for its transfer and integration and is defined only by two 25-bp direct repeats, termed T-DNA left and right borders (Zambryski et al., 1982). Thus, the entire wild-type T-DNA sequence between the borders can be replaced with a gene of interest that will be transferred to plants and integrated in the plant genome, representing the molecular basis for plant genetic engineering (for review, see Gelvin, 1998b, 2000, 2003; Potrykus et al., 1998).
Several A. tumefaciens chv (chromosomal virulence) genes and a set of vir (virulence) genes were contained on the Ti plasmid code for the protein machinery of the T-DNA transport (for review, see Sheng and Citovsky, 1996
Initially, two different models have been suggested for T-DNA integration, double-stranded break (DSB) repair and single-stranded gap repair (Mayerhofer et al., 1991
The single-stranded gap repair model was extensively revised (Tinland and Hohn, 1995
Recent studies further supported the notion that the T-strand molecule becomes double stranded before its integration (De Neve et al., 1997
Here, we present direct evidence for the role of DSBs and double-stranded T-DNA intermediates in T-DNA integration. Specifically, we used transient expression of an intron-encoded endonuclease I-SceI (Monteilhet et al., 1990
Experimental System
To investigate a role that DSB may play in T-DNA integration and to determine whether T-DNA integrates as a single- or double-stranded molecule, we utilized a genetic system for in vivo induction of DSBs (Salomon and Puchta, 1998
To determine the structure of the integrating T-DNA molecules, we designed the T-DNA to carry an I-SceI recognition sequence between the promoterless uidA gene and the T-DNA left border (Fig. 1B). If this T-DNA becomes double stranded to allow its cleavage with I-SceI before integration, and if the resulting fragment carrying the promoterless uidA integrates into the DSB created by I-SceI cleavage between the 35S promoter and the I-SceI site in the 35S-I-SceI-codA transgene, we expect to detect the uidA gene expression, i.e. GUS activity, in the transgenic plants. Thus, our experimental design aims to examine concurrently two important characteristics of the integration process: frequency of targeted integration into the specific DSB sites out of the total number of transformation events and frequency of formation of double-stranded T-DNA intermediates before integration.
Using this experimental design, we generated a total of 24 independent transgenic lines transformed with pBNE3I containing the 35S-I-SceI-codA T-DNA. Fifteen lines, showing a 3:1 kanamycin resistance segregation ratio, were analyzed by PCR to identify plants that do not contain tandem insertions of the 35S-I-SceI-codA transgene (Table I). In addition, sequence analysis of the amplified PCR fragments revealed that, in all 15 lines, the sequence of the integrated 35S-I-SceI-codA transgene was identical to the original sequence of this transgene within pBNE3I (data not shown). Plants were then tested for their cytosine deaminase activity, which indicates the degree of expression of the 35S-I-SceI-codA transgene, using leaf disc regeneration assay in the presence of 5-fluorocytosine (5-FC); 5-FC is known to select against cells that express cytosine deaminase (Stougaard, 1993
Next, these 35S-I-SceI-codA transgenic plants were transformed with a mixture of A. tumefaciens strains harboring the binary vectors pCISceI, with the I-SceIORF T-DNA, and pBIG-HYG-terGUS/I-SceI, with the I-SceI-uidA-hpt T-DNA; these vectors encode the transiently expressed I-SceI endonuclease and provide the integrating I-SceI-uidA-hpt T-DNA, respectively. A total of 620 hygromycin-resistant transgenic lines were recovered and analyzed for their sensitivity to 5-FC; the 5-FC-resistant plants were then tested for GUS activity and the nucleotide sequence of their integration junctions was determined.
From 620 hygromycin-resistant plants, 82 plants exhibited significant 5-FC resistance. We then PCR amplified the putative integration junctions around the DSB site in 68 of these plants and examined them for small deletions or insertions in the DSB site. To this end, the 35S-I-SceI-codA transgene was amplified using the 35S-F2 and codA-R primers (Table I) and a short extension time (1 min). All 68 plants exhibited sequence alterations within the codA gene, indicating approximately 11% frequency of DSB induction and improper repair and demonstrating that the 5-FC resistance of these lines was not simply and solely due to silencing of this transgene. Forty-five of 620 plants (7.25%) yielded short PCR fragments that ranged from 300 to 600 bp and, thus, could not represent the true T-DNA integration (Table II). Sequence analysis of five randomly chosen PCR fragments revealed deletions and/or insertions of short filler DNA that were most likely responsible for loss of function of the cytosine deaminase gene (data not shown).
Twenty-three of 620 plants (3.71%) did not show any visible PCR bands after short amplification, but four of them yielded longer PCR fragments, 2.2 kb, 2.8 kb, and two bands of 2.9 kb each (Table II), when longer PCR extension time (3 min) was used. Sequence analysis of these fragments revealed the integration of the full-length or truncated I-SceIORF T-DNA into DSB sites. The remaining 19 plants were subjected to a series of PCR analyses using different primer combinations shown in Table I. Table II shows that five plants contained the full-length or truncated I-SceI-uidA-hpt T-DNA integrated in their DSBs, whereas in seven plants, the integrated I-SceI-uidA-hpt T-DNA was found to have been digested by I-SceI before integration. The last seven plants did not yield discrete PCR products, potentially due to large deletions or insertions (Table II). Thus, out of 620 transgenic lines examined in this study, we observed 16 site-specific T-DNA integration events, including those of the I-SceIORF and I-SceI-uidA-hpt transgenes, corresponding to 2.58% frequency of targeted integration. Furthermore, this is the minimal estimate of the frequency of insertions into DSB sites because it assumes that transient expression of I-SceI created DSBs in all 620 transgenic lines.
Determination of the nucleotide sequence of T-DNA/DSB site integration junctions allowed their classification based on the orientation of the inserted T-DNA within the DSB site (Fig. 2). For the junctions in which the T-DNA left border was found ligated to the left side of DSB (DSB-L/LB, Fig. 2A) and the T-DNA right border was found ligated to the right side of DSB (RB/DSB-R, Fig. 2A), the sequences of the DSB site and the T-DNA were presented in sense orientation in Figures 3, 4, 5. When the T-DNA integrated in reverse orientation, we classified the junctions as DSB-L/RB and LB/DSB-R (Fig. 2B); in this case, to show the sense strand of the junction, we reversed and complemented the T-DNA sequence in Figures 3, 4, 5. Finally, when the I-SceI-digested T-DNA integrated into DSB, recreating the I-SceI site, we classified two additional types of junctions, DSB-L/I-SceI (Fig. 2C) and I-SceI/DSB-R (Fig. 2D). The T-DNA sequences of these junctions were presented in Figures 3, 4, 5 as sense (for DSB-L/I-SceI) or reversed and complemented sequences (for I-SceI/DSB-R).
In four plant lines, the full-length or truncated I-SceIORF T-DNA integrated into the DSB site (Table II). In three of these lines (Sc4-2, Sc59-5, and Sc59-9), almost the entire I-SceIORF T-DNA integrated into DSB, whereas in the fourth line (Sc70-3), a deletion of 797 bp was observed (Table III; Fig. 3). Sequence analysis of the DSB-L/LB junctions revealed that small deletions of 13 to 62 bp had occurred at the T-DNA left border. These deletions in the integrated T-DNA were accompanied by deletions, insertions, or no changes in the DSB site sequence (Sc4-2, Sc59-5, Sc70-3, and Sc59-9, Table III). The RB/DSB-R junctions were more conserved, showing no T-DNA right border alterations in three out of four cases (Sc4-2, Sc59-5, and Sc59-9) and were accompanied by small deletions or insertions in the DSB site (Table III). In the Sc59-5 line, insertion of 4 bp led to restoration of the TTAT fragment, originally located within the cleaved I-SceI sequence of DSB (Fig. 3). In the Sc59-9 line, insertion of TA nucleotides was found at the LB/DSB-R junction (Fig. 3). In the Sc70-3 line, a large T-DNA right border deletion of 797 bp occurred together with a 10-bp deletion in the flanking DSB site (Table III; Fig. 3). Sequence homologies between the integrated T-DNA and the DSB site—analyzed within 10 bp from the integration point—were small, ranging between 3 and 10 bp at the left border integration point to 0 and 2 bp at the right border integration point (Table III; Fig. 3).
In five lines (Sc1-10, Sc33-4, Sc33-7, Sc4-6, and Sc1-5), the full-length or only slightly (
Seven transgenic lines (Sc4-5, Sc4-8, Sc1-3, Sc4-12, Sc59-2, Sc33-9, and Sc70-9) contained only a part of the I-SceI-uidA-hpt T-DNA integrated into the DSB site. In all seven lines, the integrated portion of the T-DNA lacked the sequences from the T-DNA left border to the I-SceI site, probably due to cleavage of the T-DNA itself with I-SceI (Table III; Fig. 5). Also, in six of these lines, a portion of the I-SceI recognition sequence in the I-SceI-uidA-hpt T-DNA was identified within the integration junction (lines Sc4-5, Sc4-8, Sc1-3, Sc59-2, Sc33-9, and Sc70-9 in Fig. 5), whereas in the remaining Sc4-12 line, the digested I-SceI sequence was most likely deleted (see below). In lines Sc4-5, Sc4-8, and Sc1-3, the T-DNA was inserted in sense orientation to the I-SceI restriction site, producing the DSB-L/I-SceI type of the integration junction (see Fig. 2C). This resulted in a precise match between the digested I-SceI sequence of DSB and the digested I-SceI sequence of the I-SceI-uidA-hpt T-DNA, recreating the intact restriction site (Fig. 5). PCR fragments obtained from amplification of DSB-L/I-SceI junctions of all three lines were cleavable by a recombinant I-SceI enzyme (New England Biolabs, Beverly, MA) in vitro (data not shown). No homologies were observed between the I-SceI-digested end of the integrating T-DNA and the DSB site sequences in these lines (Table III). In lines Sc59-2 and Sc33-9, in which the T-DNA also integrated in sense orientation to the I-SceI site, the integration event did not recreate the restriction site due to deletions of 1 to 2 bp and 2 to 10 bp in the T-DNA and in the DSB site, respectively (Table III; Fig. 5); the amplified junction fragments from these lines failed to be digested with I-SceI in vitro (data not shown). No homology between DSB and the I-SceI-digested end of the T-DNA was observed in line Sc33-9, whereas a 5-bp homology was found in line Sc59-2 (Table III; Fig. 5). Integration of the I-SceI-digested I-SceI-uidA-hpt T-DNA in sense orientation to the I-SceI site of DSB should place the promoterless uidA gene in the T-DNA directly downstream of the 35S promoter in the DSB site (Fig. 2C). All five lines exhibiting such integration (Sc4-5, Sc4-8, Sc1-3, Sc59-2, and Sc33-9) expressed GUS activity in their tissues as detected by histochemical staining (data not shown). GUS expression was not detected in plants in which the full-length I-SceI-uidA-hpt integrated downstream of the 35S promoter (see above) because the distance between the T-DNA left border and the uidA gene (see Fig. 1) was too large to allow promoter activity. In lines Sc70-9 and Sc4-12, the T-DNA integrated in reverse orientation to the I-SceI site of DSB, producing the I-SceI/DSB-R type of the integration junction (see Fig. 2D). In Sc70-9, the integration junction contained only 2 bp of the I-SceI site located in the T-DNA and 3 bp of the I-SceI site derived from DSB. Sequence analysis of the integration junction from the Sc4-12 line showed a 17-bp deletion in the T-DNA, resulting in elimination of its entire I-SceI site and a 4-bp deletion in DSB. DSB and the I-SceI-digested end of the T-DNA showed no homologies to each other in line Sc70-9, whereas a 3-bp homology was found in line Sc4-12 (Table III; Fig. 5). The T-DNA right border of the I-SceI-digested I-SceI-uidA-hpt, which had no I-SceI sequences (see Fig. 2, C and D), integrated in a relatively conserved fashion in all lines, exhibiting deletions of 0 to 4 bp in the T-DNA (with the exception of line Sc33-9, which had a 52-bp deletion) and 7- to 36-bp deletions or 1- to 12-bp insertions in the DSB site (Table III; Fig. 5). In Sc70-9, the 30-bp deletion in DSB was also accompanied by an insertion of a 12-bp DNA fragment that did not originate from the T-DNA sequences. Homologies between the integrated T-DNA right border and the DSB site ranged from 0 to 5 bp (Table III; Fig. 5). Our sequence analysis of the RB/DSB-R integration junctions shown in Figure 5 also helped to address the likelihood of T-DNA integration in the vicinity of, rather than within, the I-SceI site followed by subsequent digestion and religation of the resulting two I-SceI sites, one within the plant DNA and the other near the T-DNA left border. In this scenario, which would give an appearance of I-SceI digestion before integration, the right border-host DNA junction is not expected to coincide with the original DSB. Our sequence data, however, did not detect such integration junctions, thus arguing against this possibility.
T-DNA Molecules Preferentially Integrate into DSBs Our findings indicate that DSBs in the host cell genome play an important role in T-DNA integration, representing the preferred sites for integration. This idea is based on the observations that, from a total number of 620 transgenic lines analyzed in this study, 16 lines contained T-DNA within the induced DSB site, corresponding to the 2.58% frequency of site-specific T-DNA integration. In reality, this number could be even higher because a sizable proportion of the 620 transgenic plants may not have contained DSBs, for example, due to insufficient levels of transient I-SceI expression and/or activity. For comparison, the probability of random T-DNA integration into a specific single DSB within a plant genome is infinitesimally small. Based on the 4.5-Gb genome size of tobacco, the plant species used in our study, the calculated frequency of random T-DNA integration into a 18-bp-long site, corresponding to the I-SceI recognition sequence, is 4 x 10–9. Importantly, the 2.58% frequency was observed for integration into the specific DSB within the 35S-I-SceI-codA transgene. Potentially, a much higher percentage of the 620 transgenic plants had derived from T-DNA integration into other DSBs, naturally occurring in the plant genome.
We also calculated the frequency of a DSB accepting a T-DNA insert instead of undergoing improper repair. From 63 lines identified to have a disrupted DSB, 16 lines verifiably contained T-DNA within their DSB site, corresponding to a 25% frequency. Previously, direct selection for a transgene insertion into DSBs was reported to result in 14% frequency of T-DNA integration in plants (Salomon and Puchta, 1998
It remains unknown how T strands are targeted to their integration sites. One obvious candidate for this "pilot" function is VirD2, which is attached to the 5' end of the T strand (Ward and Barnes, 1988
If the invading T strands are targeted to DSBs for conversion to double strands and integration, it is the frequency of DSBs in the host cell genome and the genome stability, therefore, that may represent a limiting factor for the efficiency of plant genetic transformation by A. tumefaciens. A statistical analysis of T-DNA integration sites in Arabidopsis revealed that T-DNA molecules mainly integrate in the vicinity of T-rich genomic regions (Brunaud et al., 2002
Microhomology between the T strand and the plant DNA plays a critical role in the single-stranded T-DNA integration model (Tinland and Hohn, 1995
T strands corresponding to the coding strand of a reporter gene are rapidly expressed, which requires their conversion to a double-stranded form already early in the infection process (Narasimhulu et al., 1996
Formation of double-stranded intermediates before integration may also explain the well-known conservation of the 5' end sequences of the integrated T-DNA and frequent deletions of its 3' end sequences (Matsumoto et al., 1990
It is important to emphasize that our data indicate the importance of conversion of T strands into double-stranded molecules before site-specific integration into DSBs. Although integration via double-stranded intermediates into DSBs most likely represents one of the major mechanisms of T-DNA integration, an alternative integration pathway via single-stranded intermediates into microhomology regions (Brunaud et al., 2002
DNA Constructs
Binary vectors pBNE3I and pCISceI, carrying within their T-DNA regions the cytosine deaminase (codA) and I-SceI genes, respectively, each driven by the cauliflower mosaic virus 35S promoter, were kindly provided by Dr. Holger Puchta (Puchta, 1998
Transgenic tobacco (Nicotiana tabacum cv Turk) plants carrying the I-SceI site were generated using pBNE3I and the leaf disc transformation protocol as previously described (Horsch et al., 1985
To identify transgenic lines with high cytosine deaminase activity, leaf explants from the 35S-I-SceI-codA plants were placed on regeneration medium (Horsch et al., 1985
Hygromycin-resistant transgenic lines with disrupted cytosine deaminase activity were identified by culturing their leaf explants in the presence or absence of 250 µg mL–1 5-FC as described above. To identify transgenic plants with targeted integration of the functional I-SceI-uidA-hpt T-DNA, leaf explants from lines resistant both to hygromycin and 5-FC were assayed for GUS activity by histochemical staining with chromogenic substrate 5-bromo-4-chloro-3-indolyl-
Genomic DNA was extracted from transgenic leaf tissues using the DNeasy Plant Mini DNA extraction kit (Qiagen USA, Valencia, CA) and analyzed by PCR using primer sets shown in Table I. To amplify the 35S-I-SceI-codA insertion for identification of transgenic plants from the first transformation cycle and to analyze hygromycin-resistant transgenic plants with disrupted cytosine deaminase activity from the second transformation cycle, we used the 35S-F2 and codA-R primers (Table I); amplification of undisrupted 35S-I-SceI-codA was predicted to produce a 509-kb DNA fragment (Table I). To amplify the right border integration junction of pBIG-HYG-terGUS/I-SceI T-DNA in the I-SceI cleavage site, the primer T-DNA-RB located 431 bp downstream of the T-DNA right border of pBIG-HYG-terGUS/I-SceI was used in combination with either the 35S-F2 or codA-R primers specific for the 35S promoter or the codA gene, respectively (Table I). To amplify the left border integration junction of pBIG-HYG-terGUS/I-SceI T-DNA in the I-SceI cleavage site, the primer T-DNA-LB, located 568 bp upstream of the T-DNA left border of pBIG-HYG-terGUS/I-SceI, was used in combination with either 35S-F2 or codA-R (Table I). To amplify the integration junction of I-SceI-digested T-DNA of pBIG-HYG-terGUS/I-SceI in the I-SceI cleavage site, the primer GUS-R located 310 bp upstream of the first codon of uidA in the T-DNA of pBIG-HYG-terGUS/I-SceI was used in combination of either 35S-F2 or codA-R; these combinations of primers were predicted to produce 350-bp and approximately 672-bp PCR fragments, respectively (Table I). All PCR reactions were carried out in a 50-µL volume containing 20 ng of DNA, 0.1 nM dNTP, 2.5 mM of each primer, and 2 units of TKARA EX-Taq polymerase (Pan Vera Corp., Madison, WI) with 5 µL of EX-Taq 10x reaction buffer. After 5 min of denaturation at 94°C, the junction fragment was amplified for 36 cycles of 60 s each at 94°C, 60 s at 55°C, and 1 or 3 min at 72°C. The resulting amplified junction fragments were either directly sequenced using the corresponding primers or first subcloned into pSL301 and then sequenced.
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
We thank Dr. Holger Puchta for the generous gift of the pBNE3I and pCISceI plasmids. Received August 21, 2003; returned for revision August 28, 2003; accepted August 28, 2003.
Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.032128.
1 This work was supported by the U.S.-Israel Binational Research and Development Fund (grant to T.T.), by the National Institutes of Health (grant to V.C.), by the National Science Foundation (grant to V.C.), by the U.S. Department of Agriculture (grant to V.C.), and by the U.S.-Israel Binational Science Foundation (grant to V.C.). * Corresponding author; e-mail ttzfira{at}notes.cc.sunysb.edu; fax 631–632–8575.
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657
Bako L, Umeda M, Tiburcio AF, Schell J, Koncz C (2003) The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants. Proc Natl Acad Sci USA 100: 10108–10113
Bolshoy A, McNamara P, Harrington RE, Trifonov EN (1991) Curved DNA without A-A: experimental estimation of all 16 DNA wedge angles. Proc Natl Acad Sci USA 88: 2312–2316
Breslauer KJ, Frank R, Blocker H, Marky LA (1986) Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci USA 83: 3746–3750 Brunaud V, Balzergue S, Dubreucq B, Aubourg S, Samson F, Chauvin S, Bechtold N, Cruaud C, DeRose R, Pelletier G et al. (2002) T-DNA integration into the Arabidopsis genome depends on sequences of preinsertion sites. EMBO Rep 3: 1152–1157[CrossRef][Web of Science][Medline]
Bundock P, Hooykaas PJJ (1996) Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc Natl Acad Sci USA 93: 15272–15275
Christie PJ (1997) Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria. J Bacteriol 179: 3085–3094 Christie PJ, Vogel JP (2000) Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol 8: 354–360[CrossRef][Web of Science][Medline]
Christie PJ, Ward JE, Winans SC, Nester EW (1988) The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA. J Bacteriol 170: 2659–2667
Citovsky V, De Vos G, Zambryski PC (1988) Single-stranded DNA binding protein encoded by the virE locus of Agrobacterium tumefaciens. Science 240: 501–504 Citovsky V, Guralnick B, Simon MN, Wall JS (1997) The molecular structure of Agrobacterium VirE2-single stranded DNA complexes involved in nuclear import. J Mol Biol 271: 718–727[CrossRef][Medline]
Citovsky V, Warnick D, Zambryski PC (1994) Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco. Proc Natl Acad Sci USA 91: 3210–3214
Citovsky V, Wong ML, Zambryski PC (1989) Cooperative interaction of Agrobacterium VirE2 protein with single stranded DNA: implications for the T-DNA transfer process. Proc Natl Acad Sci USA 86: 1193–1197
Citovsky V, Zupan J, Warnick D, Zambryski PC (1992) Nuclear localization of Agrobacterium VirE2 protein in plant cells. Science 256: 1802–1805
Das A (1988) Agrobacterium tumefaciens virE operon encodes a single-stranded DNA-binding protein. Proc Natl Acad Sci USA 85: 2909–2913 Das A (1998) DNA transfer from Agrobacterium to plant cells in crown gall tumor disease. Subcell Biochem 29: 343–363[Medline] De Block M, Debrouwer D (1991) Two T-DNAs co-transformed into Brassica napus by a double Agrobacterium tumefaciens infection are mainly integrated at the same locus. Theor Appl Genet 82: 257–263 De Buck S, Jacobs A, Van Montagu M, Depicker A (1999) The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration. Plant J 20: 295–304[Web of Science][Medline] De Neve M, De Buck S, Jacobs A, Van Montagu M, Depicker A (1997) T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J 11: 15–29[CrossRef][Web of Science][Medline] Filichkin SA, Gelvin SB (1993) Formation of a putative relaxation intermediate during T-DNA processing directed by the Agrobacterium tumefaciens VirD1, D2 endonuclease. Mol Microbiol 8: 915–926[Web of Science][Medline] Friesner J, Britt AB (2003) Ku80- and DNA ligase IV-deficient plants are sensitive to ionizing radiation and defective in T-DNA integration. Plant J 34: 427–440[CrossRef][Web of Science][Medline] Gaudin V, Vrain T, Jouanin L (1994) Bacterial genes modifying hormonal balances in plants. Plant Physiol Biochem 32: 11–29
Gelvin SB (1998a) Agrobacterium VirE2 proteins can form a complex with T strands in the plant cytoplasm. J Bacteriol 180: 4300–4302 Gelvin SB (1998b) The introduction and expression of transgenes in plants. Curr Opin Biotechnol 9: 227–232[CrossRef][Web of Science][Medline] Gelvin SB (2000) Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu Rev Plant Physiol Plant Mol Biol 51: 223–256[CrossRef][Web of Science]
Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiol Mol Biol Rev 67: 16–37
Gheysen G, Van Montagu M, Zambryski P (1987) Integration of Agrobacterium tumefaciens transfer DNA (T-DNA) involves rearrangements of target plant DNA. Proc Natl Acad Sci USA 84: 6169–6173
Gheysen G, Villarroel R, Van Montagu M (1991) Illegitimate recombination in plants: a model for T-DNA integration. Genes Dev 5: 287–297
Gietl C, Koukolikova-Nicola Z, Hohn B (1987) Mobilization of T-DNA from Agrobacterium to plant cells involves a protein that binds single-stranded DNA. Proc Natl Acad Sci USA 84: 9006–9010
Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227: 1229–1231 Howard E, Zupan J, Citovsky V, Zambryski PC (1992) The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells. Cell 68: 109–118[CrossRef][Web of Science][Medline] Howard EA, Citovsky V, Zambryski PC (1990) The T-complex of Agrobacterium tumefaciens. UCLA Symp Mol Cell Biol 129: 1–11
Howard EA, Winsor BA, De Vos G, Zambryski PC (1989) Activation of the T-DNA transfer process in Agrobacterium results in the generation of a T-strand protein complex: tight association of VirD2 with the 5' ends of T-strands. Proc Natl Acad Sci USA 86: 4017–4021 Janssen BJ, Gardner RC (1990) Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol 14: 61–72[CrossRef][Web of Science][Medline] Jasin M (1996) Genetic manipulation of genomes with rare-cutting endonucleases. Trends Genet 12: 224–228[CrossRef][Web of Science][Medline]
Jasper F, Koncz C, Schell J, Steinbiss H-H (1994) Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein. Proc Natl Acad Sci USA 91: 694–698 Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907[Web of Science][Medline] Kahl G (1982) Molecular biology of wound healing: the conditioning phenomenon. In G Kahl, J Schell, eds, Molecular Biology of Plant Tumors. Academic Press, New York, pp 211–268 Kohler F, Cardon G, Pohlman M, Gill R, Schider O (1989) Enhancement of transformation rates in higher plants by low-does irradiation: are DNA repair systems involved in incorporation of exogenous DNA into the plant genome? Plant Mol. Biol 12: 189–199[CrossRef] Krizkova L, Hrouda M (1998) Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions. Plant J 16: 673–680[CrossRef][Web of Science][Medline] Kumar S, Fladung M (2001) Gene stability in transgenic aspen (Populus): II. Molecular characterization of variable expression of transgene in wild and hybrid aspen. Planta 213: 731–740[CrossRef][Web of Science][Medline] Kumar S, Fladung M (2002) Transgene integratin in aspen: structures of integration sites and mechanism of T-DNA integration. Plant J 31: 543–551[Medline] Leskov KS, Criswell T, Antonio S, Li J, Yang CR, Kinsella TJ, Boothman DA (2001) When X-ray-inducible proteins meet DNA double strand break repair. Semin Radiat Oncol 11: 352–372[CrossRef][Medline] Matsumoto M, Ito Y, Hosoi T, Takahashi Y, Machida Y (1990) Integration of Agrobacterium T-DNA into a tobacco chromosome: possible involvement of DNA homology between T-DNA and plant DNA. Mol Gen Genet 224: 309–316[Web of Science][Medline] Mayerhofer R, Koncz-Kalman Z, Nawrath C, Bakkeren G, Crameri A, Angelis K, Redei GP, Schell J, Hohn B, Koncz C (1991) T-DNA integration: a mode of illegitimate recombination in plants. EMBO J 10: 697–704[Web of Science][Medline] McCormac AC, Fowler MR, Chen DF, Elliott MC (2001) Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation. Transgenic Res 10: 143–155[Medline]
Monteilhet C, Perrin A, Thierry A, Colleaux L, Dujon B (1990) Purification and characterization of the in vitro activity of I-Sce I, a novel and highly specific endonuclease encoded by a group I intron. Nucleic Acids Res 18: 1407–1413 Muller AE, Kamisugi Y, Gruneberg R, Niedenhof I, Horold RJ, Meyer P (1999) Palindromic sequences and A+T-rich DNA elements promote illegitimate recombination in Nicotiana tabacum. J Mol Biol 291: 29–46[CrossRef][Web of Science][Medline] Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473–497[CrossRef] Narasimhulu SB, Deng X-B, Sarria R, Gelvin SB (1996) Early transcription of Agrobacterium T-DNA genes in tobacco and maize. Plant Cell 8: 873–886[Abstract]
Pansegrau W, Schoumacher F, Hohn B, Lanka E (1993) Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: analogy to bacterial conjugation. Proc Natl Acad Sci USA 90: 11538–11542 Pipiras E, Coquelle A, Bieth A, Debatisse M (1998) Interstitial deletions and intrachromosomal amplification initiated from a double-strand break targeted to a mammalian chromosome. EMBO J 17: 325–333[CrossRef][Web of Science][Medline] Potrykus I, Bilang R, Futterer J, Sautter C, Schrott M, Spangenberg G (1998) Genetic Engineering of Crop Plants, Ed 1. Marcel Dekker, New York Puchta H (1998) Repair of genomic double-strand breaks in somatic plant cells by one-sided invasion of homologous sequences. Plant J 13: 77–78 Rossi L, Hohn B, Tinland B (1993) The VirD2 protein of Agrobacterium tumefaciens carries nuclear localization signals important for transfer of T-DNA to plant. Mol Gen Genet 239: 345–353[CrossRef][Web of Science][Medline] Salomon S, Puchta H (1998) Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J 17: 6086–6095[CrossRef][Web of Science][Medline] Savka MA, Dessaux Y, Oger P, Rossbach S (2002) Engineering bacterial competitiveness and persistence in the phytosphere. Mol Plant-Microbe Interact 15: 866–874[Medline]
Scheiffele P, Pansegrau W, Lanka E (1995) Initiation of Agrobacterium tumefaciens T-DNA processing: purified proteins VirD1 and VirD2 catalyze site- and strand-specific cleavage of superhelical T-border DNA in vitro. J Biol Chem 270: 1269–1276
Sen P, Pazour GJ, Anderson D, Das A (1989) Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA. J Bacteriol 171: 2573–2580 Sheng J, Citovsky V (1996) Agrobacterium-plant cell interaction: have virulence proteins, will travel. Plant Cell 8: 1699–1710[CrossRef][Web of Science][Medline] Stachel SE, Zambryski PC (1989) Generic trans-kingdom sex? Nature 340: 190–191[CrossRef][Medline] Stougaard J (1993) Substrate-dependent negative selection in plants using a bacterial cytosine deaminase gene. Plant J 3: 755–761[CrossRef] Takano M, Egawa H, Ikeda JE, Wakasa K (1997) The structures of integration sites in transgenic rice. Plant J 11: 353–361[CrossRef][Web of Science][Medline] Tinland B (1996) The integration of T-DNA into plant genomes. Trends Plant Sci 1: 178–184[CrossRef] Tinland B, Hohn B (1995) Recombination between prokaryotic and eukaryotic DNA: integration of Agrobacterium tumefaciens T-DNA into the plant genome. Genet Eng 17: 209–229
Tinland B, Hohn B, Puchta H (1994) Agrobacterium tumefaciens transfers single-stranded transferred DNA (T-DNA) into the plant cell nucleus. Proc Natl Acad Sci USA 91: 8000–8004 Tinland B, Schoumacher F, Gloeckler V, Bravo-Angel AM, Hohn B (1995) The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome. EMBO J 14: 3585–3595[Web of Science][Medline] Tzfira T, Citovsky V (2000) From host recognition to T-DNA integration: the function of bacterial and plant genes in the Agrobacterium-plant cell interaction. Mol Plant Pathol 1: 201–212 Tzfira T, Citovsky V (2002) Partners-in-infection: host proteins involved in the transformation of plant cells by Agrobacterium. Trends Cell Biol 12: 121–129[CrossRef][Web of Science][Medline] Tzfira T, Jensen CS, Wangxia W, Zuker A, Altman A, Vainstein A (1997) Transgenic Populus: a step-by-step protocol for its Agrobacterium-mediated transformation. Plant Mol Biol Rep 15: 219–235[CrossRef] Tzfira T, Rhee Y, Chen M-H, Citovsky V (2000) Nucleic acid transport in plant-microbe interactions: the molecules that walk through the walls. Annu Rev Microbiol 54: 187–219[CrossRef][Web of Science][Medline] Tzfira T, Vaidya M, Citovsky V (2001) VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity. EMBO J 20: 3596–3607[CrossRef][Web of Science][Medline] van Attikum H, Bundock P, Hooykaas PJJ (2001) Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration. EMBO J 20: 6550–6558[CrossRef][Web of Science][Medline]
van Attikum H, Bundock P, Overmeer RM, Lee LY, Gelvin SB, Hooykaas PJ (2003) The Arabidopsis AtLIG4 gene is required for the repair of DNA damage, but not for the integration of Agrobacterium T-DNA. Nucleic Acids Res 31: 4247–4255
van Attikum H, Hooykaas PJJ (2003) Genetic requirements for the targeted integration of Agrobacterium T-DNA in Saccharomyces cerevisiae. Nucleic Acids Res 31: 826–832
van der Krol AR, Chua N-H (1991) The basic domain of plant B-ZIP proteins facilitates import of a reporter protein into plant nuclei. Plant Cell 3: 667–675
Vergunst AC, Schrammeijer B, den Dulk-Ras A, de Vlaam CMT, Regensburg-Tuink TJ, Hooykaas PJJ (2000) VirB/D4-dependent protein translocation from Agrobacterium into plant cells. Science 290: 979–982
Wang K, Stachel SE, Timmerman B, Van Montagu M, Zambryski PC (1987) Site-specific nick occurs within the 25 bp transfer promoting border sequence following induction of vir gene expression in Agrobacterium tumefaciens. Science 235: 587–591 Ward E, Barnes W (1988) VirD2 protein of Agrobacterium tumefaciens very tightly linked to the 5' end of T-strand DNA. Science 242: 927–930 Winans SC, Mantis NJ, Chen CY, Chang CH, Han DC (1994) Host recognition by the VirA, VirG two-component regulatory proteins of Agrobacterium tumefaciens. Res Microbiol 145: 461–473[Medline]
Young C, Nester EW (1988) Association of the VirD2 protein with the 5' end of T-strands in Agrobacterium tumefaciens. J Bacteriol 170: 3367–3374
Yusibov VM, Steck TR, Gupta V, Gelvin SB (1994) Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells. Proc Natl Acad Sci USA 91: 2994–2998 Zambryski PC, Depicker A, Kruger K, Goodman HM (1982) Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J Mol Appl Genet 1: 361–370[Medline]
Ziemienowicz A, Merkle T, Schoumacher F, Hohn B, Rossi L (2001) Import of Agrobacterium T-DNA into plant nuclei: Two distinct functions of VirD2 and VirE2 proteins. Plant Cell 13: 369–384
Ziemienowicz A, Tinland B, Bryant J, Gloeckler V, Hohn B (2000) Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro. Mol Cell Biol 20: 6317–6322 Zupan J, Muth TR, Draper O, Zambryski PC (2000) The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J 23: 11–28[CrossRef][Web of Science][Medline] Zupan J, Zambryski PC (1997) The Agrobacterium DNA transfer complex. Crit Rev Plant Sci 16: 279–295 This article has been cited by other articles:
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ABSTRACT
RESULTS
-glucoronidase (GUS) gene; hpt, hygromycin resistance gene;NOS5', nopaline synthase promoter;NOS3', nopaline synthase terminator; pAg7, agropine synthase terminator.
29 bp) truncated I-SceI-uidA-hpt T-DNA integrated into the DSB site (
