Inhibition of cytosine methylation allows efficient cloning of T-DNA tagged plant DNA ofArabidopsis thaliana by plasmid rescue

Summary Plasmid rescue can provide an efficient way of cloning T-DNA-tagged genomic DNA of plants. However, rescue has often been hampered by extensive rearrangements in the cloned DNA. We have demonstrated using a transgenic line ofArabidopsis thaliana that the plant DNA flanking the T-DNA tag was heavily cytosine methylated. This methylation could be completely inhibited by growing the plants in the presence of azacytidine. Rescue of the T-DNA tag together with the flanking plant genomic DNA sequences from nontreated control plants into an modified cytosine restriction (mcr) proficient strain ofEscherichia coli resulted in rearrangements of the majority of the rescued plasmids. These rearrangements could be avoided if the methylation was inhibited in the transgenic plants by azacytidine treatment or by cloning into anmcr-deficient strain ofE. coli. The results indicate that cytosine methylation of the DNA in the transgenic plants is the main cause of the DNA rearrangements observed during plasmid rescue and suggest efficient strategies to eliminate such artifacts.     

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.     

Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA

We introduced a plant selection marker, nptII, to the left of border A in the Agrobacterium Ti plasmid pTiA6. Infection of tobacco leaf discs with the modified Agrobacterium strain gave rise to kanamycin-resistant calli which grew in a hormone-dependent manner. Southern hybridization analysis of DNA isolated from four transformants indicated initiation of DNA transfer at or near border A and absence of T-DNA sequences. These results demonstrate that DNA transfer events starting at a left border on a native Ti plasmid and moving away from the T-DNA region occur and that they can be detected by designing a suitable selection strategy.     

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.     

Agrobacterium T-DNA integration in Arabidopsis is correlated with DNA sequence compositions that occur frequently in gene promoter regions

Mobile insertion elements such as transposons and T-DNA generate useful genetic variation and are important tools for functional genomics studies in plants and animals. The spectrum of mutations obtained in different systems can be highly influenced by target site preferences inherent in the mechanism of DNA integration. We investigated the target site preferences of Agrobacterium T-DNA insertions in the chromosomes of the model plant Arabidopsis thaliana. The relative frequencies of insertions in genic and intergenic regions of the genome were calculated and DNA composition features associated with the insertion site flanking sequences were identified. Insertion frequencies across the genome indicate that T-strand integration is suppressed near centromeres and rDNA loci, progressively increases towards telomeres, and is highly correlated with gene density. At the gene level, T-DNA integration events show a statistically significant preference for insertion in the 5 and 3 flanking regions of protein coding sequences as well as the promoter region of RNA polymerase I transcribed rRNA gene repeats. The increased insertion frequencies in 5 upstream regions compared to coding sequences are positively correlated with gene expression activity and DNA sequence composition. Analysis of the relationship between DNA sequence composition and gene activity further demonstrates that DNA sequences with high CG-skew ratios are consistently correlated with T-DNA insertion site preference and high gene expression. The results demonstrate genomic and gene-specific preferences for T-strand integration and suggest that DNA sequences with a pronounced transition in CG- and AT-skew ratios are preferred targets for T-DNA integration.Electronic Supplementary Material Supplementary material is available for this article at .This revised version was published online in March 2005 with corrections to Dr. Tatarinovas name.     

Genetic studies on the role of octopine T-DNA border regions in crown gall tumor formation

Summary Crown gall tumors result from transfer and integration of the T-DNA from the Ti plasmid of Agrobacterium tumefaciens into plant nuclear DNA. In the present study, recombinant plasmids containing deletion and rearrangement deriviatives of the T-DNA region of the octopine Ti plasmid pTiA6 were tested in a binary tumorigenesis system (Hoekema et al. 1983) to determine the requirements for T-DNA border regions in tumor formation. Since two defined segments of the T-DNA region of octopine Ti plasmids can be detected in tumor DNA (the left (T_L-) and right (T_R-) DNA), four border regions exist in this Ti plasmid. Agrobacteria harboring plasmid constructs which contain a T-DNA gene capable of inciting tumors (gene 4, the tmr gene, which is involved in cytokinin biosynthesis) and various T-DNA border regions were tested for ability to cause tumors on Nicotiana glauca and other host plants. Such tmr constructs containing as their only border region the right border of either the T_L-DNA or the T_R-DNA are fully tumorigenic. Analogous tmr constructs containing only the T_L-DNa left border region are not tumorigenic. These results do not depend on the orientation or position of the single border with respect to the tmr gene; furthermore, the T_R-DNA right border can confer tumor-forming ability despite the presence of an intervening copy of the T_L-DNA left border.These results for relatively small plasmids are contrasted with previously determined requirements for border regions in tumorigenesis by intact Ti plasmids. A model previously proposed by Wang et al. (1984) for the role of border regions in DNA transfer to plant cells is extended in order to explain the tumor-forming ability of plasmid constructs containing a single border region. The results of this study interpreted according to the model suggest that the octopine T_L-DNA left border is defective in this DNA-transfer process.     

Improved efficiency for T-DNA-mediated transformation and plasmid rescue inArabidopsis thaliana

A vector was constructed for the isolation of gene fusions to thelacZ reporter gene following T-DNA integration into the genome ofArabidopsis thaliana. To facilitate the generation of taggedA. thaliana plants, we established a modified method for high-frequency transformation ofA. thaliana byAgrobacterium tumefaciens. The main modification required was to inhibit the methylation of T-DNA in the transformed calli. Apparently, cytosine residues of thenos-nptII gene used as a selectable marker were methylated, and the expression of this gene was suppressed. Treatment of the calli with the cytosine methylation inhibitor 5-azacytidine led to a dramatic increase (from 3% to 96%) in the regeneration of transformed (kanamycin-resistant) shoots. A total of 150 transgenic plants were isolated, and in 17 of these expression of thelacZ reporter was detected byin situ staining. The T-DNA insert together with flanking plant DNA sequences was cloned intoEscherichia coli by plasmid rescue from some of the T₃ transformants that harbored one copy of the integrated T-DNA. Comparison of the rescued DNA with the corresponding DNA of the transgenic plant showed that most of the rescued plasmids had undergone rearrangements. These rearrangements could be totally avoided if anmcrAB (modified cytosine restriction) mutant ofE. coli was used as the recipient in plasmid rescue.     

Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study

Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.     

Ars region in TL-DNA on octopine type Ti plasmids

Summary In the TL-DNA region of the octopine type Ti plasmids, an ars region was assigned as the DNA segment conferring the replicational ability to YIp5 in Saccharomyces cerevisiae. T-DNA:YIp5 hybrid plasmids containing a particular T-DNA region could transform yeast cells at a frequency of 10³–10⁴ transformants per g plasmid DNA and they were rescued in Escherichia coli, although the transformed phenotype was mitotically unstable. The instability was inferred to be caused by segregation of the plasmids due to their low efficiency of replication. The ars region was mapped on the noncoding region between the coding regions corresponding to no. 5 and no. 7 mRNA, and its minimal length determined in this experiment was about 150 bp.Abbreviations Ti plasmid tumor inducing plasmid - T-DNA transferred DNA or tumor DNA - TL-DNA left T-DNA - ars autonomously replicating sequences     

T-DNA fragments of hairy root plasmid pRi8196 are distantly related to octopine and nopaline Ti plasmid T-DNA

Agrobacterium Ti (tumor-inducing) and Ri (root-inducing) plasmids transform dicot plant cells by insertion of a specific plasmid sector called T-DNA (transferred DNA) into host plant nuclear DNA. The mannopine-type Ri plasmid pRi8196 contains four BamHI fragments that encompass core T-DNA. We report Southern hybridization studies that show that these four fragments have no strong homology to octopine-, nopaline-, or agropine-type Ti plasmids. We detected and mapped very weak homology regions, most of which are assignable to opine synthase or opine catabolic functions on the Ti plasmid. We found no homology between Ri T-DNA and the region of Ti T-DNA that encodes tumor morphology functions.     

T-DNA integration patterns in transgenic tobacco plants

To investigate the various integration patterns of T-DNA generated by infection withAgrobacterium, we developed a vector (pRCV2) for the effective T-DNA tagging and applied it to tobacco (Nicotiana tabacum cv. Havana SR1). pRCV2 was constructed for isolating not only intact T-DNA inserts containing both side borders of T-DNA, but also for partial T-DNA inserts that comprise only the right or left side. We also designed PCR confirmation primer sets that can amplify in several important regions within pRCV2 to detect various unpredictable integration patterns. These can also be used for the direct inverse PCR. Leaf disks of tobacco were transformed withAgrobacterium tumefaciens LBA4404 harboring pRCV2. PCR and Southern analysis revealed the expected 584 bp product for thehpt gene as well as one of 600 bp for thegus gene in all transformants; one or two copies were identified for these integrated genes. Flanking plant genomic DNA sequences from the transgenic tobacco were obtained via plasmid rescue and then sequenced. Abnormal integration patterns in the tobacco genome were found in many transgenic lines. Of the 17 lines examined, 11 contained intact vector backbone; a somewhat larger deletion of the left T-DNA portion was encountered in 4 lines. Because nicking sites at the right border showed irregular patterns when the T-DNA was integrated, it was difficult to predict the junction regions between the vector and the flanking plant DNA.     

Stability of the T-DNA flanking regions in transgenic Arabidopsis thaliana plants under influence of abiotic stress and cultivation practices

Genetic transformation is often associated with different rearrangements of the plant genome at the site of insertion. Therefore the question remains weather these T-DNA insertion sites are more prone to genotoxic stresses. Here, we studied the impact of propagation through generations, the influence of gene stacking and of photo oxidative stress caused by high light intensity on the stability of the transgene flanking regions in the model plant Arabidopsis thaliana. Conformational Sensitive Capillary Electrophoresis (CSCE), RFLP and sequencing were deployed in this analysis in order to study the proximal 100 bp and the long-range T-DNA flanking sequences. By screening seven transgenic lines no evidence for occurrence of mutation events were found, implying that the nucleotide sequence of the T-DNA flanking regions of the studied events is unlikely to be unstable. N. Papazova and R. Ghedira have equally contributed to the paper.     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

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

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

Rapid discrimination of sequences flanking and within T‐DNA insertions in the Arabidopsis genome

An improvement to previous methods for recovering Arabidopsis thaliana genomic DNA flanking T-DNA insertions is presented that allows for the avoidance of some of the cloning difficulties caused by the concatameric nature of T-DNA inserts. The principle of the procedure is to categorize by size restriction fragments of mutant DNA, produced in separate digestions with NdeI and Bst1107I. Given that the sites for these two enzymes are contiguous within the pGV3850:1003 T-DNA construct, the restriction fragments obtained fall into two categories: those showing identical size in both digestions, which correspond to sequences internal to T-DNA concatamers; and those of different sizes, that contain the junctions between plant DNA and the T-DNA insert. Such a criterion makes it possible to easily distinguish the digestion products corresponding to internal T-DNA parts, which do not deserve further attention, and those which presumably include a segment of the locus of interest. Discrimination between restriction fragments of genomic mutant DNA can be made on rescued plasmids, inverse PCR amplification products or bands in a genomic blot.     

SNP discovery and genetic mapping of T-DNA insertional mutants in Fragaria vesca L.

As part of a program to develop forward and reverse genetics platforms in the diploid strawberry [Fragaria vesca L.; (2n = 2x = 14)] we have generated insertional mutant lines by T-DNA mutagenesis using pCAMBIA vectors. To characterize the T-DNA insertion sites of a population of 108 unique single copy mutants, we utilized thermal asymmetric interlaced PCR (hiTAIL-PCR) to amplify the flanking region surrounding either the left or right border of the T-DNA. Bioinformatics analysis of flanking sequences revealed little preference for insertion site with regard to G/C content; left borders tended to retain more of the plasmid backbone than right borders. Primers were developed from F. vesca flanking sequences to attempt to amplify products from both parents of the reference F. vesca 815 × F. bucharica 601 mapping population. Polymorphism occurred as: presence/absence of an amplification product for 16 primer pairs and different size products for 12 primer pairs, For 46 mutants, where polymorphism was not found by PCR, the amplification products were sequenced to reveal SNP polymorphism. A cleaved amplified polymorphic sequence/derived cleaved amplified polymorphism sequence (CAPS/dCAPS) strategy was then applied to find restriction endonuclease recognition sites in one of the parental lines to map the SNP position of 74 of the T-DNA insertion lines. BLAST search of flanking regions against GenBank revealed that 46 of 108 flanking sequences were close to presumed strawberry genes related to annotated genes from other plants.     

Distribution of T-DNA carrying a Ds element on rice chromosomes

Over 3000 rice plants with T-DNA carrying a Ds element were constructed by Agrobacterium tumefaciens mediation. Using inverse PCR methodology, 590 unique right flanking sequences of T-DNA (Ds) were retrieved from independent transformants and classified into six main types on the basis of the origin of filler DNA between the right border of T-DNA and flanking sequence of rice genome. Type I sequences were the most common and showed canonical integration that T-DNA right border was followed by rice genome sequence with or without filler DNA of no more than 50 bp, while type II sequences displayed a vector-genome combination that T-DNA right border was followed by a vector fragment and then connected with rice genome sequence. The location and distribution of 340 type I and II flanking sequences on the rice chromosome were determined using BLAST analysis. The 340 Ds insertions at an average interval of 0.8 megabase (Mb) constructed a basic framework of Ds starter points on whole rice chromosomes. The frequency of T-DNA (Ds) inserted into the exons of predicted genes on chromosome one was 21%. Knowledge of T-DNA (Ds) locations on chromosomes will prove to be a useful resource for isolating rice genes by Ds transposon tagging as these Ds insertions can be used as starting lines for further mutagenesis.     

The plasmids of Acetobacter xylinum and their interaction with the host chromosome

Summary Acetobacter xylinum contains a complex system of plasmid DNA molecules. Plasmids of molecular weights or copy numbers different from the original wild-type, are found in different types of mutants. Restriction endonuclease digestion and DNA/DNA hybridization analysis, showed that the plasmids often contained partly, but not completely the same DNA sequences. Two of these plasmid classes were analysed in more detail, and could be shown to differ in size by about 5 kb. Hybridization analysis using cloned DNA fragments as probes, showed that sequences lacking in the smallest plasmid were still present in a DNA fraction co-migrating with linearized chromosomal DNA. In addition, at least part of the DNA in the smallest plasmid was present both in the plasmid and chromosomal DNA fraction. Analysis of a particular strain containing an insertion of transposon Tn1, also indicated the existence of complex interactions between plasmids and chromosomal DNA. Together with experiments on conjugative transfer and curing of the plasmids, the results indicate that at least part of the genetic system of A. xylinum is unusual when compared to that of other genetically characterized bacteria.