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

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

Structure and expression of DNA transferred to tobacco via transformation of protoplasts with Ti-plasmid DNA: co-transfer of T-DNA and non T-DNA sequences

Summary The T-DNA structure and organization in tissues obtained via transformation of tobacco protoplasts with Ti-plasmid DNA was found to be completely different from the T-DNA introduced via Agrobacterium tumefaciens. It is often fragmented. Overlapping copies of T-DNA, having various sizes, as well as separated fragments of T-DNA were detected. The border sequences of 23 basepairs (bp), flanking the T-region in the Ti-plasmid as direct repeats are not used as preferred sequences for integration. Similar results were obtained with a T-region clone lacking one of the T_L-borders. This clone, which carried the cytokinin locus and only the right border sequence of T_L and the left border sequence of T_R, still had the capacity to transform protoplasts. Also the Vir-region of the Ti-plasmid is not required for integration of foreign DNA via DNA transformation. This is demonstrated by the results with the T-region clone mentioned and by the transforming capacity of a Ti-plasmid carrying a mutated Vir-region. Nevertheless, in a number of Ti-plasmid DNA transformants Vir-region fragments were found to be stably integrated. Furthermore, it has been established that co-transformation can occur with plant cells. Besides the detection of Ti-plasmid fragments from outside the T-region also DNA sequences originating from two DNA sources, which were both independently present in transformation experiments, have been found in some DNA transformants, e.g. calf thymus DNA, which was used as carrier DNA. No expression of the co-transferred DNA was observed. In total three phenotypical classes of DNA transformants were isolated. Although the T-DNA was often scrambled, polyA⁺ mRNA studies indicated that the different phenotypes studied can be explained by the presence of active T-DNA genes with known functions.     

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.     

Use of a TR T-DNA promoter to express genes in plants and bacteria

Summary A series of plasmids have been constructed in which a promoter from the T_R region of the Agrobacterium tumefaciens Ti-plasmid has been fused to genes encoding neomycin phosphotransferase II (NPT II) or a cDNA clone encoding a 22 kd zein protein. After recombination into the Ti-plasmid pTiA6, A. tumefaciens strains harboring these plasmids were used to incite tumors on sunflower hypocotyl sections. Tumors containing the NPT II gene in the correct orientation relative to the promoter were able to grow on normally inhibitory concentrations of the antibiotic G418. Tumors with the NPT II gene in the incorrect orientation, or with the zein gene in either orientation, were killed by the antibiotic. S1 nuclease protection experiments indicated that for both the NPT II and zein genes, the T-DNA promoter was functioning correctly. The T-DNA fragment containing the promoter active in plants also contained promoter sequences active both in E. coli and in A. tumefaciens.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Formation of a putative relaxation intermediate during T-DNA processing directed by the Agrobacterium tumefaciens VirD1, D2 endonuclease

During the initial stages of crown gall tumorigenesis, the T-DNA region of the Agrobacterium tumefaciens Ti-plasmid is processed, resulting in the production of T-DNA molecules that are subsequently transferred to the plant cell. Processing of the T-DNA in the bacterium involves the nicking of T-DNA border sequences by an endonuclease encoded by the virD locus, and the subsequent tight (possibly covalent) association of the VirD2 protein with the 5′ end of the processed single-stranded or double-stranded T-DNA molecule. To investigate the interaction of the VirD1,D2 endonuclease with a right T-DNA border, a set of plasmids containing both the border and virD sequences on the same high-copy-number replicon has been constructed and introduced into Escherichia coli. In this model system a tight nucleoprotein complex is formed between the relaxed double-stranded substrate plasmid and the VirD2 protein. This putative T-DNA processing complex may be analogous to the covalent relaxation complex formed between the pilot protein and plasmid DNA during bacterial conjugation. VirD2 attachment to the relaxed substrate plasmid was resistant to denaturing agents but sensitive to S1 nuclease digestion, indicating a single-stranded region near the site of protein attachment. We speculate that this structure may be an intermediate formed prior to T-strand unwinding from the substrate plasmid in a host bacterium.     

Genetic analysis of integration mediated by single T-DNA borders.

Transformation of plant cells by the T-DNA of the Ti plasmid of Agrobacterium tumefaciens depends in part upon a sequence adjacent to the right T-DNA end. When this sequence is absent, the T-DNA is almost avirulent; when it is present, DNA between it and the left T-DNA border region becomes integrated in plants. To investigate further this process of DNA transfer and integration, we introduced the right border region and the nopaline synthase (nos) gene of plasmid pTiC58 into a variety of new positions around Ti plasmids. The border region functioned when separated from the remainder of the T-DNA by almost 50 kilobases. It also worked when placed outside of the T-DNA region where there were no known left-border sequences with which to interact. Indeed, the nos gene could be transferred to plants even when no other Ti plasmid sequences were present on the same plasmid. These results may indicate that the sequence requirements for the left borders are not as stringent as those for the right borders. In addition, mutants with an extra copy of the right border region within their T-DNA were found to transfer or integrate only parts of the bacterial T-DNA region. It is possible that abnormally placed T-DNA borders interfere with the normal process of DNA transfer, integration, or both.     

Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens.

Early studies on Agrobacterium tumefaciens showed that development of tumors on plants following infection by A. tumefaciens was optimal at temperatures around 22 degrees C and did not occur at temperatures above 29 degrees C. To assess whether this inability to induce tumors is due to a defect in the T-DNA transfer machinery, mobilization of an incompatibility group Q (IncQ) plasmid by the T-DNA transfer machinery of A. tumefaciens was tested at various temperatures. Optimal transfer occurred when matings were performed at 19 degrees C, and transfer was not seen when matings were incubated above 28 degrees C. Transfer of the IncQ plasmid was dependent upon induction of the virB and virD operons by acetosyringone but was not dependent upon induction of the tra genes by octopine. However, alterations in the level of vir gene induction could not account for the decrease in transfer with increasing temperature. A. tumefaciens did successfully mobilize IncQ plasmids at higher temperatures when alternative transfer machineries were provided. Thus, the defect in transfer at high temperature is apparently in the T-DNA transfer machinery itself. As these data correlate with earlier tumorigenesis studies, we propose that tumor suppression at higher temperatures results from a T-DNA transfer machinery which does not function properly.     

A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadoriopines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 co-inherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.     

The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA.

We used a binary-vector strategy to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. We constructed plasmids (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs in trans with complementary regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results we suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.     

Characterization of Agrobacterium tumefaciens virulence proteins induced by the plant factor acetosyringone

The Ti plasmid virulence (vir) loci encode functions essential for the transfer of the T-DNA element from Agrobacterium tumefaciens to plant cells. The expression of these loci is specifically signaled by plant phenolics such as acetosyringone. Here, we characterize the protein products that are induced in Agrobacterium grown in the presence of acetosyringone. More than 10 to 15 proteins are induced in strains harboring different Ti plasmids. Two general classes of acetosyringone-induced proteins are observed, encoded either within or outside the vir region. Synthesis of both classes of proteins requires acetosyringone and the products of the vir regulatory genes A and G. Those proteins encoded outside the vir region define a novel category of proteins, the virulence-related proteins, which are both chromosomally and Ti plasmid-encoded. The molecular weight and subcellular localization of several pTiA6 vir-induced proteins are identified. The most abundant induced protein has a molecular weight of 65,000, and is the single product of the virE locus; this protein distributes into both cell envelope and soluble fractions. Three proteins with molecular weights of approximately 33,000, 80,000 and 25,000 fractionate with the cell envelope and are encoded by genes within the 5' half of the virB locus. The envelope localization of the virB proteins suggests that they play a role in directing T-DNA transfer events that occur at the bacterial surface.     

Transient transfection of mammalian cells with DNA of the plant-pathogenic Ti-plasmid and expression of marker and resident sequences

Summary The large Ti-plasmid from Agrobacterium tumefaciens strain C58 has been used for transfection experiments with mammalian cells. In DNA from Tupaia baby fibroblasts Ti-plasmid sequences could be identified by filter hybridization as long as four weeks after transfection including two cell passages. The hybridization signals decreased rapidly after addition of the Ti-plasmid DNA-coprecipitate to the cells. The signals were often not detected any more after the first day, but were visible one week after transfection. Nuclei prepared from Ti-plasmid-transfected cells hybridized to pTi-specific RNA. With the chloramphenicol acetyl transferase-gene as marker no discrimination in DNA uptake was found between the Ti-plasmid and much smaller plasmids. According to the number of nuclei with homology to pTi-sequences it is assumed that about 0.2% of the cells carry Ti-plasmid DNA in the nucleus. Analysis of RNA isolated from cells transfected with cloned segments of the Ti-plasmid revealed that the TDNA region of the Ti-plasmid was predominantly transcribed.Abbreviations CAT Chloramphenicol Acetyl Transferase - NPT Neomycin Phosphotransferase - SDS Sodium Dodecylsulfate - TK Thymidine Kinase     

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

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

Sequence characterization of the vir region of a nopaline type Ti plasmid, pTi-SAKURA.

We isolated a crown gall tumor-inducing nopaline type Ti plasmid from Agrobacterium tumefaciens on a Sakura Japanese cherry tree, and designated it as pTi-SAKURA. By primer walking sequencing with long PCR and a newly developed PCR subcloning technique for long insert DNA, we completed DNA sequencing of the most important functional unit, the virulence (vir) region of pTi-SAKURA, which is indispensable for T-DNA transfer into the plant's chromosomes. By homology searches with the vir genes of other bacterial plasmids, we identified 11 open reading frames (orfs) and 31 genes and 11 vir box, which are 6 bp regulatory sequences. In total, 26 vir genes, including the putative virF and virK and the main vir region, were present as the vir gene cluster. The presence of vir box, GC content, codon usage and expression analysis in these genes led us to propose a new vir region.     

The interaction of Agrobacterium Ti-plasmid DNA and plant cells

The tumour-inducing plasmids of Agrobacterium tumefaciens (Ti-plasmids) reveal several interesting properties. They are catabolic plasmids, which, instead of rendering Agrobacterium strains capable of catabolizing compounds found in Nature, force a plant to synthesize these catabolites (denoted 'opines'). This situation is obtained by insertion of a segment of the Ti-plasmid (the T-DNA) into the plant nucleus, where T-DNA genes become expressed and intervene in the biosynthesis of these opines. Cells containing the T-DNA behave as neoplasms (crown gall cells). Southern blotting shows that the insertion process responsible for T-DNA transfer probably recognizes special sequences on the T-DNA since the length of the T-DNA segment observed in different, independently isolated tumour lines was found to be similar. For the nopaline Ti-plasmids both left-hand and right-hand borders were found to be constant. For the octopine plasmid the left border was constant and at least two classes of right-hand borders were found. Upon redifferentiation of the transformed plant cells, the T-DNA was found to be conserved in all somatic cells examined. However, small deletions at the border fragments of the T-DNA have been observed. The exact arrangement and copy number of the T-DNA in a nucleus is still under study, but genomic cloning has already revealed that an interspersed tandem arrangement is dominant in nopaline tumours. Clones containing both the right border of one T-DNA and the left border of the neighbouring tandem T-DNA were isolated. In order to identify the different T-plasmid encoded functions an extensive use was made of transposon insertion mutagenesis. When an antibiotic resistance transposon was inserted into the non-essential regions of the T-DNA, a linked transfer to the plant DNA of the transposon together with the T-DNA was observed. This indicates that Ti-plasmids are possible vectors for genetic engineering in plants. A strategy is described for insertion of any cloned DNA segment into the T-DNA.     

Homology mapping of T-DNA regions on three Agrobacterium rhizogenes Ri plasmids by electron microscope heteroduplex studies

Recombinant plasmids carrying segments of the Agrobacterium rhizogenes T-DNA regions of the three Ri plasmids 1855 (TL-DNA only), 8196, and 2659 were used for establishing homology maps by electron microscope examination of heteroduplexes. Plasmid DNA was linearized by digestion with suitable restriction endonucleases in order to generate large T-DNA segments. Heteroduplexes were prepared in 50% formamide and spread under standard conditions. Measurements of double and single strands allowed the drawing of homology maps. The three T-DNAs share mainly two homologous sequences of respectively about 2.5 and 1.5 kb, bracketing a largely nonhomologous central part which is about 5.5 kb long. The T-DNAs from pRi1855 and pRi2659 appear to be more related to each other than to that of pRi8196. With reference to the published nucleotide sequence of the TL-DNA of pRiA4 (probably identical to that of pRi1855), ORFs 8 and 14 seem to be the most conserved sequences of the three T-DNAs. The significance of these conserved sequences is unclear since the genetic loci involved in rhizogenicity of agropine strains identified previously are located in nonhomologous regions.