Comparison of T-DNA oncogene complements of Agrobacterium tumefaciens tumor-inducing plasmids with limited and wide host ranges.

The T-DNA oncogene complements of the limited-host-range tumor-inducing plasmid pTiAg63 and the wide-host-range plasmid pTiA6 were compared. The resulting data indicate that pTiAg63 has DNA sequences related to most of the genes encoded by the oncogene region, the TL-DNA, of pTiA6 and that these sequences are divided between two T-DNA regions, the TA-DNA, which encoded sequences related to pTiA6 genes 4 (the cytokinin independence gene) and 6a, as well as to a pTiA6 TL-DNA fragment that encoded gene 6b and a portion of gene 3, and the TB-DNA, which encoded sequences related to genes 1 and 2 (the auxin independence genes). Tumor tissues of Nicotiana rustica incited by Agrobacterium tumefaciens harboring either pTiA6 or pTiAg63 grew axenically in vitro on phytohormone-free medium. The morphologies of the tissues, however, differed; whereas those incited with pTiA6 grew as loose, friable, unorganized callus, the tumors incited by pTiAg63 grew as clumps of rootlike structures. Thus, the T-DNA oncogene complements of these plasmids were not equivalent. The results are discussed in relation to the A. tumefaciens host range.     

Hairy-root-inducing plasmid: physical map and homology to tumor-inducing plasmids.

A physical map was constructed for the 250-kilobase plasmid pRiA4b, which confers the virulence properties of a strain of Agrobacterium rhizogenes for hairy root disease in plants. The complete HindIII and KpnI restriction map was determined from a collection of overlapping HindIII partial digest clones. Homologous regions with two well-characterized plasmids that confer virulence for crown gall disease, plasmids pTiA6 and pTiT37, were mapped on pRiA4b. As much as 160 kilobases of pRiA4b had detectable homology to one or both of these crown-gall-tumor-inducing plasmids. About 33 kilobases of pRiA4b hybridized to the vir region of pTiA6, a segment of DNA required for virulence of Agrobacterium tumefaciens. Portions of pTiA6 and pTiT37 transferred into plant cells in crown gall disease (T-DNA), shared limited homology with scattered regions of pRiA4b. The tumor morphology loci tms-1 and tms-2 from the T-DNA of pTiA6 hybridized to pRiA4b. A T-DNA fragment containing the tml and tmr tumor morphology loci also hybridized to pRiA4b, but the homology has not been defined to a locus and is probably not specific to tmr. A segment of pRiA4b T-DNA which was transferred into plant cells in hairy root disease lacked detectable homology to pTiA6 and had limited homology at one end to the T-DNA of pTiT37.     

Complementation analysis of Agrobacterium tumefaciens Ti plasmid mutations affecting oncogenicity

A wide host range cosmid vector has been constructed by insertion of the lambda cos site into the plasmid pRK2501. This cosmid, which is maintained in Agrobacterium tumefaciens and is compatible with the Ti plasmid, has been used to make a clone bank of the A. tumefaciens pTiA6 plasmid. Several pTiA6 cosmids have been used to complement Tn5-induced Ti plasmid mutations. Five avirulent mutations which map outside of the region of the plasmid maintained in plant tumours (T-DNA) could be complemented in a trans orientation. Two mutations which are located on a single HpaI restriction fragment outside of the T-DNA, as well as three mutations which map within the T-DNA region, could not be complemented in a trans orientation in a REC- host.     

Physical and functional map of supervirulent Agrobacterium tumefaciens tumor-inducing plasmid pTiBo542.

Agrobacterium tumefaciens strains carrying pTiBo542 induce large, fast-appearing tumors and have an unusually wide host range. A clone bank was made from this 250-kilobase plasmid in a wide-host-range vector, and restriction maps were determined for BamHI and SalI. The virulence genes, transferred DNA genes, plasmid incompatibility region, and a region that inhibits growth of certain A. tumefaciens strains were localized. The six virulence genes and two tms genes were highly homologous to the genes of pTiA6, but the tmr gene was not. Mutations in each of the six vir loci of pTiA6 were complemented by clones from the vir region of pTiBo542.     

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.     

Physical and functional map of an Agrobacterium tumefaciens tumor-inducing plasmid that confers a narrow host range.

Agrobacterium tumefaciens Ag162 induces crown gall disease on an unusually narrow range of host plants. The 231-kilobase Ti plasmid which has been shown to determine host range, was subcloned into the vector pVCK102. By comparing overlaps of cloned insets, maps were constructed for the restriction endonucleases SalI, XhoI, EcoRI, and KpnI. Plasmid incompatibility, octopine catabolism, and at least six virulence genes were localized. Plasmid incompatibility between pTiAg162 and the wide host range plasmid pTiA6 consists of two components: mutual incompatibility and the apparent ability of pTiA6 to block RK2 replication if the pTiAg162 incompatibility locus is linked to the vector pVK102. The octopine catabolism locus maps within the 30 kilobases of DNA separating the two T-DNA regions of pTiAg162. Complementation of avirulent vir mutants of pTiA6 with clones of pTiAg162 DNA did not confer the host range of pTiAg162 but rather restored the wide host range of pTiA6. One potentially important difference between pTiA6 and pTiAg162 is that pTiAg162 T-DNA regions are widely separated.     

Host range encoded by the Agrobacterium tumefaciens tumor-inducing plasmid pTiAg63 can be expanded by modification of its T-DNA oncogene complement.

Agrobacterium tumefaciens harboring pTiA6 incite unorganized tumors on Nicotiana rustica, sunflowers, carrots, and tomatoes, whereas isogenic strains of agrobacteria harboring pTiAg63 form "rooty" tumors on N. rustica and are essentially avirulent on sunflowers, carrots, and tomatoes. In this report we show that the different host range characteristics of these two plasmids were due, in part, to differences in the T-DNA oncogene complements of the plasmids. Specifically, we constructed derivatives of pTiAg63 that contained pTiA6 oncogenes 4, 6a, and 6b inserted into the TB-DNA region and found that agrobacteria harboring these plasmids could incite unorganized tumors on N. rustica, tomatoes, carrots, and the inbred sunflower line HA202R. Undefined host factors, however, also appeared to be involved in determining A. tumefaciens host range since three inbred sunflower lines, HA303B, HA89B, and HA290B, were susceptible to tumor formation by agrobacteria harboring pTiA6 but not by strains harboring pTiAg63 or the modified pTiAg63 plasmids.     

Analysis of octopine left border-directed DNA transfer fromAgrobacterium to plants

We constructed a binary plasmid, pVR30, with a neomycin phosphotransferase II (nptII) plant expression cassette flanked by a pTiA6 left border on its right and a pTiA6 right border on its left. This plasmid was used to study transfer of DNA to plants from a left border in the presence of a right border. Infection of tobacco leaf discs with a wild type octopine strain ofAgrobacterium tumefaciens harbouring the binary plasmid resulted in the generation of kanamycin resistant calli at 18 to 26% frequency. Southern hybridization analysis of DNA isolated from eight transformed lines to different probes indicated that left border could mediate DNA transfer to plants in the presence of a right border in cis. Our results also suggest that transfer events corresponding to transfer of T- centre DNA of octopine Ti plasmid pTiA6 do occur. We have shown the relevance of left border- initiated T- DNA transfer by specifically selecting for such events and have confirmed it by Southern hybridization analysis. We also found that a border could be skipped in a few T- DNA transsfer events. This work was presented at “Plant Molecular Biology and Plant Biotechnology” symposium held in ICGEB, New Delhi during December 14–17, 1994.     

Relationship between the limited and wide host range octopine-type Ti plasmids of Agrobacterium tumefaciens.

The relationship between the limited host range octopine Ti plasmids and the wide host range octopine Ti plasmids pTiB6806 and pTiA6 was studied. The limited host range Ti plasmids shared extensive deoxyribonucleic acid homology; pTiAg63 and pTiAg162 were essentially completely homologous with pTiAg158 while pTiAg57 shared approximately 64% homology with pTiAg158. In contrast, the limited host range octopine Ti plasmids only shared 6 to 15% homology with the wide host range octopine Ti plasmid pTiB6806. Thus, limited and wide host range octopine Ti plasmids comprise distinct families of plasmids. The deoxyribonucleic acid homology shared between the limited host range Ti plasmids and pTiB6806, however, was distributed over some 50% of pTiB6806, suggesting that both families of plasmids evolved from a common progenitor plasmid. The limited host range Ti plasmids showed relatively strong homology with pTiB6806 HpaI fragment 7, a region which codes for octopine utilization by the bacterium, but showed only weak homology with pTiB6806 HpaI fragment 12, a region required for virulence. In addition, homology between the limited host range octopine Ti plasmids and the "common deoxyribonucleic acid," sequences shown to have a central role in plant cell transformation, was barely detectable when stringent hybridization conditions were used. We therefore conclude that a highly conserved version of the common deoxyribonucleic acid is not required for crown gall tumorigenesis on all plant species.     

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     

Structural Characterization of the virB Operon on the Hairy-root-inducing Plasmid A4

The hairy-root-inducing plasmid A4 (pRiA4) is capable of conferringtumorigenic symptoms on plants upon infection by its host bacterium,Agrobacterium rhizogenes. The virB operon on pRiA4 has beensequenced and found to be composed of 11 genes, virB1 to virB11,whose products mostly appear to be associated with the cellmembrane. A novel structural characteristic is frequent overlappingsbetween the translation termination and initiation codons ofadjacent genes. This is indicative of fine tuning of relativetranslation frequencies for each VirB protein. These resultssupport the view that VirB multisubunit complexes provide facilitiesfor T-DNA transfer at the bacterial cell membrane. The structuralorganization of the pRiA4 virB operon was essentially identicalto that of the previously reported virB operons of tumorinducingplasmids, pTiC58 and pTiA6, and the corresponding VirB proteinsof the three plasmids were extremely homologous to one another.On the basis of the structural similarity of each VirB protein,the phylogenetic relationship among pRiA4, pTiC58, and pTiA6is discussed.     

In vivo packaging of cosmids in transposon-mediated mutagenesis.

A technique was developed that permits the analysis of large regions of DNA by transposition mutagenesis. Large fragments of the pTiA6NC plasmid were cloned into the broad host range cosmid pHK17 and subjected to transposition mutagenesis by Tn3. Cosmids containing Tn3 insertions were selected by in vivo packaging by lambda cI857 and transduction to a new host. The insertions were localized by DNA restriction endonuclease analysis and transferred to the Ti-plasmid by marker exchange.     

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.