Functional analysis of a complex oncogene arrangement in biotype III Agrobacterium tumefaciens strains

The ubiquitous grapevine-associated octopine/cucumopine Ti plasmids of biotype III Agrobacterium tumefaciens strains carry two T regions, TA and TB, with a complex oncogene arrangement. Within the octopine/cucumopine group, two main strain types were identified: large TA strains with a TA region resembling the TL region of the biotype I octopine strain Ach5 and small TA strains with a similar T region organization as the large TA strains but with a large internal TA deletion. Structural and functional studies of the representative large TA strain Tm4 revealed six oncogenes. Each oncogene was inserted in a disarmed vector and tested for biological activity using the corresponding oncogenes of Ach5 as standards. Five Tm4 oncogenes, TA-iaaM, T-ipt, T-6b, TB-iaaH and TB-iaaM, were shown to be active, the IS-interrupted TA-iaaH gene was inactive. To study the role of each gene in the pTiTm4 context, several single and multiple pTiTm4 mutations were constructed. It was shown that whereas TA-iaaM and TB-iaaH are essential for tumour formation on grapevine, T-ipt, T-6b and TB-iaaM are not. The avirulence of the TA-iaaM ^- mutant was shown to be due to an inhibitory effect of the T-ipt gene, since a TA-iaaM ^- /T-ipt ^- double mutant was fully virulent. We conclude that the TA-iaaM gene of large TA strains is specifically required to counteract the tumour growth inhibiting activity of the T-ipt gene. Both TA-iaaM and T-ipt are absent from the small TA strains. A model on the roles and interactions of the different oncogenes in large TA and small TA strains is presented.     

Limited host range Ti plasmids: recent origin from wide host range Ti plasmids and involvement of a novel IS element, IS868.

Agrobacterium tumefaciens biotype III octopine strains have been isolated from grapevine tumors worldwide. They comprise limited and wide host range (LHR and WHR) strains that carry related tumor-inducing (Ti) plasmids with two T-regions, TA and TB. The WHR TA-region resembles the biotype I octopine region, whereas the LHR TA-region is a recent deletion derivative of the WHR TA-region, which lacks the iaa genes and part of the ipt gene. Sequencing of the TA-region of the ubiquitous LHR strain AB3 showed that the deleted region is replaced by an insertion sequence (IS) element, IS868, which resembles the IS51 element of Pseudomonas syringae subsp. savastanoi. The Ti plasmid of LHR strain Ag57 carries essentially the same iaa gene deletion as pTiAB3, but lacks IS868. We propose that the LHR Ti plasmids arose by the recent insertion of an IS868 element into the TA-region of a WHR-type Ti plasmid, followed by transposition to a nearby site. The deletion was caused during the second transposition or by later recombination between the two IS868 copies. Biotype III octopine strains also carry an IS51-like sequence close to the TB iaa genes. Our results confirm and extend earlier observations indicating that IS51-like elements in Pseudomonas and Agrobacterium are associated with iaa genes and played a major role in Ti plasmid evolution.     

Nucleotide sequence, evolutionary origin and biological role of a rearranged cytokinin gene isolated from a wide host range biotype III Agrobacterium strain

Summary A DNA fragment with homology to the cytokinin (ipt) gene from biotype I Agrobacterium tumefaciens strain Ach5 was cloned from the Ti plasmid of the wide host range biotype III Agrobacterium strain Tm-4 and sequenced. The fragment contains an intact ipt coding sequence. However, the 3 non-coding region of this ipt gene is rearranged due to a 0.9 kb deletion fusing it to the 3 coding region of the neighbouring gene 6a, most of which was found to be deleted. The Tm-4 ipt gene is strongly related to the partially deleted ipt gene of the limited host range biotype III strain Ag162. To test its biological activity, the Tm-4 ipt gene was inserted into a specially constructed, disarmed Ti vector lacking tzs and tested on tobacco, where the rearranged ipt gene induced shoot formation. The cloned Tm-4 ipt gene was mutated with Tn5 and the intact gene on the wild-type Tm-4 Ti plasmid was replaced by the mutated gene. The resulting strain was avirulent on tobacco but normally virulent on the natural host of the wild-type strain Tm-4, grapevine. As the biotype 1 6b gene diminishes the effect of a corresponding ipt gene, a larger Tm-4 fragment carrying both the ipt gene and an adjacent 6b-like gene was also tested on tobacco and compared with the Tm-4 ipt fragment alone and with an ipt and 6b/ipt fragment derived from Ach5. The Tm-4 6b gene diminishes the effect of the Tm-4 ipt gene, showing the Tm-4 6b gene to be active as well. The Tm-4 6b/ipt combination is less effective than the Ach5 combination. These results provide further insight into the molecular basis of the host range differences between limited host range and wide host range biotype III Agrobacterium strains and show that the WHR cytokinin gene, although active, does not significantly contribute to tumour formation on the natural host of the WHR biotype III strains, grapevine.Abbreviations LHR limited host range - WHR wide host range - onc oncogenicity genes - iaaH indoleacetamide hydrolase gene - iaaM tryptophan monooxygenase gene - ipt isopentenyl transferase gene - tzs transzeatin secretion gene - NAA -naphthalene acetic acid - BAP 6-benzylaminopurine - Km kanamycin - Neo neomycin - Cm chloramphenicol     

Agrobacterium vitis nopaline Ti plasmid pTiAB4: relationship to other Ti plasmids and T-DNA structure

The Ti plasmid of the Agrobacterium vitis nopaline-type strain AB4 was subcloned and mapped. Several regions of the 157 kb Ti plasmid are similar or identical to parts of the A. vitis octopine/cucumopine (o/c)-type Ti plasmids, and other regions are homologous to the nopaline-type Ti plasmid pTiC58. The T-DNA of pTiAB4 is a chimaeric structure of recent origin: the left part is 99.2% homologous to the left part of the TA-DNA of the o/c-type Ti plasmids, while the right part is 97.1 % homologous to the right part of an unusual nopaline T-DNA recently identified in strain 82.139, a biotype Il strain from wild cherry. The 3 non-coding regions of the ipt genes from pTiAB4 and pTi82.139 are different from those of other ipt genes and contain a 62 by fragment derived from the coding sequence of an ipt gene of unknown origin. A comparison of different ipt gene sequences indicates that the corresponding 62 by sequence within the coding region of the AB4 ipt gene has been modified during the course of its evolution, apparently by sequence transfer from the 62 by sequence in the 3 non-coding region. In pTi82.139 the original coding region of the ipt gene has remained largely unmodified. The pTiAB4 6b gene differs from its pTi82.139 counterpart by the lack of a 12 by repeat in the 3 part of the coding sequence. This leads to the loss of four glutamic acid residues from a series of ten. In spite of these differences, the ipt and 6b genes of pTiAB4 are functional. Our results provide new insight into the evolution of Agrobacterium Ti plasmids and confirm the remarkable plasticity of these genetic elements. Possible implications for the study of bacterial phylogeny are discussed.     

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.     

Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines.

Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid. These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A. tumefaciens strains appeared to be very similar. Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homology. Fingerprints of nopaline Ti plasmids from strains of a given biotype resemble each other but not fingerprints of Ti plasmids from strains of the other two biotypes. The wide host range octopine Ti plasmid from the biotype 3 strain Ag86 shares more DNA homology with narrow host range Ti plasmids, nopaline Ti plasmids, and octopine catabolism plasmids than with the wide host range octopine Ti plasmid from biotype 1 strain 20/1. pTiAg86 does share homology with the portion of pTi20/1 integrated and expressed in plant tumor cells. Since all wide host range Ti plasmids studied contain these sequences, we suggest that natural selection for a wide host range resulted in the presence of the common sequences in distantly related plasmids. The lack of homology between this "common DNA" and limited host range Ti plasmids shows that the DNA sequences per se are not required for tumorigenesis.     

Agrobacterium vitis nopaline Ti plasmid pTiAB4: relationship to other Ti plasmids and T-DNA structure

The Ti plasmid of the Agrobacterium vitis nopaline-type strain AB4 was subcloned and mapped. Several regions of the 157 kb Ti plasmid are similar or identical to parts of the A. vitis octopine/cucumopine (o/c)-type Ti plasmids, and other regions are homologous to the nopaline-type Ti plasmid pTiC58. The T-DNA of pTiAB4 is a chimaeric structure of recent origin: the left part is 99.2% homologous to the left part of the TA-DNA of the o/c-type Ti plasmids, while the right part is 97.1 % homologous to the right part of an unusual nopaline T-DNA recently identified in strain 82.139, a biotype Il strain from wild cherry. The 3′ non-coding regions of the ipt genes from pTiAB4 and pTi82.139 are different from those of other ipt genes and contain a 62 by fragment derived from the coding sequence of an ipt gene of unknown origin. A comparison of different ipt gene sequences indicates that the corresponding 62 by sequence within the coding region of the AB4 ipt gene has been modified during the course of its evolution, apparently by sequence transfer from the 62 by sequence in the 3′ non-coding region. In pTi82.139 the original coding region of the ipt gene has remained largely unmodified. The pTiAB4 6b gene differs from its pTi82.139 counterpart by the lack of a 12 by repeat in the 3′ part of the coding sequence. This leads to the loss of four glutamic acid residues from a series of ten. In spite of these differences, the ipt and 6b genes of pTiAB4 are functional. Our results provide new insight into the evolution of Agrobacterium Ti plasmids and confirm the remarkable plasticity of these genetic elements. Possible implications for the study of bacterial phylogeny are discussed.     

Role of T-region borders in Agrobacterium host range

The limited host range AB3 strain of Agrobacterium tumefaciens induces tumors by transferring two T-regions, TA and TB. TA is a deleted version of the well-known biotype I octopine TL-region that lacks the iaa and ipt genes, but carries an intact oncogene, gene 6b, and typical left and right border sequences. TB carries two iaa genes that together code for the synthesis of indoleacetic acid. Gene 6b and the iaa gene act synergistically when transferred in a coinoculation experiment. The TA-region of the limited host range isolate Ag57 is related to the TA-region of AB3, but differs from it at several positions. The most significant difference is the absence of the right border region. In spite of this, Ag57 and the exconjugant strain C58C9(pTiAg57) induce normal tumors on Nicotiana rustica and Vitis vinifera. Various experiments indicate that gene 6b of the Ag57 TA-region is active and transferred in spite of the absence of the right border. On N. tabacum, C58C9(pTiAg57) is nononcogenic but becomes oncogenic when the pTiAg57 TA-region is restored by the right TA border sequence of pTiAB3. Thus, the right TA border sequence of the biotype III limited host range strains is required for tumor induction on some hosts, but not on others.     

Organization and functional analysis of three T-DNAs from the vitopine Ti plasmid pTiS4

Summary The vitopine Ti plasmid pTiS4 of Agrobacterium vitis has an unusual T-DNA organization. The pTiS4 oncogenes, localized by screening selected pTiS4 clones for growth-inducing activity, are localized on three T-DNAs, whereas in all other characterized Ti plasmids one or two T-DNAs are found. The nucleotide sequences and predicted amino acid sequences of the pTiS4 oncogenes set them apart from the corresponding genes from other Ti or Ri plasmids. The oncogenes induce the same type of reaction on various test plants as the well-known pTiAch5 oncogenes but the pTiS4 ipt gene induces considerably more shoots than its Ach5 homologue. We have also identified the gene coding for vitopine synthase as well as a vitopine synthase pseudogene. Both sequences show homology to the octopine synthase gene. In terms of both nucleotide sequence and overall organization, the pTiS4 T-DNAs appear to be only distantly related to previously characterized T-DNAs.Abbreviations Ap ampicillin - IS insertion sequence - iaaH indole acetamide hydrolase - iaaM tryptophan monooxygenase - ipt isopentenyl transferase - Km kanamycin - LB Luria broth - m/a mannopine/agropine - o/c octopine/cucumopine - ocs octopine synthase     

Rapid divergence of Agrobacterium vitis octopine-cucumopine Ti plasmids from a recent common ancestor

The octopine/cucumopine (o/c) Ti plasmids of the grapevine-associated Agrobacterium vitis strains constitute a family of related DNA molecules. Restriction maps were established of two limited-host-range o/c Ti plasmids, pTiAg57 and pTiAB3, and of the wide-host-range o/c Ti plasmid pTiHml. Together with the previously obtained map of the wide-host-range o/c Ti plasmid pTiTm4, about 1000 kb were mapped with a resolution of 0.2 kb, allowing a detailed comparison of the various structures. One region of the o/c Ti plasmids is highly conserved and differs mainly by the presence or absence of relatively small DNA fragments (0.9–2.7 kb); the other region has been modified more extensively and carries large sequences specific for each Ti plasmid type. The sequence similarity within large conserved regions shows that these plasmids have diverged recently and that their evolution was driven by large-scale genetic events rather than single nucleotide changes. These results have important implications for studies on bacterial evolution.     

The limited host range of an Agrobacterium tumefaciens strain extended by a cytokinin gene from a wide host range T-region

The host range of Agrobacterium tumefaciens strain LBA649 (pTiAg57) is limited to grapevine and a few other plant species. Its host range was extended through the introduction of the T-region from the wide host range octopine plasmid pTiAch5. In contrast, R prime plasmids harboring the entire wide host range virulence region were unable to achieve this effect. Via site-directed mutagenesis a search was performed to identify the T-DNA genes which were responsible for the observed host range extension. Inactivation of one of the onc-genes (the cyt gene) was found to abolish the capacity of the T-region to extend the host range of LBA649. Therefore, we cloned the cyt gene into a disarmed T-region plant vector and used it in complementation studies with pTiAg57 via the binary vector strategy. We show that the mere presence of the cyt gene from a wide host range Ti plasmid is sufficient to extend the host range of LBA649 to certain plants. We conclude that the limited host range of LBA649 is not caused by a lack of recognition of plants but is mainly due to the absence or inactivity of a cyt gene in the T-region of pTiAg57.     

Sequence and distribution of IS866, a novel T region-associated insertion sequence from Agrobacterium tumefaciens

We have identified a new insertion sequence, IS866, located in the auxin synthesis gene TA iaaH of Tm4, a wide host range biotype III octopine/cucumopine type Agrobacterium tumefaciens strain with two T regions on its tumor-inducing (Ti) plasmid, TA, and TB. IS866 is 2716 bp long, has inverted repeats of 27 bp with three mismatches, and generates 8-bp direct repeats upon integration. In addition to IS866, pTiTm4 carries two copies of a related element, IS867, associated with TA and TB, respectively. A systematic study of 92 virulent Agrobacterium strains has shown that among the three biotypes all octopine/cucumopine and vitopine biotype III isolates contain IS866-like elements. The various octopine/cucumopine Ti plasmids always carry IS866 and IS867 at the same position as in pTiTm4. The chromosomes of the bacteria which contain these Ti plasmids also carry IS866 and IS867 copies but in varying numbers and locations.     

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.     

Agrobacterium tumefaciens 6bgenes are strain-specific and affect the activity of auxin as well as cytokinin genes

Summary The T-region located 6b gene of Agrobacterium tumefaciens has been found to interfere with cytokinin effects produced by the cotransferred ipt gene. We have compared the biological activity of three different 6b genes: A-6b from Ach5 (octopine, biotype 1), C-6b from C58 (nopaline, biotype 1) and T-6b from Tm4 (octopine, biotype III) by using different biological assays. Each 6b gene was inserted into a disarmed vector and tested on tobacco stems in coinfection experiments with the Ach5 cytokinin (ipt) gene (A-ipt). A-ipt/C-6b coinfections produced tumours with shoots, A-ipt/A-6b coinfections green tumours and A-ipt/T-6b coinfections tumours with a necrotic surface. The tumour phenotypes obtained were independent of the 6b/A-ipt coinfection ratios, indicating that the strain-specific 6b effects result from the activity of a non-diffusible 6b encoded product. Studies with ipt-less Tm4 mutants showed that 6b genes affect other tumour genes besides the ipt gene and pointed to an influence of T-6b on auxin effects resulting from the Tm4 iaa system. T-iaa/T-6b coinfection experiments showed that T-6b did indeed strongly increase tumour formation by the Tm4 iaa genes. The three 6b genes also have effects which do not require other T-region genes. The complex role of the 6b gene in crown gall induction and Agrobacterium host range will be discussed.     

Rapid divergence of Agrobacterium vitis octopine-cucumopine Ti plasmids from a recent common ancestor

The octopine/cucumopine (o/c) Ti plasmids of the grapevine-associated Agrobacterium vitis strains constitute a family of related DNA molecules. Restriction maps were established of two limited-host-range o/c Ti plasmids, pTiAg57 and pTiAB3, and of the wide-host-range o/c Ti plasmid pTiHml. Together with the previously obtained map of the wide-host-range o/c Ti plasmid pTiTm4, about 1000 kb were mapped with a resolution of 0.2 kb, allowing a detailed comparison of the various structures. One region of the o/c Ti plasmids is highly conserved and differs mainly by the presence or absence of relatively small DNA fragments (0.9–2.7 kb); the other region has been modified more extensively and carries large sequences specific for each Ti plasmid type. The sequence similarity within large conserved regions shows that these plasmids have diverged recently and that their evolution was driven by large-scale genetic events rather than single nucleotide changes. These results have important implications for studies on bacterial evolution.     

Transfer of the octopine T-DNA segment to plant cells mediated by different types of Agrobacterium tumor- or root-inducing plasmids: generality of virulence systems.

Genetic complementation studies demonstrated that the transfer to plant cells of the octopine T-DNA, entirely present as the only part of the tumor-inducing (Ti) plasmid on the plasmid pAL1050, was effected by the virulence systems from related plasmids, viz. the nopaline Ti plasmid pTiC58, the limited host range plasmid pTiAg57, and the root-inducing (Ri) plasmid pRi1855. Rhizobium symbiosis plasmids were not capable of effecting the introduction of pAL1050 into plant cells.     

Characteristics of Ti plasmids from broad-host-range and ecologically specific biotype 2 and 3 strains of Agrobacterium tumefaciens.

Agrobacterium tumefaciens strains isolated from crown gall tumors on grapevines in California were consistently of the biotype 3 group. All 11 of these strains were limited in their host range and harbored Ti plasmids with molecular masses between 119 and 142 megadaltons (Mdal) as well as a larger cryptic plasmid of greater than 200 Mdal; occasionally a smaller cryptic plasmid of 65 Mdal was also present. Ti plasmids o these strains have DNA sequences in common with Ti plasmids of octopine and nopaline strains belonging to the biotype 1 group and exhibited sequence homologies with the conserved region of the T-DNA. Ten of the 11 strains utilized octopine as a sole source of carbon and nitrogen and 3 strains catabolized both octopine and nopaline, whereas 1 strain catabolized only nopaline. All of these strains were resistant to the bacteriocin agrocin-84, except one grapevine strain that belonged to the biotype 1 group and was agrocin sensitive; it is also differed in its plasmid and virulence characteristics. Isolations from Rubus ursinus ollalieberry galls yielded exclusively biotype 2 strains. These strans were insensitive to agrocin-84, utilized nopaline as a sole carbon and nitrogen source, and were highly virulent on all host plants tested. They contained Ti plasmids ranging between 100 and 130 Mdal and occasionally a cryptic plasmid of 69 Mdal. Their Ti plasmids have DNA sequences in common with Ti plasmids of biotype 1 strains and with the conserved region of the T-DNA.     

我国葡萄根癌农杆菌Ti质粒的特征

以窄宿主葡萄农杆菌Ag162Ti质粒的T-DNA区tmr、tmsl和ocs基因座位以及T_A-DNA和T_B-DNA片段为探针,对12株我国分离的不同生物型、质粒类型和寄主范围的葡萄根癌农杆菌的引质粒转移DNA(T-DNA)进行Southern杂交分析。在9株生物3型octoplne Ti质粒菌株中,与上述探针均同源。其中窄宿主葡萄根癌农杆菌菌株杂交片段彼此较一致。广宿主葡萄根癌农杆菌菌株的杂交片段彼此差异较大。1株无致瘤能力的生物1型菌株与5个探针均不杂交。1株生物3型nopaline Ti质粒菌株及1株诱导冠瘿瘤中只合成精氨酸的菌株,杂交带的变化也大。由此可见葡萄农杆菌在生物进化过程中其转移DNA呈多态性,成为农杆菌中特殊类群。本分析对葡萄根癌农杆菌致病菌株的鉴定亦有帮助。     

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.     

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