vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens.

Mutation of the genes virA, virB, virC, and virG of the Agrobacterium tumefaciens octopine-type Ti plasmid pTiR10 was found to cause a 100- to 10,000-fold decrease in the frequency of conjugal transfer of this plasmid between Agrobacterium cells. This effect was not absolute, however, in that it occurred only during early times (18 to 24 h) of induction of the conjugal transfer apparatus by octopine. Induction of these mutant Agrobacterium strains by octopine for longer periods (48 to 72 h) resulted in a normal conjugal transfer frequency. The effect of these vir gene mutations upon conjugation could be restored by the introduction of cosmids harboring wild-type copies of the corresponding disrupted vir genes into the mutant Agrobacterium strains. In addition, transfer of the self-mobilizable plasmid pPH1JI was not impaired in any of the mutant Agrobacterium strains tested. The effect of vir gene function on the conjugal transfer of the Ti plasmid suggests that a relationship may exist between the processes that control the transfer of the T-DNA from Agrobacterium to plant cells and the conjugal transfer of the Ti plasmid between bacterial cells.     

Opines stimulate induction of the vir genes of the Agrobacterium tumefaciens Ti plasmid.

Upon incubation of Agrobacterium tumefaciens A348 with acetosyringone, the vir genes encoded by the Ti (tumor-inducing) plasmid are induced. The addition of certain opines, including octopine, nopaline, leucinopine, and succinamopine, enhanced this induction 2- to 10-fold. The compounds mannopine, acetopine, arginine, pyruvate, and leucine did not stimulate the induction of the vir genes to such an extent. The enhancement of vir gene induction by opines depended on acetosyringone and the genes virA and virG. Opines stimulated the activity of the vir genes, the double-stranded cleavage of the T (transferred)-DNA at the border repeat sequences, and the production of T-strands by the bacterium. The transformation efficiency of cotton shoot tips was markedly increased by the addition of acetosyringone and nopaline at the time of infection.     

Efficient vir gene induction in Agrobacterium tumefaciens requires virA, virG, and vir box from the same Ti plasmid

The vir genes of octopine, nopaline, and L,L-succinamopine Ti plasmids exhibit structural and functional similarities. However, we observed differences in the interactions between octopine and nopaline vir components. The induction of an octopine virE(A6)::lacZ fusion (pSM358cd) was 2.3-fold higher in an octopine strain (A348) than in a nopaline strain (C58). Supplementation of the octopine virG(A6) in a nopaline strain with pSM358 did not completely restore virE(A6) induction. However, addition of the octopine virA(A6) to the above strain increased virE(A6) induction to a level almost comparable to that in octopine strains. In a reciprocal analysis, the induction of a nopaline virE(C58)::cat fusion (pUCD1553) was two- to threefold higher in nopaline (C58 and T37) strains than in octopine (A348 and Ach5) and L,L-succinamopine (A281) strains. Supplementation of nopaline virA(C58) and virG(C58) in an octopine strain (A348) harboring pUCD1553 increased induction levels of virE(C58)::cat fusion to a level comparable to that in a nopaline strain (C58). Our results suggest that octopine and L,L-succinamopine VirG proteins induce the octopine virE(A6) more efficiently than they do the nopaline virE(C58). Conversely, the nopaline VirG protein induces the nopaline virE(C58) more efficiently than it does the octopine virE(A6). The ability of Bo542 virG to bring about supervirulence in tobacco is observed for an octopine vir helper (LBA4404) but not for a nopaline vir helper (PMP90). Our analyses reveal that quantitative differences exist in the interactions between VirG and vir boxes of different Ti plasmids. Efficient vir gene induction in octopine and nopaline strains requires virA, virG, and vir boxes from the respective Ti plasmids.     

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.     

Isolation and characterization of diverse nopaline type Ti plasmids of Agrobacterium tumefaciens from Japan

Ninety Agrobacterium strains were isolated from naturally appearing crown galls in Japan. They were classified into several groups based on opine type, biovars, tumorigenicity, and indigeneous plasmid profiles. Twenty-nine strains utilized nopaline, but none utilized octopine. Eighteen isolates were tumorigenic, nopaline type strains and thus classified as Agrobacterium tumefaciens. Some strains possessed anomalous traits such as lysine utilization, resistance to agrocin 84, and a lack of motility. Pathogenic strains contained Ti plasmids of either 200 kb or 260 kb, as identified by hybridization to T-DNA of the known Ti plasmid. However, the restriction enzyme cleavage patterns, arising from hybridization to the probe, were different from each other and indicated that nopaline type Ti plasmids possess more diverse T-DNA structures than previously reported. Five of 6 representative strains induced tumors on 6 plant species (tomato, petunia, poplar, kalanköe, apple, and grape). Among these, apple was notable, since only a few strains have been reported to be pathogenic to this plant. On petunia, 4 strains developed large tumors while 2 produced only small tumors. Teratomas were formed on poplar in a strain-dependent manner, but not on tomato. These results suggest that our isolates are wide host range strains, and that host-specificity of these strains is related to diverse T-DNA structures.     

Identification of pTi-SAKURA DNA region conferring enhancement of plasmid incompatibility and stability

In Agrobacterium tumefaciens, the stability of Ti plasmids differs depending on the strain. So far, little is known about genes that cause the difference in stability. The repABC operon is responsible for replication and incompatibility of Ti plasmids. We constructed recombinant plasmids carrying the repABC operon and different portions of pTi-SAKURA. Cells having the recombinant plasmids that harbored a 2.6-kbp NheI fragment of pTi-SAKURA were found to be transformed via conjugation 100-fold less frequently with a small incompatible repABC plasmid than cells having the recombinant plasmids lacking the 2.6-kbp NheI fragment. Since the phenomenon occurred only when the resident and incoming plasmids belonged to the same incompatibility group, it was suggested that the 2.6-kbp NheI fragment bears the potential enhancing incompatibility. The fragment contained an operon consisting of two open reading frames, tiorf24 and tiorf25. tiorf24 is an orphan gene, whereas tiorf25 is a homologue of a group of plasmid stability genes. Removal of the 2.6-kbp fragment from the resident pTi-SAKURA increased the resident plasmid ejection ratio by the incoming repABC plasmid, whereas addition of the fragment to pTiC58 decreased the ejection ratio, and the loss ratio during growth at 37 degrees C. These data suggest that tiorf24 and tiorf25 are responsible for the stability of pTi-SAKURA, and reduce, in the host bacterium, the frequency of ejection of the resident plasmid, presumably through an incompatibility mechanism.     

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants

Transposon-insertion mutants with vir^? Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir^? nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir^? octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir^? octopine Ti plasmid mutations and vir^? nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO^? mutants produced rooty tumors on Kalanchoë tubiflora.     

The right end of the vir region of an octopine-type Ti plasmid contains four new members of the vir regulon that are not essential for pathogenesis

We sequenced the virD-virE, virE-virF, and virF-T-DNA intergenic regions of an octopine Ti plasmid. Four newly described genes were induced by the vir gene inducer acetosyringone, two of which are conserved in the nopaline-type Ti plasmid pTiC58. One gene resembles a family of phosphatase genes. Each of these genes is dispensable for tumorigenesis.     

Ornithine cyclodeaminase from octopine Ti plasmid Ach5: identification, DNA sequence, enzyme properties, and comparison with gene and enzyme from nopaline Ti plasmid C58.

Octopine and nopaline are two arginine-derived opines synthesized in plant cells transformed with octopine or nopaline plasmids. Utilization in Agrobacterium tumefaciens is mediated by Ti plasmid regions called occ or noc (octopine or nopaline catabolism), and recent experiments showed that noc in pTiC58 codes for a pathway from nopaline to L-proline. The last enzyme is ornithine cyclodeaminase (OCD), an unusual protein converting L-ornithine directly into L-proline. We investigated whether octopine plasmid pTiAch5 also harbors a gene for OCD. The results revealed an ocd gene which is induced by octopine and maps in the occ region. DNA sequence analysis and comparison with the gene from pTiC58 showed that the two genes are related (69% homology in DNA and deduced amino acid sequence), and antiserum against OCD(C58) also reacted with OCD(Ach5). The enzyme activity was characterized, and a comparison with OCD(C58) showed that the properties are similar, but not identical. Differences were detected in the regulation of enzyme activity by L-arginine and L-proline and in the response to varying ratios of NAD+/NADH. It is proposed that this reflects different mechanisms for integration of opine catabolism into general metabolism.     

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.     

Molecular characterization of the virC genes of the Ti plasmid.

The virC (formerly bak) complementation group of the nopaline-type Ti plasmid pTiC58 encodes two proteins, VirC1 and VirC2. According to the primary structure of the polypeptides predicted by the nucleotide sequence, VirC1 is composed of 231 amino acids with a total molecular mass of 25.5 kilodaltons, and VirC2 is composed of 202 amino acids with a molecular mass of 22.1 kilodaltons. The pTiC58 VirC1 and VirC2 polypeptides are equal in length to VirC1 and VirC2 of the octopine-type plasmid pTiA6NC. VirC1 proteins of pTiC58 and pTiA6NC are identical at 202 (87.4%) of the amino acid residues, and this homology is distributed fairly evenly throughout the protein. VirC2 identities occur at 142 residues (70.3%), but fall predominantly into two blocks of higher homology (84.6 and 78.5%) separated by a 41-residue segment of much lower homology (29.3%). Mutations in virC resulted in attenuated virulence on all hosts tested, the severity of attenuation varying markedly depending on the type of plant inoculated. For example, the attenuation was more pronounced on Kalanchoe than on sunflower or jimson weed. Virulence was restored to normal on all hosts by in-trans complementation with corresponding nonmutant DNA fragments of pTiC58 or of the octopine-type plasmid pTi15955. Two oligopeptides from within the predicted pTiC58 VirC1 polypeptide were synthesized and used to raise antibodies. These antibodies were used to detect the VirC1 product of both pTiC58 and pTi15955. In both cases, virC was expressed constitutively in the Agrobacterium tumefaciens ros mutant. The homology between virC genes of octopine- and nopaline-type Ti plasmids thus includes a conservation of genetic regulatory control mechanisms as well as considerable conservation of the primary structure of the protein products.     

Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

A detailed physical map of the homologous and non-homologous regions between an octopine (pTiAch5) and a nopaline (pTiC58) Ti plasmid was determined by Southern type hybridization and by electron microscope heteroduplex analysis. This map was correlated with the functional maps of both plasmids. For the Southern type hybridizations, total labelled pTiAch5 DNA was hybridized to Southern blots of restriction fragments from a series of hybrid plasmids containing overlapping segments of the whole TiC58 plasmid. Reciprocal experiments were also carried out. The common sequences between the two plasmids (±30%) are restricted to four major stretches of homology. Analysis of heteroduplexes between pTiAch5 and several hybrid plasmids containing specific regions of pTiC58, and of heteroduplexes between hybrid plasmids derived from pTiC58 and pTiAch5 provided a detailed map of the fine structure of the four major homology regions. Two regions are distributed in the same relative order as compared to a common reference point, and two are inversed. Three regions contain a number of small, mostly asymmetrical substitution loops. Several regions distributed over the common DNA sequences were found to be partially homologous.