The Agrobacterium tumefaciens virD3 gene is not essential for tumorigenicity on plants.

Genetic studies indicate that three of the four polypeptides encoded within the virD operon of the Agrobacterium tumefaciens Ti plasmid are essential for virulence. In order to determine whether the fourth polypeptide, VirD3, has any role in virulence, complementation analysis was used. An A. tumefaciens strain, A348 delta D, which lacked the entire virD operon in the Ti plasmid pTiA6, was constructed. Plasmids containing defined regions of the virD operon were introduced into this strain, and virulence was tested by the strains' abilities to form tumors on Kalanchoe leaves, tomato stems, and potato tubers. As expected, deletion of the virD operon led to an avirulent phenotype. The virulence of this strain could be restored by providing virD1, virD2, and virD4 in trans. No requirement for virD3 in tumor formation was observed in these assays.     

VirD2 gene product from the nopaline plasmid pTiC58 has at least two activities required for virulence.

Virulence genes virD1 and virD2 are required for T-DNA processing in Agrobacterium tumefaciens. The regions within virD2 contributing to T-DNA processing and virulence were investigated. Some insertional mutations in virD2 prevented T-DNA border endonucleolytic cleavage and produced an avirulent phenotype. However, a non-polar insertion immediately after bp 684 of the 1344 bp open reading frame of virD2 did not inhibit endonucleolytic cleavage but still caused a loss of virulence. This suggested that in addition to T-DNA border cleaving activity, the VirD2 protein has another virulence function which resides in the C-terminal half of the protein. Comparative nucleotide sequence analyses of virD2 showed that the first 684 bp were 81% homologous to virD2 of an octopine Ti plasmid whereas the remaining 660 bp were only 44% homologous. A plasmid containing the virD region from octopine Ti plasmid could restore both virulence and processing to a nopaline virD2 mutant. No complementation resulted when a nopaline virD2 clone containing a region similar to eukaryotic nuclear envelope transport sequences was deleted from the 3' end. These results suggest that virD1 and only the first half of virD2 are required to encode for the T-DNA processing endonuclease, and that the 3'-half of virD2 encodes a function separate from endonuclease activity that is required for virulence.     

Dual control of Agrobacterium tumefaciens Ti plasmid virulence genes.

The virulence genes of nopaline (pTiC58) and octopine (pTiA6NC) Ti plasmids are similarly affected by the Agrobacterium tumefaciens ros mutation. Of six vir region complementation groups (virA, virB, virG, virC, virD, and virE) examined by using fusions to reporter genes, the promoters of only two (virC and virD) responded to the ros mutation. For each promoter that was affected by ros, the level of expression of its associated genes was substantially elevated in the mutant. This increase was not influenced by Ti plasmid-encoded factors, and the mutation did not interfere with the induction of pTiC58 vir genes by phenolic compounds via the VirA/VirG regulatory control mechanism. The effects of the ros mutation and acetosyringone were cumulative for all vir promoters examined. The pleiotropic characteristics of the ros mutant include the complete absence of the major acidic capsular polysaccharide.     

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.     

Organization and characterization of the virCD genes from Agrobacterium rhizogenes

Summary We have precisely localized virulent (vir) genes of the hairy root-inducing plasmid pRiA4b on the basis of sequence similarity with the tumor-inducing plasmid pTiA6NC, and shown that the overall organizations of vir genes in both plasmids are fairly analogous, although sizes and spacer lengths in some genes differ from each other. Among the vir genes thus mapped, the virC and virD loci were characterized in detail. Transposon insertions in virD led to loss of tumorigenicity on Kalanchoe stems and carrot discs, and one within virC exhibited an attenuated pathogenicity. The avirulent phenotype of the virD2 strain among these mutants was due to the lack of ability to recombine T-DNA border repeats in Agrobacterium cells. The nucleotide sequence of most parts of the virCD loci were similar in both plasmids. The virCD genes of these two plasmids, therefore, seem comparable both functionally and structurally. Phylogeny of pRi and pTi has also been discussed from the sequence data.     

Overexpression of virD1 and virD2 genes in Agrobacterium tumefaciens enhances T-complex formation and plant transformation.

The VirD1 and VirD2 proteins encoded by an inducible locus of the virulence (vir) region of the Agrobacterium tumefaciens Ti plasmid are required for site-specific nicking at T-DNA border sites. We have determined the nucleotide sequence of a 3.6-kilobase-pair fragment carrying the virD locus from nopaline Ti plasmid pTiC58. In contrast to the previous report (Hagiya et al., Proc. Natl. Acad. Sci. USA 82:2669-2673, 1985), we found that the first three open reading frames were capable of encoding polypeptides of 16.1, 49.7, and 21.4 kilodaltons. Deletion analysis demonstrated that the N-terminal conserved domain of VirD2 was absolutely essential for its endonuclease activity. When extra copies of the virD1 and virD2 genes were present in an A. tumefaciens strain carrying a Ti plasmid, increased amounts of T-strand and nicked molecules could be detected at early stages of vir induction. Such strains possessed the ability to transform plants with higher efficiency.     

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.     

Identification of a chromosomal tra-like region in Agrobacterium tumefaciens

The virD4 gene is one of the virulence genes present on the pTiC58 plasmid of Agrobacterium tumefaciens. Unexpectedly, we found that a pTi-free A. tumefaciens strain carried a protein of similar size to the plasmid-encoded VirD4 protein which reacted with VirD4-specific antibodies. This suggested that this strain may contain a homologue of the VirD4 protein. A chromosomal fragment encoding a protein of similar sequence to VirD4 was isolated and a 7.8 kilobase region surrounding the gene encoding this putative homologue was sequenced. This region contained four open reading frames, encoding putative proteins similar to proteins of known bacterial transfer and conjugation systems, viz., orf1 encoded a putative homologue of the TraA protein of the Rhizobium symbiosis plasmid pNGR234 and the TraA protein encoded by pTiC58 from A. tumefaciens plasmid pTiC58, orf3 encoded a protein very similar to the MobC protein encoded by the IncQ plasmid RSF1010 of E. coli and to MobS encoded by pTF1 from Thiobacillus ferrooxidans, whereas the predicted product of orf4 displayed similarity to the TraG protein encoded by the IncPalpha plasmid RP4 of E. coli, TraG and VirD4 encoded by A. tumefaciens plasmid pTiC58. The product of orf2 showed no significant similarity to any known protein. Preliminary assays with two orf4 mutants suggested that the product of this orf is involved in DNA transfer. The 7.8 kb chromosomal fragment seems to be closely related to the tra region of different conjugative plasmids and appears to be confined to Agrobacterium species, raising the question of the role of a chromosomal tra-like region during evolution.     

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.     

Octopine- and Nopaline-Inducible Proteins in Agrobacterium tumefaciens Are Also Induced by Arginine

Octopine induced the synthesis of 83, 76, 62, 58, 44, 42, 31, and 22 kDa proteins in Agrobacterium tumefaciens strains harboring the tumor-inducing (Ti) plasmids pTiA6 and pTiAch5. Nopaline induced the synthesis of 83, 76, 62, 58, 56, 44, 42, 31, and 22 kDa proteins in A. tumefaciens strains harboring the Ti plasmids pTiC58 and pTiT37. The molecular masses of proteins induced by octopine and nopaline were very similar. In accordance with the ‘opine concept’, octopine and nopaline were found to induce protein synthesis only in strains harboring the respective Ti plasmids. Arginine, a common catabolic product of octopine and nopaline, induced the synthesis of most of the proteins induced by the two opines. Our results show that only the initial step(s) of octopine and nopaline catabolism are induced by specific opines in the respective strains. The subsequent steps are likely to be regulated by arginine in both strains. Received: 5 January 1996 / Accepted: 21 February 1996     

An inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes

The 9.5kb virB operon is the largest of the six major operons in the Ti plasmid vir region. This operon contains eleven genes, the largest of which is virB4. This gene encodes an 84kDa protein whose function has not been identified. Its roles in conferring virulence on Agrobacterium tumefaciens and in the T-DNA transfer process were determined by generating non-polar mutants by using the Tn5pvirB transposon in which the virB promoter is transcribed downstream of its position of insertion. Several independent mutants were isolated and each insertion site in virB4 was confirmed by nucleotide sequence analysis. These mutants were tested for T-DNA transfer ability by agroinfection and for tumorigenicity by inoculation in Brassica and Datura. All mutants were agroinfection- and tumorigenicity-negative. These data strongly suggest that virB4 is essential for both the interkingdom transfer of the T-DNA and virulence. Furthermore, by using anti-VirB4 serum, the protein product of virB4 was localized to the inner-membrane fraction of A. tumefaciens. Purified VirB4 protein hydrolyses ATP and this activity was quenched by the anti-VirB4 serum. The energy generated by VirB4 ATPase therefore may be used to transfer T-DNA or to assemble the T-DNA transfer apparatus on the bacterial membrane. Protein sequence analyses revealed striking similarities between VirB4 protein and the proteins required for conjugative transfer, which include TraC, TrwK, and TrbE of plasmids F, R388, and RP4, repectively. These findings suggest that VirB proteins play a direct role in the assembly of a conjugative transfer apparatus required for the transfer of the T-DNA from A. tumefaciens to plant cells.     

Sequence analysis of the vir-region from Agrobacterium tumefaciens octopine Ti plasmid pTi15955

The nucleotide sequence of 42 775 bp of the vir-region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955 is reported here. Although the nucleotide sequences of several parts of this region from this or closely related plasmids have been published previously, the present work establishes for the first time the complete arrangement of all the essential virulence genes and their intergenic regions of an octopine Ti plasmid. The disruption of some of the intergenic areas by insertion (IS) elements is typical for the octopine Ti plasmids. Several new ORFs were identified, including ORFs immediately downstream of virD4 and virE2, which probably represent new genes involved in virulence.     

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.     

The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease

Tumor formation by Agrobacterium tumefaciens involves the transfer and integration of a defined segment (T-DNA) of tumor-inducing (Ti) plasmid DNA into the plant nuclear genome. A set of plasmid genes outside the T-DNA, the vir genes, are thought to mediate the transfer process. We report here that the virD operon encodes a site-specific endonuclease that cleaves at a unique site within each of the 24 bp direct repeats that flank the T-DNA. The endonuclease function was further localized to the 5' end of this operon by demonstrating that cleavage does not occur in virD mutant strains of Agrobacterium and that the 5' end of the virD operon is sufficient to direct cleavage in E. coli. Analysis of nucleotide sequence and protein data indicate that two proteins of 16.2 and 47.4 kd are involved.     

Agrobacterium plasmids encode structurally and functionally different loci for catabolism of agrocinopine-type opines

Summary Agrobacterium tumefaciens strains C58, T37, K827 and J73, A. rhizogenes strains A4 and 15834, and A. radiobacter strain K299 were all susceptible to agrocin 84 and this sensitivity was enhanced in each case by addition of agrocinopines A and B. Analysis of transconjugants showed that sensitivity of strain A4 to agrocin 84 was encoded by pArA4a and not by the rhizogenic plasmid, pRiA4. The acc region of the A. tumefaciens nopaline-type Ti plasmid pTiC58, contained on the recombinant plasmid pTHH206, hybridized strongly to restriction fragments of plasmids from strains T37, K827, J73 and K299. Hybridizing fragment patterns generated with BamHI and EcoRI were identical among the four Ti plasmids while pAtK299 showed restriction fragment length polymorphisms at acc with the two enzymes. At moderate stringency, the pTiC58 acc region hybridized weakly to a single restriction fragment from the Ar plasmid of A. rhizogenes strain A4, but not to pTiBo542, which encodes catabolism of the closely related opines agrocinopines C and D. Plasmid pAtK84b of A. radiobacter strain K84 is induced for conjugal transfer by agrocinopines A and B. However, no hybridization was detected between this plasmid and acc from pTiC58 under conditions of moderate stringency. Like pTiC58, pAtK84b conferred transport of agrocinopines A and B on its host bacteria despite the absence of detectable sequence homology with the pTiC58-derived acc probe. However, unlike pTiC58, pAtK84b failed to confer sensitivity to or uptake of agrocin 84 on its bacterial host. These results indicate that at least four distinguishable systems exist for catabolism of the two agrocinopine opine families with the prototype locus, exemplified by acc from pTiC58, being strongly conserved among nopaline-type Ti plasmids.     

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.