Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified by the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.     

Sequence of the iaa and ipt region of different Agrobacterium tumefaciens biotype III octopine strains: reconstruction of octopine Ti plasmid evolution

The TA regions of biotype III octopine/cucumopine (OC) Ti plasmids are closely related to the TL region of the biotype I octopine Ti plasmids pTiAch5 and pTi15955. Sequence analysis shows that the limited and wide host range biotype III OC TA regions are derived from a common ancestor structure which lacked the 6a gene found in the biotype I octopine TL region. The TA region of the wide host range OC Ti plasmids has conserved most of the original TL-like structure. In most wide host range OC isolates the TA-iaaH gene is inactivated by the insertion of an IS866 element. However, the TA region of the wide host range isolate Hm1 carries an intact TA-iaaH gene. This gene encodes a biologically active product, as shown by root induction tests and indole-3-acetic acid measurements.The limited host range OC Ti plasmids pTiAB3 and pTiAg57 have shorter TA regions which are derived from a wide host range TA region. The AB3 type arose by an IS868-mediated, internal TA region deletion which removed the iaa genes and part of the ipt gene and left a copy of IS868 at the position of the deleted fragment. The pTiAB3 iaa/ipt deletion was followed by insertion of a second IS element, IS869, immediately 3 of the ipt gene. pTiAg57 underwent the same iaa-ipt deletion as pTiAB3, but lacks the IS868 and IS869 elements.Analysis of the various TA region structures provides a detailed insight into the evolution of the biotype III OC strains.     

trans-Acting virulence functions of the octopine Ti plasmid from Agrobacterium tumefaciens

All Ti plasmid-encoded virulence functions that were studied act in trans. An octopine Ti plasmid-specific vir operon, called vir-O, located on an EcoRI restriction fragment has been characterized. Sequences with promoter activity in Escherichia coli were identified for a second vir operon, called vir-C, which was located close to the position of vir-O.     

Characterization of the supervirulent virG gene of the Agrobacterium tumefaciens plasmid pTiBo542.

Summary The virG gene of the Agrobacterium tumefaciens Ti plasmid pTiBo542 has previously been reported to elicit stronger vir gene expression than its counterpart in the pTiA6 plasmid, a property we call the superactivator phenotype. The DNA sequence of the pTiBo542 virG gene was determined and compared to that of the pTiA6 gene. The DNA sequences of these genes differ at 16 positions: two differences are in the promoter regions, 12 are in the coding regions, and two are in the 3 untranslated regions. The 3 end of the pTiA6 virG gene also contains a probable insertion sequence that is not found downstream of the pTiBo542 gene. The base pair differences in the two coding regions result in only two amino acid differences, both in the amino-terminal halves of the proteins. Five hybrid virG genes were constructed and used to activate the expression of a virB::lacZ gene fusion. Differences in the coding regions of these genes accounted for most of the superactivator phenotype, while differences at the promoter and 3 untranslated regions also contributed. These findings suggest that the properties of these VirG proteins and their quantities are important for vir gene induction, and also suggest a long-term selective pressure for mutations contributing to differences between these two genes.     

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.     

Conservation of IS66 homologue of octopine Ti plasmid DNA in Rhizobium fredii plasmid DNA

Summary DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the core T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the core T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.     

Function of heterologous and pseudo border repeats in T region transfer via the octopine virulence system of Agrobacterium tumefaciens

The successful transfer of the Ti plasmid T region to the plant cell is mediated by its 24 bp border repeats. Processing of the T-region prior to transfer to the plant cell is started at the right border repeat and is stimulated by a transfer enhancer sequence called overdrive. Left and right border repeats differ somewhat in nucleotide sequence; moreover, the repeats of different Ti and Ri plasmids are slightly different. Our data indicate that these differences do not have a significant influence on border activity. However, the overdrive sequence is essential for the efficient transfer of a T region via an octopine transfer system. Our data suggest that an overdrive sequence must also be present next to the right border repeats of the nopaline Ti plasmid and the agropine of octopine and nopaline Ti plasmids express some differences in T-DNA processing activities. of cotopine and nopaline Ti plasmids express some differences in T-DNA processing activities.Furthermore, we demonstrate that certain pseudo border repeats, sequences that resemble the native 24 bp border repeat and naturally occur within the octopine Ti plasmid T-region, are able to mediate T region transfer to the plant cell, albeit with much reduced efficiency as compared to wild-type border repeats.     

Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes

Abstract Efficient transformation of strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes by electroporation with binary Ti plasmid vector is reported. This procedure yields rates of transformation of 10⁶-10³ per μg DNA, which is several orders of magnitude greater than previously published procedures for this genus, the efficiency of transformation varies with the bacterial strain used. This procedure will be useful for the construction of plant DNA libraries directly in Agrobacterium .     

Functional analysis of the Agrobacterium tumefaciens octopine Ti-plasmid left and right T-region border fragments

Summary Border fragments of the octopine Ti-plasmid were tested for their ability to restore tumorigenicity of an avirulent mutant carrying a deleted right border. It was found that neither introduction of left border fragments nor that of small right border fragments at the position of the deletion resulted in a complete restoration of oncogenicity. However, insertion of a larger right border fragment in the deletion mutant gave fully oncogenic strains. In the latter case sequences to the right side of the right border repeat were found to be responsible for a complete restoration of oncogenicity. Also a left border repeat inserted together with this enhancer sequence fully restored the oncogenicity of the deletion mutant. The enhancer-sequence on itself was not able to mediate the transfer of the T-region to the plant cell. Border fragments inserted in inverted orientation in the deletion mutant were able to mediate the transfer of the T-region to the plant cell, but at a reduced frequency.     

The Agrobacterium tumefaciens T-DNA gene 6b is an onc gene

In this article it is shown that the T-DNA of Agrobacterium tumefaciens contains besides the well-known cyt and aux genes another gene with an oncogenic effect in plants. The gene in question is called 6^b and causes the formation of small tumors in plant species such as Nicotiana glauca and Kalanchoe tubiflora.     

Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation

For successful transformation of a plant by Agrobacterium tumefaciens it is essential that the explant used in cocultivation has the ability to induce Agrobacterium tumour-inducing (Ti) plasmid virulence (vir) genes. Here we report a significant variation in different tissues of Indica rice (Oryza sativa L. cv. Co43) in their ability to induce Agrobacterium tumefaciens vir genes and T-strand generation, using explants preincubated in liquid Murashige and Skoog (MS) medium. An analysis of rice leaf segments revealed that they neither induced vir genes nor inhibited vir gene induction. Of different parts of rice plants of different ages analysed only scutellum from four-day old rice seedlings induced vir genes and generation of T-strands. We observed that the physical presence of preincubated scutella is required for vir gene induction. Conditioned medium from which preincubated scutella were removed did not induce the vir genes. Scutellum-derived calli, cultured for 25 days on medium containing 2,4-D, also induced virE to an appreciable level. These results suggest that scutellum and scutellum-derived calli may be the most susceptible tissues of rice for Agrobacterium-mediated transformation.     

Double infection of tobacco plants by two complementing octopine T-region mutants of Agrobacterium tumefaciens

The molecular basis of complementation by a mixture of two different types of octopine T-region mutants (LBA4060 and LBA4210) was studied. Six randomly chosen cellular clones derived from a tumor obtained after mixed infection were analyzed for their T-DNA content via Southern blot hybridization. The clones appeared to contain T-DNA that originated from each of both mutants, indicating that they developed from doubly infected single cells. Genetic complementation, therefore, might explain at least in part the observed complementation phenomenon. However, complementation as a result of cross-feeding between separately transformed cells could not be excluded. Following protoplast isolation, small aggregates might have formed that developed into the clones analyzed.     

Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens

We have determined the complete nucleotide sequence of a 4.8 kilobase fragment encompassing the virA locus of the nopaline-type plasmid, pTiC58, of Agrobacterium tumefaciens. virA is composed of a single open reading frame of 2499 nucleotides, capable of encoding a protein of 91.3 kiloDaltons. A trpE::virA gene fusion was used to confirm the reading frame of virA. High nucleotide and amino acid sequence homologies were observed between pTiC58 virA and the virA sequences of three octopine-type plasmids. Strong homologies in amino acid sequence were observed between pTiC58 VirA and seven bacterial proteins which control various regulons. Two hydrophobic domains within VirA are also consistent with a model in which VirA acts as a membrane-bound sensor of plant signal molecules.     

Common evolutionary origin of the central portions of the Ri TL-DNA of Agrobacterium rhizogenes and the Ti T-DNAs of Agrobacterium tumefaciens

Analysis of published sequences for Ri TL-DNA (root-inducing left-hand transferred DNA) of Agrobacterium rhizogenes revealed several unsuspected structural features. First, Ri TL-DNA genes are redundant. Using redundancy as a criterion, three regions (left, middle and right) were discerned. The left one, ORFs (open reading frames) 1–7, contains no detectable redundancy. In the middle region a highly diverged gene family was detected in ORFs 8, 11, 12, 13 and 14. The right region contains an apparently recent duplication (ORF 15 =18+17). We interpret the phenomenon of redundancy, particularly in the central region that encodes the transformed phenotype, to be an adaptation that ensures function in a variety of host species. Comparison of Ri TL-DNA and Ti T-DNAs from Agrobacterium tumefaciens revealed common structures, unpredicted by previous nucleic acid hybridization studies. Ri TL-DNA ORF 8 is a diverged Ti T-DNA tms1. Both Agrobacterium genes consist of a member of the diverged gene family detected in the central part of the Ri TL-DNA, but fused to a sequence similar to iaaM of Pseudomonas savastonoi. Other members of this gene family were found scattered throughout Ti T-DNA. We argue that the central region of Ri and the part of Ti T-DNA including ORFs 5–10 evolved from a common ancestor. We present the hypothesis that the gene family encodes functions that alter developmental plasticity in higher plants.     

The promoter proximal region in the virD locus of Agrobacterium tumefaciens is necessary for the plant-inducible circularization of T-DNA

Summary The formation of crown gall tumours involves the transfer of the T-DNA region of the Ti plasmid from Agrobacterium to plant cells and its subsequent integration into plant chromosomes. When agrobacteria are incubated with plant protoplasts or exudates of plants, the T-DNA region is circularized by recombination or cleavage and rejoining between the 25 bp terminal repeats; the formation of circular T-DNAs is thought to be one step in T-DNA transfer (Koukolikova-Nicola et al. 1985; Machida et al. 1986). We previously showed that the virulence region of the Ti plasmid is required for T-DNA circularization. In the present paper, we examined the circularization event in agrobacteria harbouring octopine Ti plasmids with mutations in various loci of the virulence region. The results clearly demonstrate that the gene(s) encoded in the virD locus are necessary for T-DNA circularization. In particular, the gene(s) present in the region proximal to the virD promoter are essential. We propose that roduct(s) of this gene have recombinase or endonuclease activity which specifically recognizes the 25 bp terminal repeats of T-DNA.     

Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens

Bacteria of the genus Agrobacterium can transfer a portion of their Ti plasmid (T-DNA) in complex with the VirE2 and VirD2 proteins into the plant-cell nucleus and cause it to be integrated in the host-cell chromosomes. The mechanism of T-DNA transfer across the plant-cell membrane and cytoplasm is unknown. The aim of this study was to isolate the virulence protein VirE2 in order to explore its role in T-DNA transfer across the eukaryotic-cell membrane and cytoplasm. To obtain VirE2, we cloned the virE2 gene into plasmid pQE31 in Escherichia coli cells. VirE2 protein was isolated from E. coli XL-1 blue cells containing a recombinant plasmid, pQE31-virE2. The cells were ultrasonically disrupted, and the protein containing six histidine residues at the N-terminal end was isolated by affinity chromatography on Ni-NTA agarose. The purified preparation was tested by immunodot, by using polyclonal rabbit antibodies and miniantibodies produced toward VirE2. The capacity of the recombinant protein VirE2 for interacting with single-stranded DNA was tested by the formation of complexes, recorded by agarose-gel electrophoresis. In summary, A. tumefaciens virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into the plant cell, was isolated, purified, and partially characterized. Anti-VirE2 miniantibodies were obtained, and direct labeling of VirE2 with colloidal gold was done for the first time.     

Cloning an ipt gene from Agrobacterium tumefaciens: characterisation of cytokinins in derivative transgenic plant tissue

The isopentenyl transferase gene was isolated from Agrobacterium tumefaciens AcH5 using polymerase chain reaction and transformed into Petunia and Kalanchoë using both A. tumefaciens and A. rhizogenes transformation systems. Morphological evidence and elevated endogenous cytokinin levels indicated that the PCR product was an active gene. Accurate quantification of the cytokinins was obtained by radioimmunoassay, following purification and separation of the free bases and ribosides by HPLC. Of the six cytokinins quantified, zeatin riboside and its stabilised dihydro-derivative, dihydrozeatin riboside, showed the greatest increases in the transformed Petunia tissue (up to 600-fold). The importance of measuring changes in individual cytokinins is discussed.