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.     

Transformation of Soybean Cells Using Mixed Strains of Agrobacterium tumefaciens and Phenolic Compounds

Cotyledon explants from germinated 1-day-old soybean seedling were inoculated with single or mixed strains of Agrobacterium tumefaciens. Mixed-strain infections with the supervirulent L,L-succinamopine type strain A281 (pTiBo542) and strain LBA4404 carrying an octopine type virulence (vir) region and a binary vector (pBin6) with a chimeric gene for kanamycin detoxification gave rise to tumors of which 25% were both kanamycin resistant and capable of hormone-independent growth. Singlestrain inoculations with LBA4404 (pBin6) failed to give rise to kanamycin-resistant callus. Syringaldehyde, a compound which induces vir genes carried on the Ti plasmid, increased the number of galls incited on excised cotyledons by the weakly virulent octopine type strain A348 (pTiA6). Similar results were obtained with whole plants treated with this strain in the presence of the vir-inducing compound acetosyringone. Our results indicate that the recovery of transformed soybean cells can be enabled in some instances by coinfecting with a supervirulent strain or in other instances promoted by adding a phenolic compound to the inoculum.     

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.     

Reexamining the role of the accessory plasmid pAtC58 in the virulence of Agrobacterium tumefaciens strain C58

Isogenic strains of Agrobacterium tumefaciens carrying pTiC58, pAtC58, or both were constructed and assayed semiquantitatively and quantitatively for virulence and vir gene expression to study the effect of the large 542-kb accessory plasmid, pAtC58, on virulence. Earlier studies indicate that the att (attachment) genes of A. tumefaciens are crucial in the ability of this soil phytopathogen to infect susceptible host plants. Mutations in many att genes, notably attR and attD, rendered the strain avirulent. These genes are located on pAtC58. Previous work also has shown that derivatives of the wild-type strain C58 cured of pAtC58 are virulent as determined by qualitative virulence assays and, hence, pAtC58 was described as nonessential for virulence. We show here that the absence of pAtC58 in pTiC58-containing strains results in reduced virulence but that disruption of the attR gene does not result in avirulence or a reduction in virulence. Our studies indicate that pAtC58 has a positive effect on vir gene induction as revealed by immunoblot analysis of Vir proteins and expression of a PvirB::lacZ fusion.     

Isolation and characterization of an ipt gene from the Ti plasmid Bo542

Summary A 1.9 kb clone of the T-DNA region of the Agrobacterium tumefaciens Ti plasmid Bo542 which exhibited homology to the isopentenyl transferase (ipt) locus of pTiA6 was identified by low stringency DNA hybridization. Introduction of this segment of pTiBo542 DNA into cells of Nicotiana tabacum or N. glauca caused tumor formation in vivo, and allowed hormone independent growth in vitro. Furthermore, this DNA segment complemented ipt mutant strains of A. tumefaciens, restoring their ability to cause tumors on Kalanchöe leaves and tomato stems. The complete DNA sequence of this segment has been determined, revealing an open reading frame homologous to other known Agrobacterium ipt genes.     

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.     

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.     

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.     

Characterization of Agrobacterium tumefaciens virulence proteins induced by the plant factor acetosyringone

The Ti plasmid virulence (vir) loci encode functions essential for the transfer of the T-DNA element from Agrobacterium tumefaciens to plant cells. The expression of these loci is specifically signaled by plant phenolics such as acetosyringone. Here, we characterize the protein products that are induced in Agrobacterium grown in the presence of acetosyringone. More than 10 to 15 proteins are induced in strains harboring different Ti plasmids. Two general classes of acetosyringone-induced proteins are observed, encoded either within or outside the vir region. Synthesis of both classes of proteins requires acetosyringone and the products of the vir regulatory genes A and G. Those proteins encoded outside the vir region define a novel category of proteins, the virulence-related proteins, which are both chromosomally and Ti plasmid-encoded. The molecular weight and subcellular localization of several pTiA6 vir-induced proteins are identified. The most abundant induced protein has a molecular weight of 65,000, and is the single product of the virE locus; this protein distributes into both cell envelope and soluble fractions. Three proteins with molecular weights of approximately 33,000, 80,000 and 25,000 fractionate with the cell envelope and are encoded by genes within the 5' half of the virB locus. The envelope localization of the virB proteins suggests that they play a role in directing T-DNA transfer events that occur at the bacterial surface.     

Virulence genes promote conjugative transfer of the Ti plasmid between Agrobacterium strains.

Certain virulence region operons of the Agrobacterium tumefaciens Ti plasmid promoted conjugative Ti plasmid transfer. Mutations in the vir region of pTiC58 inhibited conjugative plasmid transfer between A. tumefaciens strains. Mutations in virA, virG, 5' virB, and virE had the greatest effect on plasmid transfer, and mutations in virC had no effect. Transfer inhibition in vir mutants occurred in the presence or absence of acetosyringone.     

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.     

Limited-host-range plasmid of Agrobacterium tumefaciens: molecular and genetic analyses of transferred DNA.

A tumor-inducing (Ti) plasmid from a strain of Agrobacterium tumefaciens that induces tumors on only a limited range of plants was characterized and compared with the Ti plasmids from strains that induce tumors on a wide range of plants. Whereas all wide-host-range Ti plasmids characterized to date contain closely linked oncogenic loci within a single transferred DNA (T-DNA) region, homology to these loci is divided into two widely separated T-DNA regions on the limited-host-range plasmid. These two plasmid regions, TA-DNA and TB-DNA, are separated by approximately 25 kilobases of DNA which is not maintained in the tumor. The TA-DNA region resembles a deleted form of the wide-host-range TL-DNA and contains a region homologous to the cytokinin biosynthetic gene. However, a region homologous to the two auxin biosynthetic loci of the wide-host-range plasmid mapped within the TB-DNA region. These latter genes play an important role in tumor formation because mutations in these loci result in a loss of virulence on Nicotiana plants. Furthermore, the TB-DNA region alone conferred tumorigenicity onto strains with an intact set of vir genes. Our results suggest that factors within both the T-DNA and the vir regions contribute to the expression of host range in Agrobacterium species. There was a tremendous variation among plants in susceptibility to tumor formation by various A. tumefaciens strains. This variation occurred not only among different plant species, but also among different varieties of plants within the same genus.     

Sequence and distribution of IS1312: evidence for horizontal DNA transfer from Rhizobium meliloti to Agrobacterium tumefaciens.

Two novel insertion sequences, IS1312 and IS1313, were found in pTiBo542, the Ti plasmid of Agrobacterium tumefaciens strains Bo542 and A281. Nucleotide sequencing and Southern hybridization revealed that IS1312 and IS1313 are homologous to Rhizobium meliloti ISRm1 and ISRm2, respectively. IS1312, ISRm1, and another Agrobacterium insertion sequence, IS426, belong to the same IS3 family of insertion sequences; however, IS1312 is more closely related to the Rhizobium ISRm1 than it is to the Agrobacterium IS426. The distribution patterns of these insertion elements and their sequence similarities suggest that IS1312 and IS1313 were horizontally transferred from R. meliloti to A. tumefaciens.     

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.