Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58.

The virulence (vir) region of pTiC58 was screened for promoter activities by using gene fusions to a promoterless lux operon in the broad-host-range vector pUCD615. Active vir fragments contained the strongly acetosyringone-inducible promoters of virB, virC, virD, and virE and the weakly inducible promoters of virA and virG. Identical induction patterns were obtained with freshly sliced carrot disks, suggesting that an inducer is released after plant tissue is wounded. Optimal conditions for vir gene induction were pH 5.7 for 50 microM acetosyringone or sinapic acid. The induction of virB and virE by acetosyringone was strictly dependent on intact virA and virG loci. An increase in the copy number of virG resulted in a proportional, acetosyringone-independent increase in vir gene expression, and a further increase occurred only if an inducing compound and virA were present.     

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.     

Characterization of the VirG binding site of Agrobacterium tumefaciens.

Expression of Agrobacterium tumefaciens virulence (vir) genes is dependent on the presence of a conserved 'vir box' sequence in their 5' nontranscribed regions. The location and number of these sequences vary considerably in different vir genes. Site-directed mutagenesis was used to identify the functional vir box(es) of virB, virC and virD. For virB expression both vir box B1 and B2 are required but only the vir box B1 is absolutely essential. Of the five vir boxes of virC and virD two are required for virC expression while only one vir box is required for virD expression. To investigate the minimum sequences necessary for vir gene induction a deletion derivative of virE that lacks the vir box region was used. This mutant is not induced by acetosyringone. The inducibility of this promoter was restored when a synthetic deoxyoligonucleotide dGTTTCAATTGAAAC was introduced at a location analogous to that of the wild type vir box sequence. Mutational analysis indicate that the functional vir box sequence is 14 residues in length, contains a dyad symmetry and has the consensus sequence d ryTncAaTTGnAaY [corrected] (r = purine, y = pyrimidine).     

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.     

Map location on Agrobacterium root-inducing plasmids of homologies with the virulence region of tumor-inducing plasmids

Southern-type hybridizations were carried out in order to identify sequence homologies with the pTi vir loci, on an agropine-type plasmid (pRiHRI) and a mannopine-type plasmid (pRi8196) of Agrobacterium rhizogenes. The localization of the sequences hybridizing with subcloned fragments containing vir A, B, G, C, and D from pTiAch5 indicated a similar linear organization of the pTi vir loci and their homologies on pRiHRI and pRi8196, though no homology was detected on both pRi with a 1.1-kb internal fragment of virD. No homology was detected either with the vir E locus on pRiHRI vir region, nor with the virF locus on both pRi vir regions. As on nopaline pTiC58, fragments bearing the homologies with virC and virG are closer together on both pRi than on octopine pTiAch5. A preliminary functional map of the pRiHRI vir region is deduced from this study.     

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.     

Negative transcriptional regulation of virulence and oncogenes of the Ti plasmid by Ros bearing a conserved C2H2-zinc finger motif

The chromosomal ros gene in Agrobacterium tumefaciens encodes a repressor of virulence and oncogenes that are located on a resident Ti plasmid. Mutational inactivation of ros de-represses the expression of the virC and virD operons, causing premature processing and accumulation of T-DNA molecules, and the premature expression of the oncogene, ipt, leading to the synthesis of cytokinin in the bacterium rather than in the plant host cell. Ros is a 15.5 kDa protein containing a novel "eukaryotic" C(2)H(2) zinc finger. Amino acid substitutions in the finger result in the loss of binding of Ros to the ros box, a 40 bp sequence within the operator of virC/D and ipt gene promoters; and the loss of binding of a zinc ion. The ros gene is highly conserved in members of the Rhizobiaceae. Evolutionary distance tree analyses revealed distant ties to the Japanese puffer fish, Fugu rupripes rather than to plants. Interestingly, ros homologues were found in microorganisms derived from marine sources, supporting the hypothesis that ros may have originated from a marine rather than a terrestrial organism.     

Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58.

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.     

Genome structure of Ri plasmid (3). Sequencing analysis of the vir region of pRi1724 in Japanese Agrobacterium rhizogenes.

The entire genome of the pRi1724 (217.6-kb) in the mikimopine type Agrobacterium rhizogenes strain MAFF03-01724 has been completely sequenced. The vir region covering 30.2-kb has found to be composed of 21 genes resembling virH1, virA, virB1-11, virG, virC1-2, and virD1-5. The structural organization of the pRi1724 vir operons in this study is exactly the same as that of the previously reported vir operons of other Ri or Ti plasmids, although the size of some ORFs showed little variations among the plasmids. We also found virE3 gene in the pRi1724 (1), but different from Ti plasmids, virE1 and virE2 that are also important for the virulence do not exist in the vir region of pRi1724.     

Regulation of Ti plasmid virulence genes by a chromosomal locus of Agrobacterium tumefaciens.

We isolated a mutant strain of Agrobacterium tumefaciens, designated Ros, that has a pleiotropic phenotype which includes elevated levels of expression of certain genes in the virulence (Vir) region of tumor-inducing plasmid pTiC58. This mutant and others were isolated by fusing the promoter of the Vir bak gene to a promoterless gene (cat) that encodes chloramphenicol acetyltransferase and then selecting for increased expression of cat in A. tumefaciens. The ros mutation is chromosomal in nature and is characterized by a more-than-300-fold increase in the level of expression of bak and a 12-fold increase in the level of expression of an adjacent divergent operon containing the hdv genes, which are involved in some aspect of host specificity. The Ros mutant is fully virulent and is typified by its unusual colony morphology; the colonies have rough surfaces, uneven edges, and a pit in the center at 24 degrees C and vary markedly in appearance from one growth temperature to another. The Ros mutant is not able to form colonies at 12 degrees C, a temperature at which the wild-type strain forms colonies in 14 days. The ros mutation occurs spontaneously with a frequency of 5 X 10(-8). Genetic and biochemical evidence indicates that the product of the ros locus is a negative regulator of Ti plasmid genes and is related to undefined chromosomally encoded functions that are involved in the mutant phenotype.     

The Agrobacterium tumefaciens rnd homolog is required for TraR-mediated quorum-dependent activation of Ti plasmid tra gene expression

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N-(3-oxo-octanoyl)-L-homoserine lactone. We isolated four Tn5-induced mutants of A. tumefaciens C58 deficient in TraR-mediated activation of tra genes on pTiC58DeltaaccR. These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing. In all four mutants Tn5 was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by rnd of Escherichia coli, an RNase known to be involved in tRNA processing. The wild-type allele of the rnd homolog cloned from C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of cya2, surround A. tumefaciens rnd, but none of these genes exerted a detectable effect on the expression of the tra reporter. In the mutant, traR was expressed from the Ti plasmid at a level about twofold lower than that in NT1. The expression of tra, but not the growth rate, was partially restored by increasing the copy number of traR or by disrupting traM, a Ti plasmid gene coding for an antiactivator specific for TraR. The mutation in rnd also slightly reduced expression of two tested vir genes but had no detectable effect on tumor induction by this mutant. Our data suggest that the defect in tra gene induction in the mutants results from lowered levels of TraR. In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D.     

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.     

Essential Components of the Ti Plasmid trb System, a Type IV Macromolecular Transporter

The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58DeltaaccR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58.     

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.     

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.     

A 3.6-kbp segment from the vir region of Ti plasmids contains genes responsible for border-sequence-directed production of T region circles in E. coli

The vir region of Ti plasmids is responsible for the transfer of the T region from Agrobacteria to plant cells; previous experiments suggested that formation of independent T region DNA circles is one step in this process. To study this step in Escherichia coli, we developed a binary vector system. One plasmid (= substrate) contains correctly oriented right and left borders from octopine plasmid pTiAch5. A gene with a counterselectable function (galK) was cloned between these borders. The galK gene is under control of the tac promoter-operator and the lac repressor with the laci gene also in the selection cassette. This construction allows determination of substrate plasmid mutants which have lost the selectable galK function. The second component of the system is one of a set of compatible plasmids harbouring various cloned parts from the vir region of nopaline plasmid pTiC58. A 3.6-kbp segment of the vir region turned out to be necessary and sufficient for production of substrate plasmid mutants which represented the equivalent of the T region containing a complete left border. From this vir region fragment four discrete proteins were expressed in minicells. The coding regions were mapped to a part conserved in nopaline and octopine plasmids; in the latter it appears to correspond to virC/D.