Mutants of the Agrobacterium tumefaciens virA gene exhibiting acetosyringone-independent expression of the vir regulon.

Hydroxylamine-induced mutations in the virA gene of Agrobacterium tumefaciens that do not require the plant phenolic-inducing compound acetosyringone for vir regulon induction were isolated. The isolation was based on the activation of both virB::lacZ and virE::cat fusions by mutant virA loci in the absence of acetosyringone. Three of these virA(Ais) (acetosyringone-independent signaling) mutants were characterized. All three mutants expressed a virB::lacZ fusion at high levels in the absence of acetosyringone. One virA (Ais) mutant, virA112, exhibited vir gene expression in the absence of inducing monosaccharides and acidic growth conditions, both of which are normally required for vir gene induction. The phenotype of the virA112 mutant resulted from a glycine to glutamic acid change near His-474, the site of VirA autophosphorylation.     

Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by using the Escherichia coli lac promoter

The Agrobacterium VirG protein is normally expressed from two promoters in response to multiple stimuli, including plant-released phenolics (at promoter P1) and acidic growth media (at promoter P2). To simplify the analysis of vir gene induction, we sought to create Agrobacterium strains in which virG could be expressed in a controllable fashion. To study the possibility of using the lac promoter and repressor, we constructed a plasmid containing the lac promoter fused to the lacZ structural gene. A derivative of this plasmid containing the lacIq gene was also constructed. The plasmid not containing lacIq expressed high levels of beta-galactosidase. The plasmid containing lacIq expressed beta-galactosidase at very low levels in the absence of o-nitrophenyl-beta-D-galactoside (IPTG) and at moderate levels in the presence of IPTG. We also fused the lac promoter to a virG::lacZ translational fusion and found that IPTG elevated expression of this translational fusion to moderate levels, though not to levels as high as from the stronger of the two native virG promoters. Finally, the lac promoter was used to express the native virG gene in strains containing a virB::lacZ translational fusion. virB expression in this strain depended on addition of IPTG as well as the vir gene inducer acetosyringone. In a similar strain lacking lacIq, virB expression was greater than in a strain in which virG was expressed from its native promoters. Expression of virG from the lac promoter did not alter the acidic pH optimum for vir gene induction, indicating that the previously observed requirement for acidic media was not due solely to the need to induce P2.     

The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation.

The virA and virG gene products are required for the regulation of the vir regulon on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. VirA is a membrane-associated protein which is homologous to the sensor molecules of other two-component regulatory systems. We overproduced truncated VirA proteins in Escherichia coli by deleting different lengths of the 5'-coding region of the virA gene and placing these genes under lacZ control. These proteins were purified from polyacrylamide gels and renatured. The renatured proteins became radiolabeled when they were incubated with [gamma-32P]ATP but not with [gamma-32P]GTP or [alpha-32P]ATP, which suggests an ATP gamma-phosphate-specific autophosphorylation. The smallest VirA protein, which retained only the C-terminal half of the protein, gave the strongest autophosphorylation signal, which demonstrates that the C-terminal domain has the autophosphorylation site. The phosphorylated amino acid was identified as phosphohistidine, and a highly conserved histidine was found in all of the VirA homologs. When this histidine was changed to glutamine, which cannot be phosphorylated, the resulting VirA protein lost both its ability to autophosphorylate and its biological function as a vir gene regulator. Results of this study indicate that VirA autophosphorylation is required for the induction of the vir regulon and subsequent tumor induction on plants by A. tumefaciens.     

Reconstitution of acetosyringone-mediated Agrobacterium tumefaciens virulence gene expression in the heterologous host Escherichia coli

The ability to utilize Escherichia coli as a heterologous system in which to study the regulation of Agrobacterium tumefaciens virulence genes and the mechanism of transfer DNA (T-DNA) transfer would provide an important tool to our understanding and manipulation of these processes. We have previously reported that the rpoA gene encoding the alpha subunit of RNA polymerase is required for the expression of lacZ gene under the control of virB promoter (virBp::lacZ) in E. coli containing a constitutively active virG gene [virG(Con)]. Here we show that an RpoA hybrid containing the N-terminal 247 residues from E. coli and the C-terminal 89 residues from A. tumefaciens was able to significantly express virBp::lacZ in E. coli in a VirG(Con)-dependent manner. Utilization of lac promoter-driven virA and virG in combination with the A. tumefaciens rpoA construct resulted in significant inducer-mediated expression of the virBp::lacZ fusion, and the level of virBp::lacZ expression was positively correlated to the copy number of the rpoA construct. This expression was dependent on VirA, VirG, temperature, and, to a lesser extent, pH, which is similar to what is observed in A. tumefaciens. Furthermore, the effect of sugars on vir gene expression was observed only in the presence of the chvE gene, suggesting that the glucose-binding protein of E. coli, a homologue of ChvE, does not interact with the VirA molecule. We also evaluated other phenolic compounds in induction assays and observed significant expression with syringealdehyde, a low level of expression with acetovanillone, and no expression with hydroxyacetophenone, similar to what occurs in A. tumefaciens strain A348 from which the virA clone was derived. These data support the notion that VirA directly senses the phenolic inducer. However, the overall level of expression of the vir genes in E. coli is less than what is observed in A. tumefaciens, suggesting that additional gene(s) from A. tumefaciens may be required for the full expression of virulence genes in E. coli.     

Glycine betaine allows enhanced induction of the Agrobacterium tumefaciens vir genes by acetosyringone at low pH.

We established growth conditions for efficient induction of the vir genes of Agrobacterium tumefaciens by acetosyringone. Optimal induction was attained at a pH below 5.2 in an AB minimal medium-derived high-osmotic-strength medium containing glycine betaine. This natural osmoprotectant accelerated the adaptation of the bacteria to these conditions. We established the kinetics of induction for virB, virD, virE, and virG by using lacZ fusions, and we found that the virB mutant strain could not adapt to this low-pH medium unless 1 mM CaCl2 was added. This pH control of vir gene expression was shown to act at the level of expression of virG, which was the limiting factor. This improved vir induction at a low pH correlated with an increase in a set of proteins which was analyzed by two-dimensional gel electrophoresis. The fact that high inducibility corresponded to a reduced growth rate and the demonstration that a set of proteins was associated with the inducible state suggest that vir gene induction is linked to the adaptation of the cells to an unfavorable environment. Hence, vir gene expression in A. tumefaciens is probably dependent upon a machinery which is specific to an adaptive response; the implications for plant transformation are discussed.     

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.     

Sequence characterization of the vir region of a nopaline type Ti plasmid, pTi-SAKURA.

We isolated a crown gall tumor-inducing nopaline type Ti plasmid from Agrobacterium tumefaciens on a Sakura Japanese cherry tree, and designated it as pTi-SAKURA. By primer walking sequencing with long PCR and a newly developed PCR subcloning technique for long insert DNA, we completed DNA sequencing of the most important functional unit, the virulence (vir) region of pTi-SAKURA, which is indispensable for T-DNA transfer into the plant's chromosomes. By homology searches with the vir genes of other bacterial plasmids, we identified 11 open reading frames (orfs) and 31 genes and 11 vir box, which are 6 bp regulatory sequences. In total, 26 vir genes, including the putative virF and virK and the main vir region, were present as the vir gene cluster. The presence of vir box, GC content, codon usage and expression analysis in these genes led us to propose a new vir region.     

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.     

Induction of a virulence response in Agrobacterium tumefaciens by exudates of Pinus pinea cotyledons

As a preliminary step in efforts to develop a successful protocol for Agrobacterium-mediated transformation of cotyledonary explants of Pinus pinea L. embryos, we tested the ability of embrionary exudates of this species to induce the expression of the virulence genes virA, virB, virC, virD, virE and virG in Agrobacterium tumefaciens containing vir: lacZ fusion constructs. The results obtained in the vir induction assay indicated the absence of bactericidal or bacteriostatic plant compounds affecting A. tumefaciens growth, and showed that cotyledonary and embrionary exudates of P. pinea are able to induce all virulence genes studied, except virG. The data suggest that A. tumefaciens can be used for gene transfer into this important forest and fruit species. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Detection of Activity Responsible for Induction of the Agrobacterium tumefaciens Virulence Genes in Bacteriological Agar

Agrobacterium tumefaciens C58 grown on acidic medium containing glucose and solidified with bacteriological agar expressed a virB::lacZ fusion. No expression of this fusion was observed on a similar medium which was solidified with purified agarose. The fraction from bacteriological agar which was responsible for vir gene induction was extracted with methanol and partially purified by preparative thin-layer chromatography.     

Structure activity Research of Signal Factors Inducing Agrobacterium vir Genes

Four rice ( Oryza sativa L. cv. IR72) signal factors with the functions of inducing Agrobaterium tumefaciens (Smith et Townsend) Corm vir genes were constructed. They were 5,7,4'-trihydmxy-3', 5'- dimethoxy-flavone (Fl) and its structure analogues F2, F3 and F4. These four signal factors were detected by Agrobacterium vir:: lacZ fusion gene system as to compare their functions of inducing virA, virB, virC, virD, virE and virG genes. The structure characters and stmcture-activity relationship of these signal factors with the best inducing effects to Agrobacterium exogenous gene transformation system were defined which may provide some basic information for foreign gene transformation in rice.     

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.     

Identification of rice (Oryza sativa L.) signal factors capable of inducing Agrobacterium vir gene expression

Two kinds of signal factors capable of inducing Agrobaorerium vir gene expression were purified and identified from leaf extracts of panicle-differentiating to flowering stage of rice (Oryza saliva L. cv. IR 72) detected by Agrobacterium vir(?) lacZ. fusion genes. The induction was similar to that observed with 5 μm actosyringone (AS). Based on the comprehensive analysis of the data by UV, IR, NMR, MS, HMQC and HMBC, the structures of these two signal factors are identified as 5, 7, 4'-trihydroxy-3', 5'-dimethoxy-flavone (named tricin) and 5, 4' -dihydroxy-3', 5' -dimethoxy-7- (β-D-glucosyloxy) -flavone, respectively. These results demonstrate that monocotyledonous plants do contain highly efficient vir gene inducing factors of Agrobacterium, and the reason why monocotyledonous plants are difficult to transform by Ayrobacterium is not due to absence of vir gene inducing factors, but due to the signal factors only produced in specific stage and tissue of monocotyledonous plants     

Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit

The entire vir regulon of Agrobacterium tumefaciens was subcloned and the complete 28.6-kbp nucleotide sequence was determined. The regulon was cloned as a single unit into two replicons, one of which replicates at a high copy number in this bacterium, and a second which has broad-host-range features to replicate in other Gram-negative bacteria. These vir region plasmids are able to confer in trans the processing and transfer activities on a second plasmid containing the T-DNA. In the high copy number vir region plasmid pUCD2614, a moderate increase in basal vir gene expression was observed as judged by virE::cat fusion expression assays relative to the wild-type control plasmid. Furthermore, higher efficiencies of tobacco leaf disk transformation were observed than with the widely used vir helper plasmid pAL4404. The nucleotide sequence studies showed that the vir region consists of 28,631 bp comprising 24 open reading frames which encode proteins involved in tumorigenicity. Two open reading frames not previously characterized, virH and ORF5, were uncovered within the virD/virE intervening spacer region. Together these studies more completely characterize the structure and function of the vir regulon.