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E W Nester

Publications and source records attributed to E W Nester.

At least 37 records · Page 2Linked to original sources

Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens.

Early studies on Agrobacterium tumefaciens showed that development of tumors on plants following infection by A. tumefaciens was optimal at temperatures around 22 degrees C and did not occur at temperatures above 29 degrees C. To assess whether this inability to induce tumors is due to a defect in the T-DNA transfer machinery, mobilization of an incompatibility group Q (IncQ) plasmid by the T-DNA transfer machinery of A. tumefaciens was tested at various temperatures. Optimal transfer occurred when matings were performed at 19 degrees C, and transfer was not seen when matings were incubated above 28 degrees C. Transfer of the IncQ plasmid was dependent upon induction of the virB and virD operons by acetosyringone but was not dependent upon induction of the tra genes by octopine. However, alterations in the level of vir gene induction could not account for the decrease in transfer with increasing temperature. A. tumefaciens did successfully mobilize IncQ plasmids at higher temperatures when alternative transfer machineries were provided. Thus, the defect in transfer at high temperature is apparently in the T-DNA transfer machinery itself. As these data correlate with earlier tumorigenesis studies, we propose that tumor suppression at higher temperatures results from a T-DNA transfer machinery which does not function properly.

Acetophenones↗

Genetic evidence for direct sensing of phenolic compounds by the VirA protein of Agrobacterium tumefaciens.

The virulence (vir) genes of Agrobacterium tumefaciens are induced by low-molecular-weight phenolic compounds and monosaccharides through a two-component regulatory system consisting of the VirA and VirG proteins. However, it is not clear how the phenolic compounds are sensed by the VirA/VirG system. We tested the vir-inducing abilities of 15 different phenolic compounds using four wild-type strains of A. tumefaciens--KU12, C58, A6, and Bo542. We analyzed the relationship between structures of the phenolic compounds and levels of vir gene expression in these strains. In strain KU12, vir genes were not induced by phenolic compounds containing 4'-hydroxy, 3'-methoxy, and 5'-methoxy groups, such as acetosyringone, which strongly induced vir genes of the other three strains. On the other hand, vir genes of strain KU12 were induced by phenolic compounds containing only a 4'-hydroxy group, such as 4-hydroxyacetophenone, which did not induce vir genes of the other three strains. The vir genes of strains KU12, A6, and Bo542 were all induced by phenolic compounds containing 4'-hydroxy and 3'-methoxy groups, such as acetovanillone. By transferring different Ti plasmids into isogenic chromosomal backgrounds, we showed that the phenolic-sensing determinant is associated with Ti plasmid. Subcloning of Ti plasmid indicates that the virA locus determines which phenolic compounds can function as vir gene inducers. These results suggest that the VirA protein directly senses the phenolic compounds for vir gene activation.

Agrobacterium tumefaciens↗

An Agrobacterium virulence factor encoded by a Ti plasmid gene or a chromosomal gene is required for T-DNA transfer into plants.

Mutagenesis of the vir region on the Ti plasmid of Agrobacterium tumefaciens revealed a new locus, virJ, that is induced by the plant-wound signal molecule, acetosyringone (AS). virJ lies between virA and virB, and is transcribed in the same direction. The amino acid sequence of virJ is similar to a region of a previously characterized chromosomal gene, acvB, required for virulence. virJ can complement the avirulent phenotype of an acvB mutant, indicating that virJ and acvB encode the same factor required for tumorigenesis. Southern analysis revealed that virJ is present on the Ti plasmid of an octopine but not a nopaline strain whereas acvB is present on the chromosomes of both octopine and nopaline strains. While virJ is regulated by AS under the control of the virA/virG two-component regulatory system, acvB is not induced by AS. VirJ possesses a putative signal peptide and was found predominantly in the periplasmic fraction. The strain lacking both acvB and virJ had an impaired ability to transfer T-DNA into plant cells, suggesting that the factor encoded by virJ or acvB is required for T-DNA transfer from A. tumefaciens to plant cells. acvB is the first chromosomal gene implicated in T-DNA transfer, but whether it functions specifically for this process is not clear. We hypothesize that virJ evolved from acvB, presumably for a more specialized role in tumorigenesis.

Acetophenones↗

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.

Agrobacterium tumefaciens↗

An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA.

The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells. This study examined one putative component of that complex, VirB4. A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with the kanamycin resistance-conferring nptII gene. The virB4 gene was found to be necessary for virulence on plants and for the transfer of IncQ plasmids to recipient cells of A. tumefaciens. Genetic complementation of the deletion strain by the virB4 gene under control of the virB promoter confirmed that the deletion was nonpolar on downstream virB genes. Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synthesis of the VirB4 protein from this promoter is far below wild-type levels. Having shown a role for the VirB4 protein in DNA transfer, lysine-439, found within the conserved mononucleotide binding domain of VirB4, was changed to a glutamic acid, methionine, or arginine by oligonucleotide-directed mutagenesis. virB4 genes bearing these mutations were unable to complement the virB4 deletion for either virulence or for IncQ transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA transfer. The necessity of the VirB4 protein with an intact mononucleotide binding site for extracellular complementation of virE2 mutants was also shown. In merodiploid studies, lysine-439 mutations present in trans decreased IncQ plasmid transfer frequencies, suggesting that VirB4 functions within a complex to facilitate DNA transfer.

Agrobacterium tumefaciens↗

The binding site of the transcriptional activator VirG from Agrobacterium comprises both conserved and specific nonconserved sequences.

Virulence genes of Agrobacterium tumefaciens are transcriptionally activated in response to phenolic compounds and certain sugars. The transcription activator VirG specifically binds to fragments containing the conserved vir box sequence present in the promoter region of all vir genes. This study shows that both the vir box as well as specific nonconserved sequences downstream of the vir box are required for VirG binding and transcriptional activation. Insertion of the identified VirG binding site into the lac promoter resulted in transcriptional activation of this heterologous promoter in response to the plant phenolic signal molecule acetosyringone.

Agrobacterium tumefaciens↗

Construction of Agrobacterium strains by electroporation of genomic DNA and its utility in analysis of chromosomal virulence mutations.

We have extended the technique of electroporation as a genetic tool for manipulating the Agrobacterium tumefaciens chromosome. We used this technique to introduce chromosomal DNA into recipient A. tumefaciens strains by electroporation and constructed isogenic chvE mutants that share the same chromosomal background but differ in their types of pTi (octopine or nopaline). Both nopaline and octopine pTi-carrying chvE mutants were deficient in vir regulon induction and exhibited similar reductions in host range.

Agrobacterium tumefaciens↗

Preformed dimeric state of the sensor protein VirA is involved in plant--Agrobacterium signal transduction.

Plant signal molecules such as acetosyringone and certain monosaccharides induce the expression of Agrobacterium tumefaciens virulence (vir) genes, which are required for the processing, transfer, and possibly integration of a piece of the bacterial plasmid DNA (T-DNA) into the plant genome. Two fo the vir genes, virA and virG, belonging to the bacterial two-component regulatory system family, control the induction of vir genes by plant signals. virA encodes a membrane-bound sensor kinase protein and virG encodes a cytoplasmic regulator protein. Although it is well established from in vitro studies that the signal transduction process involves VirA autophosphorylation and subsequent phosphate transfer to VirG, the structural state of the VirA protein involved in signal transduction is not understood. In this communication, we describe an in vivo crosslinking approach which provides physical evidence that VirA exists as a homodimer in its native configuration. The dimerization of VirA neither requires nor is stimulated by the plant signal molecule acetosyringone. We also present genetic data which support the hypothesis that VirA exists as a homodimer which is the functional state transducing the plant signal in an intersubunit mechanism. To our knowledge, this report provides the first evidence that a bacterial membrane-bound sensor kinase exists and functions as a homodimer in vivo.

Agrobacterium tumefaciens↗

VirA, the plant-signal receptor, is responsible for the Ti plasmid-specific transfer of DNA to maize by Agrobacterium.

Agrobacteria exhibit marked Ti (tumor-inducing)/Ri (root-inducing) plasmid specificity in their interaction with the Gramineae. In this study, we have used the technique of "agroinfection," in which Agrobacterium-mediated delivery of viral genomes into plants is detected by the development of viral disease symptoms, to identify the region of the Ti plasmid which is responsible for the major differences seen in the ability of nopaline- vs. octopine-type Ti plasmids to transfer maize streak virus (MSV) DNA to maize. Introduction of fragments of the C58 (nopaline-type) Ti plasmid into strains containing an octopine-type Ti plasmid showed that a fragment containing the nopaline-type virA locus was able to complement these normally non-agroinfectious strains to high levels of MSV DNA transfer. Octopine-type virA mutant strains that express vir genes at high levels in the absence of the plant inducing compound acetosyringone also efficiently transferred MSV DNA. These findings imply a functional difference between the virA gene products encoded by octopine- and nopaline-type Ti plasmids which has a profound effect on their ability to mediate DNA transfer to maize.

Arginine↗

Characterization of a virG mutation that confers constitutive virulence gene expression in Agrobacterium.

Transformation of plants by Agrobacterium tumefaciens is mediated by a set of virulence (vir) genes that are specifically induced by plant signal molecules through the VirA/VirG two-component regulatory system. The plant signal is transmitted from VirA to VirG by a cascade of phosphorylation reactions followed by the sequence-specific DNA binding of the VirG protein to the vir gene promoters which then activates their transcription. In this report, we describe a VirG mutant which is able to activate vir gene expression independently of the VirA molecule and the two plant signal molecules, acetosyringone and monosaccharides. A strain of Agrobacterium containing this virG gene but lacking a functional virA gene was able to induce tumours on all three plants that were tested. A single amino acid change of asparagine (N) to aspartate (D) at position 54, adjacent to the site of VirG phosphorylation, aspartate 52, resulted in this constitutive phenotype. In vitro phosphorylation experiments showed that the mutant protein cannot be phosphorylated by VirA, suggesting that the negative charge resulting from the N to D switch mimics the phosphorylated conformation of the VirG molecule. The same amino acid change in the virG gene of the supervirulent strain A281 also resulted in a constitutive phenotype. However, the vir genes were not induced to high levels when compared with the levels of the constitutive virG of strain A348.

Agrobacterium tumefaciens↗

The virA promoter is a host-range determinant in Agrobacterium tumefaciens.

The limited host range (LHR) Agrobacterium tumefaciens strain Ag162 is an isolate with a narrow host range. Introduction of the wide host range (WHR) virA gene is essential for extending the host range to Kalanchoë daigremontiana. In this report we show that the region upstream of the ATG start codon is responsible for the LHR phenomenon and that this is probably due to the non-inducibility of the LHRvirA promoter. By comparing the characteristics of the LHR and WHR VirA receptor proteins, it was found that the LHR VirA protein is able to activate the WHR VirG protein in the presence of acetosyringone and that this acetosyringone-dependent vir-induction is enhanced by the presence of D-glucose, as in the case of WHR VirA proteins. These results indicate that the domains, acting as receptors for sugars and phenolic signals, must be conserved between the LHR and WHR VirA receptor proteins.

Acetophenones↗

Dynamic structure of Agrobacterium tumefaciens Ti plasmids.

Agrobacterium tumefaciens C58F is a variant of strain C58 which generates a high proportion of avirulent mutants in the presence of the virulence (vir) gene inducer acetosyringone. These mutants are altered in the Ti plasmid and do not respond to the acetosyringone signal (C. Fortin, E. W. Nester, and P. Dion, J. Bacteriol. 174:5676-5685, 1992). The physical organization of the Ti plasmid was compared in strain C58 and its variant. One feature distinguishing pTiC58F from its parent plasmid was the presence of the insertion element IS426. Three copies of this element were detected in the strain C58 chromosome, whereas two additional copies were found in strain C58F, including one copy in the Ti plasmid. This particular copy of IS426 was associated with the region of arginine and nopaline catabolism of pTiC58F. Most of the avirulent mutants recovered following growth of strain C58F in the presence of acetosyringone were complemented by clones carrying either virA or virG. Element IS426 was no longer found in the arginine and nopaline catabolism region of the Ti plasmids from the virA and virG mutants, but it resided in the particular KpnI fragment containing the modified vir locus. Behavior of a strain C58F derivative, which was inactivated in a chromosomal component required for the response to acetosyringone, was consistent with the possibility that vir gene induction is essential to the massive production of avirulent mutants.

Acetophenones↗

A chromosomally encoded two-component sensory transduction system is required for virulence of Agrobacterium tumefaciens.

TnphoA mutagenesis of Agrobacterium tumefaciens identified new extracytoplasmic protein-encoding virulence loci. Mutations in these loci conferred increased sensitivity to detergents and several antibiotics. Clones carrying these loci were isolated from an A. tumefaciens cosmid library by complementation of the detergent sensitivities of the mutants. The locus on one complementing clone was delineated by Tn5 and TnphoA mutagenesis. DNA sequence analysis of the delineated region revealed that this locus is made up of two transcriptional units, chvG and chvI, which were predicted, on the basis of amino acid sequence homology, to encode the members of a two-component sensory transduction system. The membrane-spanning sensor, a histidine protein kinase, was designated ChvG, and the response regulator, presumably a transcriptional activator, was designated ChvI. Surprisingly, ChvG was also predicted to contain a Walker type A consensus nucleotide binding site, which is unusual for sensor histidine protein kinases. Site-specific insertion mutations in either chvG or chvI abolished tumor formation ability, as well as the ability to grow on complex media. Neither the genes which are regulated nor the inducing signal is known yet for this system.

Agrobacterium tumefaciens↗

The regulatory VirA protein of Agrobacterium tumefaciens does not function at elevated temperatures.

Previous studies have shown that Agrobacterium tumefaciens causes tumors on plants only at temperatures below 32 degrees C, and virulence gene expression is specifically inhibited at temperatures above 32 degrees C. We show here that this effect persists even when the virA and virG loci are expressed under the control of a lac promoter whose activity is temperature independent. This finding suggests that one or more steps in the signal transduction process mediated by the VirA and VirG proteins are temperature sensitive. Both the autophosphorylation of VirA and the subsequent transfer of phosphate to VirG are shown to be sensitive to high temperatures (> 32 degrees C), and this correlates with the reduced vir gene expression observed at these temperatures. At temperatures of 32 degrees C and higher, the VirA molecule undergoes a reversible inactivation while the VirG molecule is not affected. vir gene induction is temperature sensitive in an acetosyringone-independent virA mutant background but not in a virG constitutive mutant which is virA and acetosyringone independent. These observations all support the notion that the VirA protein is responsible for the thermosensitivity of vir gene expression. However, an Agrobacterium strain containing a constitutive virG locus still cannot cause tumors on Kalanchoe plants at 32 degrees C. This strain induces normal-size tumors at temperatures up to 30 degrees C, whereas the wild-type Agrobacterium strain produces almost no tumors at 30 degrees C. These results suggest that at temperatures above 32 degrees C, the plant becomes more resistant to infection by A. tumefaciens and/or functions of some other vir gene products are lost in spite of their normal levels of expression.

Agrobacterium tumefaciens↗

The chromosomal virulence gene, chvE, of Agrobacterium tumefaciens is regulated by a LysR family member.

Certain plant phenolic compounds and monosaccharides induce the transcription of virulence (vir) genes of Agrobacterium tumefaciens through the VirA-VirG two-component regulatory system. The product of the chromosomal virulence gene chvE is homologous to galactose-binding protein of Escherichia coli and is required for vir gene induction by sugars. Adjacent to, but divergent in transcription from, chvE is an open reading frame, now termed gbpR (galactose-binding protein regulator), that is homologous to the LysR family of transcriptional regulators. chvE::lacZ expression was induced by L-arabinose, D-galactose, and D-fucose when gbpR was present. In the absence of inducer, GbpR repressed chvE::lacZ expression. In addition, GbpR negatively regulated its own expression.

Agrobacterium tumefaciens↗

Genetic analysis of the virD operon of Agrobacterium tumefaciens: a search for functions involved in transport of T-DNA into the plant cell nucleus and in T-DNA integration.

The transferred DNA (T-DNA) is transported from Agrobacterium tumefaciens to the nucleus and is stably integrated into the genome of many plant species. It has been proposed that the VirD2 protein, tightly attached to the T-DNA, pilots the T-DNA into the plant cell nucleus and that it is involved in integration. Using agroinfection and beta-glucuronidase expression as two different very sensitive transient assays for T-DNA transfer, together with assays for stable integration, we have shown that the C-terminal half of the VirD2 protein and the VirD3 protein are not involved in T-DNA integration. However, the bipartite nuclear localization signal, which is located within the C terminus of the VirD2 protein and which has previously been shown to be able to target a foreign protein into the plant cell nucleus, was shown to be required for efficient T-DNA transfer. virD4 mutants were shown by agroinfection to be completely inactive in T-DNA transfer.

Agrobacterium tumefaciens↗

Growth inhibition and loss of virulence in cultures of Agrobacterium tumefaciens treated with acetosyringone.

Acetosyringone, a phenolic inducer of the virulence (vir) genes of Agrobacterium tumefaciens, inhibited the growth of the nopaline-type strains T37 and C58 incubated under acidic conditions. In the course of a 6-day incubation with acetosyringone, avirulent clones were produced in different proportions by strains T37 and C58 and also by a spontaneous variant of strain C58, denominated C58F. The proportion of avirulent clones in acetosyringone-treated cultures often exceeded 50% for strains T37 and C58F and was of the order of 1% for strain C58. Control cultures not exposed to acetosyringone did not yield avirulent clones. Two other vir inducers, sinapinic acid and syringaldehyde, also inhibited growth and promoted accumulation of avirulent clones in cultures of strains C58F and T37. On the other hand, various acetosyringone analogs reported not to induce the vir genes did not act as growth inhibitors. All of the T37 and most of the C58F avirulent clones examined still carried a Ti plasmid. In all instances examined, avirulent clones still carrying a Ti plasmid were mutated in this plasmid. Mutants of strain C58F lacked the capacity to induce a virB::lacZ fusion in the presence of acetosyringone.

Acetophenones↗

The ntrA gene of Agrobacterium tumefaciens: identification, cloning, and phenotype of a site-directed mutant.

A 3.6-kb EcoRI fragment containing the ntrA gene of Agrobacterium tumefaciens was cloned by using the homologous ntrA gene of Rhizobium meliloti as a probe. Construction of an ntrA mutant of A. tumefaciens by site-directed insertional mutagenesis demonstrated the requirement of the ntrA gene for nitrate utilization and C4-dicarboxylate transport but not for vir gene expression or tumorigenesis.

Agrobacterium tumefaciens↗