Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG.

Agrobacterium tumefaciens virulence genes are induced by plant signals through the VirA-VirG two-component regulatory system. The VirA protein is a membrane-spanning sensor molecule that possesses an autophosphorylating activity, and the VirG protein is a sequence-specific DNA-binding protein. In this report, we demonstrate that the VirG protein is phosphorylated by the VirA protein and that the phosphate is directly transferred from the phosphorylated VirA molecule (phosphohistidine) to the VirG protein. The chemical stability of the phospho-VirG bond suggested that the VirG protein was phosphorylated at the aspartate and/or glutamate residue. The phosphorylated VirG protein was reduced with tritiated sodium borohydride and subjected to proteolytic digestion with the Achromobacter protease I enzyme. The resulting peptide fragments were separated by C8 reversed-phase high-pressure liquid chromatography, and the tritium-labeled peptide was sequenced. Amino acid sequence data showed that the aspartate residue at position 52 was the only site phosphorylated. Changing this aspartate into asparagine resulted in a nonphosphorylatable and biologically nonfunctional gene product. As a control, a randomly chosen aspartate was changed into an asparagine (position 72), and no effect on its phosphorylation or biological activity was observed. Unlike its homologs, including CheA-CheY, EnvZ-OmpR, and NtrB-NtrC, the phospho-VirG molecule was very stable in vitro. The possible implications of these observations and the function of VirG phosphorylation in vir gene activation are discussed.     

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 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.     

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.     

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.     

The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation

This paper describes a so-called ternary transformation system for plant cells. We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of very efficient T-DNA transfer to a diverse range of plant species. For the plant species Catharanthus roseus it is shown that increased T-DNA transfer results in increased stable transformation frequencies. Analysis of stably transformed C. roseus cell lines showed that, although the T-DNA transfer frequency is greatly enhanced by addition of virGN54D, only one or a few T-DNA copies are stably integrated into the plant genome. Thus, high transformation frequencies of different plant species can be achieved by introduction of a ternary plasmid carrying a constitutive virG mutant into existing A. tumefaciens strains in combination with standard binary vectors.     

Genetic analysis of the signal-sensing region of the histidine protein kinase VirA of Agrobacterium tumefaciens

The membrane-bound sensor protein kinase VirA of Agrobacterium tumefaciens detects plant phenolic substances, which induce expression of vir genes that are essential for the formation of the crown gall tumor. VirA also responds to specific monosaccharides, which enhance vir expression. These sugars are sensed by the periplasmic domain of VirA that includes the region homologous to the chemoreceptor Trg, and the phenolics are thought to be detected by a part of the cytoplasmic linker domain, while the second transmembrane domain (TM2) is reported to be nonessential. To define regions of VirA that are essential for signal sensing, we introduced base-substitution and deletion mutations into coding regions that are conserved among the respective domains of VirA proteins from various Agrobacterium strains, and examined the effects of these mutations on vir induction and tumorigenicity. The results show that the Trg-homologous region in the periplasmic domain is not essential for the enhancement of vir gene expression by sugars. Most mutations in the TM2 domain also failed to influence enhancement by sugars and reduced the level of vir induction, but a mutation in the TM2 region adjacent to the cytoplasmic linker abolished induction of the vir genes. In the linker domain, sites essential for vir induction by phenolics were scattered over the entire region. We propose that a topological feature formed by the linker domain and at least part of the TM2 may be crucial for activation of a membrane-anchored VirA protein. Complementation analysis with two different VirA mutants suggested that intermolecular phosphorylation between VirA molecules occurs in vivo, and that two intact periplasmic regions in a VirA dimer are required for the enhancement of vir induction by sugars. Received: 14 December 1999 / Accepted: 10 April 2000     

Intersubunit complementation of sugar signal transduction in VirA heterodimers and posttranslational regulation of VirA activity in Agrobacterium tumefaciens

The VirA/VirG two-component regulatory system of Agrobacterium tumefaciens regulates expression of the virulence (vir) genes that control the infection process leading to crown gall tumor disease on susceptible plants. VirA, a membrane-bound homodimer, initiates vir gene induction by communicating the presence of molecular signals found at the site of a plant wound through phosphorylation of VirG. Inducing signals include phenols, monosaccharides, and acidic pH. While sugars are not essential for gene induction, their presence greatly increases vir gene expression when levels of the essential phenolic signal are low. Reception of the sugar signal depends on a direct interaction between ChvE, a sugar-binding protein, and VirA. Here we show that the sugar signal received in the periplasmic region of one subunit within a VirA heterodimer can enhance the kinase function of the second subunit. However, sugar enhancement of vir gene expression was vector dependent. virA alleles expressed from pSa-derived vectors inhibited signal transduction by endogenous VirA. Inhibition was conditional, depending on the induction medium and the virA allele tested. Moreover, constitutive expression of virG overcame the inhibitory effect of some but not all virA alleles, suggesting that there may be more than one inhibitory mechanism.     

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 LHR virA 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.     

High-efficiency gene transfer to recalcitrant plants by Agrobacterium tumefaciens

 Agrobacterium tumefaciens efficiently transforms most plants. A few dicotyledonous plants and most monocotyledonous plants are, however, recalcitrant to A. tumefaciens infection. We investigated whether the constitutive synthesis of a high level of the T-strand DNA intermediate can improve the transformation efficiency of plants. We previously described a mutation in the vir gene regulator virG, virGN54D, that allows constitutive expression of the vir genes. We also described the isolation of a mutant plasmid that is present at a significantly high level in A. tumefaciens. The two mutations were combined to produce an A. tumefaciens strain that synthesizes a high level of T-strand DNA in an inducer-independent manner. DNA transfer efficiency of the mutant was measured by monitoring β-glucuronidase (GUS) expression in a transient transfer assay. A significant increase in the efficiency of DNA transfer to both rice and soybean was observed with the double mutant. The presence of virGN54D had a major positive effect on transformation efficiency. Received: 4 August 2000 / Revision received: 9 October 2000 / Accepted: 12 October 2000     

Genetic analysis of the signal-sensing region of the histidine protein kinase VirA of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

The membrane-bound sensor protein kinase VirA of Agrobacterium tumefaciens detects plant phenolic substances, which induce expression of vir genes that are essential for the formation of the crown gall tumor. VirA also responds to specific monosaccharides, which enhance vir expression. These sugars are sensed by the periplasmic domain of VirA that includes the region homologous to the chemoreceptor Trg, and the phenolics are thought to be detected by a part of the cytoplasmic linker domain, while the second transmembrane domain (TM2) is reported to be nonessential. To define regions of VirA that are essential for signal sensing, we introduced base-substitution and deletion mutations into coding regions that are conserved among the respective domains of VirA proteins from various Agrobacterium strains, and examined the effects of these mutations on vir induction and tumorigenicity. The results show that the Trg-homologous region in the periplasmic domain is not essential for the enhancement of vir gene expression by sugars. Most mutations in the TM2 domain also failed to influence enhancement by sugars and reduced the level of vir induction, but a mutation in the TM2 region adjacent to the cytoplasmic linker abolished induction of the vir genes. In the linker domain, sites essential for vir induction by phenolics were scattered over the entire region. We propose that a topological feature formed by the linker domain and at least part of the TM2 may be crucial for activation of a membrane-anchored VirA protein. Complementation analysis with two different VirA mutants suggested that intermolecular phosphorylation between VirA molecules occurs in vivo, and that two intact periplasmic regions in a VirA dimer are required for the enhancement of vir induction by sugars.     

The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA protein

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. Although it is clear that the monosaccharides require binding to a periplasmic binding protein before they can interact with the sensor VirA protein, it is not certain whether the phenolic compounds also interact with a binding protein or directly interact with the sensor protein. To shed light on this question, we tested the vir-inducing abilities of several different phenolic compounds using two wild-type strains of A. tumefaciens, KU12 and A6. We found that several compounds such as 4-hydroxyacetophone and p-coumaric acid induced the vir of KU12, but not A6. On the other hand, acetosyringone and several other phenolic compounds induced the vir of A6, but not KU12. By transferring different Ti plasmids into isogenic chromosomal backgrounds, we showed that the phenolic sensing determinant is associated with the Ti plasmid. Subcloning of the Ti plasmid indicated that the virA locus determines which phenolic compounds can function as vir inducers. These results suggest that VirA directly senses the phenolic compounds for vir activation.     

Shorter T-DNA or additional virulence genes improve Agrobactrium-mediated transformation

The effect of additional virulence (vir) genes and size of plasmid T-DNA in Agrobacterium tumefa- ciens was investigated for their impact on transformation efficiency. Transformation efficiency in tobacco, cotton, and rice was increased when the T-DNA was 4.3 kb compared to 8.4 kb in size. However, when additional virG, virGN54D,virE, or virE/virG plasmids were included with the 8.4-kb T-DNA, transformation frequencies in all cases were increased over that of the shorter T-DNA without additional vir plasmids. The use of virE, virG or virGN54D copies enhanced transformation efficiency; however, the most significant increase of transformation efficiency in all three plant species was observed when the virE/virG plasmid was used for infection. The virE/virG plasmid dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer; moreover, this plasmid appears to have broad efficiency since it was consistently effective on two different dicotyledon species as well as a monocotyledon species. Received: 8 February 2000 / Accepted: 21 March 2000     

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.     

Constitutive activation of two-component response regulators: characterization of VirG activation in Agrobacterium tumefaciens

Response regulators are the ultimate modulators in two-component signal transduction pathways. The N-terminal receiver domains generally accept phosphates from cognate histidine kinases to control output. VirG for example, the response regulator of the VirA/VirG two-component system in Agrobacterium tumefaciens, mediates the expression of virulence genes in response to plant host signals. Response regulators have a highly conserved structure and share a similar conformational activation upon phosphorylation, yet the sequence and structural features that determine or perturb the cooperative activation events are ill defined. Here we use VirG and the unique features of the Agrobacterium system to extend our understanding of the response regulator activation. Two previously isolated constitutive VirG mutants, VirGN54D and VirGI77V/D52E, provide the foundation for our studies. In vivo phosphorylation patterns establish that VirGN54D is able to accumulate phosphates from small-molecule phosphate donors, such as acetyl phosphate, while the VirGI77V/D52E allele carries conformational changes mimicking the active conformation. Further structural alterations on these two alleles begin to reveal the changes necessary for response regulator activation.     

Localization of the VirA domain involved in acetosyringone-mediatedvir gene induction inAgrobacterium tumefaciens

The VirA protein ofAgrobacterium tumefaciens is thought to be a receptor for plant phenolic compounds such as acetosyringone. Although it is not known whether the interaction between VirA and the phenolics is direct or requires other phenolic-binding proteins, it is shown in this study that the first 280 amino acids of the VirA protein are not essential for the acetosyringone mediatedvir gene induction response. Considering the fact that the cytoplasmic region between the amino acids 283 and 304 is highly conserved between the different VirA proteins, and that deletion of this region abolishes VirA activity, we suggest that the acetosyringone receptor domain is located in this cytoplasmic domain of the VirA protein.