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Eugene W. Nester - One of the best experts on this subject based on the ideXlab platform.
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The Phenolic Recognition Profiles of the Agrobacterium tumefaciens VirA Protein Are Broadened by a High Level of the Sugar Binding Protein ChvE
Journal of bacteriology, 1998Co-Authors: Wen-tao Peng, Yong-woog Lee, Eugene W. NesterAbstract:The formation of crown gall tumors by Agrobacterium tumefaciens requires that the virulence (vir) genes be induced by chemical signals which consist of specific phenolic compounds and monosaccharides, synthesized at plant wound sites. Signal transduction in the activation of these genes is mediated by the VirA-VirG two-component regulatory system, together with ChvE, a glucose-galactose binding Protein which interacts with VirA. We have previously presented genetic evidence that VirA senses phenolic compounds directly (Y.-W. Lee, S. Jin, W.-S. Sim, and E. W. Nester, Proc. Natl. Acad. Sci. USA 92:12245–12249, 1995). The vir genes of strain KU12 can be induced by 4-hydroxyacetophenone, p-coumaric acid, and phenol, whereas these same phenolic compounds are weak inducers of the vir genes of strain A6. In this report, we show that a specific inducing sugar can broaden the specificity of the phenolic compound which VirA senses. 4-Hydroxyacetophenone and other related phenolic compounds function as inducing phenolic compounds with the VirA gene of A6 if arabinose replaces glucose as the inducing sugar. We further demonstrate that this broadened specificity for phenolic inducers results from the increased level of ChvE through induction by arabinose via the regulatory Protein GbpR. If high levels of ChvE are present, then poorly inducing phenolic compounds can induce the vir genes to high levels in combination with glucose. Comparing the induction response of the wild type and that of a VirA mutant with a mutation in its receiver domain revealed that the activity of the receiver domain is controlled by the periplasmic domain. We discuss these observations in terms of how VirA senses and transduces signals elicited by the two classes of plant signal molecules.
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Characterization of an unusual sensor gene (VirA) of Agrobacterium.
Gene, 1998Co-Authors: Yong-woog Lee, Woong-seop Sim, Eugene W. NesterAbstract:Abstract Previous studies have shown that the virulence(vir) genes of Agrobacterium tumefaciens strain KU12 are induced by a unique set of phenolic compounds that are non-functional in most strains of Agrobacterium. Further, strain KU12 is not induced by phenolic compounds that induce the vir genes in other strains. Previous studies have shown that these differences in inducing activity result from differences in the sensor Protein for these signal molecules, the VirA Protein. To gain some understanding of the basis for these differences in sensing ability, we sequenced the entire VirA locus of pTiKU12, including its promoter region and compared this sequence with five different published VirA sequences that respond in different ways to inducing compounds. The VirA gene of KU12 is composed of an open single reading frame coding for 851 aa. At the aa level, the VirA Protein of pTiKU12 is 45, 45, 49, 49 and 64% identical to the VirA Proteins from pTiA6, pTi15955, pRiA4, pTiC58 and pTiAg162, respectively. The transcription start sites of pTiKU12 and pTiA6 VirA genes differ significantly when mapped by primer extension. Unlike all other vir genes, except the VirA gene of pTiAg162, pTiKU12 VirA is constitutively expressed, and its synthesis is not induced by phenolic compounds. The lack of induction is accounted for by the fact that the promoter region does not have the conserved VirG-binding dodecadeoxynucleotide sequence (vir-box) that was previously identified in all promoter regions of inducible vir genes.
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The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA Protein ☆
Gene, 1996Co-Authors: Yong-woog Lee, Shouguang Jin, Woong-seop Sim, Eugene W. NesterAbstract: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.
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Mutational analysis of the input domain of the VirA Protein of Agrobacterium tumefaciens.
Journal of bacteriology, 1996Co-Authors: Sharon L. Doty, J I Lundin, Joe Don Heath, Eugene W. NesterAbstract:The transmembrane sensor Protein VirA activates VirG in response to high levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding Protein ChvE and monosaccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 VirA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated. Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain. Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2. Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 VirA sequence, caused a specific defect in the vir gene response to glucose. This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE. Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 VirA. These mutants were defective in the vir gene response to AS. These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain. Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.
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Genetic evidence for direct sensing of phenolic compounds by the VirA Protein of Agrobacterium tumefaciens.
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Yong-woog Lee, Shouguang Jin, Woong-seop Sim, Eugene W. NesterAbstract:Abstract 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.
Paul J J Hooykaas - One of the best experts on this subject based on the ideXlab platform.
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Localization of the VirA domain involved in acetosyringone-mediatedvir gene induction inAgrobacterium tumefaciens
Plant Molecular Biology, 1994Co-Authors: Stefan C. H. J. Turk, Richard P. Lange, Tonny J. G. Regensburg-tuïnk, Paul J J HooykaasAbstract:The VirA Protein of Agrobacterium 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 mediated vir 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.
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Localization of the VirA domain involved in acetosyringone-mediated vir gene induction in Agrobacterium tumefaciens.
Plant molecular biology, 1994Co-Authors: Stefan C. H. J. Turk, Tonny J. G. Regensburg-tuïnk, Richard P. Van Lange, Paul J J HooykaasAbstract: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.
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The VirA promoter is a host-range determinant in Agrobacterium tumefaciens
Molecular Microbiology, 1993Co-Authors: S. C. H. J. Turk, Eugene W. Nester, Paul J J HooykaasAbstract:: 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 Kalanchoe 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.
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The chimeric VirA-tar receptor Protein is locked into a highly responsive state.
Journal of bacteriology, 1993Co-Authors: Stefan C. H. J. Turk, R. P. Van Lange, E. Sonneveld, Paul J J HooykaasAbstract:The wild-type VirA Protein is known to be responsive not only to phenolic compounds but also to sugars via the ChvE Protein (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA 87:6708-6712, 1990, and N. Shimoda, A. Toyoda-Yamamoto, J. Nagamine, S. Usami, M. Katayama, Y. Sakagami, and Y. Machida, Proc. Natl. Acad. Sci. USA 87:6684-6688, 1990). It is shown here that the mutant VirA(Ser-44, Arg-45) Protein and the chimeric VirA-Tar Protein are no longer responsive to sugars and the ChvE Protein. However, whereas the chimeric VirA-Tar Protein was found to be locked in a highly responsive state, the VirA(Ser-44, Arg-45) mutant Protein appeared to be locked in a low responsive state. This difference turned out to be important for tumorigenicity of the host strains in virulence assays on Kalanchoe daigremontiana.
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Signal Transduction Via Vir a and Vir G in Agrobacterium
Advances in Molecular Genetics of Plant-Microbe Interactions Vol. 1, 1991Co-Authors: Paul J J Hooykaas, Leo Sjoerd Melchers, A. J. G. Regensburg-tuïnk, H. Den Dulk-ras, C. W. Rodenburg, Stefan C. H. J. TurkAbstract:The virulence genes of Agrobacterium tumefaciens are inducible by certain phenolic compounds. Regulation is mediated by the VirA and VirG gene products, which together form a two component regulatory system. Results indicate that the VirA Protein forms a sensor for phenolic compounds and that the VirG Protein becomes activated via the VirA Protein.
Stefan C. H. J. Turk - One of the best experts on this subject based on the ideXlab platform.
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Localization of the VirA domain involved in acetosyringone-mediatedvir gene induction inAgrobacterium tumefaciens
Plant Molecular Biology, 1994Co-Authors: Stefan C. H. J. Turk, Richard P. Lange, Tonny J. G. Regensburg-tuïnk, Paul J J HooykaasAbstract:The VirA Protein of Agrobacterium 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 mediated vir 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.
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Localization of the VirA domain involved in acetosyringone-mediated vir gene induction in Agrobacterium tumefaciens.
Plant molecular biology, 1994Co-Authors: Stefan C. H. J. Turk, Tonny J. G. Regensburg-tuïnk, Richard P. Van Lange, Paul J J HooykaasAbstract: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.
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The chimeric VirA-tar receptor Protein is locked into a highly responsive state.
Journal of bacteriology, 1993Co-Authors: Stefan C. H. J. Turk, R. P. Van Lange, E. Sonneveld, Paul J J HooykaasAbstract:The wild-type VirA Protein is known to be responsive not only to phenolic compounds but also to sugars via the ChvE Protein (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA 87:6708-6712, 1990, and N. Shimoda, A. Toyoda-Yamamoto, J. Nagamine, S. Usami, M. Katayama, Y. Sakagami, and Y. Machida, Proc. Natl. Acad. Sci. USA 87:6684-6688, 1990). It is shown here that the mutant VirA(Ser-44, Arg-45) Protein and the chimeric VirA-Tar Protein are no longer responsive to sugars and the ChvE Protein. However, whereas the chimeric VirA-Tar Protein was found to be locked in a highly responsive state, the VirA(Ser-44, Arg-45) mutant Protein appeared to be locked in a low responsive state. This difference turned out to be important for tumorigenicity of the host strains in virulence assays on Kalanchoe daigremontiana.
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Signal Transduction Via Vir a and Vir G in Agrobacterium
Advances in Molecular Genetics of Plant-Microbe Interactions Vol. 1, 1991Co-Authors: Paul J J Hooykaas, Leo Sjoerd Melchers, A. J. G. Regensburg-tuïnk, H. Den Dulk-ras, C. W. Rodenburg, Stefan C. H. J. TurkAbstract:The virulence genes of Agrobacterium tumefaciens are inducible by certain phenolic compounds. Regulation is mediated by the VirA and VirG gene products, which together form a two component regulatory system. Results indicate that the VirA Protein forms a sensor for phenolic compounds and that the VirG Protein becomes activated via the VirA Protein.
Stephen C. Winans - One of the best experts on this subject based on the ideXlab platform.
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Pleiotropic phenotypes caused by genetic ablation of the receiver module of the Agrobacterium tumefaciens VirA Protein.
Journal of bacteriology, 1996Co-Authors: C H Chang, Jun Zhu, Stephen C. WinansAbstract:The VirA Protein of Agrobacterium tumefaciens is a transmembrane sensory kinase that phosphorylates the VirG response regulator in response to chemical signals released from plant wound sites. VirA contains both a two-component kinase module and, at its carboxyl terminus, a receiver module. We previously provided evidence that this receiver module inhibited the activity of the kinase module and that inhibition might be neutralized by phosphorylation. In this report, we provide additional evidence for this model by showing that overexpressing the receiver module in trans can restore low-level basal activity to a VirA mutant Protein lacking the receiver module. We also show that ablation of the receiver module restores activity to the inactive VirA (delta324-413) mutant, which has a deletion within a region designated the linker module. This indicates that deletion of the linker module does not denature the kinase module, but rather locks the kinase into a phenotypically inactive conformation, and that this inactivity requires the receiver module. These data provide genetic evidence that the kinase and receiver modules of VirA attain their native conformations autonomously. The receiver module also restricts the variety of phenolic compounds that have stimulatory activity, since removal of this module causes otherwise nonstimulatory phenolic compounds such as 4-hydroxyacetophenone to stimulate vir gene expression.
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Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA Protein.
Journal of bacteriology, 1992Co-Authors: Chia-hwa Chang, Stephen C. WinansAbstract:VirA and VirG activate the Agrobacterium tumefaciens vir regulon in response to phenolic compounds, monosaccharides, and acidity released from plant wound sites. VirA contains an amino-terminal periplasmic domain and three cytoplasmic domains: a linker, a Protein kinase, and a phosphoryl receiver. We constructed internal deletions of VirA that truncate one or more domains and tested the ability of the resulting Proteins to mediate environmentally responsive vir gene activation in vivo. The periplasmic domain is required for sensing of monosaccharides (in agreement with earlier results), while the linker domain is required for sensing of phenolic compounds and acidity. The phosphoryl receiver domain of VirA plays an inhibitory role in signal transduction that may be modulated by phosphorylation. The carboxy terminus of the Protein was also dispensable for tumorigenesis, while the periplasmic domain was required. Images
Shouguang Jin - One of the best experts on this subject based on the ideXlab platform.
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The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA Protein ☆
Gene, 1996Co-Authors: Yong-woog Lee, Shouguang Jin, Woong-seop Sim, Eugene W. NesterAbstract: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.
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Genetic evidence for direct sensing of phenolic compounds by the VirA Protein of Agrobacterium tumefaciens.
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Yong-woog Lee, Shouguang Jin, Woong-seop Sim, Eugene W. NesterAbstract:Abstract 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.
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Preformed dimeric state of the sensor Protein VirA is involved in plant--Agrobacterium signal transduction.
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Sheng Pan, Shouguang Jin, Trevor C. Charles, Eugene W. NesterAbstract: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.
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The regulatory VirA Protein of Agrobacterium tumefaciens does not function at elevated temperatures.
Journal of bacteriology, 1993Co-Authors: Shouguang Jin, Milton P. Gordon, Yan-nong Song, Wanyin Deng, Eugene W. NesterAbstract:Abstract 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.
