The receiver domain of hybrid histidine kinase VirA: an enhancing factor for vir gene expression in Agrobacterium tumefaciens

Abstract

The plant pathogen Agrobacterium tumefaciens expresses virulence (vir) genes in response to chemical signals found at the site of a plant wound. VirA, a hybrid histidine kinase, and its cognate response regulator, VirG, regulate vir gene expression. The receiver domain at the carboxyl end of VirA has been described as an inhibitory element because its removal increased vir gene expression relative to that of full-length VirA. However, experiments that characterized the receiver region as an inhibitory element were performed in the presence of constitutively expressed virG. We show here that VirA's receiver domain is an activating factor if virG is expressed from its native promoter on the Ti plasmid. When virADeltaR was expressed from a multicopy plasmid, both sugar and the phenolic inducer were essential for vir gene expression. Replacement of wild-type virA on pTi with virADeltaR precluded vir gene induction, and the cells did not accumulate VirG or induce transcription of a virG-lacZ fusion in response to acetosyringone. These phenotypes were corrected if the virG copy number was increased. In addition, we show that the VirA receiver domain can interact with the VirG DNA-binding domain.

FIG. 1.

Removal of the VirA receiver domain results in defective signal-transducing activity when virG is expressed from the Ti plasmid. Strain A348-3 (ΔvirA) carried pAW16 (wild-type virA) (▪), pAW102 (virAΔ707-829) (□), pAW103 (virAΔ707-829 + P_N25-virG) (•), or pYW47 (P_N25-virG) (▴). All cells carried pSW209Ω (virB-lacZ). (A) Cells grown in AB*I medium with glycerol as the carbon source. (B) Cells grown in AB*I medium with arabinose as the carbon source.

FIG. 2.

VirAΔR requires a higher cellular concentration of VirG than wild-type VirA requires in order to activate vir gene expression. (A) A348-3/pAW118 (wild-type virG) and A136/pRG109/pAW118 (P_N25-virG) were grown in AB* medium containing glucose but no phenolic inducer. Lanes 1 and 3 were loaded with 10 μl of a cell solution, and lanes 2 and 4 were loaded with 20 μl of a cell solution. Lanes 1 through 4 are lanes from the same gel. Wt, wild type. (B) A136 carrying either wild-type virA (pAW100) (lanes 1, 3, 5, and 7) or virAΔR (pAW107) (lanes 2, 4, 6, and 8) was assayed following growth in AB*I induction medium with glycerol as the carbon source and 300 μM acetosyringone. Lanes 1 and 2 contained cells that carried the pAW10 vector, while lanes 3 to 8 contained cells that carried lacI^q on pAW106. IPTG was added at concentrations of 0, 25, and 300 μM, as indicated below the gel. The β-galactosidase assay indicates the expression of the virB-lacZ fusion. The coordinated Western blot shows the amounts of VirG in the cells used in the β-galactosidase assay. wt, wild type; ΔR, virAΔR.

FIG. 3.

VirAΔR behaves like a null mutant when it is encoded on pTi in the absence of plasmid-carried virG. (A and B) □, strain A348/pAW10 (vector); ▪, A348/pYW47 (PN25-virG); ○, AB400/pAW10; •, AB400/pYW47. (C and D) □, A348/pAW50 (low-copy-number vector); ▪, A348/pAW132 (virG); ○, AB400/pAW50; •, AB400/pAW132. In panel D the dotted line indicates data for AB400/pAW52 (wild-type virA). All cells carried pSW209Ω (virB-lacZ). Cells used for panels A and C were grown in AB*I medium with glycerol as the carbon source. Cells used for panels B and D were grown in AB*I medium with glucose.

FIG. 4.

Tumor formation on N. tabacum explants requires the VirA receiver domain. Tobacco explants were inoculated with the strains indicated. The images are representative of the results of experiments performed with 16 explants for each strain on cocultivation plates that did not contain AS. Tumor formation was observed on the 10th day after inoculation. The mean tumor counts are indicated along with standard errors (in parentheses). Tumor formation by AB400 (virAΔR) is compared with tumor formation by A348 (wild-type virA) and A348-3 (ΔvirA).

FIG. 5.

Accumulation of VirG encoded on pTi requires the receiver domain of VirA. A348 and AB400 carrying either pAW50 (low-copy-number vector) or pAW132 (virG) were grown in AB*I medium containing glucose with the concentrations of AS indicated. Lane 1, A348/pAW50; lane 2, A348/pAW132; lane 3, AB400/pAW50; lane 4, AB400/pAW132.

FIG. 6.

The VirA receiver domain is required for upregulation of virG transcription under virulence-inducing conditions. Cells were grown in AB*I medium with glucose and the amounts of AS indicated. All strains carried pSW174 (virG-lacZ). •, A348 (wild-type virA in pTi); □, AB400 (virAΔR in pTi); ♦, A136 (no pTi).

FIG. 7.

VirA receiver domain interacts with the DNA-binding domain of VirG. The organisms used were E. coli SU202 (PsulA-lacZ, op408/op⁺) carrying the pSR658 and pSR659 vectors (vectors) or the VirA, VirG, or OmpR receiver domain (indicated by R) together with the DNA-binding domain (indicated by C) of either VirG or OmpR. The repression of PsulA-lacZ fusion expression is shown.