The virA and virG two-component regulatory system is essential for transcriptional activation of virulence (vir) genes in Agrobacterium tumefaciens in the presence of inducer molecules. The VirA/VirG mediated vir gene transcription depends on a specific interaction between the C-terminal domain of the alpha subunit (RpoA) of A. tumefaciens RNA polymerase (RNAP) and N-terminal domain of the VirG. However, such interaction does not occur between RNAP of E. coli and the VirG, thus vir gene activation in E. coli requires the presence of rpoA gene from A. tumefaciens. In this report, we describe VirG mutants that are capable of activating the expression of vir genes in E. coli in the absence of A. tumefaciens RpoA. The selected 45 VirG mutants exhibited a common amino acid substitution at position 56 and additional one or more substitutions at different positions; thus the amino acid at position 56 is likely to play a key role in the interaction with the RpoA of E. coli. Furthermore, two virG mutants, with amino acid substitutions of G56V/V7I/I106N and G56V/I77V, respectively, are capable of activating vir genes in E. coli in response to inducer acetosyringone in a virA-dependent manner, demonstrating that the interaction site between VirG and RpoA is separable from that of VirG and VirA. Therefore, it is possible to establish inducer-mediated vir gene expression in heterologous hosts using virG mutants that are capable of interacting with the RpoA of the respective bacterial hosts while retaining the ability to interact with the sensor VirA.