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Expression of Agrobacterium tumefaciens virulence (vir) genes is dependent on the presence of a conserved 'vir box' sequence in their 5' nontranscribed regions. The location and number of these sequences vary considerably in different vir genes. Site-directed mutagenesis was used to identify the functional vir box(es) of virB, virC and virD. For virB expression both vir box B1 and B2 are required but only the vir box B1 is absolutely essential. Of the five vir boxes of virC and virD two are required for virC expression while only one vir box is required for virD expression. To investigate the minimum sequences necessary for vir gene induction a deletion derivative of virE that lacks the vir box region was used. This mutant is not induced by acetosyringone. The inducibility of this promoter was restored when a synthetic deoxyoligonucleotide dGTTTCAATTGAAAC was introduced at a location analogous to that of the wild type vir box sequence. Mutational analysis indicate that the functional vir box sequence is 14 residues in length, contains a dyad symmetry and has the consensus sequence d ryTncAaTTGnAaY [corrected] (r = purine, y = pyrimidine).