CHARACTERIZATION OF THE PLASMID-ENCODED VIRULENCE REGION PAT-1 OF PHYTOPATHOGENIC CLAVIBACTER-MICHIGANENSIS SUBSP MICHIGANENSIS

The tomato pathogen Clavibacter michiganensis subsp, michiganensis NCP PB382, causing bacterial wilt and canker, harbors two plasmids, pCM1 ( 27.5 kb) and pCM2 (72 kb), carrying genes involved in virulence, The r egion of plasmid pCM2 encoding the pathogenicity locus pat-1 was mappe d by deletion analysis and complementation studies to a 1.5-kb BglII/S maI DNA fragment, Introduction of the pat-1 region into endophytic, pl asmid-free isolates of C. michiganensis subsp, michiganensis converted these bacteria into virulent pathogens, Based on the nucleotide seque nce of the pat-1 region, an open reading frame (ORF1) can be predicted , coding for a protein of 280 amino acids and 29.7 kDa with homology t o serine proteases, Introduction of a frame-shift mutation in ORF1 lea ds to a loss of the pathogenic phenotype, Northern (RNA) hybridization s identified an 1.5-knt transcript of the pat-1 structural gene, The s ite of transcription initiation was mapped by primer extension and a t ypical -10/-35 region was located with significant homology to the con sensus Escherichia coli sigma(70) and Bacillus subtilis sigma(43) prom oters. Downstream of the pat-1 structural gene, a peculiar repetitive sequence motif (pat-1rep) is located, consisting of 20 direct tandem r epeats preceded by a run of 14 guanosine residues. DNA sequences homol ogous to pat-1rep were isolated and characterized from four virulent C . michiganensis subsp, michiganensis strains exhibiting a high extent of structural conservation, The deletion of this repetitive sequence r educed virulence significantly but did not lead to a complete loss of the virulence phenotype.