Molecular Monitoring of hrpB-disrupted Mutant of Ralstonia solanacearum in Tomato Plants

A nonpathogenic mutant of Ralstonia solanacearum was produced by the insertion of transposon Tn4431. The mutagenized gene was then cloned from a genomic DNA library by the gene tagging method, using the labeled lux operon located on Tn4431 of pUCD623 as a hybridization probe. From nucleotide sequence analysis of the transposon-inserted genomic clone, the hrpB gene was shown to be disrupted by the inserted transposon. Tomato plants were inoculated with the hrpB-disrupted mutant bacteria, for which multiplication and translocation were then monitored using the colony hybridization method. In addition, the original pathogenic bacteria in which the lux operon had been functionally ligated with the genomic promoter were also used for inoculation and traced by their bioluminescence. Multiplication of the hrpB-disrupted mutant was suppressed initially in the invaded root tissues and then in upper hypocotyl after translocation, suggesting that the pathogenic strain of R. solanacearum overcomes at least two steps of host responses expressed in root and hypocotyl tissues. Thus, our approach for molecular monitoring of the bacteria enabled us to precisely analyze the infection behavior of the pathogenic bacteria in planta.