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To determine whether homologous recombination could be used to inactivate selected genes inSpiroplasma citri , plasmid constructs were designed to disrupt the motility genescm1 . An internalscm1 gene fragment was inserted into plasmid pKT1, which replicates inEscherichia coli but not inS. citri , and into theS. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as inE. coli . Electrotransformation ofS. citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at theoriC region or at thescm1 gene. In the latter case, plasmid integration by a single crossover between thescm1 gene fragment carried by the plasmid and the full-lengthscm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of thescm1 -disrupted mutant to periwinkle (Catharanthus roseus ) plants through injection into the leafhopper vector (Circulifer haematoceps ) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-typeS. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, theS. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination inS. citri , a mollicute which probably lacks a functional RecA protein.

Gene Disruption through Homologous Recombination in Spiroplasma citri : an scm1 -Disrupted Motility Mutant Is Pathogenic