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

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Abstract
To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri , plasmid constructs were designed to disrupt the motility gene scm1 . An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not in S. citri , and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E. coli . Electrotransformation of S. 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 the oriC region or at the scm1 gene. In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of the scm1 -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-type S. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S. citri , a mollicute which probably lacks a functional RecA protein.