Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is pathogenic

To determine whether homologous recombination could be used to inactivate s elected genes in Spiroplasma citri, plasmid constructs were designed to dis rupt the motility gene semi. An internal scm1 gene fragment was inserted in to plasmid pKT1, which replicates in Escherichia coli but not in S. citri, and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma c ells as well as in E. coli, Electrotransformation of S. citri with the nonr eplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast , spiroplasmal transformants were obtained with the replicative, pBOT1-deri ved plasmid pCJ32, During passaging of the transformants, the plasmid was f ound to integrate into the chromosome by homologous recombination either at the oriC region or at the semi gene. In the latter case, plasmid integrati on by a single crossover between the semi gene fragment carried by the plas mid and the full-length semi gene carried by the chromosome led to a nonmot ile phenotype. Transmission of the scm1-disrupted mutant to periwinkle (Cat haranthus roseus) plants through injection into the leafhopper vector (Circ ulifer haematoceps) showed that the motility mutant multiplied in the insec ts 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.