The Gram-negative, fastidious bacterium Xylella fastidiosa was successfully transformed with two RSF1010 derivative plasmids belonging to the incompatibility group IncQ, using electroporation. These two derivative plasmids, pXF004 and pXF005, were found to be present as autonomous, structurally unchanged DNA molecules when propagated in X fastidiosa. However, neither pXF004 nor pXF005 were stably maintained in X fastidiosa without antibiotic selection. When plasmid DNAs were isolated from X fastidiosa, or plasmid DNAs isolated from E. coli were supplemented with a TypeOne(TM) inhibitor, TRI, the frequency of transformation was increased by 13- or 5-fold, respectively. Plasmid pXF005 was also used to transform one additional grapevine strain of X fastidiosa.