Submitted to: CDFA Pierce's Disease Control Program Research Symposium
Publication Type: Abstract Only
Publication Acceptance Date: 11/1/2015
Publication Date: 12/21/2015
Citation: Burbank, L.P., Stenger, D.C. 2015. Stable plasmid vectors for complementation of Xylella fastidiosa mutants in planta. CDFA Pierce's Disease Control Program Research Symposium. p. 243.
Technical Abstract: Current understanding of the mechanisms of Pierce’s Disease development has been significantly advanced by molecular genetic studies of the causal agent, Xylella fastidiosa (Xf). Plasmid vectors are an essential tool for studies of bacterial genetics and pathogenesis. However, most commonly used plasmids do not replicate in Xf, limiting the options for complementation analysis or exogenous gene expression in this bacterium. Many of the plasmids that do replicate in Xf are not stable without antibiotic selection particularly after long periods of time. Two different plasmid vectors were created (pBBR5pemIK and pXf20pemIK) utilizing different replication origins for growth in Xf. These vectors carry the PemI/PemK plasmid maintenance system for increased stability in the absence of antibiotic selection. PemK is a toxin which inhibits cell growth unless the antitoxin (PemI) is also present. Loss of the plasmid is prevented because of the need for continuous production of PemI to avoid toxic activity of PemK. Both pBBR5pemIK and pXf20pemIK are retained in Xf after more than five consecutive subcultures in vitro, as well as after 14 weeks of growth in planta. Plasmid pXf20pemIK contains a high copy number pUC origin, in addition to Xf replication elements, facilitating manipulation and propagation in Escherichia coli cloning hosts. Plasmid pBBR5pemIK is a medium-low copy number vector, but is able to replicate in a wider variety of bacterial species due to a broad host range backbone. These vectors provide a valuable tool for conducting genetic studies of Xf virulence and have the potential to be used in other bacterial species as well, particularly in situations where antibiotic selection is impractical.