Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/14/2016
Publication Date: 8/1/2016
Citation: Burbank, L.P., Stenger, D.C. 2016. Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection. Phytopathology. 106(8):928-936.
Interpretive Summary: The bacterial pathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Studies of the genetic components important for this pathogen to infect the plant host have increased understanding of the disease process. Plasmids (circular DNA molecules which replicate separately from the bacterial chromosome) are an important tool for studies of bacterial genetics. Most plasmids commonly used in other organisms do not replicate in X. fastidiosa and require the use of antibiotics so as not to be lost from successive generations of bacterial cells. Two plasmids (pXf20pemIK and pBBR5pemIK) are described here that are able to replicate in X. fastidiosa, and remain in the cell for multiple generations without antibiotics. Instead, pXf20pemIK and pBBR5pemIK are maintained in the cell by production of a toxin protein and its specific antitoxin. These plasmids are useful tools for the study of X. fastidiosa genetics, particularly for experiments conducted in the plant host where antibiotics cannot be used.
Technical Abstract: The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics, but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high copy number pUC origin for propagation in Escherichia coli cloning strains. Broad host range vector pBBR5pemIK is a medium-low copy number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.