title: Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.
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- to 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.
authors.affiliation: United States Department of Agriculture-Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, 9611 South Riverbend Ave., Parlier, CA 93648-9757.
authors.forename: Lindsey P
authors.initials: LP
authors.lastname: Burbank
authors.affiliation: United States Department of Agriculture-Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, 9611 South Riverbend Ave., Parlier, CA 93648-9757.
authors.forename: Drake C
authors.initials: DC
authors.lastname: Stenger
mesh_terms: D000992:Antitoxins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D005822:Genetic Vectors; D010957:Plasmids; D014169:Transformation, Bacterial; D027843:Vitis / Q000382:microbiology; D044167:Xylella / Q000235:genetics*
chemical_list: D000992:Antitoxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial