Christopher Van Horn; Fengnian Wu; Zheng Zheng; Zehan Dai; Jianchi Chen
Plasmids are important genetic elements contributing to bacterial evolution and environmental adaptation. Xylella fastidiosa is a nutritionally fastidious Gram-negative bacterium causing economically devastating diseases such as Pierce’s Disease (PD) of grapevine.
In this study, the plasmid status of a highly virulent PD strain, Stag’s Leap, originally isolated from Napa Valley, California, was studied using sequencing and bioinformatics tools. DNA samples extracted from a pure culture in periwinkle wilt (PW) medium (in vitro DNA) and a PD symptomatic grapevine artificially inoculated in the greenhouse (in planta DNA) were subject to next generation sequencing (NGS) analyses (Illumina MiSeq or HiSeq). Sequence analyses and PCR experiments revealed the presence of a circular plasmid, pXFSL21, of 21,665 bp. This plasmid existed as a single-copy per bacterial genome under both in vitro and in planta conditions. Two toxin-antitoxin (T-A) systems (ydcD-ydcE and higB-higA) were detected in pXFSL21, a possible mechanism for the long term survival of this single-copy plasmid in the bacterial population. BLAST searches against GenBank database (version 222) detected homologs of the two T-A systems in chromosomes/plasmids of some X. fastidiosa strains. However, double T-A systems was found only in pXFSL21. pXFSL21 was not found in the known PD strains, and, therefore, could serve as a molecular marker for strain Stag’s Leap monitoring and tracking. The NGS-based technique outlined in this paper provides an effective tool for identifying single or low copy number plasmids in fastidious prokaryotes.