Detection of a single-copied plasmid, pXFSL21, from Xylella fastidiosa strain stag’s leap with two toxin-antitoxin systems using a next generation sequencing approach

Authors: Van Horn, Christopher; WU, F - South China Agricultural University; ZHENG, Z - South China Agricultural University; DAI, Z - South China Agricultural University; Chen, Jianchi

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/24/2018
Publication Date: 1/10/2019
Citation: Van Horn, C.R., Wu, F., Zheng, Z., Dai, Z., Chen, J. 2019. Detection of a single-copied plasmid, pXFSL21, from Xylella fastidiosa strain stag’s leap with two toxin-antitoxin systems using a next generation sequencing approach. Phytopathology. 109:240-247. https://doi.org/10.1094/PHYTO-07-18-0249-FI.
DOI: https://doi.org/10.1094/PHYTO-07-18-0249-FI

Interpretive Summary: Xylella fastidiosa is a bacterial pathogen causing Pierce’s Disease (PD) of grapevine resulting in significant economic loss in California. Strain Stag’s Leap of the pathogen was originally isolated from Napa Valley, California, and is highly virulent. Little is known about the virulence mechanism(s) of this bacterium, but genome sequence analyses could provide initial clues. In this study, DNA samples of strain Stag’s Leap were extracted from a pure culture and a PD-symptomatic grapevine in the greenhouse. Next generation sequencing (NGS) and subsequent computational analyses were performed. A circular plasmid was found and designated as pXFSL21. The plasmid existed as a single-copy per bacterial cell and encoded 27 genes, including four genes forming two toxin-antitoxin (T-A) systems. The double T-A systems may facilitate maintenance of the single-copy plasmid in the bacterial population.

Technical Abstract: Plasmids are important genetic elements contributing to bacterial evolution and environmental adaptation. Xylella fastidiosa is a nutritionally fastidious Gram-negative bacterium causing economically devastating plant 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. 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 (MiSeq for in vitro DNA and HiSeq for in planta DNA). 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 environments. The plasmid had 27 open reading frames (ORFs) encoding proteins of replication/dissemination/maintenance (RDM) and conferring advantageous phenotype (CAP). Two toxin-antitoxin (T-A) systems, ydcD-ydcE and higB-higA, were detected in pXFSL21, which could be associated with the survival or consistent maintenance of this single-copy plasmid in the bacterial population. BLAST searches against GenBank database (version 222, including 46 plasmids and 41 whole genome sequences of X. fastidiosa) showed homologs of ydcD-ydcE in 28 records (whole genomes or plasmids) and higA-higB in two whole genome records. pXFSL21 was not found in PD strains Temecula1 and M23, and, therefore, could serve as a molecular marker and an initial point to study virulence of strain Stag’s Leap.