Complete sequence and detailed analysis of the first indigenous plasmid from Xanthomonas oryzae pv. oryzicola

Xiang-Na Niu, Zhi-Qiong Wei, Hai-Fan Zou, Gui-Gang Xie, Feng Wu, Kang-Jia Li, Wei Jiang, Ji-Liang Tang, Yong-Qiang He

Bacterial plasmids have a major impact on metabolic function and adaptation of their hosts. An indigenous plasmid was identified in a Chinese isolate (GX01) of the invasive phytopathogen Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of rice bacterial leaf streak (BLS). To elucidate the biological functions of the plasmid, we have sequenced and comprehensively annotated the plasmid. The plasmid DNA was extracted from Xoc strain GX01 by alkaline lysis and digested with restriction enzymes. The cloned and subcloned DNA fragments in pUC19 were sequenced by Sanger sequencing. Sequences were assembled by using Sequencher software. Gaps were closed by primer walking and sequencing, and multi-PCRs were conducted through the whole plasmid sequence for verification. BLAST, phylogenetic analysis and dinucleotide calculation were performed for gene annotation and DNA structure analysis. Transformation, transconjugation and stress tolerance tests were carried out for plasmid function assays. The indigenous plasmid from Xoc strain GX01, designated pXOCgx01, is 53,206-bp long and has been annotated to possess 64 open reading frames (ORFs), including genes encoding type IV secretion system, heavy metal exporter, plasmid stability factors, and DNA mobile factors, i.e., the Tn3-like transposon. Bioinformatics analysis showed that pXOCgx01 has a mosaic structure containing different genome contexts with distinct genomic heterogeneities. Phylogenetic analysis indicated that the closest relative of pXOCgx01 is pXAC64 from Xanthomonas axonopodis pv. citri str. 306. It was estimated that there are four copies of pXOCgx01 per cell of Xoc GX01 by PCR assay and the calculation of whole genome shotgun sequencing data. We demonstrate that pXOCgx01 is a self-transmissible plasmid and can replicate in some Xanthomonas spp. strains, but not in Escherichia coli DH5α. It could significantly enhance the tolerance of Xanthomonas oryzae pv. oryzae PXO99(A) to the stresses of heavy metal ions. The plasmid survey indicated that nine out of 257 Xoc Chinese isolates contain plasmids. pXOCgx01 is the first report of indigenous plasmid from Xanthomonas oryzae pv. oryzicola, and the first completely sequenced plasmid from Xanthomonas oryzae species. It is a self-transmissible plasmid and has a mosaic structure, containing genes for macromolecule secretion, heavy metal exportation, and DNA mobile factors, especially the Tn3-like transposon which may provide transposition function for mobile insertion cassette and play a major role in the spread of pathogenicity determinants. The results will be helpful to elucidate the biological significance of this cryptic plasmid and the adaptive evolution of Xoc.