Type VI secretion system is not required for virulence on rice but for inter-bacterial competition in Xanthomonas oryzae pv. oryzicola.

The type VI secretion system (T6SS), a multifunctional protein secretion device, plays very important roles in bacterial killing and/or virulence to eukaryotic cells. Although T6SS genes have been found in many Xanthomonas species, the biological function of T6SSs has not been elucidated in most xanthomonads. In this study, we identified two phylogenetically distinct T6SS clusters, T6SS1 and T6SS2, in a newly sequenced Chinese strain GX01 of Xanthomonas oryzea pv. oryzicola (Xoc) which causes bacterial leaf streak (BLS) of rice (Oryza sativa L.). Mutational assays demonstrated that T6SS1 and T6SS2 are not required for the virulence of Xoc GX01 on rice. Nevertheless, we found that T6SS2, but not T6SS1, played an important role in bacterial killing. Transcription and secretion analysis revealed that hcp2 gene is actively expressed and that Hcp2 protein is secreted via T6SS. Moreover, several candidate T6SS effectors were predicted by bioinformatics analysis that might play a role in the antibacterial activity of Xoc. This is the first report to investigate the type VI secretion system in Xanthomonas oryzae. We speculate that Xoc T6SS2 might play an important role in inter-bacterial competition, allowing this plant pathogen to gain niche advantage by killing other bacteria.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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 PXO99A to the stresses of heavy metal ions. The plasmid survey indicated that nine out of 257 Xoc Chinese isolates contain plasmids. CONCLUSIONS: 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.