Study of the genus Torreya (Taxaceae) based on chloroplast genomes.

BACKGROUND: Species of the genus Torreya are similar in morphology, and their morphological taxonomic characteristics are not stable because of environmentally induced changes. Therefore, morphology is insufficient for understanding their relationships. Chloroplast genome sequencing technology provides a powerful tool for molecular analysis to get more infomation for classification and identification of Torreya genus. METHODS: A total of 4 chloroplast genome of Torreya, including T. Parvifolia, T. nucifera, T. fargesii var. Yunnanensis and T. grandis var. jiulongshanensis, were sequenced and annotated. Campartive genome and phylogenetic tree were provided for variation analysis. RESULTS: The chloroplast genome size of the four samples is about 137 kb, the inverted repeat (IR) regions are identified in the genus Torreya. Genome comparison using mVISTA showed high sequence similarity among different species. Regions with divergence in exon regions include accD, ndhB, ndhF, psbA, psbJ, rpl2, rps3, rps16, rps18, ycf1, and ycf2. The phylogenetic tree based on 73 single-copy genes showed a clearer relationships among different species of Torreya. CONCLUSIONS: All genomes of the four Torreya species consist of two short IR regions, and results of the phylogenetic analysis concluded that T. parvifolia should be considered as T. fargesii var. yunnanensis or treated as a sister species. T. grandis var. jiulongshanensis should be treated as a variety of T. grandis according to molecular evidence, supporting the originally published proposal.

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.

Genome-Wide Screening for Novel Candidate Virulence Related Response Regulator Genes in Xanthomonas oryzae pv. oryzicola.

Two-component regulatory system (TCS), a major type of cellular signal transduction system, is widely used by bacteria to adapt to different conditions and to colonize certain ecological niches in response to environmental stimuli. TCSs are of distinct functional diversity, genetic diversity, and species specificity (pathovar specificity, even strain specificity) across bacterial groups. Although TCSs have been demonstrated to be crucial to the virulence of Xanthomonas, only a few researches have been reported about the studies of TCSs in Xanthomonas oryzae pathovar oryzicola (hereafter Xoc), the pathogen of rice bacterial streak disease. In the genome of Xoc strain GX01, it has been annotated 110 TCSs genes encoding 54 response regulators (RRs), 36 orthodox histidine kinase (HKs) and 20 hybrid histidine kinase (HyHKs). To evaluate the involvement of TCSs in the stress adaptation and virulence of Xoc, we mutated 50 annotated RR genes in Xoc GX01 by homologous vector integration mutagenesis and assessed their phenotypes in given conditions and tested their virulence on host rice. 17 RR genes were identified to be likely involved in virulence of Xoc, of which 10 RR genes are novel virulence genes in Xanthomonas, including three novel virulence genes for bacteria. Of the novel candidate virulence genes, some of which may be involved in the general stress adaptation, exopolysaccharide production, extracellular protease secretion and swarming motility of Xoc. Our results will facilitate further studies on revealing the biological functions of TCS genes in this phytopathogenic bacterium.

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.

Transcriptome profiling of Xanthomonas campestris pv. campestris grown in minimal medium MMX and rich medium NYG.

Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease in cruciferous plants worldwide. Although the complete genomes of several Xcc strains have been determined, the gene expression and regulation mechanisms in this pathogen are far from clear. In this work, transcriptome profiling of Xcc 8004 grown in MMX medium (minimal medium for Xanthomonas campestris) and NYG medium (peptone yeast glycerol medium) were investigated by RNA-Seq. Using the Illumina HiSeq 2000 platform, a total of 26,514,630 reads (90 nt in average) were generated, of which 15,708,478 reads mapped uniquely to coding regions of Xcc 8004 genome. Of the 4273 annotated protein-coding genes of Xcc 8004, 629 were found differentially expressed in Xcc grown in MMX and NYG. Of the differentially expressed genes, 495 were up-regulated and 134 were down-regulated in MMX. The MMX-induced genes are mainly involved in amino acid metabolism, transport systems, atypical condition adaptation and pathogenicity, especially the type III secretion system, while the MMX-repressed genes are mainly involved in chemotaxis and degradation of small molecules. The global transcriptome analyzes of Xcc 8004 grown in MMX and NYG might facilitate the gene functional characterization of this phytopathogenic bacterium.