The role of horizontal transfer in the evolution of a highly variable lipopolysaccharide biosynthesis locus in xanthomonads that infect rice, citrus and crucifers

<p>Abstract</p> <p>Background</p> <p>Lipopolysaccharide (LPS) is a pathogen associated molecular pattern (PAMP) of animal and plant pathogenic bacteria. Variation at the interstrain level is common in LPS biosynthetic gene clusters of animal pathogenic bacteria. This variation has been proposed to play a role in evading the host immune system. Even though LPS is a modulator of plant defense responses, reports of interstrain variation in LPS gene clusters of plant pathogenic bacteria are rare.</p> <p>Results</p> <p>In this study we report the complete sequence of a variant 19.9 kb LPS locus present in the BXO8 strain of <it>Xanthomonas oryzae </it>pv. <it>oryzae </it>(Xoo), the bacterial blight pathogen of rice. This region is completely different in size, number and organization of genes from the LPS locus present in most other strains of Xoo from India and Asia. Surprisingly, except for one ORF, all the other ORFs at the BXO8 LPS locus are orthologous to the genes present at this locus in a sequenced strain of <it>X. axonopodis </it>pv. <it>citri </it>(Xac; a pathogen of citrus plants). One end of the BXO8 LPS gene cluster, comprised of ten genes, is also present in the related rice pathogen, <it>X. oryzae </it>pv. <it>oryzicola </it>(Xoc). In Xoc, the remainder of the LPS gene cluster, consisting of seven genes, is novel and unrelated to LPS gene clusters of any of the sequenced xanthomonads. We also report substantial interstrain variation suggestive of very recent horizontal gene transfer (HGT) at the LPS biosynthetic locus of <it>Xanthomonas campestris </it>pv.<it> campestris </it>(Xcc), the black rot pathogen of crucifers.</p> <p>Conclusion</p> <p>Our analyses indicate that HGT has altered the LPS locus during the evolution of <it>Xanthomonas oryzae </it>pathovars and suggest that the ancestor of all <it>Xanthomonas oryzae </it>pathovars had an Xac type of LPS gene cluster. Our finding of interstrain variation in two major xanthomonad pathogens infecting different hosts suggests that the LPS locus in plant pathogenic bacteria, as in animal pathogens, is under intense diversifying selection.</p

Cloning of the rice Xo1 resistance gene and interaction of the Xo1 protein with the defense-suppressing Xanthomonas effector Tal2h

The Xo1 locus in the heirloom rice variety Carolina Gold Select confers resistance to bacterial leaf streak and bacterial blight, caused by Xanthomonas oryzae pv. oryzicola and X. oryzae pv. oryzae, respectively. Resistance is triggered by pathogen-delivered transcription activator-like effectors (TALEs) independent of their ability to activate transcription and is suppressed by truncated variants called truncTALEs, common among Asian strains. By transformation of the susceptible variety Nipponbare, we show that one of 14 nucleotide-binding, leucine-rich repeat (NLR) protein genes at the locus, with a zinc finger BED domain, is the Xo1 gene. Analyses of published transcriptomes revealed that the Xo1-mediated response is more similar to those mediated by two other NLR resistance genes than it is to the response associated with TALE-specific transcriptional activation of the executor resistance gene Xa23 and that a truncTALE dampens or abolishes activation of defense-associated genes by Xo1. In Nicotiana benthamiana leaves, fluorescently tagged Xo1 protein, like TALEs and truncTALEs, localized to the nucleus. And endogenous Xo1 specifically coimmunoprecipitated from rice leaves with a pathogen-delivered, epitope-tagged truncTALE. These observations suggest that suppression of Xo1-function by truncTALEs occurs through direct or indirect physical interaction. They further suggest that effector coimmunoprecipitation may be effective for identifying or characterizing other resistance genes

Acquisition and Evolution of Plant Pathogenesis–Associated Gene Clusters and Candidate Determinants of Tissue-Specificity in Xanthomonas

Xanthomonas is a large genus of plant-associated and plant-pathogenic bacteria. Collectively, members cause diseases on over 392 plant species. Individually, they exhibit marked host- and tissue-specificity. The determinants of this specificity are unknown.To assess potential contributions to host- and tissue-specificity, pathogenesis-associated gene clusters were compared across genomes of eight Xanthomonas strains representing vascular or non-vascular pathogens of rice, brassicas, pepper and tomato, and citrus. The gum cluster for extracellular polysaccharide is conserved except for gumN and sequences downstream. The xcs and xps clusters for type II secretion are conserved, except in the rice pathogens, in which xcs is missing. In the otherwise conserved hrp cluster, sequences flanking the core genes for type III secretion vary with respect to insertion sequence element and putative effector gene content. Variation at the rpf (regulation of pathogenicity factors) cluster is more pronounced, though genes with established functional relevance are conserved. A cluster for synthesis of lipopolysaccharide varies highly, suggesting multiple horizontal gene transfers and reassortments, but this variation does not correlate with host- or tissue-specificity. Phylogenetic trees based on amino acid alignments of gum, xps, xcs, hrp, and rpf cluster products generally reflect strain phylogeny. However, amino acid residues at four positions correlate with tissue specificity, revealing hpaA and xpsD as candidate determinants. Examination of genome sequences of xanthomonads Xylella fastidiosa and Stenotrophomonas maltophilia revealed that the hrp, gum, and xcs clusters are recent acquisitions in the Xanthomonas lineage.Our results provide insight into the ancestral Xanthomonas genome and indicate that differentiation with respect to host- and tissue-specificity involved not major modifications or wholesale exchange of clusters, but subtle changes in a small number of genes or in non-coding sequences, and/or differences outside the clusters, potentially among regulatory targets or secretory substrates

Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online

TALEN-mediated editing of the mouse Y chromosome

The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes—Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome.National Institutes of Health (U.S.) (US NIH grant R01-HG000257)National Institutes of Health (U.S.) (US NIH grant R01-CA084198)National Institutes of Health (U.S.) (US NIH grant R37-HD045022)Croucher Foundation (Scholarship)Howard Hughes Medical Institute (Investigator

Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv. oryzae PXO99A

Background: Xanthomonas oryzae pv. oryzae causes bacterial blight of rice (Oryza sativa L.), a major disease that constrains production of this staple crop in many parts of the world. We report here on the complete genome sequence of strain PXO99A and its comparison to two previously sequenced strains, KACC10331 and MAFF311018, which are highly similar to one another. Results: The PXO99 A genome is a single circular chromosome of 5,240,075 bp, considerably longer than the genomes of the other strains (4,941,439 bp and 4,940,217 bp, respectively), and it contains 5083 protein-coding genes, including 87 not found in KACC10331 or MAFF311018. PXO99A contains a greater number of virulence-associated transcription activator-like effector genes and has at least ten major chromosomal rearrangements relative to KACC10331 and MAFF311018. PXO99 A contains numerous copies of diverse insertion sequence elements, members of which are associated with 7 out of 10 of the major rearrangements. A rapidly-evolving CRISPR (clustered regularly interspersed short palindromic repeats) region contains evidence of dozens of phage infections unique to the PXO99A lineage. PXO99A also contains a unique, near-perfect tandem repeat of 212 kilobases close to the replication terminus. Conclusion: Our results provide striking evidence of genome plasticity and rapid evolution within Xanthomonas oryzae pv. oryzae. The comparisons point to sources of genomic variation and candidates for strain-specific adaptations of this pathogen that help to explain the extraordinary diversity of Xanthomonas oryzae pv. oryzae genotypes and races that have been isolated from around the world. © 2008 Salzberg et al; licensee BioMed Central Ltd

Transfer of Xanthomonas campestris pv. arecae, and Xanthomonas campestris pv. musacearum to Xanthomonas vasicola (Vauterin) as Xanthomonas vasicola pv. arecae comb. nov., and Xanthomonas vasicola pv. musacearum comb. nov. and description of Xanthomonas vasicola pv. vasculorum pv. nov.

The "Ca2+ switch" model with cultured Madin-Darby canine kidney (MDCK) cells is useful in studying the biogenesis of epithelial polarity and junction formation and provides insight into early steps in the morphogenesis of polarized epithelial tissues. When extracellular Ca2+ in the medium is changed from less than 5 microM to 1.8 mM, MDCK cells rapidly change from a nonpolarized state exhibiting little cell-cell contact (with the apical membrane and junctional proteins largely within the cell) to a polarized state with well-formed tight junctions and desmosomes. To examine the role of intracellular Ca2+ in the development of polarity and junctions, we made continuous spectrofluorimetric measurements of intracellular Ca2+ during the "switch," using the fluorescent indicator fura-2. Intracellular Ca2+ increased greater than 10-fold during the switch and gave a complex pattern of increase, decrease, and stabilization. In contrast, intracellular pH [monitored with 29,79-bis(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF)] did not change during the period studied. When intracellular Ca2+ curves in several cells were compared, considerable heterogeneity in the rate of increase of intracellular Ca2+ levels and in peak levels was evident, perhaps reflecting the heterogeneity among cells in establishing junctions and polarity. The heterogeneity of the process was confirmed by digital imaging of intracellular Ca2+ and was present even in a "clonal" line of MDCK cells, indicating the heterogeneity was intrinsic to the process and not simply a function of slight genetic variation within the population of MDCK cells. In pairs of cells that had barely established cell-cell contact, often one cell exhibited a much greater increase in intracellular Ca2+ than the other cell in the pair. At the site of cell-cell contact, an apparent localized change (an increase over the basal level) in intracellular Ca2+ was frequently present and occasionally appeared to extend beyond the point of cell-cell contact. Since the region of cell-cell contact is also the site where junctions form and where vesicles containing apical membranes fuse during the development of polarity, we postulate a role for global and local changes in intracellular Ca2+ in these events

Acquisition and Evolution of Plant Pathogenesis–Associated Gene Clusters and Candidate Determinants of Tissue-Specificity in Xanthomonas

is a large genus of plant-associated and plant-pathogenic bacteria. Collectively, members cause diseases on over 392 plant species. Individually, they exhibit marked host- and tissue-specificity. The determinants of this specificity are unknown. lineage. genome and indicate that differentiation with respect to host- and tissue-specificity involved not major modifications or wholesale exchange of clusters, but subtle changes in a small number of genes or in non-coding sequences, and/or differences outside the clusters, potentially among regulatory targets or secretory substrates

Translating the genomics revolution: the need for an international gene therapy consortium for monogenic diseases

Letter to the Edito