Morphological evidence for phages in Xylella fastidiosa

Presumptive phage particles associated with Xylella fastidiosa strain Temecula-1 grown in PW broth were observed by transmission electron microscopy (TEM) in ultrathin sections of bacterial cell-containing low speed centrifugation pellets and in partially purified preparations from CsCl equilibrium centrifugation density gradients. Ultrathin-sectioned cell pellets contained icosahedral particles of about 45 nm in diameter. Samples collected from CsCl density gradients revealed mostly non-tailed icosahedral but also tailed particles. The icosahedral particles could be divided into two types: a large type (about 45 nm) and a small type (about 30 nm). Filamentous phage-like particles (17 × 120 to 6,300 nm) were also observed. The presence of different types of phage-like particles resembling to those in several bacteriophage families provides new physical evidence, in addition to X. fastidiosa genomic information, that X. fastidiosa possesses active phages. This is the first report of phage particles released in X. fastidiosa cultures

VitisExpDB: A database resource for grape functional genomics

<p>Abstract</p> <p>Background</p> <p>The family Vitaceae consists of many different grape species that grow in a range of climatic conditions. In the past few years, several studies have generated functional genomic information on different <it>Vitis </it>species and cultivars, including the European grape vine, <it>Vitis vinifera</it>. Our goal is to develop a comprehensive web data source for Vitaceae.</p> <p>Description</p> <p>VitisExpDB is an online MySQL-PHP driven relational database that houses annotated EST and gene expression data for <it>V. vinifera </it>and non-<it>vinifera </it>grape species and varieties. Currently, the database stores ~320,000 EST sequences derived from 8 species/hybrids, their annotation (BLAST top match) details and Gene Ontology based structured vocabulary. Putative homologs for each EST in other species and varieties along with information on their percent nucleotide identities, phylogenetic relationship and common primers can be retrieved. The database also includes information on probe sequence and annotation features of the high density 60-mer gene expression chip consisting of ~20,000 non-redundant set of ESTs. Finally, the database includes 14 processed global microarray expression profile sets. Data from 12 of these expression profile sets have been mapped onto metabolic pathways. A user-friendly web interface with multiple search indices and extensively hyperlinked result features that permit efficient data retrieval has been developed. Several online bioinformatics tools that interact with the database along with other sequence analysis tools have been added. In addition, users can submit their ESTs to the database.</p> <p>Conclusion</p> <p>The developed database provides genomic resource to grape community for functional analysis of genes in the collection and for the grape genome annotation and gene function identification. The VitisExpDB database is available through our website <url>http://cropdisease.ars.usda.gov/vitis_at/main-page.htm</url>.</p

Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection

<p>Abstract</p> <p>Background</p> <p>Plant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to <it>Xylella fastidiosa </it>(<it>Xf</it>) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase <it>CYP736B </it>gene in both disease resistant and susceptible grapevines.</p> <p>Results</p> <p>Cloning of genomic DNA and cDNA revealed that the <it>CYP736B </it>gene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site. <it>CYP736B </it>transcript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks after <it>Xf </it>inoculation. However, <it>CYP736B </it>expression was very low in stem tissues at all evaluated time points. 5'RACE and 3'RACE sequence analyses revealed that there were three candidate transcription start sites (TSS) in the upstream region and three candidate polyadenylation (PolyA) sites in the downstream region of <it>CYP736B</it>. Usage frequencies of each transcription initiation site and each polyadenylation site varied depending on plant genotype, developmental stage, tissue, and treatment. These results demonstrate that expression of <it>CYP736B </it>is regulated developmentally and in response to <it>Xf </it>infection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation of <it>CYP736B </it>expression.</p> <p>Conclusions</p> <p>This report provides evidence that the cytochrome P450 monooxygenase <it>CYP736B </it>gene is involved in defense response at a specific stage of <it>Xf </it>infection in grapevines; multiple transcription initiation and polyadenylation sites exist for <it>CYP736B </it>in grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation of <it>CYP736B </it>expression during growth, development and response to <it>Xf </it>infection.</p

PrimerSNP: a web tool for whole-genome selection of allele-specific and common primers of phylogenetically-related bacterial genomic sequences

<p>Abstract</p> <p>Background</p> <p>The increasing number of genomic sequences of bacteria makes it possible to select unique SNPs of a particular strain/species at the whole genome level and thus design specific primers based on the SNPs. The high similarity of genomic sequences among phylogenetically-related bacteria requires the identification of the few loci in the genome that can serve as unique markers for strain differentiation. PrimerSNP attempts to identify reliable strain-specific markers, on which specific primers are designed for pathogen detection purpose.</p> <p>Results</p> <p>PrimerSNP is an online tool to design primers based on strain specific SNPs for multiple strains/species of microorganisms at the whole genome level. The allele-specific primers could distinguish query sequences of one strain from other homologous sequences by standard PCR reaction. Additionally, PrimerSNP provides a feature for designing common primers that can amplify all the homologous sequences of multiple strains/species of microorganisms. PrimerSNP is freely available at <url>http://cropdisease.ars.usda.gov/~primer</url>.</p> <p>Conclusion</p> <p>PrimerSNP is a high-throughput specific primer generation tool for the differentiation of phylogenetically-related strains/species. Experimental validation showed that this software had a successful prediction rate of 80.4 – 100% for strain specific primer design.</p

Comparative phylogenomics and multi-gene cluster analyses of the Citrus Huanglongbing (HLB)-associated bacterium Candidatus Liberibacter

<p>Abstract</p> <p>Background</p> <p>Huanglongbing (HLB, previously known as citrus greening), is associated with <it>Candidatus </it>Liberibacter species and is a serious threat to citrus production world-wide. The pathogen is a Gram negative, unculturable, phloem-limited bacterium with limited known genomic information. Expanding the genetic knowledge of this organism may provide better understanding of the pathogen and possibly develop effective strategies for control and management of HLB.</p> <p>Results</p> <p>Here, we report cloning and characterization of an additional 14.7 Kb of new genomic sequences from three different genomic regions of the <it>Candidatus </it>Liberibacter asiaticus (Las). Sequence variation analyses among the available <it>Ca</it>. Liberibacter species sequences as well as the newly cloned 1.5 Kb of <it>rpo</it>B gene from different <it>Ca</it>. Liberibacter strains have identified INDELs and SNPs. Phylogenetic analysis of the deduced protein sequences from the cloned regions characterizes the HLB-associated <it>Candidatus </it>Liberibacter as a new clade in the sub-division of the α-proteobacteria.</p> <p>Conclusion</p> <p>Comparative analyses of the cloned gene regions of <it>Candidatus </it>Liberibacter with members of the order Rhizobiales suggest overall gene structure and order conservation, albeit with minor variations including gene decay due to the identified pseudogenes. The newly cloned gene regions contribute to our understanding of the molecular aspects of genomic evolution of <it>Ca</it>. Liberibacter.</p

Xylella fastidiosa comparative genomic database is an information resource to explore the annotation, genomic features, and biology of different strains

The Xylella fastidiosa comparative genomic database is a scientific resource with the aim to provide a user-friendly interface for accessing high-quality manually curated genomic annotation and comparative sequence analysis, as well as for identifying and mapping prophage-like elements, a marked feature of Xylella genomes. Here we describe a database and tools for exploring the biology of this important plant pathogen. The hallmarks of this database are the high quality genomic annotation, the functional and comparative genomic analysis and the identification and mapping of prophage-like elements. It is available from web site http://www.xylella.lncc.br

Origins of the Xylella fastidiosa Prophage-Like Regions and Their Impact in Genome Differentiation

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes

The Complete Genome Sequence of ‘Candidatus Liberibacter solanacearum’, the Bacterium Associated with Potato Zebra Chip Disease

Zebra Chip (ZC) is an emerging plant disease that causes aboveground decline of potato shoots and generally results in unusable tubers. This disease has led to multi-million dollar losses for growers in the central and western United States over the past decade and impacts the livelihood of potato farmers in Mexico and New Zealand. ZC is associated with ‘Candidatus Liberibacter solanacearum’, a fastidious alpha-proteobacterium that is transmitted by a phloem-feeding psyllid vector, Bactericera cockerelli Sulc. Research on this disease has been hampered by a lack of robust culture methods and paucity of genome sequence information for ‘Ca. L. solanacearum’. Here we present the sequence of the 1.26 Mbp metagenome of ‘Ca. L. solanacearum’, based on DNA isolated from potato psyllids. The coding inventory of the ‘Ca. L. solanacearum’ genome was analyzed and compared to related Rhizobiaceae to better understand ‘Ca. L. solanacearum’ physiology and identify potential targets to develop improved treatment strategies. This analysis revealed a number of unique transporters and pathways, all potentially contributing to ZC pathogenesis. Some of these factors may have been acquired through horizontal gene transfer. Taxonomically, ‘Ca. L. solanacearum’ is related to ‘Ca. L. asiaticus’, a suspected causative agent of citrus huanglongbing, yet many genome rearrangements and several gene gains/losses are evident when comparing these two Liberibacter. species. Relative to ‘Ca. L. asiaticus’, ‘Ca. L. solanacearum’ probably has reduced capacity for nucleic acid modification, increased amino acid and vitamin biosynthesis functionalities, and gained a high-affinity iron transport system characteristic of several pathogenic microbes