Taisei Kikuchi

Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

REAPR: a universal tool for genome assembly evaluation.

Methods to reliably assess the accuracy of genome sequence data are lacking. Currently completeness is only described qualitatively and mis-assemblies are overlooked. Here we present REAPR, a tool that precisely identifies errors in genome assemblies without the need for a reference sequence. We have validated REAPR on complete genomes or de novo assemblies from bacteria, malaria and Caenorhabditis elegans, and demonstrate that 86% and 82% of the human and mouse reference genomes are error-free, respectively. When applied to an ongoing genome project, REAPR provides corrected assembly statistics allowing the quantitative comparison of multiple assemblies. REAPR is available at http://www.sanger.ac.uk/resources/software/reapr/.

A family of glycosyl hydrolase family 45 cellulases from the pine wood nematode Bursaphelenchus xylophilus

We have characterized a family of GHF45 cellulases from the pine wood nematode Bursaphelenchus xylophilus. The absence of such genes from other nematodes and their similarity to fungal genes suggests that they may have been acquired by horizontal gene transfer (HGT) from fungi. The cell wall degrading enzymes of other plant parasitic nematodes may have been acquired by HGT from bacteria. B. xylophilus is not directly related to other plant parasites and our data therefore suggest that horizontal transfer of cell wall degrading enzymes has played a key role in evolution of plant parasitism by nematodes on more than one occasion.

The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery

BackgroundThe small ruminant parasite Haemonchus contortus is the most widely used parasitic nematode in drug discovery, vaccine development and anthelmintic resistance research. Its remarkable propensity to develop resistance threatens the viability of the sheep industry in many regions of the world and provides a cautionary example of the effect of mass drug administration to control parasitic nematodes. Its phylogenetic position makes it particularly well placed for comparison with the free-living nematode Caenorhabditis elegans and the most economically important parasites of livestock and humans.ResultsHere we report the detailed analysis of a draft genome assembly and extensive transcriptomic dataset for H. contortus. This represents the first genome to be published for a strongylid nematode and the most extensive transcriptomic dataset for any parasitic nematode reported to date. We show a general pattern of conservation of genome structure and gene content between H. contortus and C. elegans, but also a dramatic expansion of important parasite gene families. We identify genes involved in parasite-specific pathways such as blood feeding, neurological function, and drug metabolism. In particular, we describe complete gene repertoires for known drug target families, providing the most comprehensive understanding yet of the action of several important anthelmintics. Also, we identify a set of genes enriched in the parasitic stages of the lifecycle and the parasite gut that provide a rich source of vaccine and drug target candidates.ConclusionsThe H. contortus genome and transcriptome provide an essential platform for postgenomic research in this and other important strongylid parasites.

The genome and life-stage specific transcriptomes of Globodera pallida elucidate key aspects of plant parasitism by a cyst nematode

BackgroundGlobodera pallida is a devastating pathogen of potato crops, making it one of the most economically important plant parasitic nematodes. It is also an important model for the biology of cyst nematodes. Cyst nematodes and root-knot nematodes are the two most important plant parasitic nematode groups and together represent a global threat to food security.ResultsWe present the complete genome sequence of G. pallida, together with transcriptomic data from most of the nematode life cycle, particularly focusing on the life cycle stages involved in root invasion and establishment of the biotrophic feeding site. Despite the relatively close phylogenetic relationship with root-knot nematodes, we describe a very different gene family content between the two groups and in particular extensive differences in the repertoire of effectors, including an enormous expansion of the SPRY domain protein family in G. pallida, which includes the SPRYSEC family of effectors. This highlights the distinct biology of cyst nematodes compared to the root-knot nematodes that were, until now, the only sedentary plant parasitic nematodes for which genome information was available. We also present in-depth descriptions of the repertoires of other genes likely to be important in understanding the unique biology of cyst nematodes and of potential drug targets and other targets for their control.ConclusionsThe data and analyses we present will be central in exploiting post-genomic approaches in the development of much-needed novel strategies for the control of G. pallida and related pathogens.

A Rapid and Precise Diagnostic Method for Detecting the Pinewood Nematode Bursaphelenchus xylophilus by Loop-Mediated Isothermal Amplification

ABSTRACT Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which is a major forest disease in Japan, Korea, China, Taiwan, and Portugal. A diagnostic method which is rapid, precise, and simple could greatly help the proper management of this disease. Here, we present a novel detection method using loop-mediated isothermal amplification (LAMP) targeting the internal transcribed spacer region of ribosomal DNA of the nematode. Specificity of the primers and LAMP was confirmed using DNA from various nematode species related to B. xylophilus. Our experimental results suggest that LAMP can detect B. xylophilus faster and with higher sensitivity than the traditional diagnostic method. Moreover, because it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions.

Molecular and biochemical characterization of an endo-β-1,3-glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria

We report the cloning and functional characterization of an endo-beta-1,3-glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria. This is the first gene of this type from any nematode species. We show that a similar cDNA is also present in another closely related species B. mucronatus, but that similar sequences are not present in any other nematode studied to date. The B. xylophilus gene is expressed solely in the oesophageal gland cells of the nematode and the protein is present in the nematodes secretions. The deduced amino acid sequence of the gene is very similar to glycosyl hydrolase family 16 proteins. The recombinant protein, expressed in Escherichia coli, preferentially hydrolysed the beta-1,3-glucan laminarin, and had very low levels of activity on beta-1,3-1,4-glucan, lichenan and barley beta-glucan. Laminarin was degraded in an endoglucanase mode by the enzyme. The optimal temperature and pH for activity of the recombinant enzyme were 65 degrees C and pH 4.9. The protein is probably important in allowing the nematodes to feed on fungi. Sequence comparisons suggest that the gene encoding the endo-beta-1,3-glucanase was acquired by horizontal gene transfer from bacteria. B. xylophilus therefore contains genes that have been acquired by this process from both bacteria and fungi. These findings support the idea that multiple independent horizontal gene transfer events have helped in shaping the evolution of several different life strategies in nematodes.

A complete view of the genetic diversity of the Escherichia coli O-antigen biosynthesis gene cluster

The O antigen constitutes the outermost part of the lipopolysaccharide layer in Gram-negative bacteria. The chemical composition and structure of the O antigen show high levels of variation even within a single species revealing itself as serological diversity. Here, we present a complete sequence set for the O-antigen biosynthesis gene clusters (O-AGCs) from all 184 recognized Escherichia coli O serogroups. By comparing these sequences, we identified 161 well-defined O-AGCs. Based on the wzx/wzy or wzm/wzt gene sequences, in addition to 145 singletons, 37 serogroups were placed into 16 groups. Furthermore, phylogenetic analysis of all the E. coli O-serogroup reference strains revealed that the nearly one-quarter of the 184 serogroups were found in the ST10 lineage, which may have a unique genetic background allowing a more successful exchange of O-AGCs. Our data provide a complete view of the genetic diversity of O-AGCs in E. coli showing a stronger association between host phylogenetic lineage and O-serogroup diversification than previously recognized. These data will be a valuable basis for developing a systematic molecular O-typing scheme that will allow traditional typing approaches to be linked to genomic exploration of E. coli diversity.

Horizontal gene transfer from bacteria and fungi as a driving force in the evolution of plant parasitism in nematodes

Phylogenetic studies have shown that parasitism of plants by nematodes has arisen independently on at least three separate occasions. We argue that horizontal gene transfer has played a critical role in the evolution of plant parasitism on each occasion. In addition, we discuss evidence that suggests this process has driven the evolution of other life strategies within the Nematoda and that it may be considerably more common within the Phylum than commonly thought. We review recent literature that shows horizontal gene transfer to nematodes has occurred from both bacteria and fungi.

The genomic basis of parasitism in the Strongyloides clade of nematodes

Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families—families encoding astacin-like and SCP/TAPS proteins—is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism.