Signatures of adaptation to plant parasitism in nematode genomes.

Plant-parasitic nematodes cause considerable damage to global agriculture. The ability to parasitize plants is a derived character that appears to have independently emerged several times in the phylum Nematoda. Morphological convergence to feeding style has been observed, but whether this is emergent from molecular convergence is less obvious. To address this, we assess whether genomic signatures can be associated with plant parasitism by nematodes. In this review, we report genomic features and characteristics that appear to be common in plant-parasitic nematodes while absent or rare in animal parasites, predators or free-living species. Candidate horizontal acquisitions of parasitism genes have systematically been found in all plant-parasitic species investigated at the sequence level. Presence of peptides that mimic plant hormones also appears to be a trait of plant-parasitic species. Annotations of the few genomes of plant-parasitic nematodes available to date have revealed a set of apparently species-specific genes on every occasion. Effector genes, important for parasitism are frequently found among those species-specific genes, indicating poor overlap. Overall, nematodes appear to have developed convergent genomic solutions to adapt to plant parasitism.

Computational and phylogenetic validation of nematode horizontal gene transfer.

Sequencing of expressed genes has shown that nematodes, particularly the plant-parasitic nematodes, have genes purportedly acquired from other kingdoms by horizontal gene transfer. The prevailing orthodoxy is that such transfer has been a driving force in the evolution of niche specificity, and a recent paper in BMC Evolutionary Biology that presents a detailed phylogenetic analysis of cellulase genes in the free-living nematode Pristionchus pacificus at the species, genus and family levels substantiates this hypothesis.

The genomes of root-knot nematodes.

Plant-parasitic nematodes are the most destructive group of plant pathogens worldwide and are extremely challenging to control. The recent completion of two root-knot nematode genomes opens the way for a comparative genomics approach to elucidate the success of these parasites. Sequencing revealed that Meloidogyne hapla, a diploid that reproduces by facultative, meiotic parthenogenesis, encodes approximately 14,200 genes in a compact, 54 Mpb genome. Indeed, this is the smallest metazoan genome completed to date. By contrast, the 86 Mbp Meloidogyne incognita genome encodes approximately 19,200 genes. This species reproduces by obligate mitotic parthenogenesis and exhibits a complex pattern of aneuploidy. The genome includes triplicated regions and contains allelic pairs with exceptionally high degrees of sequence divergence, presumably reflecting adaptations to the strictly asexual reproductive mode. Both root-knot nematode genomes have compacted gene families compared with the free-living nematode Caenorhabditis elegans, and both encode large suites of enzymes that uniquely target the host plant. Acquisition of these genes, apparently via horizontal gene transfer, and their subsequent expansion and diversification point to the evolutionary history of these parasites. It also suggests new routes to their control.

Proteomic and bioinformatic analysis of the root-knot nematode Meloidogyne hapla: the basis for plant parasitism.

On the basis of the complete genome sequence of the root-knot nematode Melodogyne hapla, we have deduced and annotated the entire proteome of this plant-parasite to create a database of 14,420 proteins. We have made this database, termed HapPep3, available from the Superfamily repository of model organism proteomes (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY). To experimentally confirm the HapPep3 assignments using proteomics, we applied a data-independent LC/MS(E) analysis to M. hapla protein extracts fractionated by SDS-PAGE. A total of 516 nonredundant proteins were identified with an average of 9 unique peptides detected per protein. Some proteins, including examples with complex gene organization, were defined by more than 20 unique peptide matches, thus, providing experimental confirmation of computational predictions of intron/exon structures. On the basis of comparisons of the broad physicochemical properties of the experimental and computational proteomes, we conclude that the identified proteins reflect a true and unbiased sampling of HapPep3. Conversely, HapPep3 appears to broadly cover the protein space able to be experimentally sampled. To estimate the false discovery rate, we queried human, plant, and bacterial databases for matches to the LC/MS(E)-derived peptides, revealing fewer than 1% of matches, most of which were to highly conserved proteins. To provide a functional comparison of the acquired and deduced proteomes, each was subjected to higher order annotation, including comparisons of Gene Ontology, protein domains, signaling, and localization predictions, further indicating concordance, although those proteins that did deviate seem to be highly significant. Approximately 20% of the experimentally sampled proteome was predicted to be secreted, and thus potentially play a role at the host-parasite interface. We examined reference pathways to determine the extent of proteome similarity of M. hapla to that of the free-living nematode, Caenorhabditis elegans, revealing significant similarities and differences. Collectively, the analyzed protein set provides an initial foundation to experimentally dissect the basis of plant parasitism by M. hapla.

A Single, Shared Triploidy in Three Species of Parasitic Nematodes.

The root-knot nematodes of the genus Meloidogyne are important and damaging parasites capable of infecting most flowering plants. Within this genus, several species of the Meloidogyne incognita group show evidence of paleopolyploidy in their genomes. We used our software tool POInT, the Polyploidy Orthology Inference Tool, to phylogenetically model the gene losses that followed that polyploidy. These models, and simulations based on them, show that three of these species (M. incognita, M. arenaria and M. javanica) descend from a single common hybridization event that yielded triplicated genomes with three distinguishable subgenomes. While one of the three subgenomes shows elevated gene loss rates relative to the other two, this subgenome does not show elevated sequence divergence. In all three species, ancestral loci where two of the three gene copies have been lost are less likely to have orthologs in Caenorhabditis elegans that are lethal when knocked down than are ancestral loci with surviving duplicate copies.

Infection by cyst nematodes induces rapid remodelling of developing xylem vessels in wheat roots.

Cyst nematodes induce host-plant root cells to form syncytia from which the nematodes feed. Comprehensive histological investigation of these feeding sites is complicated by their variable shape and their positions deep within root tissue. Using tissue clearing and confocal microscopy, we examined thick (up to 150 µm) sections of wheat roots infected by cereal cyst nematodes (Heterodera avenae). This approach provided clear views of feeding sites and surrounding tissues, with resolution sufficient to reveal spatial relationships among nematodes, syncytia and host vascular tissues at the cellular level. Regions of metaxylem vessels near syncytia were found to have deviated from classical developmental patterns. Xylem vessel elements in these regions had failed to elongate but had undergone radial expansion, becoming short and plump rather than long and cylindrical. Further investigation revealed that vessel elements cease to elongate shortly after infection and that they later experience delays in secondary thickening (lignification) of their outer cell walls. Some of these elements were eventually incorporated into syncytial feeding sites. By interfering with a developmental program that normally leads to programmed cell death, H. avenae may permit xylem vessel elements to remain alive for later exploitation by the parasite.

Bacterial Community Structure Dynamics in Meloidogyne incognita-Infected Roots and Its Role in Worm-Microbiome Interactions.

Plant parasitic nematodes such as Meloidogyne incognita have a complex life cycle, occurring sequentially in various niches of the root and rhizosphere. They are known to form a range of interactions with bacteria and other microorganisms that can affect their densities and virulence. High-throughput sequencing can reveal these interactions in high temporal and geographic resolutions, although thus far we have only scratched the surface. In this study, we have carried out a longitudinal sampling scheme, repeatedly collecting rhizosphere soil, roots, galls, and second-stage juveniles from 20 plants to provide a high-resolution view of bacterial succession in these niches, using 16S rRNA metabarcoding. Our findings indicate that a structured community develops in the root, in which gall communities diverge from root segments lacking a gall, and that this structure is maintained throughout the crop season. We describe the successional process leading toward this structure, which is driven by interactions with the nematode and later by an increase in bacteria often found in hypoxic and anaerobic environments. We present evidence that this structure may play a role in the nematode's chemotaxis toward uninfected root segments. Finally, we describe the J2 epibiotic microenvironment as ecologically deterministic, in part, due to the active bacterial attraction of second-stage juveniles.IMPORTANCE The study of high-resolution successional processes within tightly linked microniches is rare. Using the power and relatively low cost of metabarcoding, we describe the bacterial succession and community structure in roots infected with root-knot nematodes and in the nematodes themselves. We reveal separate successional processes in galls and adjacent non-gall root sections, which are driven by the nematode's life cycle and the progression of the crop season. With their relatively low genetic diversity, large geographic range, spatially complex life cycle, and the simplified agricultural ecosystems they occupy, root-knot nematodes can serve as a model organism for terrestrial holobiont ecology. This perspective can improve our understanding of the temporal and spatial aspects of biological control efficacy.

SILIP: a novel stable isotope labeling method for in planta quantitative proteomic analysis.

Due to ease of manipulation, metabolic isotope coding of samples for proteomic analysis is typically performed in cell culture, thus preventing an accurate in vivo quantitative analysis, which is only achievable in intact organisms. To address this issue in plant biology, we developed SILIP (stable isotope labeling in planta) using tomato plants (Solanum lycopersicum cv. Rutgers) as a method that allows soil-grown plants to be efficiently labeled using a 14N/15N isotope coding strategy. After 2 months of growth on 14N- and 15N-enriched nitrogen sources, proteins were extracted from four distinct tomato tissues (roots, stems, leaves and flowers), digested, and analyzed by LC/MS/MS (data-dependent acquisition, DDA) and alternating low- and elevated-energy MS scans (data-independent acquisition, MS(E)). Using a derived relationship to generate a theoretical standard curve, the measured ratio of the M (monoisotopic) and M-1 isotopologues of 70 identified 15N-labeled peptides from 16 different proteins indicated that 15N incorporation was almost 99%, which is in excellent agreement with the 99.3% 15N-enriched nitrate used in the soil-based medium. Values for the various tissues ranged from 98.2 +/- 0.3% 15N incorporation in leaves to 98.8 2 +/- 0.2% in stems, demonstrating uniform labeling throughout the plant. In addition, SILIP is compatible with root-knot nematode (Meloidogyne spp.) development, and thus provides a new quantitative proteomics tool to study both plant and plant-microorganism systems.

Networks Underpinning Symbiosis Revealed Through Cross-Species eQTL Mapping.

Organisms engage in extensive cross-species molecular dialog, yet the underlying molecular actors are known for only a few interactions. Many techniques have been designed to uncover genes involved in signaling between organisms. Typically, these focus on only one of the partners. We developed an expression quantitative trait locus (eQTL) mapping-based approach to identify cause-and-effect relationships between genes from two partners engaged in an interspecific interaction. We demonstrated the approach by assaying expression of 98 isogenic plants (Medicago truncatula), each inoculated with a genetically distinct line of the diploid parasitic nematode Meloidogyne hapla With this design, systematic differences in gene expression across host plants could be mapped to genetic polymorphisms of their infecting parasites. The effects of parasite genotypes on plant gene expression were often substantial, with up to 90-fold (P = 3.2 × 10-52) changes in expression levels caused by individual parasite loci. Mapped loci included a number of pleiotropic sites, including one 87-kb parasite locus that modulated expression of >60 host genes. The 213 host genes identified were substantially enriched for transcription factors. We distilled higher-order connections between polymorphisms and genes from both species via network inference. To replicate our results and test whether effects were conserved across a broader host range, we performed a confirmatory experiment using M. hapla-infected tomato. This revealed that homologous genes were similarly affected. Finally, to validate the broader utility of cross-species eQTL mapping, we applied the strategy to data from a Salmonella infection study, successfully identifying polymorphisms in the human genome affecting bacterial expression.

Signaling between nematodes and plants.

After hatching in the soil, root-knot nematodes must locate and penetrate a root, migrate into the vascular cylinder, and establish a permanent feeding site. Presumably, these events are accompanied by extensive signaling between the nematode parasite and the host. Hence, much emphasis has been placed on identifying proteins that are secreted by the nematode during the migratory phase. Further progress in understanding the signaling events has been made recently by studying the host response. Striking parallels can be drawn between the nematode-plant interaction and plant symbioses with other microorganisms, and evidence is emerging to suggest that nematodes acquired components of their parasitic armory from those microbes.

A Method for Isolation of Pasteuria penetrans Endospores for Bioassay and Genomic Studies.

A rapid method for collection of Pasteuria penetrans endospores was developed. Roots containing P. penetrans-infected root-knot nematode females were softened by pectinase digestion, mechanically processed, and filtered to collect large numbers of viable endospores. This method obviates laborious handpicking of Pasteuria-infected females and yields endospores competent to attach to and infect nematodes. Endospores are suitable for morphology studies and DNA preparations.

A Method for Generating Meloidogyne incognita Males.

A method for producing mass quantities of Meloidogyne incognita males free from other developmental stages was developed. Host plants were grown hydroponically to facilitate nematode harvest. Pruning stress was shown to cause a higher percentage of juveniles to develop as males vs. a no-stress control. Application of pruning stress in the first 48 hr post-inoculation was also shown to be more effective at driving male development than at later times.

Optimizing k-mer size using a variant grid search to enhance de novo genome assembly.

Largely driven by huge reductions in per-base costs, sequencing nucleic acids has become a near-ubiquitous technique in laboratories performing biological and biomedical research. Most of the effort goes to re-sequencing, but assembly of de novogenerated, raw sequence reads into contigs that span as much of the genome as possible is central to many projects. Although truly complete coverage is not realistically attainable, maximizing the amount of sequence that can be correctly assembled into contigs contributes to coverage. Here we compare three commonly used assembly algorithms (ABySS, Velvet and SOAPdenovo2), and show that empirical optimization of k-mer values has a disproportionate influence on de novo assembly of a eukaryotic genome, the nematode parasite Meloidogynechitwoodi. Each assembler was challenged with about 40 million Iluumina II paired-end reads, and assemblies performed under a range of k-mer sizes. In each instance, the optimal k-mer was 127, although based on N50 values,ABySS was more efficient than the others. That the assembly was not spurious was established using the "Core Eukaryotic Gene Mapping Approach", which indicated that 98.79% of the M. chitwoodi genome was accounted for by the assembly. Subsequent gene finding and annotation are consistent with this and suggest that k-mer optimization contributes to the robustness of assembly.

A white paper on nematode comparative genomics.

In response to the new opportunities for genome sequencing and comparative genomics, the Society of Nematology (SON) formed a committee to develop a white paper in support of the broad scientific needs associated with this phylum and interests of SON members. Although genome sequencing is expensive, the data generated are unique in biological systems in that genomes have the potential to be complete (every base of the genome can be accounted for), accurate (the data are digital and not subject to stochastic variation), and permanent (once obtained, the genome of a species does not need to be experimentally re-sampled). The availability of complete, accurate, and permanent genome sequences from diverse nematode species will underpin future studies into the biology and evolution of this phylum and the ecological associations (particularly parasitic) nematodes have with other organisms. We anticipate that upwards of 100 nematode genomes will be solved to varying levels of completion in the coming decade and suggest biological and practical considerations to guide the selection of the most informative taxa for sequencing.

Interactions between bacteria and plant-parasitic nematodes: now and then.

Based on genome-to-genome analyses of gene sequences obtained from plant-parasitic, root-knot nematodes (Meloidogyne spp.), it seems likely that certain genes have been derived from bacteria by horizontal gene transfer. Strikingly, a common theme underpinning the function of these genes is their apparent direct relationship to the nematodes' parasitic lifestyle. Phylogenetic analyses implicate rhizobacteria as the predominant group of 'gene donor' bacteria. Root-knot nematodes and rhizobia occupy similar niches in the soil and in roots, and thus the opportunity for genetic exchange may be omnipresent. Further, both organisms establish intimate developmental interactions with host plants, and mounting evidence suggests that the mechanisms for these interactions are shared too. We propose that the origin of parasitism in Meloidogyne may have been facilitated by acquisition of genetic material from soil bacteria through horizontal transfer, and that such events represented key steps in speciation of plant-parasitic nematodes. To further understand the mechanisms of horizontal gene transfer, and also to provide experimental tools to manipulate this promising bio-control agent, we have initiated a genomic sequence of the bacterial hyper-parasite of plant parasitic nematodes, Pasteuria penetrans. Initial data have established that P. penetrans is closely related to Bacillus spp., to the extent that considerable genome synteny is apparent. Hence, Bacillus serves as a model for Pasteuria, and vice versa.

Improved structural annotation of protein-coding genes in the Meloidogyne hapla genome using RNA-Seq.

As high-throughput cDNA sequencing (RNA-Seq) is increasingly applied to hypothesis-driven biological studies, the prediction of protein coding genes based on these data are usurping strictly in silico approaches. Compared with computationally derived gene predictions, structural annotation is more accurate when based on biological evidence, particularly RNA-Seq data. Here, we refine the current genome annotation for the Meloidogyne hapla genome utilizing RNA-Seq data. Published structural annotation defines 14 420 protein-coding genes in the M. hapla genome. Of these, 25% (3751) were found to exhibit some incongruence with RNA-Seq data. Manual annotation enabled these discrepancies to be resolved. Our analysis revealed 544 new gene models that were missing from the prior annotation. Additionally, 1457 transcribed regions were newly identified on the ends of as-yet-unjoined contigs. We also searched for trans-spliced leaders, and based on RNA-Seq data, identified genes that appear to be trans-spliced. Four 22-bp trans-spliced leaders were identified using our pipeline, including the known trans-spliced leader, which is the M. hapla ortholog of SL1. In silico predictions of trans-splicing were validated by comparison with earlier results derived from an independent cDNA library constructed to capture trans-spliced transcripts. The new annotation, which we term HapPep5, is publically available at www.hapla.org.

A sequence-anchored linkage map of the plant-parasitic nematode Meloidogyne hapla reveals exceptionally high genome-wide recombination.

Root-knot nematodes (Meloidogyne spp.) cause major yield losses to many of the world's crops, but efforts to understand how these pests recognize and interact with their hosts have been hampered by a lack of genetic resources. Starting with progeny of a cross between inbred strains (VW8 and VW9) of Meloidogyne hapla that differed in host range and behavioral traits, we exploited the novel, facultative meiotic parthenogenic reproductive mode of this species to produce a genetic linkage map. Molecular markers were derived from SNPs identified between the sequenced and annotated VW9 genome and de novo sequence of VW8. Genotypes were assessed in 183 F2 lines. The colinearity of the genetic and physical maps supported the veracity of both. Analysis of local crossover intervals revealed that the average recombination rate is exceptionally high compared with that in other metazoans. In addition, F2 lines are largely homozygous for markers flanking crossover points, and thus resemble recombinant inbred lines. We suggest that the unusually high recombination rate may be an adaptation to generate within-population genetic diversity in this organism. This work presents the most comprehensive linkage map of a parasitic nematode to date and, together with genomic and transcript sequence resources, empowers M. hapla as a tractable model. Alongside the molecular map, these progeny lines can be used for analyses of genome organization and the inheritance of phenotypic traits that have key functions in modulating parasitism, behavior, and survival and for the eventual identification of the responsible genes.

RNAi effector diversity in nematodes.

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes.