Phased chromosome-scale genome assembly of an asexual, allopolyploid root-knot nematode reveals complex subgenomic structure.

We present the chromosome-scale genome assembly of the allopolyploid root-knot nematode Meloidogyne javanica. We show that the M. javanica genome is predominantly allotetraploid, comprising two subgenomes, A and B, that most likely originated from hybridisation of two ancestral parental species. The assembly was annotated using full-length non-chimeric transcripts, comparison to reference databases, and ab initio prediction techniques, and the subgenomes were phased using ancestral k-mer spectral analysis. Subgenome B appears to show fission of chromosomal contigs, and while there is substantial synteny between subgenomes, we also identified regions lacking synteny that may have diverged in the ancestral genomes prior to or following hybridisation. This annotated and phased genome assembly forms a significant resource for understanding the origins and genetics of these globally important plant pathogens.

Root-knot nematodes produce functional mimics of tyrosine-sulfated plant peptides.

Root-knot nematodes (Meloidogyne spp.) are highly evolved obligate parasites threatening global food security. These parasites have a remarkable ability to establish elaborate feeding sites in roots, which are their only source of nutrients throughout their life cycle. A wide range of nematode effectors have been implicated in modulation of host pathways for defense suppression and/or feeding site development. Plants produce a diverse array of peptide hormones including PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY)-family peptides, which promote root growth via cell expansion and proliferation. A sulfated PSY-like peptide RaxX (required for activation of XA21 mediated immunity X) produced by the biotrophic bacterial pathogen (Xanthomonas oryzae pv. oryzae) has been previously shown to contribute to bacterial virulence. Here, we report the identification of genes from root-knot nematodes predicted to encode PSY-like peptides (MigPSYs) with high sequence similarity to both bacterial RaxX and plant PSYs. Synthetic sulfated peptides corresponding to predicted MigPSYs stimulate root growth in Arabidopsis. MigPSY transcript levels are highest early in the infection cycle. Downregulation of MigPSY gene expression reduces root galling and egg production, suggesting that the MigPSYs serve as nematode virulence factors. Together, these results indicate that nematodes and bacteria exploit similar sulfated peptides to hijack plant developmental signaling pathways to facilitate parasitism.

Plant metabolism of nematode pheromones mediates plant-nematode interactions.

Microorganisms and nematodes in the rhizosphere profoundly impact plant health, and small-molecule signaling is presumed to play a central role in plant rhizosphere interactions. However, the nature of the signals and underlying mechanisms are poorly understood. Here we show that the ascaroside ascr#18, a pheromone secreted by plant-parasitic nematodes, is metabolized by plants to generate chemical signals that repel nematodes and reduce infection. Comparative metabolomics of plant tissues and excretions revealed that ascr#18 is converted into shorter side-chained ascarosides that confer repellency. An Arabidopsis mutant defective in two peroxisomal acyl-CoA oxidases does not metabolize ascr#18 and does not repel nematodes, indicating that plants, like nematodes, employ conserved peroxisomal ß-oxidation to edit ascarosides and change their message. Our results suggest that plant-editing of nematode pheromones serves as a defense mechanism that acts in parallel to conventional pattern-triggered immunity, demonstrating that plants may actively manipulate chemical signaling of soil organisms.

Homologs of Caenorhabditis elegans Chemosensory Genes Have Roles in Behavior and Chemotaxis in the Root-Knot Nematode Meloidogyne incognita.

Nematode chemosensation is a vital component of their host-seeking behavior. The globally important phytonematode Meloidogyne incognita perceives and responds (via sensory organs such as amphids and phasmids) differentially to various chemical cues emanating from the rhizosphere during the course of host finding. However, compared with the free-living worm Caenorhabditis elegans, the molecular intricacies behind the plant nematode chemotaxis are a yet-unexploited territory. In the present study, four putative chemosensory genes of M. incognita, namely, Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 were molecularly characterized. Mi-odr-1 mRNA was found to be expressed in the cell bodies of amphidial neurons and phasmids of M. incognita. Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 transcripts were highly expressed in early life stages of M. incognita, consistent with a role of these genes in host recognition. Functional characterization of Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 via RNA interference revealed behavioral defects in M. incognita and perturbed attraction to host roots in Pluronic gel medium. Knockdown of Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 resulted in defective chemotaxis of M. incognita to various volatile compounds (alcohol, ketone, aromatic compound, ester, thiazole, pyrazine), nonvolatiles of plant origin (carbohydrate, phytohormone, organic acid, amino acid, phenolic), and host root exudates in an agar-Pluronic gel-based assay plate. In addition, ascaroside-mediated signaling was impeded by downregulation of chemosensory genes. This new information that behavioral response in M. incognita is modulated by specific olfactory genes can be extended to understand chemotaxis in other nematodes.

Potent Attractant for Root-Knot Nematodes in Exudates from Seedling Root Tips of Two Host Species.

Root-knot nematodes (RKN; Meloidogyne spp.) can parasitize over 2,000 plant species and are generally considered to be the most agriculturally damaging group of plant-parasitic nematodes worldwide. Infective juveniles (J2) are non-feeding and must locate and invade a host before their reserves are depleted. However, what attracts J2 to appropriate root entry sites is not known. An aim of this research is to identify semiochemicals that attract RKN to roots. J2 of the three RKN species tested are highly attracted to root tips of both tomato and Medicago truncatula. For both hosts, mutants defective in ethylene signaling were found to be more attractive than those of wild type. We determined that cell-free exudates collected from tomato and M. truncatula seedling root tips were highly attractive to M. javanica J2. Using a pluronic gel-based microassay to monitor chemical fractionation, we determined that for both plant species the active component fractionated similarly and had a mass of ~400 based on size-exclusion chromatography. This characterization is a first step toward identification of a potent and specific attractant from host roots that attracts RKN. Such a compound is potentially a valuable tool for developing novel and safe control strategies.

Comparative Genomics of Apomictic Root-Knot Nematodes: Hybridization, Ploidy, and Dynamic Genome Change.

The root-knot nematodes (genus Meloidogyne) are important plant parasites causing substantial agricultural losses. The Meloidogyne incognita group (MIG) of species, most of which are obligatory apomicts (mitotic parthenogens), are extremely polyphagous and important problems for global agriculture. While understanding the genomic basis for their variable success on different crops could benefit future agriculture, analyses of their genomes are challenging due to complex evolutionary histories that may incorporate hybridization, ploidy changes, and chromosomal fragmentation. Here, we sequence 19 genomes, representing five species of key root-knot nematodes collected from different geographic origins. We show that a hybrid origin that predated speciation within the MIG has resulted in each species possessing two divergent genomic copies. Additionally, the apomictic MIG species are hypotriploids, with a proportion of one genome present in a second copy. The hypotriploid proportion varies among species. The evolutionary history of the MIG genomes is revealed to be very dynamic, with noncrossover recombination both homogenizing the genomic copies, and acting as a mechanism for generating divergence between species. Interestingly, the automictic MIG species M. floridensis differs from the apomict species in that it has become homozygous throughout much of its genome.

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.

Assessing Attraction of Nematodes to Host Roots Using Pluronic Gel Medium.

Nematodes and other organisms perceive and respond to plant root exudates. These exudates are affected by the condition and genetic makeup of the plant. Attraction of the root-knot nematode Meloidogyne hapla to roots is altered in plants with mutations affecting ethylene signaling, suggesting that the root exudates to which the nematode responds are modulated by ethylene signaling. Nematode interactions with roots have been difficult to observe directly due to the opaqueness of soil. A medium based on the block copolymer Pluronic F-127 has been useful for studying these interactions. Here, we present protocols for culturing root-knot nematodes, isolating infective juveniles, and measuring their attraction to Arabidopsis seedling roots in this medium.

Ethylene response pathway modulates attractiveness of plant roots to soybean cyst nematode Heterodera glycines.

Plant parasitic nematodes respond to root exudates to locate their host roots. In our studies second stage juveniles of Heterodera glycines, the soybean cyst nematode (SCN), quickly migrated to soybean roots in Pluronic F-127 gel. Roots of soybean and non-host Arabidopsis treated with the ethylene (ET)-synthesis inhibitor aminoethoxyvinylglycine (AVG) were more attractive to SCN than untreated roots, and significantly more nematodes penetrated into roots. Moreover, Arabidopsis ET insensitive mutants (ein2, ein2-1, ein2-5, ein3-1, ein5-1, and ein6) were more attractive than wild-type plants. Conversely, the constitutive triple-response mutant ctr1-1, was less attractive to SCN. While ET receptor gain-of-function mutant ein4-1 attracted more SCN than the wild-type, there were no significant differences in attractiveness between another gain-of-function ET receptor mutant, etr1-3, or the loss-of-function mutants etr1-7 and ers1-3 and the wild type. Expression of the reporter construct EBS: ß-glucuronidase (GUS) was detected in Arabidopsis root tips as early as 6 h post infection, indicating that ET signaling was activated in Arabidopsis early by SCN infection. These results suggest that an active ET signaling pathway reduces root attractiveness to SCN in a way similar to that reported for root-knot nematodes, but opposite to that suggested for the sugar beet cyst nematode Heterodera schachtii.

Conserved nematode signalling molecules elicit plant defenses and pathogen resistance.

Plant-defense responses are triggered by perception of conserved microbe-associated molecular patterns (MAMPs), for example, flagellin or peptidoglycan. However, it remained unknown whether plants can detect conserved molecular patterns derived from plant-parasitic animals, including nematodes. Here we show that several genera of plant-parasitic nematodes produce small molecules called ascarosides, an evolutionarily conserved family of nematode pheromones. Picomolar to micromolar concentrations of ascr#18, the major ascaroside in plant-parasitic nematodes, induce hallmark defense responses including the expression of genes associated with MAMP-triggered immunity, activation of mitogen-activated protein kinases, as well as salicylic acid- and jasmonic acid-mediated defense signalling pathways. Ascr#18 perception increases resistance in Arabidopsis, tomato, potato and barley to viral, bacterial, oomycete, fungal and nematode infections. These results indicate that plants recognize ascarosides as a conserved molecular signature of nematodes. Using small-molecule signals such as ascarosides to activate plant immune responses has potential utility to improve economic and environmental sustainability of agriculture.

Mitochondrial haplotype-based identification of ethanol-preserved root-knot nematodes from Africa.

The asexual root-knot nematodes (RKNs) (Meloidogyne spp.) exemplified by Meloidogyne incognita are widespread and damaging pests in tropical and subtropical regions worldwide. Comparison of amplification products of two adjacent polymorphic regions of the mitochondrial genome using DNA extracts of characterized RKN strains, including 15 different species, indicate that several species are derived from the same or closely related female lineages. Nevertheless, M. javanica, M. enterolobii, M. incognita, and other key species could each be assigned unique mitochondrial haplotypes based on polymerase chain reaction fragment size and restriction cleavage patterns. M. arenaria isolates did not group as a single haplotype, consistent with other reports of diversity within this species. To test the utility of this assay, we characterized ethanol-preserved samples from 103 single-species isolates from four countries in sub-Saharan Africa (Benin, Nigeria, Kenya, and Tanzania). Mitochondrial haplotypes corresponding to M. javanica and M. incognita were the most prevalent. Samples from western Africa included several instances of M. enterolobii but this species was not detected in samples from East Africa. This protocol provides progress toward a standardized strategy for identification of RKN species from small, preserved samples and a rational starting point for classifying species present in regions where previous knowledge has been limited.

Segregation and mapping in the root-knot nematode Meloidogyne hapla of quantitatively inherited traits affecting parasitism.

The root-knot nematode Meloidogyne hapla can reproduce on a wide range of crop species but there is variability in host range and pathogenicity both within and between isolates. The inbred strain VW9 causes galling but does not reproduce on Solanum bulbocastanum clone SB22 whereas strain VW8 causes little galling and reproduces poorly on this host. Comparison of reproduction on SB22 of nematode F2 lines generated from hybrids between strains VW8 and VW9 revealed that, whereas over half the lines produced no progeny, some lines reproduced to higher levels than did either parental strain. Using a genetic map previously generated using the same set of F2 lines, three quantitative trait loci (QTLs) were identified and positioned on linkage groups. A combination of two QTL alleles from one parent and one from the other was highly represented in F2 lines that exhibited higher reproduction than either parental strain but was absent from lines that failed to reproduce on SB22. This result suggests that sexual hybridization and assortment of opposing alleles leads to segregation of individuals with improved reproductive ability on a particular host. The genome sequence and integrated genetic and physical linkage map of M. hapla provide resources for identification of genes responsible for the identified QTL.

Ethylene signaling pathway modulates attractiveness of host roots to the root-knot nematode Meloidogyne hapla.

Infective juveniles of the root-knot nematode Meloidogyne hapla are attracted to the zone of elongation of roots where they invade the host but little is known about what directs the nematode to this region of the root. We found that Arabidopsis roots exposed to an ethylene (ET)-synthesis inhibitor attracted significantly more nematodes than control roots and that ET-overproducing mutants were less attractive. Arabidopsis seedlings with ET-insensitive mutations were generally more attractive whereas mutations resulting in constitutive signaling were less attractive. Roots of the ET-insensitive tomato mutant Never ripe (Nr) were also more attractive, indicating that ET signaling also modulated attraction of root-knot nematodes to this host. ET-insensitive mutants have longer roots due to reduced basipetal auxin transport. However, assessments of Arabidopsis mutants that differ in various aspects of the ET response suggest that components of the ET-signaling pathway directly affecting root length are not responsible for modulating root attractiveness and that other components of downstream signaling result in changes in levels of attractants or repellents for M. hapla. These signals may aid in directing this pathogen to an appropriate host and invasion site for completing its life cycle.

An Aegilops ventricosa Translocation Confers Resistance Against Root-knot Nematodes to Common Wheat.

Root knot nematodes (RKN; Meloidogyne spp.) cause severe losses worldwide to a wide range of crops. Crop rotations with resistant hosts can be used to control losses, but the wide host range of RKN limits this option. In this study, we found that the wheat cultivar Lassik is resistant to several isolates of the RKN species M. incognita and M. javanica, including those that can reproduce on tomato with the resistance gene Mi-1. Comparison of near-isogenic lines of wheat showed that the wheat resistance gene(s) is localized within a segment of the short arm of chromosome 2N from Aegilops ventricosa (Zhuk.) Chennav translocated into common wheat (Triticum aestivum L.) chromosome arm 2AS and is associated with a highly significant decrease in RKN eggs in the roots. This RKN resistance gene has been assigned the name Rkn3. While wheat itself is tolerant of RKN infection, a microplot experiment coupled with tomato bioassays showed less RKN root galling in the tomato samples grown in soil from the previous microplots including RKN resistant wheat varieties than in those including a susceptible wheat isogenic line. This result suggests that rotation with Rkn3 resistant wheat cultivars has the potential to be a valuable component of nematode management for crops that are highly susceptible to nematode damage and for which alternative strategies are limited.

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.

Tm1: a mutator/foldback transposable element family in root-knot nematodes.

Three closely related parthenogenetic species of root-knot nematodes, collectively termed the Meloidogyne incognita-group, are economically significant pathogens of diverse crop species. Remarkably, these asexual root-knot nematodes are capable of acquiring heritable changes in virulence even though they lack sexual reproduction and meiotic recombination. Characterization of a near isogenic pair of M. javanica strains differing in response to tomato with the nematode resistance gene Mi-1 showed that the virulent strain carried a deletion spanning a gene called Cg-1. Herein, we present evidence that the Cg-1 gene lies within a member of a novel transposable element family (Tm1; Transposon in Meloidogyne-1). This element family is defined by composite terminal inverted repeats of variable lengths similar to those of Foldback (FB) transposable elements and by 9 bp target site duplications. In M. incognita, Tm1 elements can be classified into three general groups: 1) histone-hairpin motif elements; 2) MITE-like elements; 3) elements encoding a putative transposase. The predicted transposase shows highest similarity to gene products encoded by aphids and mosquitoes and resembles those of the Phantom subclass of the Mutator transposon superfamily. Interestingly, the meiotic, sexually-reproducing root-knot nematode species M. hapla has Tm1 elements with similar inverted repeat termini, but lacks elements with histone hairpin motifs and contains no elements encoding an intact transposase. These Tm1 elements may have impacts on root-knot nematode genomes and contribute to genetic diversity of the asexual species.

Root-knot nematodes exhibit strain-specific clumping behavior that is inherited as a simple genetic trait.

Root-knot nematodes are obligate parasites of a wide range of plant species and can feed only on the cytoplasm of living plant cells. In the absence of a suitable plant host, infective juveniles of strain VW9 of the Northern root-knot nematode, Meloidogyne hapla, when dispersed in Pluronic F-127 gel, aggregate into tight, spherical clumps containing thousands of worms. Aggregation or clumping behavior has been observed in diverse genera in the phylum Nematoda spanning free-living species such as Caenorhabditis elegans as well as both plant and animal parasites. Clumping behavior differs between strains of M. hapla and occurs with other species within this genus where strain-specific differences in clumping ability are also apparent. Exposure of M. hapla juveniles to a gradient formed using low levels of cyanide promotes formation of clumps at a preferred cyanide level. Analysis of F2 lines from a cross of M. hapla strains that differ in clump-forming behavior reveals that the behavior segregates as a single, major locus that can be positioned on the genetic map of this nematode. Clumping behavior may be a survival strategy whose importance and function depend on the niche of the nematode strain or species.

Determination of preferred pH for root-knot nematode aggregation using pluronic F-127 gel.

Root-knot nematodes (Meloidogyne spp.) are obligate endoparasites of a wide range of plant species. The infective stage is attracted strongly to and enters host roots at the zone of elongation, but the compounds responsible for this attraction have not been identified. We developed a simple assay to investigate nematode response to chemical gradients that uses Pluronic F-127, a synthetic block copolymer that, as a 23% aqueous solution, forms a liquid at low temperature and a gel at room temperature. Test chemicals are put into a modified pipette tip, or 'chemical dispenser,' and dispensers are inserted into the gel in which nematodes have been dispersed. Meloidogyne hapla is attracted to pH gradients formed by acetic acid and several other Brønsted acids and aggregates between pH 4.5 and 5.4. While this pH range was attractive to all tested root-knot nematode strains and species, the level of aggregation depended on the species/strain assessed. For actively growing roots, the pH at the root surface is most acidic at the zone of elongation. This observation is consistent with the idea that low pH is an attractant for nematodes. Root-knot nematodes have been reported to be attracted to carbon dioxide, but our experiments suggest that the observed attraction may be due to acidification of solutions by dissolved CO(2) rather than to CO(2) itself. These results suggest that Pluronic F-127 gel will be broadly applicable for examining responses of a range of organisms to chemical gradients or to each other.

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.