The root-knot nematode effector Mi2G02 hijacks a host plant trihelix transcription factor to promote nematode parasitism.

Root-knot nematodes (RKNs) cause huge agricultural losses every year. They secrete a repertoire of effectors to facilitate parasitism through the induction of plant-derived giant feeding cells, which serve as their sole source of nutrients. However, the mode of action of these effectors and their targeted host proteins remain largely unknown. In this study, we investigated the role of the effector Mi2G02 in Meloidogyne incognita parasitism. Host-derived Mi2G02 RNA interference in Arabidopsis thaliana affected giant cell development, whereas ectopic expression of Mi2G02 promoted root growth and increased plant susceptibility to M. incognita. We used various combinations of approaches to study the specific interactions between Mi2G02 and A. thaliana GT-3a, a trihelix transcription factor. GT-3a knockout in A. thaliana affected feeding-site development, resulting in production of fewer egg masses, whereas GT-3a overexpression in A. thaliana increased susceptibility to M. incognita and also root growth. Moreover, we demonstrated that Mi2G02 plays a role in maintaining GT-3a protein stabilization by inhibiting the 26S proteasome-dependent pathway, leading to suppression of TOZ and RAD23C expression and thus promoting nematode parasitism. This work enhances our understanding of how a pathogen effector manipulates the role and regulation of a transcription factor by interfering with a proteolysis pathway to reprogram gene expression for development of nematode feeding cells.

Meloidogyne enterolobii MeMSP1 effector targets the glutathione-S-transferase phi GSTF family in Arabidopsis to manipulate host metabolism and promote nematode parasitism.

Meloidogyne enterolobii is an emerging root-knot nematode species that overcomes most of the nematode resistance genes in crops. Nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we show the MeMSP1 effector is secreted into giant cells and promotes M. enterolobii parasitism. Using co-immunoprecipitation and bimolecular fluorescent complementation assays, we identified glutathione-S-transferase phi GSTFs as host targets of the MeMSP1 effector. This protein family plays important roles in plant responses to abiotic and biotic stresses. We demonstrate that MeMSP1 interacts with all Arabidopsis GSTF. Moreover, we confirmed that the N-terminal region of AtGSTF9 is critical for its interaction, and atgstf9 mutant lines are more susceptible to root-knot nematode infection. Combined transcriptome and metabolome analyses showed that MeMSP1 affects the metabolic pathways of Arabidopsis thaliana, resulting in the accumulation of amino acids, nucleic acids, and their metabolites, and organic acids and the downregulation of flavonoids. Our study has shed light on a novel effector mechanism that targets plant metabolism, reducing the production of plant defence-related compounds while favouring the accumulation of metabolites beneficial to the nematode, and thereby promoting parasitism.

AUXIN RESPONSIVE FACTOR8 regulates development of the feeding site induced by root-knot nematodes in tomato.

Root-knot nematodes (RKN) from the genus Meloidogyne induce the dedifferentiation of root vascular cells into giant multinucleate feeding cells. These feeding cells result from an extensive reprogramming of gene expression, and auxin is known to be a key player in their development. However, little is known about how the auxin signal is transmitted during giant cell development. Integrative analyses combining transcriptome and small non-coding RNA datasets with the specific sequencing of cleaved transcripts identified genes targeted by miRNAs in tomato (Solanum lycopersicum) galls. The two auxin-responsive transcription factors ARF8A and ARF8B, and their miRNA167 regulators, were identified as robust gene-miRNA pair candidates to be involved in the tomato response to M. incognita. Spatiotemporal expression analysis using promoter-ß-glucuronidase (GUS) fusions showed the up-regulation of ARF8A and ARF8B in RKN-induced feeding cells and surrounding cells. The generation and phenotyping of CRISPR (clustered regularly interspaced palindromic repeats) mutants demonstrated the role of ARF8A and ARF8B in giant cell development and allowed the characterization of their downstream regulated genes.

Copper microRNAs modulate the formation of giant feeding cells induced by the root knot nematode Meloidogyne incognita in Arabidopsis thaliana.

Root-knot nematodes (RKNs) are root endoparasites that induce the dedifferentiation of a few root cells and the reprogramming of their gene expression to generate giant hypermetabolic feeding cells. We identified two microRNA families, miR408 and miR398, as upregulated in Arabidopsis thaliana and Solanum lycopersicum roots infected by RKNs. In plants, the expression of these two conserved microRNA families is known to be activated by the SPL7 transcription factor in response to copper starvation. By combining functional approaches, we deciphered the network involving these microRNAs, their regulator and their targets. MIR408 expression was located within nematode-induced feeding cells like its regulator SPL7 and was regulated by copper. Moreover, infection assays with mir408 and spl7 knockout mutants or lines expressing targets rendered resistant to cleavage by miR398 demonstrated the essential role of the SPL7/MIR408/MIR398 module in the formation of giant feeding cells. Our findings reveal how perturbation of plant copper homeostasis, via the SPL7/MIR408/MIR398 module, modulates the development of nematode-induced feeding cells.

Silencing the conserved small nuclear ribonucleoprotein SmD1 target gene alters susceptibility to root-knot nematodes in plants.

Root-knot nematodes (RKNs) are among the most damaging pests of agricultural crops. Meloidogyne is an extremely polyphagous genus of nematodes that can infect thousands of plant species. A few genes for resistance (R-genes) to RKN suitable for use in crop breeding have been identified, but virulent strains and species of RKN have emerged that render these R-genes ineffective. Secretion of RKN effectors targeting plant functions mediates the reprogramming of root cells into specialized feeding cells, the giant cells, essential for RKN development and reproduction. Conserved targets among plant species define the more relevant strategies for controlling nematode infection. The EFFECTOR18 (EFF18) protein from M. incognita interacts with the spliceosomal small nuclear ribonucleoprotein D1 (SmD1) in Arabidopsis (Arabidopsis thaliana), disrupting its function in alternative splicing regulation and modulating the giant cell transcriptome. We show here that EFF18 is a conserved RKN-specific effector that targets this conserved spliceosomal SmD1 protein in Solanaceae. This interaction modulates alternative splicing events produced by tomato (Solanum lycopersicum) in response to M. incognita infection. The alteration of SmD1 expression by virus-induced gene silencing in Solanaceae affects giant cell formation and nematode development. Thus, our work defines a promising conserved SmD1 target gene to develop broad resistance for the control of Meloidogyne spp. in plants.

Chromatin Landscape Dynamics in the Early Development of the Plant Parasitic Nematode Meloidogyne incognita.

In model organisms, epigenome dynamics underlies a plethora of biological processes. The role of epigenetic modifications in development and parasitism in nematode pests remains unknown. The root-knot nematode Meloidogyne incognita adapts rapidly to unfavorable conditions, despite its asexual reproduction. However, the mechanisms underlying this remarkable plasticity and their potential impact on gene expression remain unknown. This study provides the first insight into contribution of epigenetic mechanisms to this plasticity, by studying histone modifications in M. incognita. The distribution of five histone modifications revealed the existence of strong epigenetic signatures, similar to those found in the model nematode Caenorhabditis elegans. We investigated their impact on chromatin structure and their distribution relative to transposable elements (TE) loci. We assessed the influence of the chromatin landscape on gene expression at two developmental stages: eggs, and pre-parasitic juveniles. H3K4me3 histone modification was strongly correlated with high levels of expression for protein-coding genes implicated in stage-specific processes during M. incognita development. We provided new insights in the dynamic regulation of parasitism genes kept under histone modifications silencing. In this pioneering study, we establish a comprehensive framework for the importance of epigenetic mechanisms in the regulation of the genome expression and its stability in plant-parasitic nematodes.

A Meloidogyne incognita C-type lectin effector targets plant catalases to promote parasitism.

Root-knot nematodes, Meloidogyne spp., secrete effectors to modulate plant immune responses and establish a parasitic relationship with host plants. However, the functions and plant targets of C-type lectin (CTL)-like effectors of Meloidogyne incognita remain unknown. Here, we characterized a CTL-like effector of M. incognita, MiCTL1a, and identified its target and role in nematode parasitism. In situ hybridization demonstrated the expression of MiCTL1 in the subventral glands; and in planta, immunolocalization showed its secretion during M. incognita parasitism. Virus-induced gene silencing of the MiCTL1 reduced the infection ability of M. incognita in Nicotiana benthamiana. The ectopic expression in Arabidopsis not only increased susceptibility to M. incognita but also promoted root growth. Yeast two-hybrid and co-immunoprecipitation assays revealed that MiCTL1a interacts with Arabidopsis catalases, which play essential roles in hydrogen peroxide homeostasis. Knockout or overexpression of catalases showed either increased or reduced susceptibility to M. incognita, respectively. Moreover, MiCTL1a not only reduced catalase activity in vitro and in planta but also modulated stress-related gene expressions in Arabidopsis. Our data suggest that MiCTL1a interacts with plant catalases and interferes with catalase activity, allowing M. incognita to establish a parasitic relationship with its host by fine-tuning responses mediated by reactive oxygen species.

The Meloidogyne incognita Nuclear Effector MiEFF1 Interacts With Arabidopsis Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenases to Promote Parasitism.

Root-knot nematodes are obligate endoparasites that maintain a biotrophic relationship with their hosts over a period of several weeks. They induce the differentiation of root cells into specialized multinucleate hypertrophied feeding cells known as giant cells. Nematode effectors synthesized in the esophageal glands and injected into the plant tissue through the syringe-like stylet play a key role in giant cell ontogenesis. The Meloidogyne incognita MiEFF1 is one of the rare effectors of phytopathogenic nematodes to have been located in vivo in feeding cells. This effector specifically targets the giant cell nuclei. We investigated the Arabidopsis functions modulated by this effector, by using a yeast two-hybrid approach to identify its host targets. We characterized a universal stress protein (USP) and cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs) as the targets of MiEFF1. We validated the interaction of MiEFF1 with these host targets in the plant cell nucleus, by bimolecular fluorescence complementation (BiFC). A functional analysis with Arabidopsis GUS reporter lines and knockout mutant lines showed that GAPCs were induced in giant cells and that their non-metabolic functions were required for root-knot nematode infection. These susceptibility factors are potentially interesting targets for the development of new root-knot nematode control strategies.

Toward genetic modification of plant-parasitic nematodes: delivery of macromolecules to adults and expression of exogenous mRNA in second stage juveniles.

Plant-parasitic nematodes are a continuing threat to food security, causing an estimated 100 billion USD in crop losses each year. The most problematic are the obligate sedentary endoparasites (primarily root knot nematodes and cyst nematodes). Progress in understanding their biology is held back by a lack of tools for functional genetics: forward genetics is largely restricted to studies of natural variation in populations and reverse genetics is entirely reliant on RNA interference. There is an expectation that the development of functional genetic tools would accelerate the progress of research on plant-parasitic nematodes, and hence the development of novel control solutions. Here, we develop some of the foundational biology required to deliver a functional genetic tool kit in plant-parasitic nematodes. We characterize the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimize various methods for the delivery, expression, and/or detection of exogenous nucleic acids in plant-parasitic nematodes. We demonstrate that delivery of macromolecules to cyst and root knot nematode male germlines is difficult, but possible. Similarly, we demonstrate the delivery of oligonucleotides to root knot nematode gametes. Finally, we develop a transient expression system in plant-parasitic nematodes by demonstrating the delivery and expression of exogenous mRNA encoding various reporter genes throughout the body of H. schachtii juveniles using lipofectamine-based transfection. We anticipate these developments to be independently useful, will expedite the development of genetic modification tools for plant-parasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security.

The root-knot nematode effector MiEFF18 interacts with the plant core spliceosomal protein SmD1 required for giant cell formation.

The root-knot nematode Meloidogyne incognita secretes specific effectors (MiEFF) and induces the redifferentiation of plant root cells into enlarged multinucleate feeding 'giant cells' essential for nematode development. Immunolocalizations revealed the presence of the MiEFF18 protein in the salivary glands of M. incognita juveniles. In planta, MiEFF18 localizes to the nuclei of giant cells demonstrating its secretion during plant-nematode interactions. A yeast two-hybrid approach identified the nuclear ribonucleoprotein SmD1 as a MiEFF18 partner in tomato and Arabidopsis. SmD1 is an essential component of the spliceosome, a complex involved in pre-mRNA splicing and alternative splicing. RNA-seq analyses of Arabidopsis roots ectopically expressing MiEFF18 or partially impaired in SmD1 function (smd1b mutant) revealed the contribution of the effector and its target to alternative splicing and proteome diversity. The comparison with Arabidopsis galls data showed that MiEFF18 modifies the expression of genes important for giant cell ontogenesis, indicating that MiEFF18 modulates SmD1 functions to facilitate giant cell formation. Finally, Arabidopsis smd1b mutants exhibited less susceptibility to M. incognita infection, and the giant cells formed on these mutants displayed developmental defects, suggesting that SmD1 plays an important role in the formation of giant cells and is required for successful nematode infection.

Gall-Inducing Parasites: Convergent and Conserved Strategies of Plant Manipulation by Insects and Nematodes.

Gall-inducing insects and nematodes engage in sophisticated interactions with their host plants. These parasites can induce major morphological and physiological changes in host roots, leaves, and other tissues. Sedentary endoparasitic nematodes, root-knot and cyst nematodes in particular, as well as gall-inducing and leaf-mining insects, manipulate plant development to form unique organs that provide them with food from feeding cells. Sometimes, infected tissues may undergo a developmental switch resulting in the formation of aberrant and spectacular structures (clubs or galls). We describe here the complex interactions between these plant-reprogramming sedentary endoparasites and their infected hosts, focusing on similarities between strategies of plant manipulation. We highlight progress in our understanding of the host plant response to infection and focus on the nematode and insect molecules secreted in planta. We suggest thatlooking at similarities may identify convergent and conserved strategies and shed light on the promise they hold for the development of new management strategies in agriculture and forestry.

The root-knot nematode effector MiPDI1 targets a stress-associated protein (SAP) to establish disease in Solanaceae and Arabidopsis.

Large amounts of effectors are secreted by the oesophageal glands of plant-parasitic nematodes, but their molecular mode of action remains largely unknown. We characterized a Meloidogyne incognita protein disulphide isomerase (PDI)-like effector protein (MiPDI1) that facilitates nematode parasitism. In situ hybridization showed that MiPDI1 was expressed specifically in the subventral glands of M. incognita. It was significantly upregulated during parasitic stages. Immunolocalization demonstrated MiPDI1 secretion in planta during nematode migration and within the feeding cells. Host-induced silencing of the MiPDI1 gene affected the ability of the nematode to infect the host, whereas MiPDI1 expression in Arabidopsis increased susceptibility to M. incognita, providing evidence for a key role of MiPDI1 in M. incognita parasitism. Yeast two-hybrid, bimolecular fluorescence complementation and coimmunoprecipitation assays showed that MiPDI1 interacted with a tomato stress-associated protein (SlSAP12) orthologous to the redox-regulated AtSAP12, which plays an important role in plant responses to abiotic and biotic stresses. SAP12 silencing or knocking out in Nicotiana benthamiana and Arabidopsis increased susceptibility to M. incognita. Our results suggest that MiPDI1 acts as a pathogenicity factor promoting disease by fine-tuning SAP-mediated responses at the interface of redox signalling, defence and stress acclimation in Solanaceae and Arabidopsis.

A MIF-like effector suppresses plant immunity and facilitates nematode parasitism by interacting with plant annexins.

Plant-parasitic nematodes secrete numerous effectors to facilitate parasitism, but detailed functions of nematode effectors and their plant targets remain largely unknown. Here, we characterized four macrophage migration inhibitory factors (MIFs) in Meloidogyne incognita resembling the MIFs secreted by human and animal parasites. Transcriptional data showed MiMIFs are up-regulated in parasitism. Immunolocalization provided evidence that MiMIF proteins are secreted from the nematode hypodermis to the parasite surface, detected in plant tissues and giant cells. In planta MiMIFs RNA interference in Arabidopsis decreased infection and nematode reproduction. Transient expression of MiMIF-2 could suppress Bax- and RBP1/Gpa2-induced cell death. MiMIF-2 ectopic expression led to higher levels of Arabidopsis susceptibility, suppressed immune responses triggered by flg22, and impaired [Ca2+]cyt influx induced by H2O2. The immunoprecipitation of MiMIF-2-interacting proteins, followed by co-immunoprecipitation and bimolecular fluorescence complementation validations, revealed specific interactions between MiMIF-2 and two Arabidopsis annexins, AnnAt1 and AnnAt4, involved in the transport of calcium ions, stress responses, and signal transduction. Suppression of expression or overexpression of these annexins modified nematode infection. Our results provide functional evidence that nematode effectors secreted from hypodermis to the parasite cuticle surface target host proteins and M. incognita uses MiMIFs to promote parasitism by interfering with the annexin-mediated plant immune responses.

Plant Proteins and Processes Targeted by Parasitic Nematode Effectors.

Sedentary endoparasitic nematodes, such as root-knot nematodes (RKN; Meloidogyne spp.) and cyst nematodes (CN; Heterodera spp. and Globodera spp.) cause considerable damage to agricultural crops. RKN and CN spend most of their life cycle in plant roots, in which they induce the formation of multinucleate hypertrophied feeding cells, called "giant cells" and "syncytia," respectively. The giant cells result from nuclear divisions of vascular cells without cytokinesis. They are surrounded by small dividing cells and they form a new organ within the root known as a root knot or gall. CN infection leads to the fusion of several root cells into a unique syncytium. These dramatically modified host cells act as metabolic sinks from which the nematode withdraws nutrients throughout its life, and they are thus essential for nematode development. Both RKN and CN secrete effector proteins that are synthesized in the oesophageal glands and delivered to the appropriate cell in the host plant via a syringe-like stylet, triggering the ontogenesis of the feeding structures. Within the plant cell or in the apoplast, effectors associate with specific host proteins, enabling them to hijack important processes for cell morphogenesis and physiology or immunity. Here, we review recent findings on the identification and functional characterization of plant targets of RKN and CN effectors. A better understanding of the molecular determinants of these biotrophic relationships would enable us to improve the yields of crops infected with parasitic nematodes and to expand our comprehension of root development.

Gene copy number variations as signatures of adaptive evolution in the parthenogenetic, plant-parasitic nematode Meloidogyne incognita.

Adaptation to changing environmental conditions represents a challenge to parthenogenetic organisms, and until now, how phenotypic variants are generated in clones in response to the selection pressure of their environment remains poorly known. The obligatory parthenogenetic root-knot nematode species Meloidogyne incognita has a worldwide distribution and is the most devastating plant-parasitic nematode. Despite its asexual reproduction, this species exhibits an unexpected capacity of adaptation to environmental constraints, for example, resistant hosts. Here, we used a genomewide comparative hybridization strategy to evaluate variations in gene copy numbers between genotypes of M. incognita resulting from two parallel experimental evolution assays on a susceptible vs. resistant host plant. We detected gene copy number variations (CNVs) associated with the ability of the nematodes to overcome resistance of the host plant, and this genetic variation may reflect an adaptive response to host resistance in this parthenogenetic species. The CNV distribution throughout the nematode genome is not random and suggests the occurrence of genomic regions more prone to undergo duplications and losses in response to the selection pressure of the host resistance. Furthermore, our analysis revealed an outstanding level of gene loss events in nematode genotypes that have overcome the resistance. Overall, our results support the view that gene loss could be a common class of adaptive genetic mechanism in response to a challenging new biotic environment in clonal animals.

Characterization of siRNAs clusters in Arabidopsis thaliana galls induced by the root-knot nematode Meloidogyne incognita.

BACKGROUND: Root-knot nematodes (RKN), genus Meloidogyne, are plant parasitic worms that have the ability to transform root vascular cylinder cells into hypertrophied, multinucleate and metabolically over-active feeding cells. Redifferentiation into feeding cells is the result of a massive transcriptional reprogramming of root cells targeted by RKN. Since RKN are able to induce similar feeding cells in roots of thousands of plant species, these worms are thought to manipulate essential and conserved plant molecular pathways. RESULTS: Small non-coding RNAs of uninfected roots and infected root galls induced by M. incognita from Arabidopsis thaliana were sequenced by high throughput sequencing. SiRNA populations were analysed by using the Shortstack algorithm. We identified siRNA clusters that are differentially expressed in infected roots and evidenced an over-representation of the 23-24 nt siRNAs in infected tissue. This size corresponds to heterochromatic siRNAs (hc-siRNAs) which are known to regulate expression of transposons and genes at the transcriptional level, mainly by inducing DNA methylation. CONCLUSIONS: Correlation of siRNA clusters expression profile with transcriptomic data identified several protein coding genes that are candidates to be regulated by siRNAs at the transcriptional level by RNA directed DNA methylation (RdDM) pathway either directly or indirectly via silencing of neighbouring transposable elements.

Genome-wide expert annotation of the epigenetic machinery of the plant-parasitic nematodes Meloidogyne spp., with a focus on the asexually reproducing species.

BACKGROUND: The renewed interest in epigenetics has led to the understanding that both the environment and individual lifestyle can directly interact with the epigenome to influence its dynamics. Epigenetic phenomena are mediated by DNA methylation, stable chromatin modifications and non-coding RNA-associated gene silencing involving specific proteins called epigenetic factors. Multiple organisms, ranging from plants to yeast and mammals, have been used as model systems to study epigenetics. The interactions between parasites and their hosts are models of choice to study these mechanisms because the selective pressures are strong and the evolution is fast. The asexually reproducing root-knot nematodes (RKN) offer different advantages to study the processes and mechanisms involved in epigenetic regulation. RKN genomes sequencing and annotation have identified numerous genes, however, which of those are involved in the adaption to an environment and potentially relevant to the evolution of plant-parasitism is yet to be discovered. RESULTS: Here, we used a functional comparative annotation strategy combining orthology data, mining of curated genomics as well as protein domain databases and phylogenetic reconstructions. Overall, we show that (i) neither RKN, nor the model nematode Caenorhabditis elegans possess any DNA methyltransferases (DNMT) (ii) RKN do not possess the complete machinery for DNA methylation on the 6th position of adenine (6mA) (iii) histone (de)acetylation and (de)methylation pathways are conserved between C. elegans and RKN, and the corresponding genes are amplified in asexually reproducing RKN (iv) some specific non-coding RNA families found in plant-parasitic nematodes are dissimilar from those in C. elegans. In the asexually reproducing RKN Meloidogyne incognita, expression data from various developmental stages supported the putative role of these proteins in epigenetic regulations. CONCLUSIONS: Our results refine previous predictions on the epigenetic machinery of model species and constitute the most comprehensive description of epigenetic factors relevant to the plant-parasitic lifestyle and/or asexual mode of reproduction of RKN. Providing an atlas of epigenetic factors in RKN is an informative resource that will enable researchers to explore their potential role in adaptation of these parasites to their environment.

A root-knot nematode small glycine and cysteine-rich secreted effector, MiSGCR1, is involved in plant parasitism.

Root-knot nematodes, Meloidogyne spp., are obligate endoparasites that maintain a biotrophic relationship with their hosts. They infect roots as microscopic vermiform second-stage juveniles, and establish specialized feeding structures called 'giant-cells', from which they withdraw water and nutrients. The nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we compared Illumina RNA-seq transcriptomes for M. incognita obtained at various points in the lifecycle, and identified 31 genes more strongly expressed in parasitic stages than in preparasitic juveniles. We then selected candidate effectors for functional characterization. Quantitative real-time PCR and in situ hybridizations showed that the validated differentially expressed genes are predominantly specifically expressed in oesophageal glands of the nematode. We also soaked the nematodes in siRNA to silence these genes and to determine their role in pathogenicity. The silencing of the dorsal gland specific-Minc18876 and its paralogues resulted in a significant, reproducible decrease in the number of mature females with egg masses, demonstrating a potentially important role for the small glycine- and cysteine-rich effector MiSGCR1 in early stages of plant-nematode interaction. Finally, we report that MiSGCR1 suppresses plant cell death induced by bacterial or oomycete triggers of plant defense.

Characterization of microRNAs from Arabidopsis galls highlights a role for miR159 in the plant response to the root-knot nematode Meloidogyne incognita.

Root knot nematodes (RKN) are root parasites that induce the genetic reprogramming of vascular cells into giant feeding cells and the development of root galls. MicroRNAs (miRNAs) regulate gene expression during development and plant responses to various stresses. Disruption of post-transcriptional gene silencing in Arabidopsis ago1 or ago2 mutants decrease the infection rate of RKN suggesting a role for this mechanism in the plant-nematode interaction. By sequencing small RNAs from uninfected Arabidopsis roots and from galls 7 and 14 d post infection with Meloidogyne incognita, we identified 24 miRNAs differentially expressed in gall as putative regulators of gall development. Moreover, strong activity within galls was detected for five miRNA promoters. Analyses of nematode development in an Arabidopsis miR159abc mutant had a lower susceptibility to RKN, suggesting a role for the miR159 family in the plant response to M. incognita. Localization of mature miR159 within the giant and surrounding cells suggested a role in giant cell and gall. Finally, overexpression of miR159 in galls at 14 d post inoculation was associated with the repression of the miR159 target MYB33 which expression is restricted to the early stages of infection. Overall, these results implicate the miR159 in plant responses to RKN.

Hybridization and polyploidy enable genomic plasticity without sex in the most devastating plant-parasitic nematodes.

Root-knot nematodes (genus Meloidogyne) exhibit a diversity of reproductive modes ranging from obligatory sexual to fully asexual reproduction. Intriguingly, the most widespread and devastating species to global agriculture are those that reproduce asexually, without meiosis. To disentangle this surprising parasitic success despite the absence of sex and genetic exchanges, we have sequenced and assembled the genomes of three obligatory ameiotic and asexual Meloidogyne. We have compared them to those of relatives able to perform meiosis and sexual reproduction. We show that the genomes of ameiotic asexual Meloidogyne are large, polyploid and made of duplicated regions with a high within-species average nucleotide divergence of ~8%. Phylogenomic analysis of the genes present in these duplicated regions suggests that they originated from multiple hybridization events and are thus homoeologs. We found that up to 22% of homoeologous gene pairs were under positive selection and these genes covered a wide spectrum of predicted functional categories. To biologically assess functional divergence, we compared expression patterns of homoeologous gene pairs across developmental life stages using an RNAseq approach in the most economically important asexually-reproducing nematode. We showed that >60% of homoeologous gene pairs display diverged expression patterns. These results suggest a substantial functional impact of the genome structure. Contrasting with high within-species nuclear genome divergence, mitochondrial genome divergence between the three ameiotic asexuals was very low, signifying that these putative hybrids share a recent common maternal ancestor. Transposable elements (TE) cover a ~1.7 times higher proportion of the genomes of the ameiotic asexual Meloidogyne compared to the sexual relative and might also participate in their plasticity. The intriguing parasitic success of asexually-reproducing Meloidogyne species could be partly explained by their TE-rich composite genomes, resulting from allopolyploidization events, and promoting plasticity and functional divergence between gene copies in the absence of sex and meiosis.