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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.

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.

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.

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.

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.

Description du sujet. Une approche système basée sur la co-conception et l’évaluation expérimentale in situ de prototypes de systèmes de culture (SDC) a été mise en œuvre dans le projet INRA « GeDuNem » pour une gestion durable des nématodes à galles (NG) dans les systèmes maraîchers sous abris. Objectifs. Il s’agissait (i) d’évaluer pendant quatre ans diverses stratégies de culture combinant résistances génétiques et pratiques culturales (rotations culturales incluant des plantes sensibles, résistantes et non-hôtes, gestion de l’interculture avec couvert végétal nématicide ou solarisation) pour réduire les populations de NG dans le sol et augmenter la durabilité des résistances variétales à ces bioagresseurs, (ii) d’étudier leur impact sur les communautés de nématodes rencontrées et (iii) d’évaluer l’acceptabilité des nouveaux SDC par les agriculteurs. Méthode. Trois prototypes de SDC, co-conçus entre acteurs de la recherche et ceux du développement, ont été comparés à des SDC appliqués classiquement en région méditerranéenne et évalués grâce à des dispositifs complémentaires : (i) expérimentations-système sur trois sites de producteurs du Sud de la France ; (ii) expérimentations analytiques pour approfondir les mécanismes d’action des leviers mobilisés ; (iii) enquêtes pour évaluer l’acceptabilité des prototypes par les agriculteurs. Résultats. Les trois SDC se sont révélés efficaces (90 % de réduction des NG, protection des Solanaceae à résistance partielle, pas d’effet négatif sur les nématodes non phytoparasites) et durables lorsque les conditions d’application et les équilibres biologiques du sol étaient favorables (nématofaune totale diversifiée et abondante). Leur degré d’acceptabilité dépendait du type d’exploitation et de la sensibilité des agriculteurs face à l’innovation. Conclusions. Ces nouveaux SDC doivent encore être améliorés, en interaction avec les producteurs, tant en termes d’efficacité, par l’introduction de nouveaux leviers agroécologiques, que de cout. Les recherches futures devront aussi s’ouvrir à la gestion plus globale de la santé des sols. Design and assessment of innovative Mediterranean vegetable cropping systems to manage root-knot nematodes Description of the subject. A system approach based on co-design and experimental field evaluation of cropping systems (CSs), combining technical and varietal innovations, has been implemented for sustainable management of root-knot nematodes (RKN) in Mediterranean sheltered vegetable systems. Objectives. Cropping systems combining genetic resistance and cultural practices (crop rotations including susceptible, resistant, and non-host plants; intercropping management with nematicidal cover crops or soil solarization) were assessed over a period of 4 years (i) to reduce RKN populations and increase the durability of varietal resistances, (ii) to study the impact of these systems on soil ecology (plant-parasitic and free-living nematode communities), and (iii) to evaluate their acceptability by farmers. Method. Three CS prototypes, resulting from a co-design process with research and development stakeholders, were compared with CSs conventionally implemented in the Mediterranean region. The three prototypes were also evaluated using complementary methods: (i) system experiments in three commercial farms in Southern France; (ii) analytical experiments to decipher the mechanisms of action for some [agroecological??] levers; (iii) surveys to evaluate the acceptability of the prototypes by farmers. Results. All three CSs were found to be effective (90% RKN decrease, protection of partially resistant Solanaceae, no negative effect on non-phytoparasitic nematodes) and sustainable, when application conditions and soil biological equilibrium were favorable (global soil nematofauna diversified and abundant). The acceptability of the three systems depended on the type of farm where they were implemented and the attitude of the farmers towards innovation. Conclusions. These three CSs still need to be improved, in terms of their efficiency, in consultation with participating farmers, by introducing new agroecological levers, as well as innovation costs. Future research will also need to open up to a more comprehensive management of soil health.

KEY MESSAGE : The plant genetic background influences the efficiency of major resistance genes to root-knot nematodes in pepper and has to be considered in breeding strategies. Root-knot nematodes (RKNs), Meloidogyne spp., are extremely polyphagous plant parasites worldwide. Since the use of most chemical nematicides is being prohibited, genetic resistance is an efficient alternative way to protect crops against these pests. However, nematode populations proved able to breakdown plant resistance, and genetic resources in terms of resistance genes (R-genes) are limited. Sustainable management of these valuable resources is thus a key point of R-gene durability. In pepper, Me1 and Me3 are two dominant major R-genes, currently used in breeding programs to control M. arenaria, M. incognita and M. javanica, the three main RKN species. These two genes differ in the hypersensitive response induced by nematode infection. In this study, they were introgressed in either a susceptible or a partially resistant genetic background, in either homozygous or heterozygous allelic status. Challenging these genotypes with an avirulent M. incognita isolate demonstrated that (1) the efficiency of the R-genes in reducing the reproductive potential of RKNs is strongly affected by the plant genetic background, (2) the allelic status of the R-genes has no effect on nematode reproduction. These results highlight the primary importance of the choice of both the R-gene and the genetic background into which it is introgressed during the selection of new elite cultivars by plant breeders.

The emergence of virulent root-knot nematode populations, able to overcome the resistance conferred by some of the resistance genes (R-genes) in Solanaceous crops, i.e., Mi (s) in tomato, Me (s) in pepper, may constitute a severe limitation to their use in the field. Research has been conducted to evaluate the durability of these R-genes, by comparing the reproduction of several laboratory-selected and wild virulent Meloidogyne incognita isolates, on both susceptible and resistant tomatoes and peppers. We first show that the Me1 R-gene in pepper behaves as a robust R-gene controlling avirulent and virulent Me3, Me7 or Mi-1 isolates. Although the reproductive potential of the virulent isolates was highly variable on susceptible and resistant plants, we also confirm that virulence is highly specific to a determined R-gene on which selection has occurred. Another significant experimental result is the observation that a reproductive fitness cost is associated with nematode virulence against Mi-1 in tomato and Me3 and Me7 in pepper. The adaptative significance of trade-offs between selected characters and fitness-related traits, suggests that, although the resistance can be broken, it may be preserved in some conditions if the virulent nematodes are counter-selected in susceptible plants. All these results have important consequences for the management of plant resistance in the field.

Root-knot nematodes (RKNs, Meloidogyne spp.) are major plant pathogens of vegetables in most production areas, including the Solanaceous crops tomato and pepper. Due to the banning of chemical nematicides, current control strategies are mainly based on the deployment of resistance genes (R-genes), i.e., Mi(s) in tomato, Me(s) in pepper. These genes are effective against a wide range of RKN species, including M. arenaria, M. incognita and M. javanica, the most common species in temperate and tropical areas. However, the recent emergence of virulent populations able to overcome the resistance conferred by some of these R-genes may constitute a severe limitation to their use in the field. Research has been conducted to evaluate the durability of the Mi(s) and Me(s) R-genes, by comparing the reproduction of several laboratory-selected and wild virulent M. incognita isolates on both susceptible and resistant tomatoes and peppers. We first showed that the Me1 R-gene in pepper behaves as a robust R-gene controlling avirulent and virulent Me3 or Mi-1 isolates. We also confirm that virulence is highly specific to a determined R-gene on which selection has occurred, thus allowing the alternance of R-genes in the rotation as an effective mean to improve soil health. Another significant experimental result is the observation that a reproductive fitness cost is associated to nematode virulence. The adaptative significance of trade-offs between selected characters and fitness-related traits suggests that, although the resistance can be broken, it might prove durable in some conditions if the virulent nematodes are counterselected in susceptible plants, which has important consequences for the management of plant resistance in the field.

With the banning of most chemical nematicides, the control of root-knot nematodes (RKNs) in vegetable crops is now based essentially on the deployment of single, major resistance genes (R-genes). However, these genes are rare and their efficacy is threatened by the capacity of RKNs to adapt. In pepper, several dominant R-genes are effective against RKNs, and their efficacy and durability have been shown to be greater in a partially resistant genetic background. However, the genetic determinants of this partial resistance were unknown. Here, a quantitative trait loci (QTL) analysis was performed on the F2:3 population from the cross between Yolo Wonder, an accession considered partially resistant or resistant, depending on the RKN species, and Doux Long des Landes, a susceptible cultivar. A genetic linkage map was constructed from 130 F2 individuals, and the 130 F3 families were tested for resistance to the three main RKN species, Meloidogyne incognita, M. arenaria, and M. javanica. For the first time in the pepper-RKN pathosystem, four major QTLs were identified and mapped to two clusters. The cluster on chromosome P1 includes three tightly linked QTLs with specific effects against individual RKN species. The fourth QTL, providing specific resistance to M. javanica, mapped to pepper chromosome P9, which is known to carry multiple NBS-LRR repeats, together with major R-genes for resistance to nematodes and other pathogens. The newly discovered cluster on chromosome P1 has a broad spectrum of action with major additive effects on resistance. These data highlight the role of host QTLs involved in plant-RKN interactions and provide innovative potential for the breeding of new pepper cultivars or rootstocks combining quantitative resistance and major R-genes, to increase both the efficacy and durability of RKN control by resistance genes.