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Globodera ellingtonae was originally described from populations collected in the United States. In the original description, ribosomal DNA loci from Globodera sp. collected in Chile and Argentina were similar to G. ellingtonae, suggesting this nematode originated in this region of South America. In an effort to find additional populations of G. elllingtonae, collection trips were conducted in 2017 and 2020 in the Antofagasta and Arica y Parinacota Regions in Northern Chile, respectively. Globodera sp. were more prevalent in Antofagasta (17 samples collected, 53% positive for Globodera sp.) than in Arica y Parincota (16 samples collected, 13% positive for Globodera sp.). The genomes of single cysts (N ≥ 3) from four fields were sequenced. Additionally, the genomes of the G. ellingtonae population from Oregon and a Globodera sp. population originally collected in Antofagasta Region but maintained in culture in France were also sequenced. Based upon a HSP90 sequenced data mined from WSG data, all of the populations from the Antofagasta Region were G. ellingtonae and grouped in a monophyletic clade. A population collected from the Arica y Parincota Region was identified as G. rostochiensis based upon HSP90 data. Genome-wide SNP patterns of the G. ellingtonae populations showed strong clustering based on geographic location indicating that G. ellingtonae has high genetic diversity within Chile. A phylogenetic tree derived from 168,354 binary SNPs in the nuclear genome showed separate but distinct clustering of the Oregon population and the population from Antofagasta maintained in France. The Oregon G. ellingtonae population subtended the Chilean clades and placed on a long branch representing approximately twice the genetic variation observed among all Chilean G. ellingtonae populations. The possibility remains that G. ellingtonae from Chile may be sufficiently diverged to constitute a new species from G. ellingtonae originally described from a population collected in Oregon.

International audience; Management of plant pests is probably the most serious challenge in sustainable food production and the maintenance of food security. Due to the strict regulation of or ban on major categories of pesticide, the potato cyst nematode Globodera pallida has been managed by a combination of crop rotation and the potato resistance locus Grp1, a relatively narrow range resistance gene which was introgressed into a range of commercial potato cultivars in Europe. However, in 2014, G. pallida populations were described that can no longer be controlled by Grp1. Most likely similar highly virulent populations will also emerge in all major potato growing areas in North Western Europe where production practices are very similar. Except for laborious, costly and often moderately accurate pot experiments, there is currently no rapid and reliable method to identify virulent populations. This represents a strong limitation and prevents an accurate and durable management of infestations. The PalAdapt project funded by EFSA represents the first step of a European battle plan against the emergence of virulent G. pallida populations and aims at improving the methods and tools for a fast identification of virulence outbreaks. Four main research questions were investigated during the project: (i) Do resistance breaking populations correspond to novel introductions into Europe? (ii) Can miniaturized in vitro tests be used to get more rapidly an accurate identification of the virulence status?, (iii) Is cyst size a life history trait useful to estimate the virulence status of a population?, (iv) Can we identify polymorphism to design molecular tools for an accurate virulence monitoring? The EFSA partnering grants initiative was an accurate way to improve the EU risk assessment capacity through a knowledge exchange among partners having complementary resources and expertise.

International audience; Plant-parasite coevolution has generated much interest and studies to understand and manage diseases in agriculture. Such a reciprocal evolutionary process could lead to a pattern of local adaptation between plants and parasites. Based on the phylogeography of each partner, the present study tested the hypothesis of local adaptation between the potato cyst nematode Globodera pallida and wild potatoes in Peru. The measured fitness trait was the hatching of cysts which is induced by host root exudates. Using a cross-hatching assay between 13 populations of G. pallida and root exudates from 12 wild potatoes, our results did not show a strong pattern of local adaptation of the parasite but the sympatric combinations induced better hatching of cysts than allopatric combinations, and there was a negative relationship between the hatching percentage and the geographical distance between nematode populations and wild potatoes. Moreover, a strong effect of the geographic origin of root exudates was found, with root exudates from south of Peru inducing better hatching than root exudates from north of Peru. These results could be useful to develop new biocontrol products or potato cultivars to limit damages caused by G. pallida.

International audience; Evidence of strong gene flow among French populations of the carrot cyst nematode [i]Heterodera carotae[/i]

The effective size of a population is the size of an ideal population which would undergo genetic drift at the same rate as the real population. The balance between selection and genetic drift depends on the effective population size (Ne), rather than the real numbers of individuals in the population (N). The objectives of the present study were to estimateNein the potato cyst nematodeGlobodera pallidaand to explore the causes of a lowNe/Nratio in cyst nematodes using artificial populations. Using a temporal analysis of 24 independent populations, the medianNewas 58 individuals (minNe= 25 and maxNe= 228).Neis commonly lower thanNbut in the case of cyst nematodes, theNe/Nratio was extremely low. Using artificial populations showed that this low ratio did not result from migration, selection and overlapping generations, but could be explain by the fact thatG. pallidapopulations deviate in structure from the assumptions of the ideal population by having unequal sex ratios, high levels of inbreeding and a high variance in family sizes. The consequences of a lowNe, resulting in a strong intensity of genetic drift, could be important for their control becauseG. pallidapopulations will have a low capacity to adapt to changing environments.

Background In hyperspecialized parasites, the ability to grow on a particular host relies on specific virulence factors called effectors. These excreted proteins are involved in the molecular mechanisms of parasitism and distinguish virulent pathogens from non-virulent related species. The potato cyst nematodes (PCN) Globodera rostochiensis and G. pallida are major plant-parasitic nematodes developing on numerous solanaceous species including potato. Their close relatives, G. tabacum and G. mexicana are stimulated by potato root diffusate but unable to establish a feeding site on this plant host. Results RNA sequencing was used to characterize transcriptomic differences among these four Globodera species and to identify genes associated with host specificity. We identified seven transcripts that were unique to PCN species, including a protein involved in ubiquitination. We also found 545 genes that were differentially expressed between PCN and non-PCN species, including 78 genes coding for effector proteins, which represent more than a 6-fold enrichment compared to the whole transcriptome. Gene polymorphism analysis identified 359 homozygous non-synonymous variants showing a strong evidence for selection in PCN species. Conclusions Overall, we demonstrated that the determinant of host specificity resides in the regulation of essential effector gene expression that could be under the control of a single or of very few regulatory genes. Such genes are therefore promising targets for the development of novel and more sustainable resistances against potato cyst nematodes. Electronic supplementary material The online version of this article (10.1186/s12864-019-5853-4) contains supplementary material, which is available to authorized users.

International audience; In this article, we describe the analysis of over 9000 expressed sequence tags (ESTs) from cDNA libraries obtained from various life cycle stages of Globodera pallida. We have identified over 50 G. pallida effectors from this dataset using bioinformatics analysis, by screening clones in order to identify secreted proteins up-regulated after the onset of parasitism and using in situ hybridization to confirm the expression in pharyngeal gland cells. A substantial gene family encoding G. pallida SPRYSEC proteins has been identified. The expression of these genes is restricted to the dorsal pharyngeal gland cell. Different members of the SPRYSEC family of proteins from G. pallida show different subcellular localization patterns in plants, with some localized to the cytoplasm and others to the nucleus and nucleolus. Differences in subcellular localization may reflect diverse functional roles for each individual protein or, more likely, variety in the compartmentalization of plant proteins targeted by the nematode. Our data are therefore consistent with the suggestion that the SPRYSEC proteins suppress host defences, as suggested previously, and that they achieve this through interaction with a range of host targets.

Native to South America, the potato cyst nematode Globodera pallida is one of the principal pests of Andean potato crops and is also an important global pest following its introduction to Europe, Africa, North America, Asia and Oceania. Building on earlier work showing a clear south to north phylogeographic pattern in Peruvian populations, we have been able to identify the origin of Western European populations with high accuracy. They are all derived from a single restricted area in the extreme south of Peru, located between the north shore of the Lake Titicaca and Cusco. Only four cytochrome b haplotypes are found in Western Europe, one of them being also found in some populations of this area of southern Peru. The allelic richness at seven microsatellite loci observed in the Western European populations, although only one-third of that observed in this part of southern Peru, is comparable to the allelic richness observed in the northern region of Peru. This result could be explained by the fact that most of the genetic variability observed at the scale of a field or even of a region is already observed at the scale of a single plant within a field. Thus, even introduction via a single infected potato plant could result in the relatively high genetic variability observed in Western Europe. This finding has important consequences for the control of this pest and the development of quarantine measures.

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall-degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies

Using a complementary (c)DNA-amplified fragment length polymorphism (AFLP) approach, we investigated differential gene expression linked to resistance mechanisms during the incompatible potato – Globodera pallida interaction. Expression was compared between a resistant and a susceptible potato clone, inoculated or not inoculated with G. pallida. These clones were issued from a cross between the resistant Solanum sparsipilum spl329.18 accession and the susceptible dihaploid S. tuberosum Caspar H3, and carried, respectively, resistant and susceptible alleles at the resistance quantitative trait loci (QTLs). Analysis was done on root fragments picked up at 4 time points, during a period of 6 days after infection, from penetration of the nematode in the root to degradation of the feeding site in resistant plants. A total of 2560 transcript-derived fragments (TDFs) were analyzed, resulting in the detection of 46 TDFs that were up- or downregulated. The number of TDFs that were up- or downregulated increased with time after inoculation. The majority of TDFs were upregulated at only 1 or 2 time points in response to infection. After isolation and sequencing of the TDFs of interest, a subset of 36 sequences were identified, among which 22 matched plant sequences and 2 matched nematode sequences. Some of the TDFs that matched plant genes showed clear homologies to genes involved in cell-cycle regulation, transcription regulation, resistance downstream signalling pathways, and defense mechanisms. Other sequences with homologies to plant genes of unknown function or without any significant similarity to known proteins were also found. Although not exhaustive, these results represent the most extensive list of genes with altered RNA levels after the incompatible G. pallida–potato interaction that has been published to date. The function of these genes could provide insight into resistance or plant defense mechanisms during incompatible potato-cyst nematode interactions.

International audience; Deviations of genotypic frequencies from Hardy-Weinberg equilibrium (HWE) expectations could reveal important aspects of the biology of populations. Deviations from HWE due to heterozygote deficits have been recorded for three plant-parasitic nematode species. However, it has never been determined whether the observed deficits were due (i) to the presence of null alleles, (ii) to a high level of consanguinity and/or (iii) to a Wahlund effect. The aim of the present work was, while taking into the possible confounding effect of null alleles, to disentangle consanguinity and Wahlund effect in natural populations of those three economically important cyst nematodes using microsatellite markers: Globodera pallida, G. tabacum and Heterodera schachtii, pests of potato, tobacco and sugar beet, respectively. The results show a consistent pattern of heterozygote deficiency in the three nematode species sampled at the spatial scale of the host plant. We demonstrate that the prevalence of null alleles is weak and that heterozygote deficits do not have a single origin. Our results suggested that it is restricted dispersal that leads to heterozygote deficits through both consanguinity and substructure, which effects can be linked to soil movement, cyst density, and the number of generations per year. We discuss potential implications for the durability of plant resistances that are used to protect crops against parasites in which mating between relatives occur. While consanguineous mating leads to homozygosity at all loci, including loci governing avirulence/virulence, which favours the expression of virulence when recessive, the Wahlund effect is expected to have no particular effect on the adaptation of nematodes to resistances.

A homologue of a Ran binding protein to microtubules ( ranbpm ) gene, initially known as IC5, was identified and characterised in second-stage juveniles (J2s) of the potato cyst nematode Globodera pallida. The full-length cDNA (937 bp) was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE) and specific primers were designed to amplify the genomic sequences of 2396 bp containing six introns. The ORF (798 bp) encodes a putative 265 amino acid sequence with a predicted SPRY domain and a signal peptide of 23 amino acids on the N-terminal part of the protein. In situ hybridisation experiments showed that the transcript is located in the dorsal gland of the J2s, suggesting that the encoded protein has an extracellular function and can be involved in the late stages of parasitism such as feeding site establishment. This gene was specifically over-expressed in the juveniles before and during parasitism, but not in adult developmental stages. As this gene is presumed to be involved in plant–nematode interaction, particularly in the development and maintenance of the feeding structure that allows the nematode to achieve parasitic development, homologous genes were sought in other cyst nematode species. One of them was cloned and sequenced in the closely related species Globodera ‘ mexicana ’.

Peruvian potato cyst nematode populations were analysed to assess both their inter- and intraspecific similarities. ITS--RFLP and two satellite DNA sequences were used as taxonomic tools. Both techniques have confirmed that the Peruvian populations have as their closest relatives the European Globodera pallida, despite the detection of clear differences that prevents us from assigning these South American populations unambiguously to any Globodera species. A more precise study of the variability of these Peruvian populations was investigated and they were compared with the imported European populations using protein (2-DGE) and DNA (RAPD) datasets. The clear distinction between the Peruvian and the European populations was confirmed and, inside each group, no correlation was found between the pathotype classification and the observed clustering of the populations. Surprisingly, while RAPDs revealed a higher variability in the Peruvian group than in the European one, some characteristic proteins were found by 2-DGE in some European populations, whereas it was impossible to find any in the Peruvian populations. It is concluded that the primary founders of the European populations may have an origin other than that of the Peruvian populations involved in this study.

For the first time, mariner elements were found in insect parasitic nematodes. Full-length elements were isolated from the rhabditid Heterorhabditis bacteriophora. They were 1279 bp long, flanked by two 30-bp inverted repeats, and were able to encode a putative 358-amino acid transposase. These elements were present in about 30 copies in the H. bacteriophora genome, but their distribution among closely related Heterorhabditis and Steinernema genera was patchy. DNA and encoded peptide sequences of H. bacteriophora mariners showed greater similarity to the mariner of the coleopteran Carpelimus sp. than to the mariners of the rhabditid Caenorhabditis elegans. The possibility of horizontal transfer was investigated by examination of a host for Heterorhabditis nematodes, a beetle of the Phyllophaga sp. Mariner elements were found in this insect, but they were not very similar to the H. bacteriophora elements. Finally, the H. bacteriophora mariners formed a group with those of invertebrates, suggesting vertical transmission from a common ancestor.

International audience; The golden cyst nematode (Globodera rostochiensis), native to South America, has been introduced in many parts of the world, including Europe and North America. Recently, it was found for the first time in the province of Quebec, Canada in the locality of St. Amable near Montreal. To date, very few studies have examined the population genetics of this pest. Consequently, there is a lack of knowledge about the genetic structure and evolution of this nematode. In this study, twelve new microsatellite markers were developed in order to explore these questions. These markers were used to genotype fifteen populations originating from different regions of the world, including five from Canada. Within populations, the highest genetic diversity was consistently observed in the populations from Bolivia, the postulated region of origin of the golden nematode, and the lowest in populations from British Columbia (Canada) and New York (USA). The two Quebec populations were very similar to each other and to the population found in Newfoundland, but surprisingly, they were significantly different from three other North American populations including those from New York and British Columbia. Based on our results, we conclude that the golden cyst nematode has been introduced in North America at least twice from distinct regions of the world. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved.

Background The Ran GTPase Activating Protein 2 (RanGAP2) was first described as a regulator of mitosis and nucleocytoplasmic trafficking. It was then found to interact with the Coiled-Coil domain of the Rx and GPA2 resistance proteins, which confer resistance to Potato Virus X (PVX) and potato cyst nematode Globodera pallida, respectively. RanGAP2 is thought to mediate recognition of the avirulence protein GP-RBP-1 by GPA2. However, the Gpa2-induced hypersensitive response appears to be relatively weak and Gpa2 is limited in terms of spectrum of efficiency as it is effective against only two nematode populations. While functional and evolutionary analyses of Gp-Rbp-1 and Gpa2 identified key residues in both the resistance and avirulence proteins that are involved in recognition determination, whether variation in RanGAP2 also plays a role in pathogen recognition has not been investigated. Results We amplified a total of 147 RanGAP2 sequences from 55 accessions belonging to 18 different di-and tetraploid Solanum species from the section Petota. Among the newly identified sequences, 133 haplotypes were obtained and 19.1% of the nucleotide sites were found to be polymorphic. The observed intra-specific nucleotide diversity ranges from 0.1 to 1.3%. Analysis of the selection pressures acting on RanGAP2 suggests that this gene evolved mainly under purifying selection. Nonetheless, we identified polymorphic positions in the protein sequence at the intra-specific level, which could modulate the activity of RanGAP2. Two polymorphic sites and a three amino-acid deletion in RanGAP2 were found to affect the timing and intensity of the Gpa2-induced hypersensitive response to avirulent GP-RBP-1 variants even though they did not confer any gain of recognition of virulent GP-RBP-1 variants. Conclusions Our results highlight how a resistance gene co-factor can manage in terms of evolution both an established role as a cell housekeeping gene and an implication in plant parasite interactions. StRanGAP2 gene appears to evolve under purifying selection. Its variability does not seem to influence the specificity of GPA2 recognition but is able to modulate this activity by enhancing the defence response. It seems therefore that the interaction with the plant resistance protein GPA2 (and/or Rx) rather than with the nematode effector was the major force in the evolution of the RanGAP2 locus in potato. From a mechanistic point of view these results are in accordance with a physical interaction of RanGAP2 with GPA2 and suggest that RBP-1 would rather bind the RanGAP2-GPA2 complex than the RanGAP2 protein alone.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; The potato cyst nematodes Globodera pallida and G. rostochiensis are listed in the EU Plant Health Directive 2000/29/EC and are also subject to the new EU Council Directive 2007/33/EC on the control of potato cyst nematodes, requiring unilateral suppression of these pests in Europe. At the same time there is also pressure to increase world trade in potatoes. Such pressure has to be balanced by the risks involved in the associated spread of these pests and subsequent problems in management. Populations of the potato cyst nematodes from outside Europe, in particular South America, which is considered the origin of G. pallida and G. rostochiensis, pose a risk to those European countries where limited genetic variability of these nematode species has been recorded. The development and usage of resistant cultivars under such conditions has formed a pivotal role in integrated management programmes in Europe. Molecular studies have shown that populations of G. pallida and G. rostochiensis from South America have a different genetic composition from those in Europe. The introduction of such populations would pose a threat to the use of resistant cultivars as a major tool in reducing the potential spread and damage caused by these species. At present, an inability to link precisely genetic variability to the virulence characteristics of a specific nematode population, and quickly identify the virulence status of intercepted populations for inspection purposes, strengthens the case for using plant health legislation to prevent their introduction.

The Globodera pallida SPRYSEC Gp-Rbp-1 gene encodes a secreted protein which induces effector-triggered immunity (ETI) mediated by the Solanum tuberosum disease resistance gene Gpa2. Nonetheless, it is not known how the Andes orogeny, the richness in Solanum species found along the Cordillera or the introduction of the nematode into Europe have affected the diversity of Gp-Rbp-1 and its recognition by Gpa2. We generated a dataset of 157 highly polymorphic Gp-Rbp-1 sequences and identified three Gp-Rbp-1 evolutionary pathways: the ‘Northern Peru’, ‘Peru clade I/European’ and ‘Chilean’ paths. These may have been shaped by passive dispersion of the nematode and by climatic variations that have influenced the nature and diversity of wild host species. We also confirmed that, by an analysis of the selection pressures acting on Gp-Rbp-1, this gene has evolved under positive/diversifying selection, but differently among the three evolutionary pathways described. Using this extended sequence dataset, we were able to detect eight sites under positive selection. Six sites appear to be of particular interest because of their predicted localization to the extended loops of the B30.2 domain and/or support by several computational methods. The P/S 187 position was previously identified for its effect on the interaction with GPA2. The functional importance of the other five amino acid polymorphisms observed was investigated using Agrobacterium transient transformation assays. None of these new residues, however, appears to be directly involved in Gpa2-mediated plant defence mechanisms. Thus, the P/S polymorphism observed at position 187 remains the sole variation sufficient to explain the recognition of Gp-Rbp-1 by Gpa2.

International audience; Like microbial pathogens, nematodes secrete a repertoire of effectors with diverse functions. In the case of sedentary nematodes, completion of the life cycle requires the maintenance of the biotrophic interaction for several weeks and the induction of a permanent feeding site. These essential processes require fine tuning of plant cell responses. About a hundred candidate effectors have been isolated from nematodes to date that have roles in the invasion of host tissues, evasion of plant defences, suppression of plant defences or manipulation of plant cell fate. Evolutionary studies suggest that several distinct mechanisms have led to the acquisition of these effectors during the evolution of plant parasitism. In addition, since any compound secreted by the parasite can potentially be perceived as nonself by the plant, nematode effectors are subject to selective pressure to escape plant immunity. This chapter reviews the latest findings on nematode effector functions and evolutionary traits.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; While pectate lyases are major parasitism factors in plant-parasitic nematodes, there is little information on the variability of these genes within species and their utility as pathotype or host range molecular markers. We have analysed polymorphisms of pectate lyase 2 (pel-2) gene, which degrades the unesterified polygalacturonate (pectate) of the host cell-wall, in the genus Globodera. Molecular variability of the pel-2 gene and the predicted protein was evaluated in populations of G. rostochiensis, G. pallida, G. "mexicana" and G. tabacum. Seventy eight pel-2 sequences were obtained and aligned. Point mutations were observed at 373 positions, 57% of these affect the coding part of the gene and produce 129 aa replacements. The observed polymorphism does not correlate either to the pathotypes proposed in potato cyst nematodes (PCN) or the subspecies described in tobacco cyst nematodes. The trees reveal a topology different from the admitted species topology as G. rostochiensis and G. pallida sequences are more similar to each other than to G. tabacum. Species-specific sites, potentially applicable for identification, and sites distinguishing PCN from tobacco cyst nematodes, were identified. As both G. rostochiensis and G. pallida display the same host range, but distinct from G. tabacum, which cannot parasitize potato plants, it is tempting to speculate that pel-2 genes polymorphism may be implicated in this adaptation, a view supported by the fact that no active pectate lyase 2 was found in G. "mexicana", a close relative of G. pallida that is unable to develop on cultivated potato varieties

Plant NB-LRR proteins confer robust protection against microbes and metazoan parasites by recognizing pathogen-derived avirulence (Avr) proteins that are delivered to the host cytoplasm. Microbial Avr proteins usually function as virulence factors in compatible interactions; however, little is known about the types of metazoan proteins recognized by NB-LRR proteins and their relationship with virulence. In this report, we demonstrate that the secreted protein RBP-1 from the potato cyst nematode Globodera pallida elicits defense responses, including cell death typical of a hypersensitive response (HR), through the NB-LRR protein Gpa2. Gp-Rbp-1 variants from G. pallida populations both virulent and avirulent to Gpa2 demonstrated a high degree of polymorphism, with positive selection detected at numerous sites. All Gp-RBP-1 protein variants from an avirulent population were recognized by Gpa2, whereas virulent populations possessed Gp-RBP-1 protein variants both recognized and non-recognized by Gpa2. Recognition of Gp-RBP-1 by Gpa2 correlated to a single amino acid polymorphism at position 187 in the Gp-RBP-1 SPRY domain. Gp-RBP-1 expressed from Potato virus X elicited Gpa2-mediated defenses that required Ran GTPase-activating protein 2 (RanGAP2), a protein known to interact with the Gpa2 N terminus. Tethering RanGAP2 and Gp-RBP-1 variants via fusion proteins resulted in an enhancement of Gpa2-mediated responses. However, activation of Gpa2 was still dependent on the recognition specificity conferred by amino acid 187 and the Gpa2 LRR domain. These results suggest a two-tiered process wherein RanGAP2 mediates an initial interaction with pathogen-delivered Gp-RBP-1 proteins but where the Gpa2 LRR determines which of these interactions will be productive., Author Summary Biotrophic plant pathogens produce effector proteins that are delivered to the host cytoplasm where they alter defense responses and metabolism to favor pathogen colonization. In turn, plants have evolved intra-cellular proteins to recognize pathogen effector proteins, known as NB-LRR proteins, which are similar in structure to animal NOD-LRR immune receptors. While effector proteins recognized by NB-LRR proteins have been identified from many organisms, the identification of such proteins from metazoan plant parasites has presented unique challenges due to the lack of genetically tractable model species. The potato Gpa2 protein confers resistance to some isolates of the potato pale cyst nematode, Globodera pallida. In this report, we show that Gpa2 recognizes certain variants of the G. pallida protein, Gp-RBP-1, which is highly polymorphic both within and between populations. This recognition in turn induces defense responses, including a form of programmed cell death characteristic of plant immune receptor activation. Moreover, we show that a Gpa2-interacting protein, RanGAP2, is required for Gpa2 function and that activation of Gpa2 is enhanced when Gp-RBP-1 is artificially tethered to RanGAP2. Thus, our findings suggest that RanGAP2 acts as a recognition co-factor for Gpa2, and have important implications for our understanding of the mechanisms and evolution of pathogen recognition by NB-LRR proteins.

National audience; Inscrits parmi les parasites de quarantaine, les nématodes à kyste, Globodera rostochiensis et Globodera pallida sont présents dans de nombreux bassins de production de pomme de terre où les dégâts sont parfois considérables. La lutte contre ces ravageurs est préventive dans les zones de culture indemnes, condition indispensable à la production de plant de pomme de terre. Des pratiques agronomiques adéquates et l’utilisation de variétés résistantes conduisent à la réduction du taux d’infestation des terrains contaminés. Dans certaines situations géographiques, le traitement du sol par solarisation peut remplacer efficacement l’emploi de nématicides destinés à être interdits. La résistance génétique représente le moyen de lutte le plus efficace. L’introgression de nouvelles formes de résistance a été entreprise à partir d’autres espèces tubéreuses, notamment Solanum vernei. La récente caractérisation moléculaire de nouveaux facteurs génétiques de la résistance chez les espèces Solanum spegazzinii et Solanum sparsipilum et l’étude de leur association devraient faire progresser le travail de sélection variétale. La combinaison des différents moyens de lutte présentés en fonction des situations devrait aboutir à la mise en pratique d’une véritable lutte intégrée en culture de pomme de terre de consommation.

International audience; Ditylenchus dipsaci, the stem nematode of alfalfa (Medicago sativa), Mycosphaerella pinodes, cause of Ascochyta blight in pea (Pisum sativum) and Aphanomyces euteiches, cause of pea root rot, result in major yield losses in French alfalfa and pea crops. These diseases are difficult to control and the partial resistances currently available are not effective enough. Medicago truncatula, the barrel medic, is the legume model for genetic studies, which should lead to the identification and characterization of new resistance genes for pathogens. We evaluated a collection of 34 accessions of M. truncatula and nine accessions from three other species (two from M. italica, six from M. littoralis and one from M. polymorpha) for resistance to these three major diseases. We developed screening tests, including standard host references, for each pathogen. Most of the accessions tested were resistant to D. dipsaci, with only three accessions classified as susceptible. A very high level of resistance to M. pinodes was observed among the accessions, none of which was susceptible to this pathogen. Conversely, a high level of variation, from resistant to susceptible accessions, was identified in response to infection by A. euteiches

International audience; In order to identify genes involved in pathogenicity, we compared the closely related species Globodera pallida (GP) and Globodera "mexicana" (GM) that have different host ranges and are able to produce viable and fertile hybrids. Three pioneer genes were previously identified as differentially expressed between GP and GM: GPLIA7 and GPLIB3 were found to be more highly expressed in GP, whereas GMLIVG9 was found more highly expressed in GM. In this study, we showed that Ia7 and IVg9 genes probably encode products secreted by the subventral oesophageal glands and the dorsal oesophageal gland, respectively. No Blast homolog was found in the databases, but a metridin-like ShK (Stichodactyla helianthus) toxin domain was identified in the Ia7 sequence. Analysis of the full-length sequences of these 2 genes between GP and GM revealed a high level of interspecies variability (8% for the Ia7 transcript and 17% for the IVg9 transcript) and a high proportion (90%) of nonsynonymous mutations among the substitutions observed. This suggested that these 2 pioneer genes are under strong diversifying selection pressures and therefore may be involved in pathogenicity. Further investigations of the sequence polymorphism of Ia7 and IVg9 genes were conducted in GP x GM hybrid lines that were selected in laboratory conditions for their different ability to develop on potato and black nightshade. As similar sequences were obtained for all the hybrid lines tested independently of their pathogenicity status, no correlation could be established between IA7 and IVG9 amino acid changes and the host range differences observed between GP and GM

Amplified fragment length polymorphism (AFLP) was used to obtain information on the within-species geneticvariability of the tobacco cyst – nematode (TCN) complex. AFLP was found to be well suited to this type ofstudy. The current classification of TCN was confirmed. Results indicate that the Globodera tabacum solanacearumgroup, believed to be restricted to the U.S.A., also occurs in Mexico. The within-species variability of TCN is considerable.Populations from Mexico may form a new subgroup. AFLP group-specific markers were identified for two ofthe TCN subgroups: Globodera tabacum tabacum and Globodera tabacum solanacearum.; La technique dite de l'AFLP a été utilisée sur les nématodes à kystes du tabac (TCN) afin d'acquérir des connaissances sur leur polymorphisme moléculaire intra-spécifique. L'efficience de la technique AFLP pour ce type d'études a été réaffirmée. La classification intra-spécifique des TCN communément admise est confirmée. Les résultats indiquent que le groupe Globodera tabacum solanacearum, jusqu'à présent décrit uniquement aux États-Unis, existe également au Mexique. Une variabilité intra-spécifique élargie est mise en évidence au sein des TCN. Des populations originaires du Mexique pourraient former un nouveau sous-groupe. Des marqueurs AFLP spécifiques pour deux des sous-groupes du complexe des TCN, Globodera tabacum tabacum et Globodera tabacum solanacearum, sont mis en évidence.Mots clés : Heteroderinae, classification, marqueurs, diversité génétique, distribution géographique.

There is currently no information on the genome size and complexity in the entomoparasitic nematodes Steinernema and Heterorhabditis. DNA reassociation kinetics were used to determine the genome size and complexity in 2 species: Steinernema carpocapsae and Heterorhabditis bacteriophora. C0t curves derived from renaturation kinetics of denatured DNA indicate that the genomes of these entomoparasitic rhabditids are different both in size and complexity. Genome sizes were estimated at 2·3 × 108 bp for S. carpocapsae and 3·9 × 107 bp for H. bacteriophora and repetitive DNA contents were found to represent 39% and 51% of these respective genomes.

The success of alternative crop protection practices against plant-parasitic nematodes using host resistance genes depends fundamentally upon identification of the species and pathotypes effectively controlled by these genes. In the same way, biological control of insects by entomopathogenic nematodes will work only if the nematode strains used are indeed active against the pests to be eliminated. For these applications, the accurate interspecific and/or intraspecific identification of nematodes is thus of outstanding importance. Here, Eric Grenier, Philippe Castagnone-Sereno and Pierre Abad discuss the recent use of satellite DNA sequences in nematode taxonomic diagnostics.

SUMMARYThree satellite DNAs previously isolated from the entomopathogenic nematodesSteinernema carpocapsae, Heterorhabditis bacteriophoraandHeterorhabditis indicusgive hybridization signals only with theS. carpocapsae, H. bacteriophoraandH. indicuspopulations tested, indicating that these satellite sequences are species-specific. Because of their reiteration and their variabilities, we have shown that these sequences are able to discriminate at the interspecific level between theSteinernemaandHeterorhabditisspecies, but also at the intraspecific level betweenS. carpocapsaestrains. Furthermore, in simple squashed nematode experiments, we are able to unambiguously identifyS. carpocapsae,H. bacteriophoraandH. indicuspopulations. This last procedure is effective even on a single infective juvenile, with the main advantage that it avoids time-consuming DNA extractions.

Two AluI tandemly repeated DNAs were cloned from two entomopathogenic nematodes: the first one from Steinernema glaseri and the second one from Heterorhabditis bacteriophora. The monomeric units of these two satellite DNAs have a repeat length of 174 and 168 bp, respectively. These AluI repeated element families appear to constitute 5.5% of the S. glaseri genome and 5% of the H. bacteriophora genome. Their A + T contents were estimated at 55% and 57%. Moreover, the monomers of these two families are quite homogeneous in sequence, showing, on average, 3.9% and 2.7% divergence from their respective consensus sequence. These results suggest that some mechanism is acting to maintain the homogeneity of these repeated DNAs despite their abundance. We have also shown that these two DNA families are species-specific and therefore could be used for the identification of Steinernema and Heterorhabditis entomopathogenic nematode species.

An HaeIII satellite DNA family has been cloned from the entomopathogenic nematode Steinernema carpocapsae. This repeated sequence appears to be an unusually abundant satellite DNA, since it constitutes about 62% of the S. carpocapsae genome. The nucleotide sequences of 13 monomers have been determined. This satellite DNA family is represented by two sub-families: one with monomeric units of 170 bp and the other with monomeric units of 182 bp. These monomers are quite homogeneous in sequence, showing an average intermonomer variability of 6% from the consensus sequence. These results suggest that some homogenizing mechanism is acting to maintain the homogeneity of this satellite DNA. After hybridization with the genomic DNA of several other Steinernema species, this DNA sequence appears to be specific to the S. carpocapsae genome. Therefore, the species specificity and the high copy number of the HaeIII satellite DNA sequence should provide a rapid and powerful tool which could contribute to the identification of Steinernema species.