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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.

International audience; 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.

Funding: Work on plant-parasitic nematodes at the University of Cambridge is supported by DEFRA licence 125034/359149/3, and was funded by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/R011311/1, BB/N021908/1, and BB/S006397/1, a Synthego Genome Engineer grant, and a Genewiz grant. A.A. was supported by a Wellcome/Newton trust Institutional Strategic Support Fund grant and a UKRI Future Leaders Fellowship MR/S033769/1. C.J.L. was supported by BBSRC grant BB/N016866/1. J.J. receives funding from the Scottish Government Rural and Environmental Science and Analytical Services division. T.R.M. and T.J.B. were supported by grants from the Iowa Soybean Association and by State of Iowa and Hatch funds. A.P.S. and S.A. were supported by grants RPG-2016-089 and BB/L019884/1. 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. Publisher PDF

SummaryRoot-knot nematodes (RKNs) are among the most damaging pests of agricultural crops. Indeed, Meloidogyne is an extremely polyphagous genus of nematodes that can infect thousands of plant species. A few genes for resistance (R-genes) to RKNs suitable for use in crop breeding have been identified, and new virulent strains and species of nematode emerge rendering these R-genes ineffective. Effective parasitism is dependent on the secretion, by the RKN, of effectors targeting plant functions, which mediate the reprogramming of root cells into specialised feeding cells. These cells, the giant cells, are essential for RKN development and reproduction. The EFFECTOR 18 protein (EFF18) from M. incognita interacts with the spliceosomal protein SmD1 in Arabidopsis, disrupting its function in alternative splicing regulation and modulating the giant cell transcriptome. We show here that EFF18 is a conserved RKN-specific effector. We also show here that EFF18 effectors also target SmD1 in Nicotiana benthamiana and Solanum lycopersicum. The alteration of SmD1 expression by virus-induced gene silencing (VIGS) in Solanaceae affects giant cell formation and nematode development. Thus, SmD1 is a susceptibility gene and a promising target for the development of broad resistance, especially in Solanaceae, for the control of Meloidogyne spp.

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. Electronic supplementary material The online version of this article (10.1186/s12864-018-5296-3) contains supplementary material, which is available to authorized users.

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., SCOPUS: ar.j, info:eu-repo/semantics/published

The establishment of galls and syncytia as feeding sites induced by root-knot and cyst nematodes, respectively, involves a progressive increase in nuclear and cellular size. Here we describe the functional characterization of endocycle activators CCS52A, CCS52B and a repressor of the endocycle, DEL1, during two types of nematode feeding site development in Arabidopsis thaliana. In situ hybridization analysis showed that expression of CCS52A1 and CCS52B was strongly induced in galls and syncytia and DEL1 was stably but weakly expressed throughout feeding site development. Down-regulation and over-expression of CCS52 and DEL1 in Arabidopsis drastically affected giant cell and syncytium growth, resulting in restrained nematode development, illustrating the need for mitotic activity and endo-reduplication for feeding site maturation. Exploiting the mechanism of endo-reduplication may be envisaged as a strategy to control plant-parasitic nematodes.

L'article original est publié par The American Society of Plant Biologists 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; Root-knot nematode (RKN) Meloidogyne species are major polyphagous pests of most crops worldwide, and cultivars with durable resistance are urgently needed because of nematicide bans. The Ma gene from the Myrobalan plum (Prunus cerasifera) confers complete-spectrum, heat-stable, and high-level resistance to RKN, which is remarkable in comparison with the Mi-1 gene from tomato (Solanum lycopersicum), the sole RKN resistance gene cloned. We report here the positional cloning and the functional validation of the Ma locus present at the heterozygous state in the P.2175 accession. High-resolution mapping totaling over 3,000 segregants reduced the Ma locus interval to a 32-kb cluster of three Toll/Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat (LRR) genes (TNL1–TNL3), including a pseudogene (TNL2) and a truncated gene (TNL3). The sole complete gene in this interval (TNL1) was validated as Ma, as it conferred the same complete-spectrum and high-level resistance (as in P.2175) using its genomic sequence and native promoter region in Agrobacterium rhizogenes-transformed hairy roots and composite plants. The full-length cDNA (2,048 amino acids) of Ma is the longest of all Resistance genes cloned to date. Its TNL structure is completed by a huge post-LRR (PL) sequence (1,088 amino acids) comprising five repeated carboxyl-terminal PL exons with two conserved motifs. The amino-terminal region (213 amino acids) of the LRR exon is conserved between alleles and contrasts with the high interallelic polymorphisms of its distal region (111 amino acids) and of PL domains. The Ma gene highlights the importance of these uncharacterized PL domains, which may be involved in pathogen recognition through the decoy hypothesis or in nuclear signaling.

During a compatible interaction, root-knot nematodes (Meloidogyne spp.) induce the redifferentiation of root cells into multinucleate nematode feeding cells (giant cells). Hyperplasia and hypertrophy of the surrounding cells leads to the formation of a root gall. We investigated the plant response to root-knot nematodes by carrying out a global analysis of gene expression during gall formation in Arabidopsis, using giant cell-enriched root tissues. Among 22 089 genes monitored with the complete Arabidopsis transcriptome microarray gene-specific tag, we identified 3373 genes that display significant differential expression between uninfected root tissues and galls at different developmental stages. Quantitative PCR analysis and the use of promoter GUS fusions confirmed the changes in mRNA levels observed in our microarray analysis. We showed that a comparable number of genes were found to be up- and downregulated, indicating that gene downregulation might be essential to allow proper gall formation. Moreover, many genes belonging to the same family are differently regulated in feeding cells. This genome-wide overview of gene expression during plant-nematode interaction provides new insights into nematode feeding-cell formation, and highlights that the suppression of plant defence is associated with nematode feeding-site development.

A cDNA-amplification fragment length polymorphism (AFLP)-based strategy has been used to identify genes differentially expressed between two pairs of near-isogenic lines (NIL) of the root-knot nematode Meloidogyne incognita either avirulent or virulent against the tomato Mi resistance gene. Gene expression profiles from infective second-stage juveniles (J2) were compared, and 22 of the 24,025 transcript-derived fragments (TDF) generated proved to be differential, i.e., present in both avirulent NIL and absent in both virulent NIL. Fourteen of the TDF sequences did not show any significant similarity to known proteins, while eight matched reported sequences from nematodes and other invertebrates. The differential expression of nine genes was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) experiments. In situ hybridization conducted with five of the sequences showed that two were specifically expressed in the intestinal cells (HM10 and PM1), one in the subventral esophageal glands (HM1), and two in the dorsal esophageal gland of J2 (HM7 and HM12). Analysis of full-length cDNA sequences revealed the presence of a signal peptide for HM1, HM10, and HM12, indicating that the encoded proteins are putatively secreted. Since secreted products in general and esophageal gland secretions in particular are thought to be among the main M. incognita pathogenicity factors, this result suggests a possible dual role for some of the genes encoding such secretions, i.e., they could be involved in both pathogenicity and virulence or avirulence of these biotrophic parasites.

Two copies of a new mariner-like element (MLE) presenting unusual inverted terminal repeats (ITRs), Mcmar1-1 and Mcmar1-2, were cloned and sequenced in the genome of the phytoparasitic nematode Meloidogyne chitwoodi. Although the sequence features of these Mcmar1 transposons are commonplace and link them to the mariner family, at their extremities they have large 355-pb long inverted terminal repeats that are perfectly conserved. This characteristic distinguishes them from all the other MLEs so far described that have imperfectly conserved ITRs of about 26–30 bp. In consequence, the sequenced full-length Mcmar1-1 element is 2000 bp long, and comprises an uninterrupted open reading frame (ORF) that encodes a putatively active transposase with 340 amino acid residues. The Mcmar1-2 element is a deleted form of Mcmar1-1 that contains a deletion overlapping most of the internal region of the 5′ITR and the 5′ region of the transposase ORF. The presence of large ITRs in different transposons related to the Tc1-mariner super-family is discussed.

The first animal polygalacturonase (PG, EC 2.1.15) encoding cDNA, Mi-pg-1, was cloned from the plant parasitic nematode Meloidogyne incognita. The enzymatic activity of MI-PG-1 was confirmed after heterologous expression in Escherichia coli. The presence of a predicted signal peptide on the MI-PG-1 sequence together with the specific localization of the transcripts of the Mi-pg-1 gene in the oesophageal glands of infective juveniles imply that MI-PG-1 could be secreted into plant tissues. The potential role of MI-PG-1 in parasitism is discussed.

We have analyzed the sequences of mariner-like element (MLE) transposases, in order to obtain a clearer picture of their phylogenetic relationships. In particular, we have considered their two known structural domains, as well as the nucleic acid sequences of the MLE inverted terminal repeats (ITR). The most consistent tree was obtained using sequences of the catalytic domain of the transposase. The trees obtained with the amino acid sequences of the ITR-binding domain and the ITR sequences themselves were similar to that obtained with the catalytic domain. However, a major difference indicated that the cecropia sub-family is divided into two sub-groups. These new trees were used to examine the evolutionary divergence of mariner-like transposable elements, with particular reference to the possibility that recombination events or gene conversions created mosaic elements during the evolution of transposons.

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.

An AluI satellite DNA family has been isolated in the genome of the root-knot nematode Meloidogyne chitwoodi. This repeated sequence was shown to be present at approximately 11,400 copies per haploid genome, and represents about 3.5% of the total genomic DNA. Nineteen monomers were cloned and sequenced. Their length ranged from 142 to 180 bp, and their A + T content was high (from 65.7 to 79.1%), with frequent runs of As and Ts. An unexpected heterogeneity in primary structure was observed between monomers, and multiple alignment analysis showed that the 19 repeats could be unambiguously clustered in six subfamilies. A consensus sequence has been deduced for each subfamily, within which the number of positions conserved is very high, ranging from 86.7% to 98.6%. Even though blocks of conserved regions could be observed, multiple alignment of the six consensus sequences did not enable the establishment of a general unambiguous consensus sequence. Screening of the six consensus sequences for evidence of internal repeated subunits revealed a 6-bp motif (AAATTT), present in both direct and inverted orientation. This motif was found up to nine times in the consensus sequences, also with the occurrence of degenerated subrepeats. Along with the meiotic parthenogenetic mode of reproduction of this nematode, such structural features may argue for the evolution of this satellite DNA family either (1) from a common ancestral sequence by amplification followed by mechanisms of sequence divergence, or (2) through independent mutations of the ancestral sequence in isolated amphimictic nematode populations and subsequent hybridization events. Overall, our results suggest the ancient origin of this satellite DNA family, and may reflect for M. chitwoodi a phylogenetic position close to the ancestral amphimictic forms of root-knot nematodes.

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria, M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.

Root-knot nematodes (RKNs) (Meloidogyne spp.) are obligate endoparasites of major worldwide economic importance. They exhibit a wide continuum of variation in their reproductive strategies, ranging from amphimixis to obligatory mitotic parthenogenesis. Molecular phylogenetic studies have highlighted divergence between mitotic and meiotic parthenogenetic RKN species and probable interspecific hybridization as critical steps in their speciation and diversification process. The recent completion of the genomes of two RKNs, Meloidogyne hapla and Meloidogyne incognita, that exhibit striking differences in their mode of reproduction (with and without sex, respectively), their geographic distribution, and their host range has opened the way for deciphering the evolutionary significance of (a)sexual reproduction in these parasites. Accumulating evidence suggests that whole-genome duplication (in M. incognita) and horizontal gene transfers (HGTs) represent major forces that have shaped the genome of current RKN species and may account for the extreme adaptive capacities and parasitic success of these nematodes.

Root-knot nematodes are globally the most aggressive and damaging plant-parasitic nematodes. Chemical nematicides have so far constituted the most efficient control measures against these agricultural pests. Because of their toxicity for the environment and danger for human health, these nematicides have now been banned from use. Consequently, new and more specific control means, safe for the environment and human health, are urgently needed to avoid worldwide proliferation of these devastating plant-parasites. Mining the genomes of root-knot nematodes through an evolutionary and comparative genomics approach, we identified and analyzed 15,952 nematode genes conserved in genomes of plant-damaging species but absent from non target genomes of chordates, plants, annelids, insect pollinators and mollusks. Functional annotation of the corresponding proteins revealed a relative abundance of putative transcription factors in this parasite-specific set compared to whole proteomes of root-knot nematodes. This may point to important and specific regulators of genes involved in parasitism. Because these nematodes are known to secrete effector proteins in planta, essential for parasitism, we searched and identified 993 such effector-like proteins absent from non-target species. Aiming at identifying novel targets for the development of future control methods, we biologically tested the effect of inactivation of the corresponding genes through RNA interference. A total of 15 novel effector-like proteins and one putative transcription factor compatible with the design of siRNAs were present as non-redundant genes and had transcriptional support in the model root-knot nematode Meloidogyne incognita. Infestation assays with siRNA-treated M. incognita on tomato plants showed significant and reproducible reduction of the infestation for 12 of the 16 tested genes compared to control nematodes. These 12 novel genes, showing efficient reduction of parasitism when silenced, constitute promising targets for the development of more specific and safer control means., Author Summary Plant-parasitic nematodes are annually responsible for more than $100 billion crop yield loss worldwide and those considered as causing most of the damages are root-knot nematodes. These nematodes used to be controlled by chemicals that are now banned from use because of their poor specificity and high toxicity for the environment and human health. In the absence of sustainable alternative solutions, new control means, more specifically targeted against these nematodes and safe for the environment are needed. We searched in root-knot nematode genomes, genes conserved in various plant-damaging species while otherwise absent from the genomes of non target species such as those of chordates, plants, annelids, insect pollinators and mollusks. These genes are probably important for plant parasitism and their absence from non-target species make them interesting candidates for the development of more specific and safer control means. Further bioinformatics pruning of this set of genes yielded 16 novel candidates that could be biologically tested. Using RNA interference, we knocked down each of these 16 genes in a root-knot nematode and tested the effect on plant parasitism efficiency. Out of the 16 tested genes, 12 showed a significant and reproducible diminution of infestation when silenced and are thus particularly promising.

International audience; Tandemly arrayed non-coding sequences or satellite DNAs (satDNAs) are rapidly evolving segments of eukaryotic genomes, including the centromere, and may raise a genetic barrier that leads to speciation. However, determinants and mechanisms of satDNA sequence dynamics are only partially understood. Sequence analyses of a library of five satDNAs common to the root-knot nematodes Meloidogyne chitwoodi and M. fallax together with a satDNA, which is specific for M. chitwoodi only revealed low sequence identity (32-64%) among them. However, despite sequence differences, two conserved motifs were recovered. One of them turned out to be highly similar to the CENP-B box of human alpha satDNA, identical in 10-12 out of 17 nucleotides. In addition, organization of nematode satDNAs was comparable to that found in alpha satDNA of human and primates, characterized by monomers concurrently arranged in simple and higher-order repeat (HOR) arrays. In contrast to alpha satDNA, phylogenetic clustering of nematode satDNA monomers extracted either from simple or from HOR array indicated frequent shuffling between these two organizational forms. Comparison of homogeneous simple arrays and complex HORs composed of different satDNAs, enabled, for the first time, the identification of conserved motifs as obligatory components of monomer junctions. This observation highlights the role of short motifs in rearrangements, even among highly divergent sequences. Two mechanisms are proposed to be involved in this process, i.e., putative transposition-related cut-and-paste insertions and/or illegitimate recombination. Possibility for involvement of the nematode CENP-B box-like sequence in the transposition-related mechanism and together with previously established similarity of the human CENP-B protein and pogo-like transposases implicate a novel role of the CENP-B box and related sequence motifs in addition to the known function in centromere protein binding.

International audience; Lateral gene transfers (LGT), species to species transmission of genes by means other than direct inheritance from a common ancestor, have played significant role in shaping prokaryotic genomes and are involved in gain or transfer of important biological processes. Whether LGT significantly contributed to the composition of an animal genome is currently unclear. In nematodes, multiple LGT are suspected to have favored emergence of plant-parasitism. With the availability of whole genome sequences it is now possible to assess whether LGT have significantly contributed to the composition of an animal genome and to establish a comprehensive list of these events. We generated clusters of homologous genes and automated phylogenetic inference, to detect LGT in the genomes of root-knot nematodes and found that up to 3.34% of the genes originate from LGT of non-metazoan origin. After their acquisition, the majority of genes underwent series of duplications. Compared to the rest of the genes in these species, several predicted functional categories showed a skewed distribution in the set of genes acquired via LGT. Interestingly, functions related to metabolism, degradation or modification of carbohydrates or proteins were substantially more frequent. This suggests that genes involved in these processes, related to a parasitic lifestyle, have been more frequently fixed in these parasites after their acquisition. Genes from soil bacteria, including plant-pathogens were the most frequent closest relatives, suggesting donors were preferentially bacteria from the rhizosphere. Several of these bacterial genes are plasmid-borne, pointing to a possible role of these mobile genetic elements in the transfer mechanism. Our analysis provides the first comprehensive description of the ensemble of genes of non-metazoan origin in an animal genome. Besides being involved in important processes regarding plant-parasitism, genes acquired via LGT now constitute a substantial proportion of protein-coding genes in these nematode genomes.

Capsicum annuum L. has resistance to root-knot nematodes (RKN) (Meloidogyne spp.), severe polyphagous pests that occur world-wide. Several single dominant genes confer this resistance. Some are highly specific, whereas others are effective against a wide range of species. The spectrum of resistance to eight clonal RKN populations of the major Meloidogyne species, M. arenaria (2 populations), M. incognita (2 populations), M. javanica (1 population), and M. hapla (3 populations) was studied using eight lines of Capsicum annuum. Host susceptibility was determined by counting the egg masses (EM) on the roots. Plants were classified into resistant (R; EM ≤ 5) or susceptible (H; EM5) classes. The french cultivar Doux Long des Landes was susceptible to all nematodes tested. The other seven pepper lines were highly resistant to M. arenaria, M. javanica and one population of M. hapla. Variability in resistance was observed for the other two populations of M. hapla. Only lines PM687, PM217, Criollo de Morelos 334 and Yolo NR were resistant to M. incognita. To investigate the genetic basis of resistance in the highly resistant line PM687, the resistance of two progenies was tested with the two populations of M. incognita: 118 doubled-haploid (DH) lines obtained by androgenesis from F(1) hybrids of the cross between PM687 and the susceptible cultivar Yolo Wonder, and 163 F(2) progenies. For both nematodes populations, the segregation patterns 69 R / 49 S for DH lines and 163 R / 45 S for F(2) progenies were obtained at 22°C and at high temperatures (32°C and 42°C). The presence of a single dominant gene that totally prevented multiplication of M. incognita was thus confirmed and its stability at high temperature was demonstrated. This study confirmed the value of C. annuum as a source of complete spectrum resistance to the major RKN.

International audience

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes., Author Summary Parasitic nematodes are microscopic worms that cause major diseases of plants, animals and humans. During compatible interactions, root-knot nematodes (RKN) induce the formation of galls in which redifferentiation of root cells into multinucleate and hypertrophied giant cells is essential for nematode growth and reproduction. The importance of glutathione (GSH), a major antioxidant molecule involved in plant development, in plant microbe interaction and in abiotic stress response, was analyzed during the plant-RKN interaction. Our analyses demonstrated that the gall development and functioning are characterized by an adapted GSH metabolism and that depletion of GSH content impairs nematode reproduction and modified sex ratio. This phenotype is linked to specific modifications of carbon metabolism which do not occur in uninfected roots indicating a peculiar metabolism of this neoformed organ. This first metabolomic analysis during the plant-RKN interaction highlights the regulatory role played by GSH in this pathogenic interaction and completes our vision of the role of GSH during plant-pathogen interactions. RKN sex ratio modification has previously been observed under unfavorable nematode feeding conditions suggesting that the GSH-redox system could be a general sensor of gall fitness in natural conditions.

Expanding genomic data on plant pathogens open new perspectives for the development of specific and environment friendly pest management strategies based on the inhibition of parasitism genes that are essential for the success of infection. Identifying such genes relies on accurate reverse genetics tools and the screening of pathogen knock-down phenotypes. Root-knot nematodes are major cosmopolitan crop pests that feed on a wide range of host plants. Small interfering RNAs (siRNAs) would provide a powerful tool for reverse genetics of nematode parasitism genes provided that they could (1) target genes expressed in inner tissues of infective nematodes and (2) target genes expressed during parasitism. In this study, we show that siRNAs can access inner tissues of the infective juveniles during soaking and accumulate in the esophagus, amphidial pouches and related neurons of the nematode. We provide evidence that siRNAs can trigger knock-down of the parasitism gene Mi-CRT, a calreticulin gene expressed in the esophageal glands of Meloidogyne incognita. Mi-CRT knock-down in infective juveniles affected nematode virulence. However, Mi-CRT knock-down was not persistent after plant infection, indicating that siRNA-mediated RNAi is best suited for functional analysis of genes involved in pre-parasitic stages or in the early steps of infection.

Two highly reiterated StyI satellite DNAs have been cloned from two nematode species: one from Meloidogyne hapla and another from M. incognita. The monomeric units of these two satellites have a repeat length of 169 and 295 bp, respectively. These StyI repeated element families constitute 5% of the M. hapla and 2.5% of the M. incognita haploid genomes. The A + T content is elevated in both families (i.e., 68% and 77%, respectively). Nucleotide methylation and transcriptional activity are negative. No similarity was found between the two satellites, nor to other known highly repetitive elements. These StyI satellite DNAs are quite homogenous in sequence, showing on average 3% and 3.5% divergence from their respective calculated consensus sequence. An internal subrepeating unit of about 11 bp is observed in the StyI satellite monomer sequences of M. hapla, suggesting that it could have evolved from a shorter sequence. Because of the small size of the Meloidogyne genome (51 Mb) and the abundance of repeated sequences, this genus approaches a limit in terms of coding fraction.

Reorganization of the microtubule network is important for the fast isodiametric expansion of giant-feeding cells induced by root-knot nematodes. The efficiency of microtubule reorganization depends on the nucleation of new microtubules, their elongation rate and activity of microtubule severing factors. New microtubules in plants are nucleated by cytoplasmic or microtubule-bound γ-tubulin ring complexes. Here we investigate the requirement of γ-tubulin complexes for giant feeding cells development using the interaction between Arabidopsis and Meloidogyne spp. as a model system. Immunocytochemical analyses demonstrate that γ-tubulin localizes to both cortical cytoplasm and mitotic microtubule arrays of the giant cells where it can associate with microtubules. The transcripts of two Arabidopsis γ-tubulin (TUBG1 and TUBG2) and two γ-tubulin complex proteins genes (GCP3 and GCP4) are upregulated in galls. Electron microscopy demonstrates association of GCP3 and γ-tubulin as part of a complex in the cytoplasm of giant cells. Knockout of either or both γ-tubulin genes results in the gene dose-dependent alteration of the morphology of feeding site and failure of nematode life cycle completion. We conclude that the γ-tubulin complex is essential for the control of microtubular network remodelling in the course of initiation and development of giant-feeding cells, and for the successful reproduction of nematodes in their plant hosts., Author Summary Among plant pathogens, sedentary endoparasitic nematodes are one of the most damaging pests in global agriculture. Nematodes are greatly resistant due to a broad physiological variability, consequently difficult to fight against. The use of pesticides is highly pollutant to the environment and therefore new strategies must be envisaged. As nematodes induce fragmentation and long-term rearrangements of the plant cytoskeleton during infection, manipulation of cytoskeleton components necessary for parasitism could be used as targets for the development of resistant plants provoking the awareness of biotechnology companies and crop breeders in developing new strategies for the control of pathogen infection. Here we report the first stable γ-tubulin-GFP expressing plant line and provide compelling evidence for the physical interaction between components of the γTuRC, γ-tubulin and γ-tubulin-complex protein 3 (GCP3) as part of free cytoplasmic and microtubules associated complexes. We show here that γTuRC is an essential component of the microtubule nucleation machinery during giant cell development. The reduction of γ-tubulin and GCP4 levels compromises γTuRC functioning and affects microtubule nucleation in giant-feeding cells, delaying their development and affecting nematode reproduction. We conclude that upregulation of microtubule nucleation induced by γTuRC is essential for the nematode parasitism and this process can be targeted in order to protect plants against nematode infection.

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes., Author Summary Many organisms regulate gene expression through an RNA interference (RNAi) pathway, first characterized in the nematode Caenorhabditis elegans. This pathway can be triggered experimentally using double-stranded (ds)RNA to selected gene targets, thereby allowing researchers to ‘silence’ individual genes and so investigate their function. It is hoped that this technology will facilitate gene silencing in important parasitic nematodes that impose a considerable health and economic burden on mankind. Unfortunately, differences in RNAi susceptibility have been observed between species. Here we investigated the possibility that differences in the complement of effector proteins involved in the RNAi pathway are responsible for these differences in susceptibility. Our data revealed that most facets of the RNAi pathway are well represented across parasitic nematodes, although there were fewer pathway proteins in other nematodes compared to C. elegans. In contrast, the proteins responsible for uptake and spread of dsRNA are not well represented in parasitic nematodes. However, the importance of these differences is undermined by our observation that the protein complements in all the parasites were qualitatively similar, regardless of RNAi-susceptibility. Clearly, differences in the RNAi pathway of parasitic nematodes do not explain the variations in susceptibility to experimental RNAi.

SummaryI factors are transposable elements ofDrosophila melanogaster similarto mammalian LINEs, that transpose by reverse transcription of an RNA intermediate and are responsible for the I–R system of hybrid dysgenesis. There are two categories of strains in this species: inducer, that contain about 15 I elements at the various sites on chromosomal arms, and reactive, that lack active I factors. I elements occur in variousDrosophilaspecies. Potentially functional I factors fromDrosophila teissierican transpose when introduced by P-element-mediated transformation in a reactive strain ofDrosophila melanogaster. We have studied the properties ofDrosophila melanogasterstrains into which such an I factor fromDrosophila teissieri, namedItei, was introduced. Typical hybrid dysgenesis is produced when males carryingIteiare crossed with reactive females. However, more than one copy of the element seems necessary to produce dysgenic traits, whereas only one I factor ofDrosophila melanogasterseems to be sufficient. The copy number ofIteiin transformed lines maintained by endogamous crosses increases rapidly and stabilizes at values similar to those observed in inducer strains. AsDrosophila teissiericontains much fewer copies than theDrosophila melanogasterstrains, this suggests that the copy number of I elements is not simply regulated by sequences present in the element itself.

Plant-parasitic nematodes of the genus Meloidogyne are known to reproduce either by cross-fertilization (amphimixis), facultative meiotic parthenogenesis or obligatory mitotic parthenogenesis. Among them, M. incognita, M. arenaria and M. javanica are obligatory mitotic parthenogenetic species, while M. hapla can reproduce by both cross-fertilization and meiotic parthenogenesis. Phylogenetic relationships in this genus have been investigated by hybridization of BamHI-digested genomic DNAs of 18 geographical isolates belonging to six species with three homologous repeated DNA probes cloned at random from a genomic library of one population of M. incognita. Due to the repetitive nature of the probes, the autoradiograms exhibited extensive restriction fragment length polymorphisms (RFLPs) both between and within nematode species. Genetic distance values estimated from hybridization patterns were analysed by two phylogenetic tree-building distance methods, respectively based on constant (UPGMA) and varying (FITCH) rates of nucleotide substitution, and the resulting dendrograms showed a very similar clustering of species and populations. Comparison of these results with the other sources of phylogenetic data available for this genus, i.e. cytogenetic, isoenzymatic and mitochondrial DNA (mtDNA) data, revealed consistency with all but the mtDNA phylogeny. Due to the maternal inheritance of mtDNA, and the parthenogenetic reproductive mode of these organisms, which excludes any possibility of horizontal transfer, we conclude that nuclear DNA phylogeny should represent a more likely evolutionary history of this particular genus, and that interspecific hybridizations between sexual ancestors may account for the results with mtDNA. Thus the early split off of the mitotically parthenogenetic species cluster and M. hapla confirms the amphimictic ancestral mode of reproduction of root-knot nematodes. Moreover, the existence of polymorphism within each species at the repeated DNA level is discussed in relation to the adaptative evolution of these parthenogenetic species.

Background Microsatellites are the most popular source of molecular markers for studying population genetic variation in eukaryotes. However, few data are currently available about their genomic distribution and abundance across the phylum Nematoda. The recent completion of the genomes of several nematode species, including Meloidogyne incognita, a major agricultural pest worldwide, now opens the way for a comparative survey and analysis of microsatellites in these organisms. Results Using MsatFinder, the total numbers of 1-6 bp perfect microsatellites detected in the complete genomes of five nematode species (Brugia malayi, Caenorhabditis elegans, M. hapla, M. incognita, Pristionchus pacificus) ranged from 2,842 to 61,547, and covered from 0.09 to 1.20% of the nematode genomes. Under our search criteria, the most common repeat motifs for each length class varied according to the different nematode species considered, with no obvious relation to the AT-richness of their genomes. Overall, (AT)n, (AG)n and (CT)n were the three most frequent dinucleotide microsatellite motifs found in the five genomes considered. Except for two motifs in P. pacificus, all the most frequent trinucleotide motifs were AT-rich, with (AAT)n and (ATT)n being the only common to the five nematode species. A particular attention was paid to the microsatellite content of the plant-parasitic species M. incognita. In this species, a repertoire of 4,880 microsatellite loci was identified, from which 2,183 appeared suitable to design markers for population genetic studies. Interestingly, 1,094 microsatellites were identified in 801 predicted protein-coding regions, 99% of them being trinucleotides. When compared against the InterPro domain database, 497 of these CDS were successfully annotated, and further assigned to Gene Ontology terms. Conclusions Contrasted patterns of microsatellite abundance and diversity were characterized in five nematode genomes, even in the case of two closely related Meloidogyne species. 2,245 di- to hexanucleotide loci were identified in the genome of M. incognita, providing adequate material for the future development of a wide range of microsatellite markers in this major plant parasite.

International audience; Lateral gene transfer from prokaryotes to animals is poorly understood, and the scarce documented examples generally concern genes of uncharacterized role in the receiver organism. In contrast, in plant-parasitic nematodes, several genes, usually not found in animals and similar to bacterial homologs, play essential roles for successful parasitism. Many of these encode plant cell wall-degrading enzymes that constitute an unprecedented arsenal in animals in terms of both abundance and diversity. Here we report that independent lateral gene transfers from different bacteria, followed by gene duplications and early gain of introns, have shaped this repertoire. We also show protein immunolocalization data that suggest additional roles for some of these cell wall-degrading enzymes in the late stages of these parasites' life cycle. Multiple functional acquisitions of exogenous genes that provide selective advantage were probably crucial for the emergence and proficiency of plant parasitism in nematodes

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

SUMMARYPine wilt is the most serious disease of native pines in Japan and potentially the most important nematode disease of conifers in the world. The pinewood nematode Bursaphelenchus xylophilus was found to be the causal agent. Difficulties arose with respect to the precise identity of some isolates of B. xylophilus and of similar species B. mucronatus and B. fraudulentus. Restriction enzyme analyses of repetitive DNA revealed bands specific for the species B. xylophilus, B. mucronatus and B. fraudulentus. Hybridization patterns obtained with unc-22 gene of C. elegans, clearly identified B. xylophilus, B. mucronatus and B. fraudulentus as well as the different geographic isolates of these species. Furthermore, it is possible to define the phylogenetic relationships between the different populations constituting the ‘pine wood nematode’ complex.

The root knot nematode, Meloidogyne incognita, is an obligate parasite that causes significant damage to a broad range of host plants. Infection is associated with secretion of proteins surrounded by proliferating cells. Many parasites are known to secrete effectors that interfere with plant innate immunity, enabling infection to occur; they can also release pathogen-associated molecular patterns (PAMPs, e.g., flagellin) that trigger basal immunity through the nematode stylet into the plant cell. This leads to suppression of innate immunity and reprogramming of plant cells to form a feeding structure containing multinucleate giant cells. Effectors have generally been discovered using genetics or bioinformatics, but M. incognita is non-sexual and its genome sequence has not yet been reported. To partially overcome these limitations, we have used mass spectrometry to directly identify 486 proteins secreted by M. incognita. These proteins contain at least segmental sequence identity to those found in our 3 reference databases (published nematode proteins; unpublished M. incognita ESTs; published plant proteins). Several secreted proteins are homologous to plant proteins, which they may mimic, and they contain domains that suggest known effector functions (e.g., regulating the plant cell cycle or growth). Others have regulatory domains that could reprogram cells. Using in situ hybridization we observed that most secreted proteins were produced by the subventral glands, but we found that phasmids also secreted proteins. We annotated the functions of the secreted proteins and classified them according to roles they may play in the development of root knot disease. Our results show that parasite secretomes can be partially characterized without cognate genomic DNA sequence. We observed that the M. incognita secretome overlaps the reported secretome of mammalian parasitic nematodes (e.g., Brugia malayi), suggesting a common parasitic behavior and a possible conservation of function between metazoan parasites of plants and animals., Author Summary Parasitic nematodes are microscopic worms that cause major diseases of plants, animals, and humans. Infection is associated with secretion of proteins by the parasite; these proteins suppress the immune system and cause other changes to host cells that are required for infection. Identification of secreted proteins has been difficult because they are released only in trace amounts. We have developed very sensitive methods that enabled the discovery of 486 proteins secreted by the root knot nematode, Meloidogyne incognita; prior to this, only a handful of secreted proteins were known. Several secreted proteins appear to mimic normal plant proteins, and they may participate in the process by which the nematode hijacks the plant cell for its own purposes. Meloidogyne species infect many crops, including corn, soybean, cotton, rice, tomato, carrots, alfalfa, and tobacco. The discovery of these secreted proteins could lead to new methods for protecting these important crops from nematode damage. We observed that the secretome of the human pathogen, Brugia malayi, overlaps that of M. incognita, suggesting a common parasitic behavior between pathogens of plants and animals.

The root-knot nematode (Meloidogyne spp.) is a major plant pathogen, affecting several solanaceous crops worldwide. In Capsicum annuum, resistance to this pathogen is controlled by several independent dominant genes--the Me genes. Six Me genes have previously been shown to be stable at high temperature in three highly resistant and genetically distant accessions: PI 322719, PI 201234, and CM334 (Criollo de Morelos 334). Some genes (Me4, Mech1, and Mech2) are specific to certain Meloidogyne species or populations, whereas others (Me1, Me3, and Me7) are effective against a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita, the most common species in Mediterranean and tropical areas. These genes direct different response patterns in root cells depending on the pepper line and nematode species. Allelism tests and fine mapping using the BSA-AFLP approach showed these genes to be different but linked, with a recombination frequency of 0.02-0.18. Three of the PCR-based markers identified in several genetic backgrounds were common to the six Me genes. Comparative mapping with CarthaGene software indicated that these six genes clustered in a single genomic region within a 28 cM interval. Four markers were used to anchor this cluster on the P9 chromosome on an intraspecific reference map for peppers. Other disease resistance factors have earlier been mapped in the vicinity of this cluster. This genomic area is colinear to chromosome T12 of tomato and chromosome XII of potato. Four other nematode resistance genes have earlier been identified in this area, suggesting that these nematode resistance genes are located in orthologous genomic regions in Solanaceae.

Cellulases from plant parasitic nematodes are encoded by multiple gene families and are thought to originate from horizontal gene transfer. Unraveling the evolution of these genes in the phylum will help understanding the evolution of plant parasitism in nematodes. Here we describe a new gene, named MI-eng-2 , that encodes a family 5 glycosyl hydrolase (GHF5) with a predicted signal peptide and devoid of linker domain and cellulose-binding domain. The β-1,4-endoglucanase activity of the protein MI-ENG-2 was confirmed in vitro and the transcription of the gene was localized in the secretory oesophageal glands of infective juveniles, suggesting that MI-ENG-2 is involved in plant cell wall degradation during parasitism. Phylogenetic and exon/intron structure analyses of β-1,4-endoglucanase genes in the order Tylenchida strengthen the hypothesis that nematode GHF5 genes result from horizontal gene transfer of a bacterial gene with a cellulose-binding domain. GHF5 gene families in Tylenchida result from gene duplications associated with occasional loss of the cellulose-binding domain and the linker domain during their evolution.

Two satellite DNAs have been characterized in the mitotic parthenogenetic root-knot nematodes Meloidogyne javanica and M. paranaensis, agriculturally important phytoparasitic species. The satellite repeat variants cloned from M. javanica could not be resolved from those described earlier in M. arenaria [Castagnone-Sereno, P., Leroy, F., Abad, P., 2000. Cloning and characterization of an extremely conserved satellite DNA family from the root-knot nematode Meloidogyne arenaria. Genome 43, 346-353] and are therefore classified as a single satellite named MARJA. However, this satellite shows 34.3% sequence divergence in comparison with the MPA1 satellite characterized in M. paranaensis, and monomer variants of both satellites are clearly distinguished by homogenized nucleotide substitutions. Nucleotide variability analysis revealed in one segment of the satellite monomer domains of high and low variability, conserved both within and between monomer variants of the two satellites. Intersatellite conservation of these domains indicates evolution of satellite sequence under different constraints, probably due to some functional interactions. In addition, high intrasatellite homogeneity, presence of ancestral mutations in groups of MARJA monomers in both M. javanica and M. arenaria and highly homogenized divergent positions in comparison with the MPA1 indicate similar sequence dynamics in mitotic parthenogenetic taxa to that observed in amphimictic species.

Among plant pathogens, sedentary endoparasitic nematodes are one of the most damaging pests in global agriculture. These obligate parasites interact with their hosts in a quite unique and intriguing way. They induce the redifferentiation of root cells into specialized feeding cells essential for nematode growth and reproduction; thus, nematodes have evolved the ability to exploit plant genes and hijack host functions for their own requirements. Various approaches to engineer plants with resistance to parasitic nematodes have been pursued, most focusing on the introduction of resistance genes. An alternative strategy to achieve resistance is to exploit the susceptibility of plant disease. Better knowledge of the plant response during the compatible interaction should allow the identification of targets to engineer resistance to parasitic nematodes in crop species.

A new cDNA, named Mi-ace-2, encoding an acetylcholinesterase, was isolated from Meloidogyne incognita. The full-length cDNA, carrying the trans-spliced SL1 leader sequence, was 2122 bp long with an open reading frame of 2058 bp. The predicted protein shared 32.3% identity with the previously identified MI ACHE and 49.1% identity with the C. elegans ACE-2. The conserved motifs involved in the catalytic triad, the choline binding site and 11 aromatic residues lining the catalytic gorge were present in the MI-ACE-2 deduced protein. RT-PCR analysis showed that Mi-ace-2 is transcribed in second stage juveniles before and after hatching, in females and in males. Phylogenetic analysis showed that MI-ACE-2 and ACE-2 were clustered in a distinct group closely related to the acetylcholinesterases secreted by animal-parasitic nematodes.

A cathepsin L protease full-length cDNA (named Mi-cpl-1 ) was characterised from infective second-stage juveniles of the root-knot nematode Meloidogyne incognita by SL1-primed PCR and 3′ rapid amplification of cDNA ends (3′-RACE). A single open reading frame was identified, encoding a putative 383-amino acid protein predicted to contain a putative N-terminal short secretion signal peptide, which suggests that Mi-CPL-1 should function as an extracellular protein. The putative mature enzyme included several conserved amino acid residues/motives, among which the typical catalytic triad of the active site. In situ mRNA hybridization analyses showed that transcripts of Mi-cpl-1 accumulated specifically in the intestinal cells of specimens, which suggests that the protein could be involved in the digestive processes of the nematode. However, expression of Mi-cpl-1 was shown to occur exclusively in the developmental stages which are in close interaction with the root tissues (i.e. second-stage juveniles and females). This may indicate that some function of the cathepsin L cysteine protease in M. incognita is more directly related to the parasitic aspects of the plant-nematode relationship, e.g. pathogenicity and/or evasion of primary host plant defence systems.

International audience; Arabidopsis thaliana transgenic plants expressing the mannose binding lectin GNA from snowdrop (Galanthus nivalis), under the control of the constitutive 35S promoter of the Cauliflower Mosaic Virus (CaMV) with an enhancer sequence were produced. They were used to investigate the potential of this lectin to control the root-knot nematode Meloidogyne incognita. Homozygous lines expressing GNA in roots at an estimated range of 0.1–1.3% of total soluble proteins were selected. Protection against root-knot nematodes was investigated in an in vitro test. Some lines had a significant decrease (20–50%) in root gall number. However, one line showed a higher gall number compared to the control line. This effect, which is not correlated to GNA protein root level, suggests a complex action mechanism of lectin against nematodes. Nevertheless, GNA seems to have a strong potential against M. incognita and could be used as a component of integrated pest management programs

The species X. index, X. diversicaudatum, X. vuittenezi, and X. italiae are established (E) or putative (P) vectors of Grapevine fanleaf virus (GFLV) (E), Arabis mosaic virus (E), Grapevine chrome mosaic virus (P), and GFLV (P) nepoviruses of grapevine, respectively. All four species are very closely related taxonomically and their low field densities make them difficult to identify from morphological and morphometrical diagnostic characters when only single or few individuals are detected. To improve diagnostic accuracy, a simple method was developed. The internal transcribed spacer 1 (ITS1) region spanning the 18S and 5.8S ribosomal genes was sequenced in one population of each species using two conserved primers from these genes. The ITS1 fragments were 1,132 bp (X. vuittenezi), 1,153 bp (X. index), 1,175 bp (X. diversicaudatum), and 1,190 bp (X. italiae), i.e., a difference of over 5% between the extremes. The sequence variability made it possible to design species-specific internal sense primers that amplified, in combination with the same antisense ITS1 primer, a single signature fragment (340 bp for X. index, 414 bp for X. italiae, 591 bp for X. vuittenezi, and 813 bp for X. diversicaudatum). Tests with DNA from a single adult or juvenile nematode confirmed the specificity of the primers from diverse isolates or populations. The primers were successfully used in a multiplex test for the reliable detection of two to four mixed species, each represented by a single individual. This multiplex-based diagnostic tool will be particularly useful for successful nematode management practices in vineyards.

Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.

International audience