Publications by authors named "Martine Da Rocha"

24 Publications

  • Page 1 of 1

Movements of transposable elements contribute to the genomic plasticity and species diversification in an asexually reproducing nematode pest.

Evol Appl 2021 Jul 15;14(7):1844-1866. Epub 2021 May 15.

Université Côte d'Azur INRAE CNRS ISA Sophia Antipolis France.

Despite reproducing without sexual recombination, is an adaptive and versatile phytoparasitic nematode. This species displays a global distribution, can parasitize a large range of plants, and can overcome plant resistance in a few generations. The mechanisms underlying this adaptability remain poorly known. At the whole-genome level, only a few single nucleotide variations have been observed across different geographical isolates with distinct ranges of compatible hosts. Exploring other factors possibly involved in genomic plasticity is thus important. Transposable elements (TEs), by their repetitive nature and mobility, can passively and actively impact the genome dynamics. This is particularly expected in polyploid hybrid genomes such as the one of . . Here, we have annotated the TE content of . , analyzed the statistical properties of this TE landscape, and used whole-genome pool-seq data to estimate the mobility of these TEs across twelve geographical isolates, presenting variations in ranges of compatible host plants. DNA transposons are more abundant than retrotransposons, and the high similarity of TE copies to their consensus sequences suggests they have been at least recently active. We have identified loci in the genome where the frequencies of presence of a TE showed substantial variations across the different isolates. Overall, variations in TE frequencies across isolates followed their phylogenetic divergence, suggesting TEs participate in the species diversification. Compared with the .  reference genome, we detected isolate and lineage-specific de novo insertion of some TEs, including within genic regions or in the upstream regulatory regions. We validated by PCR the insertion of some of these TEs inside genic regions, confirming TE movements have possible functional impacts. Overall, we show DNA transposons can drive genomic plasticity in .  and their role in genome evolution of other parthenogenetic animal deserves further investigation.
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http://dx.doi.org/10.1111/eva.13246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8288018PMC
July 2021

Genome Expression Dynamics Reveal the Parasitism Regulatory Landscape of the Root-Knot Nematode and a Promoter Motif Associated with Effector Genes.

Genes (Basel) 2021 05 18;12(5). Epub 2021 May 18.

INRAE, Institut Sophia Agrobiotech, Université Côte d'Azur, CNRS, 06903 Sophia Antipolis, France.

Root-knot nematodes (genus ) are the major contributor to crop losses caused by nematodes. These nematodes secrete effector proteins into the plant, derived from two sets of pharyngeal gland cells, to manipulate host physiology and immunity. Successful completion of the life cycle, involving successive molts from egg to adult, covers morphologically and functionally distinct stages and will require precise control of gene expression, including effector genes. The details of how root-knot nematodes regulate transcription remain sparse. Here, we report a life stage-specific transcriptome of . Combined with an available annotated genome, we explore the spatio-temporal regulation of gene expression. We reveal gene expression clusters and predicted functions that accompany the major developmental transitions. Focusing on effectors, we identify a putative cis-regulatory motif associated with expression in the dorsal glands, providing an insight into effector regulation. We combine the presence of this motif with several other criteria to predict a novel set of putative dorsal gland effectors. Finally, we show this motif, and thereby its utility, is broadly conserved across the genus, and we name it Mel-DOG. Taken together, we provide the first genome-wide analysis of spatio-temporal gene expression in a root-knot nematode and identify a new set of candidate effector genes that will guide future functional analyses.
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http://dx.doi.org/10.3390/genes12050771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158474PMC
May 2021

The molecular signatures of compatible and incompatible pollination in Arabidopsis.

BMC Genomics 2021 Apr 14;22(1):268. Epub 2021 Apr 14.

Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, Inria, F-69342, Lyon, France.

Background: Fertilization in flowering plants depends on the early contact and acceptance of pollen grains by the receptive papilla cells of the stigma. Deciphering the specific transcriptomic response of both pollen and stigmatic cells during their interaction constitutes an important challenge to better our understanding of this cell recognition event.

Results: Here we describe a transcriptomic analysis based on single nucleotide polymorphisms (SNPs) present in two Arabidopsis thaliana accessions, one used as female and the other as male. This strategy allowed us to distinguish 80% of transcripts according to their parental origins. We also developed a tool which predicts male/female specific expression for genes without SNP. We report an unanticipated transcriptional activity triggered in stigma upon incompatible pollination and show that following compatible interaction, components of the pattern-triggered immunity (PTI) pathway are induced on the female side.

Conclusions: Our work unveils the molecular signatures of compatible and incompatible pollinations both at the male and female side. We provide invaluable resource and tools to identify potential new molecular players involved in pollen-stigma interaction.
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http://dx.doi.org/10.1186/s12864-021-07503-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048354PMC
April 2021

Genome assembly and annotation of Meloidogyne enterolobii, an emerging parthenogenetic root-knot nematode.

Sci Data 2020 10 5;7(1):324. Epub 2020 Oct 5.

Université Côte d'Azur, INRAE, CNRS, Institut Sophia Agrobiotech, Sophia Antipolis, France.

Root-knot nematodes (genus Meloidogyne) are plant parasites causing huge economic loss in the agricultural industry and affecting severely numerous developing countries. Control methods against these plant pests are sparse, the preferred one being the deployment of plant cultivars bearing resistance genes against Meloidogyne species. However, M. enterolobii is not controlled by the resistance genes deployed in the crop plants cultivated in Europe. The recent identification of this species in Europe is thus a major concern. Here, we sequenced the genome of M. enterolobii using short and long-read technologies. The genome assembly spans 240 Mbp with contig N50 size of 143 kbp, enabling high-quality annotations of 59,773 coding genes, 4,068 non-coding genes, and 10,944 transposable elements (spanning 8.7% of the genome). We validated the genome size by flow cytometry and the structure, quality and completeness by bioinformatics metrics. This ensemble of resources will fuel future projects aiming at pinpointing the genome singularities, the origin, diversity, and adaptive potential of this emerging plant pest.
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http://dx.doi.org/10.1038/s41597-020-00666-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536185PMC
October 2020

Characterization of Two Satellite DNA Families in the Genome of the Oomycete Plant Pathogen .

Front Genet 2020 5;11:557. Epub 2020 Jun 5.

INRAE, UCA, CNRS, ISA Sophia Antipolis, Biot, France.

Satellite DNA is a class of repetitive sequences that are organized in long arrays of tandemly repeated units in most eukaryotes. Long considered as selfish DNA, satellite sequences are now proposed to contribute to genome integrity. Despite their potential impact on the architecture and evolution of the genome, satellite DNAs have not been investigated in oomycetes due to the paucity of genomic data and the difficulty of assembling highly conserved satellite arrays. Yet gaining knowledge on the structure and evolution of genomes of oomycete pathogens is crucial to understanding the mechanisms underlying adaptation to their environment and to proposing efficient disease control strategies. A assembly of the genome of , an important oomycete plant pathogen, led to the identification of several families of tandemly repeated sequences varying in size, copy number, and sequence conservation. Among them, two abundant families, designated as and , displayed typical features of satellite DNA and were collectively designated as . These two satellite families differ by their length, sequence, organization, genomic environment, and evolutionary dynamics. , but not , presented homologs among oomycetes. This observation, as well as the characterization of transcripts of families, suggested that these satellite DNA families likely play a conserved role within this important group of pathogens.
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http://dx.doi.org/10.3389/fgene.2020.00557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290008PMC
June 2020

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

Mol Ecol 2019 05 29;28(10):2559-2572. Epub 2019 Apr 29.

INRA, Université Côte d'Azur, CNRS, ISA, Sophia Antipolis, France.

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.
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http://dx.doi.org/10.1111/mec.15095DOI Listing
May 2019

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

BMC Genomics 2018 Dec 18;19(1):943. Epub 2018 Dec 18.

INRA, Université Côte d'Azur, CNRS, ISA, Paris, France.

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.
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http://dx.doi.org/10.1186/s12864-018-5296-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297998PMC
December 2018

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

BMC Genomics 2018 May 3;19(1):321. Epub 2018 May 3.

Université Côte d'Azur, INRA, ISA, Sophia Antipolis, France.

Background: The renewed interest in epigenetics has led to the understanding that both the environment and individual lifestyle can directly interact with the epigenome to influence its dynamics. Epigenetic phenomena are mediated by DNA methylation, stable chromatin modifications and non-coding RNA-associated gene silencing involving specific proteins called epigenetic factors. Multiple organisms, ranging from plants to yeast and mammals, have been used as model systems to study epigenetics. The interactions between parasites and their hosts are models of choice to study these mechanisms because the selective pressures are strong and the evolution is fast. The asexually reproducing root-knot nematodes (RKN) offer different advantages to study the processes and mechanisms involved in epigenetic regulation. RKN genomes sequencing and annotation have identified numerous genes, however, which of those are involved in the adaption to an environment and potentially relevant to the evolution of plant-parasitism is yet to be discovered.

Results: Here, we used a functional comparative annotation strategy combining orthology data, mining of curated genomics as well as protein domain databases and phylogenetic reconstructions. Overall, we show that (i) neither RKN, nor the model nematode Caenorhabditis elegans possess any DNA methyltransferases (DNMT) (ii) RKN do not possess the complete machinery for DNA methylation on the 6th position of adenine (6mA) (iii) histone (de)acetylation and (de)methylation pathways are conserved between C. elegans and RKN, and the corresponding genes are amplified in asexually reproducing RKN (iv) some specific non-coding RNA families found in plant-parasitic nematodes are dissimilar from those in C. elegans. In the asexually reproducing RKN Meloidogyne incognita, expression data from various developmental stages supported the putative role of these proteins in epigenetic regulations.

Conclusions: Our results refine previous predictions on the epigenetic machinery of model species and constitute the most comprehensive description of epigenetic factors relevant to the plant-parasitic lifestyle and/or asexual mode of reproduction of RKN. Providing an atlas of epigenetic factors in RKN is an informative resource that will enable researchers to explore their potential role in adaptation of these parasites to their environment.
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http://dx.doi.org/10.1186/s12864-018-4686-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934874PMC
May 2018

Transcriptome Profiling of Neurosensory Perception Genes in Wing Tissue of Two Evolutionary Distant Insect Orders: Diptera (Drosophila melanogaster) and Hemiptera (Acyrthosiphon pisum).

J Mol Evol 2017 Dec 26;85(5-6):234-245. Epub 2017 Oct 26.

Université Côte d'Azur, INRA, CNRS, ISA, Institut Agrobiotech, 400 route des Chappes, 06903, Sophia Antipolis, France.

The neurogenesis and neuronal functions in insect wing have been understudied mainly due to technical hindrances that have prevented electrophysiology studies for decades. The reason is that the nano-architecture of the wing chemosensory bristles hampers the receptors accessibility of odorants/tastants to receptors in fixed setup, whereas in nature, the wing flapping mixes these molecules in bristle lymph. In this report, we analyzed the transcriptome of the wing tissue of two species phylogenetically strongly divergent: Drosophila melanogaster a generic model for diptera order (complete metamorphosis) and the aphid acyrthosiphon pisum, representative of hemiptera order (incomplete metamorphosis) for which a conditional winged/wingless polyphenism is under control of population density and resources. The transcriptome shows that extensive gene networks involved in chemosensory perception are active in adult wing for both species. Surprisingly, the specific transcripts of genes that are commonly found in eye were present in Drosophila wing but not in aphid. The analysis reveals that in the aphid conditional wing, expressed genes show strong similarities with those in the gut epithelia. This suggests that the epithelial cell layer between the cuticle sheets is persistent at least in young aphid adult, whereas it disappears after emergence in Drosophila. Despite marked differences between the two transcriptomes, the results highlight the probable universalism of wing chemosensory function in the holometabolous and hemimetabolous orders of winged insects.
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http://dx.doi.org/10.1007/s00239-017-9814-8DOI Listing
December 2017

The Transcriptomes of Xiphinema index and Longidorus elongatus Suggest Independent Acquisition of Some Plant Parasitism Genes by Horizontal Gene Transfer in Early-Branching Nematodes.

Genes (Basel) 2017 Oct 23;8(10). Epub 2017 Oct 23.

Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

Nematodes have evolved the ability to parasitize plants on at least four independent occasions, with plant parasites present in Clades 1, 2, 10 and 12 of the phylum. In the case of Clades 10 and 12, horizontal gene transfer of plant cell wall degrading enzymes from bacteria and fungi has been implicated in the evolution of plant parasitism. We have used ribonucleic acid sequencing (RNAseq) to generate reference transcriptomes for two economically important nematode species, and , representative of two genera within the early-branching Clade 2 of the phylum Nematoda. We used a transcriptome-wide analysis to identify putative horizontal gene transfer events. This represents the first in-depth transcriptome analysis from any plant-parasitic nematode of this clade. For each species, we assembled ~30 million Illumina reads into a reference transcriptome. We identified 62 and 104 transcripts, from and , respectively, that were putatively acquired via horizontal gene transfer. By cross-referencing horizontal gene transfer prediction with a phylum-wide analysis of Pfam domains, we identified Clade 2-specific events. Of these, a GH12 cellulase from was analysed phylogenetically and biochemically, revealing a likely bacterial origin and canonical enzymatic function. Horizontal gene transfer was previously shown to be a phenomenon that has contributed to the evolution of plant parasitism among nematodes. Our findings underline the importance and the extensiveness of this phenomenon in the evolution of plant-parasitic life styles in this speciose and widespread animal phylum.
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http://dx.doi.org/10.3390/genes8100287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664137PMC
October 2017

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

New Phytol 2018 01 16;217(2):687-699. Epub 2017 Oct 16.

INRA, Université Côte d'Azur, CNRS, ISA, 400 route des Chappes, 06903, Cedex Sophia-Antipolis, France.

Root-knot nematodes, Meloidogyne spp., are obligate endoparasites that maintain a biotrophic relationship with their hosts. They infect roots as microscopic vermiform second-stage juveniles, and establish specialized feeding structures called 'giant-cells', from which they withdraw water and nutrients. The nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we compared Illumina RNA-seq transcriptomes for M. incognita obtained at various points in the lifecycle, and identified 31 genes more strongly expressed in parasitic stages than in preparasitic juveniles. We then selected candidate effectors for functional characterization. Quantitative real-time PCR and in situ hybridizations showed that the validated differentially expressed genes are predominantly specifically expressed in oesophageal glands of the nematode. We also soaked the nematodes in siRNA to silence these genes and to determine their role in pathogenicity. The silencing of the dorsal gland specific-Minc18876 and its paralogues resulted in a significant, reproducible decrease in the number of mature females with egg masses, demonstrating a potentially important role for the small glycine- and cysteine-rich effector MiSGCR1 in early stages of plant-nematode interaction. Finally, we report that MiSGCR1 suppresses plant cell death induced by bacterial or oomycete triggers of plant defense.
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http://dx.doi.org/10.1111/nph.14837DOI Listing
January 2018

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

New Phytol 2017 Nov 14;216(3):882-896. Epub 2017 Sep 14.

INRA, Université Côte d'Azur, CNRS, ISA, 400 route des Chappes, BP167, 06903, Sophia Antipolis, France.

Root knot nematodes (RKN) are root parasites that induce the genetic reprogramming of vascular cells into giant feeding cells and the development of root galls. MicroRNAs (miRNAs) regulate gene expression during development and plant responses to various stresses. Disruption of post-transcriptional gene silencing in Arabidopsis ago1 or ago2 mutants decrease the infection rate of RKN suggesting a role for this mechanism in the plant-nematode interaction. By sequencing small RNAs from uninfected Arabidopsis roots and from galls 7 and 14 d post infection with Meloidogyne incognita, we identified 24 miRNAs differentially expressed in gall as putative regulators of gall development. Moreover, strong activity within galls was detected for five miRNA promoters. Analyses of nematode development in an Arabidopsis miR159abc mutant had a lower susceptibility to RKN, suggesting a role for the miR159 family in the plant response to M. incognita. Localization of mature miR159 within the giant and surrounding cells suggested a role in giant cell and gall. Finally, overexpression of miR159 in galls at 14 d post inoculation was associated with the repression of the miR159 target MYB33 which expression is restricted to the early stages of infection. Overall, these results implicate the miR159 in plant responses to RKN.
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http://dx.doi.org/10.1111/nph.14717DOI Listing
November 2017

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

PLoS Genet 2017 06 8;13(6):e1006777. Epub 2017 Jun 8.

INRA, Université Côte d'Azur, CNRS, ISA, France.

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.
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http://dx.doi.org/10.1371/journal.pgen.1006777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465968PMC
June 2017

Nod Factor Effects on Root Hair-Specific Transcriptome of Medicago truncatula: Focus on Plasma Membrane Transport Systems and Reactive Oxygen Species Networks.

Front Plant Sci 2016 7;7:794. Epub 2016 Jun 7.

Biochimie and Physiologie Moléculaire des Plantes, UMR 5004 Centre National de la Recherche Scientifique/386 Institut National de la Recherche Agronomique/SupAgro Montpellier/Université de Montpellier, Campus SupAgro-Institut National de la Recherche Agronomique Montpellier, France.

Root hairs are involved in water and nutrient uptake, and thereby in plant autotrophy. In legumes, they also play a crucial role in establishment of rhizobial symbiosis. To obtain a holistic view of Medicago truncatula genes expressed in root hairs and of their regulation during the first hours of the engagement in rhizobial symbiotic interaction, a high throughput RNA sequencing on isolated root hairs from roots challenged or not with lipochitooligosaccharides Nod factors (NF) for 4 or 20 h was carried out. This provided a repertoire of genes displaying expression in root hairs, responding or not to NF, and specific or not to legumes. In analyzing the transcriptome dataset, special attention was paid to pumps, transporters, or channels active at the plasma membrane, to other proteins likely to play a role in nutrient ion uptake, NF electrical and calcium signaling, control of the redox status or the dynamic reprogramming of root hair transcriptome induced by NF treatment, and to the identification of papilionoid legume-specific genes expressed in root hairs. About 10% of the root hair expressed genes were significantly up- or down-regulated by NF treatment, suggesting their involvement in remodeling plant functions to allow establishment of the symbiotic relationship. For instance, NF-induced changes in expression of genes encoding plasma membrane transport systems or disease response proteins indicate that root hairs reduce their involvement in nutrient ion absorption and adapt their immune system in order to engage in the symbiotic interaction. It also appears that the redox status of root hair cells is tuned in response to NF perception. In addition, 1176 genes that could be considered as "papilionoid legume-specific" were identified in the M. truncatula root hair transcriptome, from which 141 were found to possess an ortholog in every of the six legume genomes that we considered, suggesting their involvement in essential functions specific to legumes. This transcriptome provides a valuable resource to investigate root hair biology in legumes and the roles that these cells play in rhizobial symbiosis establishment. These results could also contribute to the long-term objective of transferring this symbiotic capacity to non-legume plants.
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http://dx.doi.org/10.3389/fpls.2016.00794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894911PMC
July 2016

The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the basis of parasitism and virulence.

Genome Biol 2016 Jun 10;17(1):124. Epub 2016 Jun 10.

USDA-ARS Horticultural Crops Research Laboratory, Corvallis, OR, USA.

Background: The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes.

Results: We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative 'effector islands' in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking.

Conclusions: These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.
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http://dx.doi.org/10.1186/s13059-016-0985-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901422PMC
June 2016

Environmentally selected aphid variants in clonality context display differential patterns of methylation in the genome.

PLoS One 2014 31;9(12):e115022. Epub 2014 Dec 31.

Institute Sophia Agrobiotech, INRA/CNRS/UNS, University Nice Sophia Antipolis, Sophia Antipolis, France.

Heritability of acquired phenotypic traits is an adaptive evolutionary process that appears more complex than the basic allele selection guided by environmental pressure. In insects, the trans-generational transmission of epigenetic marks in clonal and/or sexual species is poorly documented. Aphids were used as a model to explore this feature because their asexual phase generates a stochastic and/or environment-oriented repertoire of variants. The a priori unchanged genome in clonal individuals prompts us to hypothesize whether covalent methyl DNA marks might be associated to the phenotypic variability and fitness selection. The full differential transcriptome between two environmentally selected clonal variants that originated from the same founder mother was mapped on the entire genomic scaffolds, in parallel with the methyl cytosine distribution. Data suggest that the assortments of heavily methylated DNA sites are distinct in these two clonal phenotypes. This might constitute an epigenetic mechanism that confers the robust adaptation of insect species to various environments involving clonal reproduction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115022PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281257PMC
September 2015

Identification of novel target genes for safer and more specific control of root-knot nematodes from a pan-genome mining.

PLoS Pathog 2013 Oct 31;9(10):e1003745. Epub 2013 Oct 31.

INRA, UMR 1355 ISA, Institut Sophia Agrobiotech, Sophia-Antipolis, France ; CNRS, UMR 7254 ISA, Institut Sophia Agrobiotech, Sophia-Antipolis, France ; Université de Nice Sophia-Antipolis, UMR ISA, Institut Sophia Agrobiotech, Sophia-Antipolis, France.

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.
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http://dx.doi.org/10.1371/journal.ppat.1003745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814813PMC
October 2013

Genomic evidence for ameiotic evolution in the bdelloid rotifer Adineta vaga.

Nature 2013 Aug 21;500(7463):453-7. Epub 2013 Jul 21.

University of Namur, Department of Biology, URBE, Laboratory of Evolutionary Genetics and Ecology, 5000 Namur, Belgium.

Loss of sexual reproduction is considered an evolutionary dead end for metazoans, but bdelloid rotifers challenge this view as they appear to have persisted asexually for millions of years. Neither male sex organs nor meiosis have ever been observed in these microscopic animals: oocytes are formed through mitotic divisions, with no reduction of chromosome number and no indication of chromosome pairing. However, current evidence does not exclude that they may engage in sex on rare, cryptic occasions. Here we report the genome of a bdelloid rotifer, Adineta vaga (Davis, 1873), and show that its structure is incompatible with conventional meiosis. At gene scale, the genome of A. vaga is tetraploid and comprises both anciently duplicated segments and less divergent allelic regions. However, in contrast to sexual species, the allelic regions are rearranged and sometimes even found on the same chromosome. Such structure does not allow meiotic pairing; instead, we find abundant evidence of gene conversion, which may limit the accumulation of deleterious mutations in the absence of meiosis. Gene families involved in resistance to oxidation, carbohydrate metabolism and defence against transposons are significantly expanded, which may explain why transposable elements cover only 3% of the assembled sequence. Furthermore, 8% of the genes are likely to be of non-metazoan origin and were probably acquired horizontally. This apparent convergence between bdelloids and prokaryotes sheds new light on the evolutionary significance of sex.
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http://dx.doi.org/10.1038/nature12326DOI Listing
August 2013

Contribution of lateral gene transfers to the genome composition and parasitic ability of root-knot nematodes.

PLoS One 2012 30;7(11):e50875. Epub 2012 Nov 30.

Aix-Marseille Université, Centre National de la Recherche Scientifique, LATP, UMR 7353, Evolution Biologique et Modélisation, Marseille, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050875PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511272PMC
May 2013

A root-knot nematode-secreted protein is injected into giant cells and targeted to the nuclei.

New Phytol 2012 Jun 27;194(4):924-931. Epub 2012 Apr 27.

INRA, Université Aix-Marseille, UMR1163 Biotechnologie des Champignons Filamenteux, F-13288 Marseille, France.

Root-knot nematodes (RKNs) are obligate endoparasites that maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors synthesized in the oesophageal glands and injected into the plant tissue through the syringe-like stylet certainly play a central role in these processes. In a search for nematode effectors, we used comparative genomics on expressed sequence tag (EST) datasets to identify Meloidogyne incognita genes encoding proteins potentially secreted upon the early steps of infection. We identified three genes specifically expressed in the oesophageal glands of parasitic juveniles that encode predicted secreted proteins. One of these genes, Mi-EFF1 is a pioneer gene that has no similarity in databases and a predicted nuclear localization signal. We demonstrate that RKNs secrete Mi-EFF1 within the feeding site and show Mi-EFF1 targeting to the nuclei of the feeding cells. RKNs were previously shown to secrete proteins in the apoplasm of infected tissues. Our results show that nematodes sedentarily established at the feeding site also deliver proteins within plant cells through their stylet. The protein Mi-EFF1 injected within the feeding cells is targeted at the nuclei where it may manipulate nuclear functions of the host cell.
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http://dx.doi.org/10.1111/j.1469-8137.2012.04164.xDOI Listing
June 2012

Polycalin (chlorophyllid A binding protein): a novel, very large fluorescent lipocalin from the midgut of the domestic silkworm Bombyx mori L.

Insect Biochem Mol Biol 2006 Aug 20;36(8):623-33. Epub 2006 May 20.

Unité Nationale Séricicole/INRA, 25 quai J.J. Rousseau, 69350 La Mulatière, France.

We studied a protein from the midgut of the silkworm Bombyx mori characterized by its ability to bind the prosthetic group of chlorophyll, that confers fluorescent properties to this protein. Several techniques, 2D electrophoresis purification, MS-MS and Maldi-TOF peptide sequencing, RT-PCR and nucleotide sequencing were used to obtain the nucleotide sequence and the deduced amino acid sequence. The coding sequence was compared to the gene sequence to define the number and size of introns and exons. The gene spanned 45.5 kb of DNA and consisted of 46 exons. The cDNA encoded a protein of 2721 amino acids. The protein was identified as a lipocalin with novel features. Most lipocalins are proteins with high affinity to small lipophilic molecules, with a molecular size in the 25 kDa range and a well conserved tertiary structure. The apoprotein described here revealed 15 lipocalin like structures, in line. We called this protein a polycalin (pentadecacalin).
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http://dx.doi.org/10.1016/j.ibmb.2006.05.006DOI Listing
August 2006

Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms.

Transgenic Res 2005 Aug;14(4):463-72

Unité Nationale Séricicole, INRA, 25 quai J-J Rousseau 69350 La Mulatière, France.

A gene construct was made by fusing the coding sequence of the red fluorescent protein (DsRed) to the exon 2 of the fibrohexamerin gene (fhx), that encodes a subunit of fibroin, the major silk protein of the silkworm Bombyx mori. The fusion gene was inserted into a piggyBac vector to establish a series of transgenic lines. The expression of the transgene was monitored during the course of larval life and was found restricted to the posterior silk gland cells as the endogenous fhx gene, in all the selected transgenic lines. The exogenous polypeptide was secreted into the lumen of the posterior silk gland together with fibroin, and further exported with the silk proteins as a foreign constituent of the cocoon fiber. The capacity of DsRed to emit fluorescence in the air-dried silk thread led to show that the recombinant protein was distributed over the whole length of the fiber. A remarkable property of the system lies in the localization of the globular protein at the periphery of the silk thread, allowing its rapid and easy recovery in aqueous solutions, without dissolving fibroin. The procedure represents a novel and promising strategy for the production of massive recombinant proteins of biomedical and pharmaceutical interest, with reduced cost.
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http://dx.doi.org/10.1007/s11248-005-4351-4DOI Listing
August 2005

Artificial parthenogenesis and control of voltinism to manage transgenic populations in Bombyx mori.

J Insect Physiol 2004 Aug;50(8):751-60

Unité Nationale Séricicole, INRA, 25 Quai Jean-Jacques Rousseau, 69350 La Mulatière, France.

In order to improve the management of transformed populations in a routine application of transgenesis technology in Bombyx mori, we modified its mode of reproduction and its voltinism. On one hand, after a stable integration of the gene of interest by transgenesis, it is preferable to maintain this gene in an identical genomic context through successive generations. This can be obtained by artificial parthenogenetic reproduction (ameiotic parthenogenesis) giving isogenic females identical to their transformed mother. On the other hand, it is essential to obtain continuous generations (polyvoltinism) after microinjection, in order to screen positive transgenic insects and study genetics and insertion of the transgene. Thereafter, it is more convenient to store these populations, as diapause eggs before their use in biotechnology application. We obtained such polyvoltine parthenoclones, first by selection for a parthenogenetic character in polyvoltine races, and second, by selection for a polyvoltine character in a parthenogenetic, but diapausing clone of B. mori. As diapause was directly under the control of diapause hormone (DH), we also tested direct injection of DH in female pupae of polyvoltine strains, as well as anti-DH antibody treatment to eliminate diapause in univoltine strains. We discussed the advantages and limitations of these methods and proved the feasibility in obtaining polyvoltine parthenoclones and determining the voltinism in B. mori. These methods would permit us to improve the management of populations used in transgenesis technology.
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http://dx.doi.org/10.1016/j.jinsphys.2004.06.002DOI Listing
August 2004
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