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

Danchin EG, Arguel MJ, Campan-Fournier A, Perfus-Barbeoch L, Magliano M, Rosso MN, Da Rocha M, Da Silva C, Nottet N, Labadie K, Guy J, Artiguenave F, Abad P - PLoS Pathog. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
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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Related in: MedlinePlus

Phylogenetic tree of selected species for OrthoMCL comparison.The relative phylogenetic position and simplified taxonomy of the 25 species included in the OrthoMCL comparison of whole proteomes. The topology is according to the NCBI's taxonomy, except within the nematode and insecta lineages which are according to ref [45] and to ref [46], respectively. Species that are known plant-parasites plant-pathogens or phytophagous are highlighted in green and with a plant symbol. The animal-parasitic nematode B. malayi is highlighted in red and with a dog symbol. All the other species, in black, with a “wrong way” sign, are blacklisted.
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ppat-1003745-g002: Phylogenetic tree of selected species for OrthoMCL comparison.The relative phylogenetic position and simplified taxonomy of the 25 species included in the OrthoMCL comparison of whole proteomes. The topology is according to the NCBI's taxonomy, except within the nematode and insecta lineages which are according to ref [45] and to ref [46], respectively. Species that are known plant-parasites plant-pathogens or phytophagous are highlighted in green and with a plant symbol. The animal-parasitic nematode B. malayi is highlighted in red and with a dog symbol. All the other species, in black, with a “wrong way” sign, are blacklisted.

Mentions: To select RKN proteins without predicted homologs in non-target species, we first performed an OrthoMCL [9] analysis comparing all predicted proteins in M. incognita and M. hapla (34,780 proteins) with the whole proteomes of 23 other species (Figure 2). This step was aimed at eliminating RKN proteins having evident orthologs in fully-sequenced non-target genomes and to substantially reduce the number of proteins that will be subsequently compared against the NCBI's nr library. We selected, in priority, species whose whole genomes have been annotated to a quality level allowing a reliable prediction of the ensemble of protein-coding genes. Our selection of species comprised 4 other nematodes, 5 insects, 9 vertebrates (including mammals, ray-finned fishes, amphibian and sauropsida), 4 fungi and 1 plant. Among selected species, we included two plant-pathogenic fungi (Magnaporthe grisea and Fusarium graminearum), one nematode parasite of animals (Brugia malayi) and two insects that feed on living plant tissue (Acyrthosiphon pisum and Bombyx mori). The 18 other species were blacklisted and whenever a RKN protein had a predicted ortholog in these blacklisted species, the protein was discarded from the rest of the analysis. According to OrthoMCL, a total of 15,181 RKN proteins had a predicted ortholog in at least one blacklisted species and were thus eliminated. The rest of RKN proteins (19,599) had no predicted ortholog in any of the blacklisted species and passed this first filter. Among these proteins, a total of 2,446 were redundant between M. incognita and M. hapla. To avoid redundancy, and because subsequent biological assays will be performed in M. incognita, we kept as representative the M. incognita versions. At the end of this first filtering step, a total of 17,153 Meloidogyne proteins were kept for further analysis.


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

Danchin EG, Arguel MJ, Campan-Fournier A, Perfus-Barbeoch L, Magliano M, Rosso MN, Da Rocha M, Da Silva C, Nottet N, Labadie K, Guy J, Artiguenave F, Abad P - PLoS Pathog. (2013)

Phylogenetic tree of selected species for OrthoMCL comparison.The relative phylogenetic position and simplified taxonomy of the 25 species included in the OrthoMCL comparison of whole proteomes. The topology is according to the NCBI's taxonomy, except within the nematode and insecta lineages which are according to ref [45] and to ref [46], respectively. Species that are known plant-parasites plant-pathogens or phytophagous are highlighted in green and with a plant symbol. The animal-parasitic nematode B. malayi is highlighted in red and with a dog symbol. All the other species, in black, with a “wrong way” sign, are blacklisted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814813&req=5

ppat-1003745-g002: Phylogenetic tree of selected species for OrthoMCL comparison.The relative phylogenetic position and simplified taxonomy of the 25 species included in the OrthoMCL comparison of whole proteomes. The topology is according to the NCBI's taxonomy, except within the nematode and insecta lineages which are according to ref [45] and to ref [46], respectively. Species that are known plant-parasites plant-pathogens or phytophagous are highlighted in green and with a plant symbol. The animal-parasitic nematode B. malayi is highlighted in red and with a dog symbol. All the other species, in black, with a “wrong way” sign, are blacklisted.
Mentions: To select RKN proteins without predicted homologs in non-target species, we first performed an OrthoMCL [9] analysis comparing all predicted proteins in M. incognita and M. hapla (34,780 proteins) with the whole proteomes of 23 other species (Figure 2). This step was aimed at eliminating RKN proteins having evident orthologs in fully-sequenced non-target genomes and to substantially reduce the number of proteins that will be subsequently compared against the NCBI's nr library. We selected, in priority, species whose whole genomes have been annotated to a quality level allowing a reliable prediction of the ensemble of protein-coding genes. Our selection of species comprised 4 other nematodes, 5 insects, 9 vertebrates (including mammals, ray-finned fishes, amphibian and sauropsida), 4 fungi and 1 plant. Among selected species, we included two plant-pathogenic fungi (Magnaporthe grisea and Fusarium graminearum), one nematode parasite of animals (Brugia malayi) and two insects that feed on living plant tissue (Acyrthosiphon pisum and Bombyx mori). The 18 other species were blacklisted and whenever a RKN protein had a predicted ortholog in these blacklisted species, the protein was discarded from the rest of the analysis. According to OrthoMCL, a total of 15,181 RKN proteins had a predicted ortholog in at least one blacklisted species and were thus eliminated. The rest of RKN proteins (19,599) had no predicted ortholog in any of the blacklisted species and passed this first filter. Among these proteins, a total of 2,446 were redundant between M. incognita and M. hapla. To avoid redundancy, and because subsequent biological assays will be performed in M. incognita, we kept as representative the M. incognita versions. At the end of this first filtering step, a total of 17,153 Meloidogyne proteins were kept for further analysis.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus