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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

Transcript abundance percentage change in siRNA soaked J2s relative to control.siRNAs induced significant change in the targeted transcripts expression level. Transcript level for each of the targeted gene was measured by qPCR 24M. incognita genome (siRNA random).
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ppat-1003745-g005: Transcript abundance percentage change in siRNA soaked J2s relative to control.siRNAs induced significant change in the targeted transcripts expression level. Transcript level for each of the targeted gene was measured by qPCR 24M. incognita genome (siRNA random).

Mentions: Six siRNAs induced significant reduction in the corresponding targeted transcripts (Minc00801, Minc01632, Minc02483, Minc08335, Minc09526, Minc17987) 24 h after soaking treatment, compared to their expression level in control samples (Table 2, Figure 5). On the other hand, Minc08013, Minc08014 and Minc12224 siRNAs induced a diminution that was not reproducible between independent qPCR replicate experiments (data not shown). Surprisingly, 7 siRNAs (Minc03313, Minc03866, Minc05001, Minc10706, Minc14652, Minc17713, Minc07817) induced significant and reproducible increase in transcript abundance when qPCR was performed 24 h after soaking. A similar ‘bounce’ effect, that could be due to a response of the nematode RNAi machinery to increased siRNA quantities within the cells, had already been reported in PPN after gene silencing [21], [22], [23]. To test this ‘bounce’ effect, we performed qPCR analysis 16 h after soaking on Minc07817, Minc03866 and Minc05001. Confirming siRNA knockdown and bounce effect, Minc03866 was significantly silenced 16 h after soaking. However, Minc05001 expression was reduced but not significantly and Minc07817 expression was still higher than control (Figure S1). Overall, our qPCR results indicate that the designed siRNAs, were efficient to knockdown 7 out of the 16 target genes at the tested time points.


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)

Transcript abundance percentage change in siRNA soaked J2s relative to control.siRNAs induced significant change in the targeted transcripts expression level. Transcript level for each of the targeted gene was measured by qPCR 24M. incognita genome (siRNA random).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003745-g005: Transcript abundance percentage change in siRNA soaked J2s relative to control.siRNAs induced significant change in the targeted transcripts expression level. Transcript level for each of the targeted gene was measured by qPCR 24M. incognita genome (siRNA random).
Mentions: Six siRNAs induced significant reduction in the corresponding targeted transcripts (Minc00801, Minc01632, Minc02483, Minc08335, Minc09526, Minc17987) 24 h after soaking treatment, compared to their expression level in control samples (Table 2, Figure 5). On the other hand, Minc08013, Minc08014 and Minc12224 siRNAs induced a diminution that was not reproducible between independent qPCR replicate experiments (data not shown). Surprisingly, 7 siRNAs (Minc03313, Minc03866, Minc05001, Minc10706, Minc14652, Minc17713, Minc07817) induced significant and reproducible increase in transcript abundance when qPCR was performed 24 h after soaking. A similar ‘bounce’ effect, that could be due to a response of the nematode RNAi machinery to increased siRNA quantities within the cells, had already been reported in PPN after gene silencing [21], [22], [23]. To test this ‘bounce’ effect, we performed qPCR analysis 16 h after soaking on Minc07817, Minc03866 and Minc05001. Confirming siRNA knockdown and bounce effect, Minc03866 was significantly silenced 16 h after soaking. However, Minc05001 expression was reduced but not significantly and Minc07817 expression was still higher than control (Figure S1). Overall, our qPCR results indicate that the designed siRNAs, were efficient to knockdown 7 out of the 16 target genes at the tested time points.

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