Combinatorial in vitro RNAi of two neuropeptide genes and a pharyngeal gland gene on Meloidogyne incognita

in Nematology
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Root-knot nematodes are the most economically important group of plant-parasitic nematodes. In the present study, functional validation using in vitro RNAi was carried out on Meloidogyne incognita with two FMRFamide-like peptide genes, flp-14 and flp-18, and a subventral pharyngeal gland specific gene, 16D10. It was found that RNAi silencing of each gene reduced the attraction of M. incognita at different time intervals both in combination and individually. Silencing of the genes reduced nematode infection by 23-30% and development as indicated by a reduction in the number of females by 26-62%. Reproduction was decreased by 27-73% and fecundity was decreased by 19-51%. In situ hybridisation revealed the expression of flp-18 in cells associated with the ventral and retro vesicular ganglia of the central nervous system. qRT-PCR supported the correlation between phenotypic effects of silencing with that of transcript quantification.

Nematology

International Journal of Fundamental and Applied Nematological Research

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References

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Figures

  • Multiple sequence alignment of flp-18 showing similarity among plant-parasitic (Meloidogyne chitwoodi, M. javanica, M. hapla, M. incognita), animal-parasitic (Ascaris suum) and free-living (Caenorhabditis elegans) nematodes.

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  • Phylogenetic tree showing the relationship of flp-18 sequence among Meloidogyne spp., Ascaris suum and Caenorhabditis elegans. Numbers in the figure indicate distance.

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  • Multiple sequence alignment of 16D10. The signal peptide is marked in blue, CLE3 domain marked in red. MEJA: Meloidogyne javanica; MELIC: M. incognita; MELHA: M. hapla; MELAR: M. arenaria; HETGL: Heterodera glycines. This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/15685411.

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  • Images showing in situ hybridisation of digoxigenin-labelled DNA probes to the flp-18 gene in second-stage juveniles of Meloidogyne incognita. A, C: No colour was detected in the nerve ring with sense strand probes; B, D: Nematode nerve ring region showing flp-18 gene expression in vesicular and retro-vesicular ganglion of the nervous system.

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  • Effect of gene silencing on the attraction of second-stage juveniles of Meloidogyne incognita towards host tomato roots in Pluronic gel assays after 2, 4, 8, 24 and 72 h. Bars = standard error. Duncan’s multiple range test (P<0.01) was used to determine significant differences indicated by different lower case letters. GFP = green fluorescent protein.

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  • Effect of gene silencing on the penetration of second-stage juveniles of Meloidogyne incognita into host tomato roots in Pluronic gel assays. Bars = standard error. Duncan’s multiple range test (P<0.01) was used to determine significant differences indicated by different lower case letters. GFP = green fluorescent protein.

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  • Quantitative real time PCR to find the mRNA levels of target gene silenced in second-stage juveniles of Meloidogyne incognita. A: Single silenced nematodes; B: Silenced with combination of three genes simultaneously; C: Silenced with different combination of two genes at a time. Cytochrome c oxidase was used as reference gene and fold change was calculated by using the 2ΔΔCT method. The fold change values were transformed to personate with percentage values. Error bars = ±SD among the biological replicates; ∗∗ P<0.01. GFP = green fluorescent protein.

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