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Silencing of Mg - pat-10 and Mg - unc-87 in the Plant Parasitic Nematode Meloidogyne graminicola Using siRNAs

Author

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  • Joseph Nsengimana

    (Department of Molecular Biotechnology, Ghent University, 653 Coupure links, 9000 Gent, Belgium
    Current Address: Department of Applied Biology, Kigali Institute of Science and Technology (KIST), 3900 Avenue de la Paix, 250 Kigali, Rwanda;)

  • Lander Bauters

    (Department of Molecular Biotechnology, Ghent University, 653 Coupure links, 9000 Gent, Belgium)

  • Annelies Haegeman

    (Department of Molecular Biotechnology, Ghent University, 653 Coupure links, 9000 Gent, Belgium)

  • Godelieve Gheysen

    (Department of Molecular Biotechnology, Ghent University, 653 Coupure links, 9000 Gent, Belgium)

Abstract

Until recently, the standard method for RNA interference (RNAi)-based reverse genetics in plant parasitic nematodes (PPNs) was based on the use of long double-stranded RNA (dsRNA). This increased the chance of off-target gene silencing through interactions between different short interfering RNAs (siRNAs) and non-cognate mRNA targets. In this work, we applied gene-specific knockdown of Mg-pat-10 and Mg-unc-87 of the root knot nematode Meloidogyne graminicola , using discrete 21 bp siRNAs. The homologue of Mg-pat-10 in C. elegans encodes body wall troponin C, which is essential for muscle contraction, whereas the homologue of Mg-unc-87 encodes two proteins involved in maintenance of the structure of myofilaments in the body wall muscle of C. elegans . The knockdown at the transcript level, as seen by semi-quantitative RT-PCR analysis, indicates that the Mg-pat-10 gene was silenced after soaking the nematodes in a specific siRNA for 48 h. At 72 h post-soaking, the Mg-pat-10 mRNA level was similar to the control, indicating the recovery of expression between 48 h and 72 h post-soaking. For Mg-unc-87 the nematodes started to recover from siRNA silencing 24 h after thorough washing. A migration assay showed that for the nematodes that were soaked in the control (siRNA of β-1,4-endoglucanase), 77% of the nematodes completed migration through the column in a 12 h period. By comparison with the control, nematodes incubated in the siRNA of pat-10 or unc-87 were significantly inhibited in their motility. After 12 h, only 6.3% of the juveniles incubated in the Mg-pat-10 siRNA and 9.3% of those incubated in Mg-unc-87 siRNA had migrated through the column, representing 91.8% and 87.9% inhibition respectively compared to the control. In the present work, we demonstrated that M. graminicola is readily susceptible to siRNAs of two genes involved in nematode motility. This is an important contribution to the progressive use of siRNA for functional analysis. Moreover, the application of RNAi in PPNs opens the way for environmentally friendly control of M. graminicola .

Suggested Citation

  • Joseph Nsengimana & Lander Bauters & Annelies Haegeman & Godelieve Gheysen, 2013. "Silencing of Mg - pat-10 and Mg - unc-87 in the Plant Parasitic Nematode Meloidogyne graminicola Using siRNAs," Agriculture, MDPI, vol. 3(3), pages 1-12, September.
    • Handle: RePEc:gam:jagris:v:3:y:2013:i:3:p:567-578:d:28897
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    References listed on IDEAS

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    1. Andrew Fire & SiQun Xu & Mary K. Montgomery & Steven A. Kostas & Samuel E. Driver & Craig C. Mello, 1998. "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans," Nature, Nature, vol. 391(6669), pages 806-811, February.
    2. Sayda M. Elbashir & Jens Harborth & Winfried Lendeckel & Abdullah Yalcin & Klaus Weber & Thomas Tuschl, 2001. "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells," Nature, Nature, vol. 411(6836), pages 494-498, May.
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    1. Satish Kumar Rajasekharan & Chaitany Jayprakash Raorane & Jintae Lee, 2021. "A Facile and Modified Scheme for Synchronization and Isolation of Nematode Eggs," Agriculture, MDPI, vol. 11(7), pages 1-7, July.

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