Agriculture 2013, 3(3), 567-578; doi:10.3390/agriculture3030567
Article

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

email, email, email and * email
Received: 27 April 2013; in revised form: 6 August 2013 / Accepted: 22 August 2013 / Published: 18 September 2013
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.
Keywords: short interfering RNA (siRNA); Meloidogyne graminicola (Mg); Mg-pat-10; Mg-unc-87; migration assay; expression analysis
PDF Full-text Download PDF Full-Text [480 KB, uploaded 18 September 2013 09:24 CEST]

Export to BibTeX |
EndNote


MDPI and ACS Style

Nsengimana, J.; Bauters, L.; Haegeman, A.; Gheysen, G. Silencing of Mg-pat-10 and Mg-unc-87 in the Plant Parasitic Nematode Meloidogyne graminicola Using siRNAs. Agriculture 2013, 3, 567-578.

AMA Style

Nsengimana J, Bauters L, Haegeman A, Gheysen G. Silencing of Mg-pat-10 and Mg-unc-87 in the Plant Parasitic Nematode Meloidogyne graminicola Using siRNAs. Agriculture. 2013; 3(3):567-578.

Chicago/Turabian Style

Nsengimana, Joseph; Bauters, Lander; Haegeman, Annelies; Gheysen, Godelieve. 2013. "Silencing of Mg-pat-10 and Mg-unc-87 in the Plant Parasitic Nematode Meloidogyne graminicola Using siRNAs." Agriculture 3, no. 3: 567-578.

Agriculture EISSN 2077-0472 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert