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.
Highlights
Plant-parasitic nematodes (PPNs), which include the genus Meloidogyne, are responsible for some of the most damaging biotic stresses on crops [1]
The two target genes for M. graminicola were selected because they have similarity with their homologues in C. elegans: Meloidogyne graminicola (Mg)-pat-10 is 91% similar, (Evalue = 2−23) while Mg-unc-87 is 88%
SiRNAs designed against β-1,4-endoglucanase sequence of M. graminicola were used as a control during the investigation on the effect of short interfering RNAs (siRNAs) of Mg-pat-10 and Mg-unc-87 in
Summary
Plant-parasitic nematodes (PPNs), which include the genus Meloidogyne, are responsible for some of the most damaging biotic stresses on crops [1]. The current practice operating to control PPNs is through the integrated application of several procedures; nematicides, resistant crop varieties, and biological control strategies such as the use of trap crops, natural enemies and cultural practice [3]. These approaches are becoming increasingly unsatisfactory due to the proliferation of PPNs through the continuous and intensive cultivation of susceptible varieties. Significant progress has been made with the use of RNAi in PPNs and this may be advantageous for parasite control through plants engineered to express PPN-specific transcripts. In contrast to C. elegans, PPNs rarely feed outside of their host
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