RNA interference in Pratylenchus coffeae: knock down of Pc-pat-10 and Pc-unc-87 impedes migration

Many of the currently available nematicides used in nematode control are hazardous to the user, environment and beneficial non-target organisms. Therefore the need to develop alternative methods for nematode control such as the development of nematode-resistant crops through RNA-mediated interference (RNAi) holds great promise. The Caenorhabditis elegans genes unc-87 and pat-10 are essential components of the body wall muscle and are thus required for nematode movement. The Pratylenchus coffeae orthologs of these two genes, namely Pc-pat-10 and Pc-unc-87 were cloned and used to test RNAi in this migratory nematode. RNAi was performed by soaking P. coffeae in a solution containing dsRNA of either Pc-unc-87 or Pc-pat-10. The levels of both Pc-unc-87 and Pc-pat-10 mRNAs were significantly reduced in a sequence-specific manner in nematodes soaked for 24h. Nematodes incubated in Pc-pat-10 dsRNA appeared straight and rigid while Pc-unc-87 resulted in nematodes that were coiled, in contrast to the regular sinusoidal movement of the control nematodes. While 88.4 ± 3.9% of the control nematodes successfully migrated to the bottom of the sand column in 12h, only 6 ± 1.3% and 7 ± 2.3%, respectively, of the Pc-pat-10 (RNAi) and Pc-unc-87 (RNAi) nematodes successfully migrated to the bottom. However a recovery in movement as well as transcript level was observed in both treatments when the nematodes were incubated in distilled water for 24h following the dsRNA soaking. The recovery rate was slower in Pc-unc-87 when compared to Pc-pat-10. In summary, this study demonstrates the existence of the RNAi phenomenon in P. coffeae and shows that the function of unc-87 and pat-10 genes has been evolutionarily conserved among free-living and plant parasitic nematodes.