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

Abstract

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 24 h. 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 12 h, 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 24 h 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.