RNAi delivery by feeding in the pinewood nematode Bursaphelenchus xylophilus

R interference (RNAi) is a valuable tool for studying gene function in vivo and provides a functional genomics platform in a wide variety of organisms. The pinewood nematode, Bursaphelenchus xylophilus, is a prominent invasive plant-parasitic nematode and has become a serious worldwide threat to forest ecosystems. Currently, we constructed an effective silencing vector, pDH-RH, which contains a transcriptional unit for a hairpin loop structure. Utilizing this vector, double-stranded (ds) RNAs with sequences homologous to the target genes can be expressed in a transformed filamentous fungus via Agrobacterium tumefaciens mediated transformation technology and can subsequently induce the knockdown of target gene mRNA expression in B. xylophilus by allowing the nematode to feed on the fungal transformants. Dumpy related genes were used as targets to detect RNAi efficiency. By allowing the nematode to feed on target gene transformed Fusarium oxysporum strains, target transcripts were knocked down significantly compared with those feeding on the wild type strain as determined by real-time quantitative PCR (RT-qPCR). Morphological RNAi phenotypes were observed, displaying obviously reduced body length; weak dumpy or small (short and thin) body size or general abnormalities. Moreover, compensatory regulation and non specific silencing of dpy genes were found in B. xylophilus. Our results indicate that RNAi delivery by feeding in B. xylophilus is a successful technique. This platform may provide a new opportunity for undertaking RNAi-based, genome wide gene functional studies in vitro in B. xylophilus. This platform may also be applicable to other parasitic nematodes that have a facultative, fungivorous habit.