Application of dsRNA in the Pine Wood Nematode, Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant parasitic nematodes through conventional forward genetics and transgenic methods. RNA interference (RNAi), as a reverse genetics technology, offers great convenience for studying the functional genes of nematodes, including B. xylophilus. We here outline a protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5'-terminal of the target fragment by polymerase chain reaction (PCR), and then double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking nematodes with the dsRNA solution mixed with synthetic neurostimulants. Synchronized eggs, juveniles, and adults of B. xylophilus (approximately 20,000 individuals of each stage) were washed and soaked in dsRNA (0.8 μg/mL) with the soaking buffer for 24 h in the dark at 25 °C. The same quantity of nematodes was placed in the soaking buffer without dsRNA or with green fluorescent protein dsRNA as a control. After soaking, the expression level of the target transcripts was determined by real-time quantitative PCR. The effects of RNAi were then confirmed by microscopic observation of the phenotypes and comparison of the body size of adults among groups. The current protocol can help to progress research to understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.