Molecular characterization and functional analysis of multidrug resistance-associated genes of Pinewood nematode (Bursaphelenchus xylophilus) for nematicides.

Bursaphelenchus xylophilus (Pinewood nematode, PWN) is the causative agent of pine wilt disease (PWD) which caused serious threat to pine forests in the world, especially in East Asia and Western Europe. At present, the control of PWD mainly rely on the massive use of pesticide despite the damage to human health and environmental safety. Developing novel drug targets is the optimized strategy for developing new method to control PWN. In this study, four multidrug resistance-associated protein (MRP) genes containing highly conserved MRP-associated domains were cloned from PWN. The expression patterns of the four Bx-mrps under three different nematicides treatments were studied by quantitative reverse transcription PCR (qRT-PCR) and the function of the four genes in multidrug resistance were also validated by RNA interference (RNAi). Results showed that the expression of Bx-mrp1, Bx-mrp2, Bx-mrp3, and Bx-mrp4 were significantly increased when exposed to different nematicides, wherein, Bx-mrp4 exposed by 4.0 mg/mL of matrine own the highest expression level. The mortality rates of Bx-mrps silenced nematodes revealed significant increase(P < 0.05)under matrine, avermectin, and emamectin benzoate exposure. Specially, Bx-mrp4 exposed with 4.0 mg/mL matrine for 24 h own the highest mortality increase by 18.34%. After RNAi of Bx-mrps, feeding ability of the nematodes were also significantly decreased. These results demonstrate that Bx-mrps were linked to the detoxification process and feeding behavior of PWN. Silencing of Bx-mrps can lead to increased sensitivity of PWN to nematicides and decrease its feeding ability. Bx-mrps are potential new PWN control targets in the future.