Plant Biotechnology Research Group, School of Biological Sciences and Biotechnology, WA State Agricultural Biotechnology Centre, Murdoch University, Perth, WA 6150, Australia.
Exp Parasitol. 2013 Feb;133(2):166-78. doi: 10.1016/j.exppara.2012.11.011. Epub 2012 Nov 29.
Root lesion nematodes (RLNs, Pratylenchus species) are a group of economically important migratory endoparasitic plant pathogens that attack host roots of major crops such as wheat and sugarcane, and can reduce crop yields by 7-15%. Pratylenchus thornei and Pratylenchus zeae were treated with double stranded RNA (dsRNA) to study gene silencing, (RNA interference, RNAi), as a potential strategy for their control. Mixed stages of nematodes of both species ingested dsRNA when incubated in a basic soaking solution in the presence of the neurostimulant octopamine. Incubation for up to 16 h in soaking solutions containing 10-50 mM octopamine, 0.1-1.0 mg/mL FITC, and 0.5-6 mM spermidine did not affect vitality. Spermidine phosphate salt hexahydrate rather than spermidine or spermidine trihydrochloride increased uptake of FITC by nematodes, and this resulted in more effective gene silencing. Silencing pat-10 and unc-87 genes of P. thornei and P. zeae resulted in paralysis and uncoordinated movements in both species, although to a higher degree in P. thornei. There was also a greater reduction in transcript of both genes in P. thornei indicating that it may be more susceptible to RNAi. For P. thornei treated with dsRNA of pat-10 and unc-87 there was a significant reduction (77-81%) in nematode reproduction on carrot mini discs over a 5 week period. The results show that RLNs are clearly amenable to gene silencing, and that in planta delivery of dsRNA to target genes in these nematodes should confer host resistance. Moreover, for the two genes, dsRNA derived from either nematode species silenced the corresponding gene in both species. This implies cross-species control of nematodes via RNAi is possible.
根结线虫（RLNs，滑刃线虫属）是一组具有经济重要性的迁移内寄生植物病原体，攻击小麦和甘蔗等主要作物的宿主根系，并可使作物减产 7-15%。使用双链 RNA（dsRNA）处理螺旋线虫和玉米根结线虫，以研究基因沉默（RNA 干扰，RNAi），作为其控制的一种潜在策略。当在存在神经兴奋剂章鱼胺的碱性浸泡溶液中孵育时，两种线虫的混合阶段都摄取了 dsRNA。在含有 10-50mM 章鱼胺、0.1-1.0mg/mL FITC 和 0.5-6mM 亚精胺的浸泡溶液中孵育长达 16 小时不会影响活力。亚精胺磷酸盐六水合物而不是亚精胺或亚精胺三盐酸盐增加了线虫对 FITC 的摄取，从而导致更有效的基因沉默。沉默 P. thornei 和 P. zeae 的 pat-10 和 unc-87 基因导致两种线虫麻痹和不协调运动，尽管在 P. thornei 中更为严重。P. thornei 中这两个基因的转录水平也有更大程度的降低，表明它可能更容易受到 RNAi 的影响。用 pat-10 和 unc-87 的 dsRNA 处理 P. thornei 后，在 5 周的时间内，胡萝卜迷你圆盘上线虫的繁殖显著减少（77-81%）。结果表明 RLNs 明显易受基因沉默的影响，并且将 dsRNA 递送到这些线虫中的靶基因应该赋予宿主抗性。此外，对于用 pat-10 和 unc-87 的 dsRNA 处理的 P. thornei，两种线虫的相应基因都被沉默。这意味着通过 RNAi 对线虫进行跨物种控制是可能的。
