Wang Meng, Wang Diandong, Zhang Xi, Wang Xu, Liu Wencui, Hou Xiaomeng, Huang Xiaoyin, Xie Bingyan, Cheng Xinyue
College of Life Sciences, Beijing Normal University, Beijing 100875, China.
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China; Yangtze Normal University, Chongqing 408100, China.
Int J Parasitol. 2016 May;46(5-6):351-60. doi: 10.1016/j.ijpara.2016.01.008. Epub 2016 Mar 4.
RNA 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. Presently, the complete genome sequence of B. xylophilus has been published, and research involving genome-wide functional analyses is likely to increase. In this study, we describe the construction of an effective silencing vector, pDH-RH, which contains a transcriptional unit for a hairpin loop structure. Utilising 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. Four dumpy genes (Bx-dpy-2, 4, 10 and 11) 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 34-87% 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. Moreover, as B. xylophilus feeds on endophytic fungi when a host has died, RNAi feeding technology will offer the prospect for developing a novel control strategy for the nematode. Furthermore, this platform may also be applicable to other parasitic nematodes that have a facultative, fungivorous habit.
RNA干扰（RNAi）是研究体内基因功能的一种有价值的工具，并在多种生物体中提供了一个功能基因组学平台。松材线虫（Bursaphelenchus xylophilus）是一种著名的入侵性植物寄生线虫，已成为对森林生态系统的严重全球威胁。目前，松材线虫的完整基因组序列已公布，涉及全基因组功能分析的研究可能会增加。在本研究中，我们描述了一种有效的沉默载体pDH-RH的构建，其包含一个用于发夹环结构的转录单元。利用该载体，与靶基因序列同源的双链（ds）RNA可通过根癌农杆菌介导的转化技术在转化的丝状真菌中表达，并随后通过让线虫取食真菌转化体来诱导松材线虫中靶基因mRNA表达的敲低。四个短粗基因（Bx-dpy-2、4、10和11）用作靶标以检测RNAi效率。通过让线虫取食靶基因转化的尖孢镰刀菌菌株，与取食野生型菌株的线虫相比，通过实时定量PCR（RT-qPCR）测定，靶转录本被敲低了34-87%。观察到了形态学RNAi表型，表现为体长明显缩短；短粗或弱小（短而细）的体型；或普遍异常。此外，在松材线虫中发现了dpy基因的补偿性调控和非特异性沉默。我们的结果表明，通过取食在松材线虫中进行RNAi传递是一种成功的技术。该平台可能为在体外对松材线虫进行基于RNAi的全基因组基因功能研究提供新机会。此外，由于松材线虫在宿主死亡时以内生真菌为食，RNAi取食技术将为开发线虫的新型控制策略提供前景。此外，该平台也可能适用于其他具有兼性食真菌习性的寄生线虫。
