title: Double-stranded RNA-mediated interference of dumpy genes in Bursaphelenchus xylophilus by feeding on filamentous fungal transformants.
abstract: 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.
authors.affiliation: College of Life Sciences, Beijing Normal University, Beijing 100875, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Wang
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China; Yangtze Normal University, Chongqing 408100, China.
authors.forename: Diandong
authors.initials: D
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Beijing Normal University, Beijing 100875, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Zhang
authors.affiliation: College of Life Sciences, Beijing Normal University, Beijing 100875, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Beijing Normal University, Beijing 100875, China.
authors.forename: Wencui
authors.initials: W
authors.lastname: Liu
authors.affiliation: College of Life Sciences, Beijing Normal University, Beijing 100875, China.
authors.forename: Xiaomeng
authors.initials: X
authors.lastname: Hou
authors.affiliation: College of Life Sciences, Beijing Normal University, Beijing 100875, China.
authors.forename: Xiaoyin
authors.initials: X
authors.lastname: Huang
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China. Electronic address: xiebingyan@caas.cn.
authors.forename: Bingyan
authors.initials: B
authors.lastname: Xie
authors.affiliation: College of Life Sciences, Beijing Normal University, Beijing 100875, China; Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Beijing 100875, China. Electronic address: chengxy@bnu.edu.cn.
authors.forename: Xinyue
authors.initials: X
authors.lastname: Cheng
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000818:Animals; D005260:Female; D005670:Fusarium / Q000235:genetics; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D015801:Helminth Proteins / Q000235:genetics; D008297:Male; D010641:Phenotype; D028223:Pinus / Q000469:parasitology*; D010935:Plant Diseases / Q000469:parasitology*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000494:pharmacology*; D014170:Transformation, Genetic; D017187:Tylenchida / Q000033:anatomy & histology / Q000235:genetics*
chemical_list: D015801:Helminth Proteins; D012330:RNA, Double-Stranded
keywords: Agrobacterium tumefaciens-mediated transformation (ATMT); Bursaphelenchus xylophilus; Collagen; Dumpy gene; Filamentous fungal transformant; Pinewood nematode; RNA interference (RNAi) feeding; Silencing vector pDH-RH