Double-stranded RNA-mediated interference of dumpy genes in Bursaphelenchus xylophilus by feeding on filamentous fungal transformants

文献类型： 外文期刊

第一作者： Wang, Meng

作者： Wang, Meng;Zhang, Xi;Wang, Xu;Liu, Wencui;Hou, Xiaomeng;Huang, Xiaoyin;Cheng, Xinyue;Wang, Diandong;Xie, Bingyan;Cheng, Xinyue;Wang, Diandong

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关键词： Pinewood nematode;Bursaphelenchus xylophilus;Silencing vector pDH-RH;RNA interference (RNAi) feeding;Dumpy gene;Collagen;Filamentous fungal transformant;Agrobacterium tumefaciens-mediated transformation (ATMT)

期刊名称：INTERNATIONAL JOURNAL FOR PARASITOLOGY （ 影响因子：3.981； 五年影响因子：4.214 ）

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收录情况： SCI

摘要： 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. (C) 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

分类号： Q958.9

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