Systemic RNAi mediated gene silencing in the anhydrobiotic nematode Panagrolaimus superbus.

Adam J Shannon,Jacqueline Boyd,Ann M Burnell,Ilona Dix,Trevor Tyson

doi:10.1186/1471-2199-9-58

Abstract

BackgroundGene silencing by RNA interference (RNAi) is a powerful tool for functional genomics. Although RNAi was first described in Caenorhabditis elegans, several nematode species are unable to mount an RNAi response when exposed to exogenous double stranded RNA (dsRNA). These include the satellite model organisms Pristionchus pacificus and Oscheius tipulae. Available data also suggest that the RNAi pathway targeting exogenous dsRNA may not be fully functional in some animal parasitic nematodes. The genus Panagrolaimus contains bacterial feeding nematodes which occupy a diversity of niches ranging from polar, temperate and semi-arid soils to terrestrial mosses. Thus many Panagrolaimus species are adapted to tolerate freezing and desiccation and are excellent systems to study the molecular basis of environmental stress tolerance. We investigated whether Panagrolaimus is susceptible to RNAi to determine whether this nematode could be used in large scale RNAi studies in functional genomics.ResultsWe studied two species: Panagrolaimus sp. PS1159 and Panagrolaimus superbus. Both nematode species displayed embryonic lethal RNAi phenotypes following ingestion of Escherichia coli expressing dsRNA for the C. elegans embryonic lethal genes Ce-lmn-1 and Ce-ran-4. Embryonic lethal RNAi phenotypes were also obtained in both species upon ingestion of dsRNA for the Panagrolaimus genes ef1b and rps-2. Single nematode RT-PCR showed that a significant reduction in mRNA transcript levels occurred for the target ef1b and rps-2 genes in RNAi treated Panagrolaimus sp. 1159 nematodes. Visible RNAi phenotypes were also observed when P. superbus was exposed to dsRNA for structural genes encoding contractile proteins. All RNAi phenotypes were highly penetrant, particularly in P. superbus.ConclusionThis demonstration that Panagrolaimus is amenable to RNAi by feeding will allow the development of high throughput methods of RNAi screening for P. superbus. This greatly enhances the utility of this nematode as a model system for the study of the molecular biology of anhydrobiosis and cryobiosis and as a possible satellite model nematode for comparative and functional genomics. Our data also identify another nematode infraorder which is amenable to RNAi and provide additional information on the diversity of RNAi phenotypes in nematodes.

Highlights

Gene silencing by RNA interference (RNAi) is a powerful tool for functional genomics
This demonstration that Panagrolaimus is amenable to RNAi by feeding will allow the development of high throughput methods of RNAi screening for P. superbus and further enhance the utility of this nematode as a model system for the study of the molecular biology of anhydrobiosis, and as a possible satellite model nematode for comparative and functional genomics
Panagrolaimus fed with E. coli expressing double stranded RNA (dsRNA) for C. elegans embryonic lethal genes give rise to progeny with highly penetrant embryonic lethal phenotypes We tested two species of Panagrolaimus for RNAi effects using E. coli clones from a C. elegans feeding library [59]

Summary

Introduction

Gene silencing by RNA interference (RNAi) is a powerful tool for functional genomics. RNA interference (RNAi) was first described in the nematode Caenorhabditis elegans [1,2], when it was found that the injection of double-stranded RNA (dsRNA) into a hermaphrodite worm resulted in the degradation of endogenous mRNA corresponding in sequence to the injected dsRNA This resulted in a loss of function phenotype for the target gene. The potential for RNAi by feeding to be used in a large scale manner for functional genomics was demonstrated by the construction of a feeding library corresponding to 86% of the 19,000 predicted genes in C. elegans [5] This library has already been screened for genes involved in a wide range of biological processes including development [6,7], ageing [8,9,10], cell biology [11,12] and neurobiology [13]. RNAi-related mechanisms are involved in heterochromatin formation [20,21]

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