Faculty Opinions recommendation of A rapid and scalable system for studying gene function in mice using conditional RNA interference.

The origin of RNA interference (RNAi), the cell sentinel system widely shared among eukaryotes that recognizes RNAs and specifically degrades or prevents their translation in cells, is suggested to predate the last eukaryote common ancestor ( 138 ). Of particular relevance to plant pathology is that in plants, but also in some fungi, insects, and lower eukaryotes, RNAi is a primary and effective antiviral defense, and recent studies have revealed that small RNAs (sRNAs) involved in RNAi play important roles in other plant diseases, including those caused by cellular plant pathogens. Because of this, and because RNAi can be manipulated to interfere with the expression of endogenous genes in an intra- or interspecific manner, RNAi has been used as a tool in studies of gene function but also for plant protection. Here, we review the discovery of RNAi, canonical mechanisms, experimental and translational applications, and new RNA-based technologies of importance to plant pathology.

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Analysis of Gene Function in Trypanosoma brucei Using RNA Interference

RNA Interference, Editing, and Modification ◽

10.1385/1-59259-775-0:073 ◽

2004 ◽

pp. 73-83 ◽

Cited By ~ 1

Author(s):

Appolinaire Djikeng ◽

Shuiyuan Shen ◽

Christian Tschudi ◽

Elisabetta Ullu

Keyword(s):

Rna Interference ◽

Trypanosoma Brucei ◽

Gene Function

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Comprehensive Analysis of Gene Function: RNA interference and Chemical Genomics

Genomic and Personalized Medicine ◽

10.1016/b978-0-12-369420-1.00016-0 ◽

2009 ◽

pp. 193-203

Author(s):

Bjorn T. Gjertsen ◽

James B. Lorens

Keyword(s):

Rna Interference ◽

Gene Function ◽

Comprehensive Analysis ◽

Chemical Genomics

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Considering RNAi experimental design in parasitic helminths

Parasitology ◽

10.1017/s0031182011001946 ◽

2012 ◽

Vol 139 (5) ◽

pp. 589-604 ◽

Cited By ~ 25

Author(s):

JOHNATHAN J. DALZELL ◽

NEIL D. WARNOCK ◽

PAUL MCVEIGH ◽

NIKKI J. MARKS ◽

ANGELA MOUSLEY ◽

...

Keyword(s):

Rna Interference ◽

Experimental Design ◽

Gene Function ◽

Experimental Validation ◽

Parasite Species ◽

Model Organisms ◽

Helminth Parasites ◽

Parasitic Helminth ◽

Parasitic Helminths ◽

Parasite Biology

SUMMARYAlmost a decade has passed since the first report of RNA interference (RNAi) in a parasitic helminth. Whilst much progress has been made with RNAi informing gene function studies in disparate nematode and flatworm parasites, substantial and seemingly prohibitive difficulties have been encountered in some species, hindering progress. An appraisal of current practices, trends and ideals of RNAi experimental design in parasitic helminths is both timely and necessary for a number of reasons: firstly, the increasing availability of parasitic helminth genome/transcriptome resources means there is a growing need for gene function tools such as RNAi; secondly, fundamental differences and unique challenges exist for parasite species which do not apply to model organisms; thirdly, the inherent variation in experimental design, and reported difficulties with reproducibility undermine confidence. Ideally, RNAi studies of gene function should adopt standardised experimental design to aid reproducibility, interpretation and comparative analyses. Although the huge variations in parasite biology and experimental endpoints make RNAi experimental design standardization difficult or impractical, we must strive to validate RNAi experimentation in helminth parasites. To aid this process we identify multiple approaches to RNAi experimental validation and highlight those which we deem to be critical for gene function studies in helminth parasites.

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Developments in RNA interference and genetic transformation to define gene function in parasitic helminths

Parasitology ◽

10.1017/s0031182012000108 ◽

2012 ◽

Vol 139 (5) ◽

pp. 557-559 ◽

Cited By ~ 2

Author(s):

David P. Knox

Keyword(s):

Rna Interference ◽

Genetic Transformation ◽

Gene Function ◽

Parasitic Helminths

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RNA Interference in Protozoan Parasites: Achievements and Challenges

Eukaryotic Cell ◽

10.1128/ec.05114-11 ◽

2011 ◽

Vol 10 (9) ◽

pp. 1156-1163 ◽

Cited By ~ 85

Author(s):

Nikolay G. Kolev ◽

Christian Tschudi ◽

Elisabetta Ullu

Keyword(s):

Rna Interference ◽

Gene Function ◽

Current Status ◽

Diverse Group ◽

Protozoan Parasites ◽

Parasitic Protozoa ◽

Content Type ◽

Classical Genetic

ABSTRACT Protozoan parasites that profoundly affect mankind represent an exceptionally diverse group of organisms, including Plasmodium , Toxoplasma , Entamoeba , Giardia , trypanosomes, and Leishmania . Despite the overwhelming impact of these parasites, there remain many aspects to be discovered about mechanisms of pathogenesis and how these organisms survive in the host. Combined with the ever-increasing availability of sequenced genomes, RNA interference (RNAi), discovered a mere 13 years ago, has enormously facilitated the analysis of gene function, especially in organisms that are not amenable to classical genetic approaches. Here we review the current status of RNAi in studies of parasitic protozoa, with special emphasis on its use as a postgenomic tool.

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Antisense RNA Modulation of Alkyl Hydroperoxide Reductase Levels in Helicobacter pylori Correlates with Organic Peroxide Toxicity but Not Infectivity

Journal of Bacteriology ◽

10.1128/jb.00012-07 ◽

2007 ◽

Vol 189 (9) ◽

pp. 3359-3368 ◽

Cited By ~ 18

Author(s):

Matthew A. Croxen ◽

Peter B. Ernst ◽

Paul S. Hoffman

Keyword(s):

Helicobacter Pylori ◽

Rna Interference ◽

Gene Function ◽

Antisense Rna ◽

In Vitro Growth ◽

Protein Levels ◽

Alkyl Hydroperoxide ◽

Gastric Colonization ◽

H Pylori

ABSTRACT Much of the gene content of the human gastric pathogen Helicobacter pylori (∼1.7-Mb genome) is considered essential. This view is based on the completeness of metabolic pathways, infrequency of nutritional auxotrophies, and paucity of pathway redundancies typically found in bacteria with larger genomes. Thus, genetic analysis of gene function is often hampered by lethality. In the absence of controllable promoters, often used to titrate gene function, we investigated the feasibility of an antisense RNA interference strategy. To test the antisense approach, we targeted alkyl hydroperoxide reductase (AhpC), one of the most abundant proteins expressed by H. pylori and one whose function is essential for both in vitro growth and gastric colonization. Here, we show that antisense ahpC (as-ahpC) RNA expression from shuttle vector pDH37::as-ahpC achieved an ∼72% knockdown of AhpC protein levels, which correlated with increased susceptibilities to hydrogen peroxide, cumene, and tert-butyl hydroperoxides but not with growth efficiency. Compensatory increases in catalase levels were not observed in the knockdowns. Expression of single-copy antisense constructs (expressed under the urease promoter and containing an fd phage terminator) from the rdxA locus of mouse-colonizing strain X47 achieved a 32% knockdown of AhpC protein levels (relative to wild-type X47 levels), which correlated with increased susceptibility to organic peroxides but not with mouse colonization efficiency. Our studies indicate that high levels of AhpC are not required for in vitro growth or for primary gastric colonization. Perhaps AhpC, like catalase, assumes a greater role in combating exogenous peroxides arising from lifelong chronic inflammation. These studies also demonstrate the utility of antisense RNA interference in the evaluation of gene function in H. pylori.

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