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      Silencing of Aphid Genes by dsRNA Feeding from Plants

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          Abstract

          Background

          RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid ( Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.

          Methodology/Principal Findings

          In this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 ( MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.

          Conclusions/Significance

          Similar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.

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          Most cited references58

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          Mechanisms of gene silencing by double-stranded RNA.

          Double-stranded RNA (dsRNA) is an important regulator of gene expression in many eukaryotes. It triggers different types of gene silencing that are collectively referred to as RNA silencing or RNA interference. A key step in known silencing pathways is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. These short dsRNAs guide RNA silencing by specific and distinct mechanisms. Many components of the RNA silencing machinery still need to be identified and characterized, but a more complete understanding of the process is imminent.
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            Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review.

            RNA interference already proved its usefulness in functional genomic research on insects, but it also has considerable potential for the control of pest insects. For this purpose, the insect should be able to autonomously take up the dsRNA, for example through feeding and digestion in its midgut. In this review we bring together current knowledge on the uptake mechanisms of dsRNA in insects and the potential of RNAi to affect pest insects. At least two pathways for dsRNA uptake in insects are described: the transmembrane channel-mediated uptake mechanism based on Caenorhabditis elegans' SID-1 protein and an 'alternative' endocytosis-mediated uptake mechanism. In the second part of the review dsRNA feeding experiments on insects are brought together for the first time, highlighting the achievement of implementing RNAi in insect control with the first successful experiments in transgenic plants and the diversity of successfully tested insect orders/species and target genes. We conclude with points of discussion and concerns regarding further research on dsRNA uptake mechanisms and the promising application possibilities for RNAi in insect control. Copyright (c) 2009 Elsevier Ltd. All rights reserved.
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              Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate.

              Duplexes of 21-23 nucleotide (nt) RNAs are the sequence-specific mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3' overhangs were the most efficient triggers of sequence-specific mRNA degradation. Substitution of one or both siRNA strands by 2'-deoxy or 2'-O-methyl oligonucleotides abolished RNAi, although multiple 2'-deoxynucleotide substitutions at the 3' end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5' end of the guide siRNA rather than its 3' end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2011
                5 October 2011
                : 6
                : 10
                : e25709
                Affiliations
                [1 ]Department of Disease and Stress Biology, The John Innes Centre, Norwich Research Park, Norwich, United Kingdom
                [2 ]Plant Physiology Unit, Department of Plant Biology, Innovation Centre, University of Turin, Turin, Italy
                University of Miami, United States of America
                Author notes

                Conceived and designed the experiments: MP AC SH. Performed the experiments: MP AC. Analyzed the data: MP AC SH. Contributed reagents/materials/analysis tools: SH CR MM. Wrote the paper: MP AC MM CR SH.

                Article
                PONE-D-11-15361
                10.1371/journal.pone.0025709
                3187792
                21998682
                beb08f67-cf7d-445a-bbbc-73b981ef0c1d
                Pitino et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 10 August 2011
                : 8 September 2011
                Page count
                Pages: 8
                Categories
                Research Article
                Agriculture
                Agricultural Biotechnology
                Genetically Modified Organisms
                Agricultural Production
                Environmental Impacts
                Crops
                Crop Management
                Pest Control
                Integrated Control
                Biology
                Biotechnology
                Plant Biotechnology
                Transgenic Plants
                Genetics
                Gene Expression
                RNA interference
                Model Organisms
                Plant and Algal Models
                Arabidopsis Thaliana
                Zoology
                Entomology

                Uncategorized
                Uncategorized

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