RNA interference in the Colorado potato beetle,Leptinotarsa decemlineata: Identification of key contributors

RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. RNAi works very well in coleopteran insects including the Colorado potato beetle (CPB), Leptinotarsa decemlineata. We used a cell line (Lepd-SL1) developed from CPB to identify genes that play key roles in RNAi. We screened 50 genes with potential functions in RNAi by exposing Lepd-SL1 cells to dsRNA targeting one of the potential RNAi pathway genes followed by incubation with dsRNA targeting inhibitor of apoptosis (IAP, silencing of this gene induces apoptosis). Out of 50 genes tested, silencing of 29 genes showed an effect on RNAi. Silencing of five genes (Argonaute-1, Argonaute-2a, Argonaute-2b, Aubergine and V-ATPase 16 kDa subunit 1, Vha16) blocked RNAi suggesting that these genes are essential for functioning of RNAi in Lepd-SL1 cells. Interestingly, Argonaute-1 and Aubergine which are known to function in miRNA and piRNA pathways respectively are also critical to siRNA pathway. Using ³²P labeled dsRNA, we showed that these miRNA and piRNA Argonautes but not Argonaute-2 are required for processing of dsRNA to siRNA. Transfection of pIZT/V5 constructs containing these five genes into Sf9 cells (the cells where RNAi does not work well) showed that expression of all genes tested, except the Argonaute-2a, improved RNAi in these cells. Results from Vha16 gene silencing and bafilomycin-A1 treatment suggest that endosomal escape plays an important role in dsRNA-mediated RNAi in Lepd-SL1 cells.     

Search for limiting factors in the RNAi pathway in silkmoth tissues and the Bm5 cell line: the RNA-binding proteins R2D2 and Translin

RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.     

Gene silencing in tick cell lines using small interfering or long double-stranded RNA

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system.     

The effectiveness of double-stranded short inhibitory RNAs (siRNAs) may depend on the method of transfection

RNA interference (RNAi) is a recently described powerful experimental tool that can cause sequence-specific gene silencing, thereby facilitating functional analysis of gene function. Consequently, we became interested in using RNAi to determine the function of aberrantly expressed ErbB3 in the KAS-6/1 human myeloma cell line. Despite the wealth of information available on the use of RNAi, dsRNA target design, and the transfection of dsRNA in vitro, little information is available for transfecting dsRNA into nonadherent cells from any species. In the present study, we report that gene silencing of ErbB3 was not observed in myeloma cells when dsRNA targeting ErbB3 was introduced using conventional transfection agents and protocols that have proved successful for several adherent cell lines. Silencing of ErbB3, however, was observed in T47D cells, an adherent breast carcinoma cell line, using the same transfection methods, indicating that our target sequence was functional for gene silencing of ErbB3. Interestingly, ErbB3 was silenced in myeloma cells when the dsRNA target was introduced by electroporation. Thus, our studies illustrate the striking dependence of dsRNA-mediated gene silencing in some cells on the methods of dsRNA transfection.     

A Transient RNA Interference Assay System Using Arabidopsis Protoplasts

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.     

In C. elegans,High Levels of dsRNA Allow RNAi in the Absence of RDE-4

C. elegans Dicer requires an accessory double-stranded RNA binding protein, RDE-4, to enact the first step of RNA interference, the cleavage of dsRNA to produce siRNA. While RDE-4 is typically essential for RNAi, we report that in the presence of high concentrations of trigger dsRNA, rde-4 deficient animals are capable of silencing a transgene. By multiple criteria the silencing occurs by the canonical RNAi pathway. For example, silencing is RDE-1 dependent and exhibits a decrease in the targeted mRNA in response to an increase in siRNA. We also find that high concentrations of dsRNA trigger lead to increased accumulation of primary siRNAs, consistent with the existence of a rate-limiting step during the conversion of primary to secondary siRNAs. Our studies also revealed that transgene silencing occurs at low levels in the soma, even in the presence of ADARs, and that at least some siRNAs accumulate in a temperature-dependent manner. We conclude that an RNAi response varies with different conditions, and this may allow an organism to tailor a response to specific environmental signals.     

Suppression of RNA interference pathway <Emphasis Type="Italic">in vitro</Emphasis> by Grass carp reovirus

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined.The present study aimed to investigate the effect of Grass carp reovirus...     

Enhancement of Larval RNAi Efficiency by Over-expressing Argonaute2 in Bombyx mori

RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.     

Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm,Spodoptera frugiperda

RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda cells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells and Spodoptera frugiperda larvae treated with CFII conjugated ³²P-UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues.     

Chemically modified symmetric and asymmetric duplex RNAs: an enhanced stability to nuclease degradation and gene silencing effect

The present study accents on the relationship between dicing, nuclease stability, and RNAi activity of various types of chemically modified symmetric and asymmetric dsRNAs, covalently bound with amino-groups or cholesterol at one or both terminals. All modified dsRNAs were subjected to cleavage by recombinant Dicer enzyme. They possessed a high resistance to nuclease degradation in cell cultured medium and an excellent RNAi activity in viable cells. The best stability and RNAi activity was detected for 5′-sense amino-modified RNAs. These modifications manifested also a high long-term gene silencing effect within seven days post-transfection, while the RNAi activity of the native 21nt siRNA expired within two days. The conjugation of dsRNA with cholesterol at 5′-sense end resulted in easy intracellular delivery without transfection reagents. After a direct transfection in cells, the cholesterol-conjugated 27nt dsRNA possessed a higher RNAi activity than cholesterol-conjugated 21nt siRNA.     

A transient RNA interference assay system using Arabidopsis protoplasts

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.     

Characterization of Argonaute cDNA from Penaeus monodon and implication of its role in RNA interference

RNA interference (RNAi) has recently become a promising strategy for therapeutic of several viral diseases including those in the black tiger shrimp Penaeus monodon. However, the protein components that play role in RNAi in P. monodon have not yet been identified. Here, we report the cloning and functional characterization of a cDNA encoding Argonaute, a principal constituent of RNAi pathway in P. monodon. P. monodon’s Argonaute (Pem-AGO) exhibited the two signature domains, PAZ and PIWI. Substantial level of Pem-ago expression could be suppressed by double-stranded RNA (dsRNA) that targeted PAZ coding sequence in shrimp primary culture of Oka cells. The Pem-ago depleted cells showed impaired RNAi as the expression of an endogenous gene was rescued from the dsRNA-mediated silencing in these cells. Our results imply that Pem-ago is required for effective RNAi in P. monodon and thus identify the first protein constituent of RNAi machinery in penaeid shrimp.     

SID-1 protein of Caenorhabditis elegans mediates uptake of dsRNA into Bombyx cells

RNA interference is one of the most revolutionary tools in the study of gene function, particularly in non-model systems. However, in Bombyx mori, as with many lepidopteran species, attempts at systemic RNAi have had mixed success. Gene identification and phylogenetic analyses suggest that Bombyx has the core RNAi machinery, which is necessary to undergo RNAi as a cellular response. We introduced sid genes from Caenorhabditis elegans into Bombyx BmN4 cells to enhance the uptake of dsRNA and revealed that the SID-1 protein, but not SID-2, has the ability to endow the RNAi effect with the addition of dsRNA to the medium. Observed RNAi effect was dependent on both the levels of sid-1 expression and the concentration of the dsRNA. These results suggest that SID-1 promotes the uptake of dsRNA from the medium into Bombyx cells. We generated transgenic animals that express sid-1 but have not detected significant enhancements of in vivo phenotype in response to the injection of the dsRNA into hemocoel.     

A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response

Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.     

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.