Silencing of aphid genes by dsRNA feeding from plants

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.     

Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans

Background  

In Caenorhabditis elegans, injection of double-stranded RNA (dsRNA) results in the specific inactivation of genes containing homologous sequences, a technique termed RNA-mediated interference (RNAi). It has previously been shown that RNAi can also be achieved by feeding worms Escherichia coli expressing dsRNA corresponding to a specific gene; this mode of dsRNA introduction is conventionally considered to be less efficient than direct injection, however, and has therefore seen limited use, even though it is considerably less labor-intensive.     

Identification of the <Emphasis Type="Italic">C. elegans</Emphasis>anaphase promoting complex subunit Cdc26 by phenotypic profiling and functional rescue in yeast

Background  

RNA interference coupled with videorecording of C. elegans embryos is a powerful method for identifying genes involved in cell division processes. Here we present a functional analysis of the gene B0511.9, previously identified as a candidate cell polarity gene in an RNAi videorecording screen of chromosome I embryonic lethal genes.     

Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans

Background

In Caenorhabditis elegans, injection of double-stranded RNA (dsRNA) results in the specific inactivation of genes containing homologous sequences, a technique termed RNA-mediated interference (RNAi). It has previously been shown that RNAi can also be achieved by feeding worms Escherichia coli expressing dsRNA corresponding to a specific gene; this mode of dsRNA introduction is conventionally considered to be less efficient than direct injection, however, and has therefore seen limited use, even though it is considerably less labor-intensive.

Results

Here we present an optimized feeding method that results in phenotypes at least as strong as those produced by direct injection of dsRNA for embryonic lethal genes, and stronger for genes with post-embryonic phenotypes. In addition, the interference effect generated by feeding can be titrated to uncover a series of hypomorphic phenotypes informative about the functions of a given gene. Using this method, we screened 86 random genes on consecutive cosmids and identified functions for 13 new genes. These included two genes producing an uncoordinated phenotype (a previously uncharacterized POU homeodomain gene, ceh-6, and a gene encoding a MADS-box protein) and one gene encoding a novel protein that results in a high-incidence-of-males phenotype.

Conclusions

RNAi by feeding can provide significant information about the functions of an individual gene beyond that provided by injection. Moreover, it can be used for special applications for which injection or the use of mutants is sometimes impracticable (for example, titration, biochemistry and large-scale screening). Thus, RNAi by feeding should make possible new experimental approaches for the use of genomic sequence information.     

Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference

Background

Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms.

Results

We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain.

Conclusion

A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.     

RNAi Experiments in D. melanogaster: Solutions to the Overlooked Problem of Off-Targets Shared by Independent dsRNAs

Background

RNAi technology is widely used to downregulate specific gene products. Investigating the phenotype induced by downregulation of gene products provides essential information about the function of the specific gene of interest. When RNAi is applied in Drosophila melanogaster or Caenorhabditis elegans, often large dsRNAs are used. One of the drawbacks of RNAi technology is that unwanted gene products with sequence similarity to the gene of interest can be down regulated too. To verify the outcome of an RNAi experiment and to avoid these unwanted off-target effects, an additional non-overlapping dsRNA can be used to down-regulate the same gene. However it has never been tested whether this approach is sufficient to reduce the risk of off-targets.

Methodology

We created a novel tool to analyse the occurance of off-target effects in Drosophila and we analyzed 99 randomly chosen genes.

Principal Findings

Here we show that nearly all genes contain non-overlapping internal sequences that do show overlap in a common off-target gene.

Conclusion

Based on our in silico findings, off-target effects should not be ignored and our presented on-line tool enables the identification of two RNA interference constructs, free of overlapping off-targets, from any gene of interest.     

In Vivo RNAi Rescue in Drosophila melanogaster with Genomic Transgenes from Drosophila pseudoobscura

Background

Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.

Methodology/Principal Findings

We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.

Conclusions/Significance

The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.     

Protein interaction network topology uncovers melanogenesis regulatory network components within functional genomics datasets

Background  

RNA-mediated interference (RNAi)-based functional genomics is a systems-level approach to identify novel genes that control biological phenotypes. Existing computational approaches can identify individual genes from RNAi datasets that regulate a given biological process. However, currently available methods cannot identify which RNAi screen "hits" are novel components of well-characterized biological pathways known to regulate the interrogated phenotype. In this study, we describe a method to identify genes from RNAi datasets that are novel components of known biological pathways. We experimentally validate our approach in the context of a recently completed RNAi screen to identify novel regulators of melanogenesis.     

The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides

Background

RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants.

Results

Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway.

Conclusion

This work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1443-2) contains supplementary material, which is available to authorized users.     

A Pre- and Co-Knockdown of RNAseT Enzyme,Eri-1, Enhances the Efficiency of RNAi Induced Gene Silencing in Caenorhabditis elegans

Background

The approach of RNAi mediated gene knockdown, employing exogenous dsRNA, is being beneficially exploited in various fields of functional genomics. The immense utility of the approach came to fore from studies with model system C. elegans, but quickly became applicable with varied research models ranging from in vitro to various in vivo systems. Previously, there have been reports on the refractoriness of the neuronal cells to RNAi mediated gene silencing following which several modulators like eri-1 and lin-15 were described in C. elegans which, when present, would negatively impact the gene knockdown.

Methodology/Principal Findings

Taking a clue from these findings, we went on to screen hypothesis-driven- methodologies towards exploring the efficiency in the process of RNAi under various experimental conditions, wherein these genes would be knocked down preceding to, or concurrently with, the knocking down of a gene of interest. For determining the efficiency of gene knockdown, we chose to study visually stark phenotypes of uncoordinated movement, dumpy body morphology and blistered cuticle obtained by knocking down of genes unc-73, dpy-9 and bli-3 respectively, employing the RNAi-by-feeding protocol in model system C. elegans.

Conclusions/Significance

Our studies led to a very interesting outcome as the results reveal that amongst various methods tested, pre-incubation with eri-1 dsRNA synthesizing bacteria followed by co-incubation with eri-1 and gene-of-interest dsRNA synthesizing bacteria leads to the most efficient gene silencing as observed by the analysis of marker phenotypes. This provides an approach for effectively employing RNAi induced gene silencing while working with different genetic backgrounds including transgenic and mutant strains.     

Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

Background  

The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently.     

A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

Background  

Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available.     

Simultaneous analysis of large-scale RNAi screens for pathogen entry

Background

Large-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in human host cells for eight pathogens using image-based kinome-wide siRNA screens with siRNAs from three vendors. We propose a Parallel Mixed Model (PMM) approach that simultaneously analyzes several non-identical screens performed with the same RNAi libraries.

Results

We show that PMM gains statistical power for hit detection due to parallel screening. PMM allows incorporating siRNA weights that can be assigned according to available information on RNAi quality. Moreover, PMM is able to estimate a sharedness score that can be used to focus follow-up efforts on generic or specific gene regulators. By fitting a PMM model to our data, we found several novel hit genes for most of the pathogens studied.

Conclusions

Our results show parallel RNAi screening can improve the results of individual screens. This is currently particularly interesting when large-scale parallel datasets are becoming more and more publicly available. Our comprehensive siRNA dataset provides a public, freely available resource for further statistical and biological analyses in the high-content, high-throughput siRNA screening field.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1162) contains supplementary material, which is available to authorized users.     

Control of brown patch (<Emphasis Type="Italic">Rhizoctonia solani</Emphasis>) in tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) by host induced gene silencing

Key message

Transgenic tall fescue plants expressing RNAi constructs of essential genes of Rhizoctonia solani were resistant to R. solani.

Abstract

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species widely used for home lawns and on golf courses in North Carolina and other transition zone states in the US. The most serious and frequently occurring disease of tall fescue is brown patch, caused by a basidiomycete fungus, Rhizoctonia solani. This research demonstrates resistance to brown patch disease achieved by the application of host induced gene silencing. We transformed tall fescue with RNAi constructs of four experimentally determined “essential” genes from R. solani (including genes encoding RNA polymerase, importin beta-1 subunit, Cohesin complex subunit Psm1, and a ubiquitin E3 ligase) to suppress expression of those genes inside the fungus and thus inhibit fungal infection. Four gene constructs were tested, and 19 transgenic plants were obtained, among which 12 plants had detectable accumulation of siRNAs of the target genes. In inoculation tests, six plants displayed significantly improved resistance against R. solani. Lesion size was reduced by as much as 90 %. Plants without RNAi accumulation did not show resistance. To our knowledge, this is the first case that RNAi constructs of pathogen genes introduced into a host plant can confer resistance against a necrotrophic fungus.

     

Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium

Background

RNA interference (RNAi) is a highly conserved cellular mechanism. In some organisms, such as Caenorhabditis elegans, the RNAi response can be transmitted systemically. Some insects also exhibit a systemic RNAi response. However, Drosophila, the leading insect model organism, does not show a robust systemic RNAi response, necessitating another model system to study the molecular mechanism of systemic RNAi in insects.

Results

We used Tribolium, which exhibits robust systemic RNAi, as an alternative model system. We have identified the core RNAi genes, as well as genes potentially involved in systemic RNAi, from the Tribolium genome. Both phylogenetic and functional analyses suggest that Tribolium has a somewhat larger inventory of core component genes than Drosophila, perhaps allowing a more sensitive response to double-stranded RNA (dsRNA). We also identified three Tribolium homologs of C. elegans sid-1, which encodes a possible dsRNA channel. However, detailed sequence analysis has revealed that these Tribolium homologs share more identity with another C. elegans gene, tag-130. We analyzed tag-130 mutants, and found that this gene does not have a function in systemic RNAi in C. elegans. Likewise, the Tribolium sid-like genes do not seem to be required for systemic RNAi. These results suggest that insect sid-1-like genes have a different function than dsRNA uptake. Moreover, Tribolium lacks homologs of several genes important for RNAi in C. elegans.

Conclusion

Although both Tribolium and C. elegans show a robust systemic RNAi response, our genome-wide survey reveals significant differences between the RNAi mechanisms of these organisms. Thus, insects may use an alternative mechanism for the systemic RNAi response. Understanding this process would assist with rendering other insects amenable to systemic RNAi, and may influence pest control approaches.     

Monitoring immediate-early gene expression through firefly luciferase imaging of HRS/J hairless mice

Background  

Gene promoters fused to the firefly luciferase gene (luc) are useful for examining gene regulation in live transgenic mice and they provide unique views of functioning organs. The dynamics of gene expression in cells and tissues expressing luciferase can be observed by imaging this enzyme's bioluminescent oxidation of luciferin. Neural pathways involved in specific behaviors have been identified by localizing expression of immediate-early genes such as c-fos. A transgenic mouse line with luc controlled by the human c-fos promoter (fos::luc) has enabled gene expression imaging in brain slice cultures. To optimize imaging of immediate-early gene expression throughout intact mice, the present study examined fos::luc mice and a second transgenic mouse containing luc controlled by the human cytomegalovirus immediate-early gene 1 promoter and enhancer (CMV::luc). Because skin pigments and hair can significantly scatter light from underlying structures, the two transgenic lines were crossed with a hairless albino mouse (HRS/J) to explore which deep structures could be imaged. Furthermore, live anesthetized mice were compared with overdosed mice.