Molecular mechanisms of dsRNA uptake and intracellular trafficking in the fat body of Locusta migratoria.

RNA interference (RNAi) technologies have recently been developed to control a growing number of agronomically significant fungal phytopathogens, including the white mold pathogen, Sclerotinia sclerotiorum. Exposure of this fungus to exogenous double-stranded RNA (dsRNA) results in potent RNAi-mediated knockdown of target genes’ transcripts, but it is unclear how the dsRNA can enter the fungal cells. In nematodes, specialized dsRNA transport proteins such as SID-1 facilitate dsRNA uptake, but for many other eukaryotes in which the dsRNA uptake mechanisms have been examined, endocytosis appears to mediate the uptake process. In this study, using live cell imaging, transgenic fungal cultures and endocytic inhibitors, we determined that the uptake mechanism in S. sclerotiorum occurs through clathrin-mediated endocytosis. RNAi-mediated knockdown of several clathrin-mediated endocytic genes’ transcripts confirmed the involvement of this cellular uptake process in facilitating RNAi in this fungus. Understanding the mode of dsRNA entry into the fungus will prove useful in designing and optimizing future dsRNA-based control methods and in anticipating possible mechanisms by which phytopathogens may develop resistance to this novel category of fungicides.

Systemic RNA interference deficient-1-like (SIL1) is considered a core component in dsRNA uptake in some insect species. Investigation related to the potential function of SIL1 in dsRNA uptake can contribute to a further understanding of RNA interference (RNAi) mechanisms in insects and agricultural pest control. However, the role of SIL1 in dsRNA uptake in insects such as aphids remains controversial. We have thoroughly analyzed the role of SIL1 from the model aphid Acyrthosiphon pisum (ApSIL1) in cellular dsRNA to clarify its function. First, the induced expression of ApSIL1 upon dsRNA oral exposure provided a vital clue for the possible involvement of ApSIL1 in cellular dsRNA uptake. Subsequent in vivo experiments using the RNAi-of-RNAi approach for ApSIL1 supported our hypothesis that the silencing efficiencies of reporter genes were reduced after inhibition of ApSIL1 expression. The impaired biological phenotypes of aphids, including cumulative average offspring, deformities of the nymph, and mortality upon pathogen infection, were then observed in the treatment group. Thereafter, in vitro dual-luciferase reporter assay showed compelling evidence that the luciferin signal was significantly attenuated when dsluciferase or dsGFP was transferred into ApSIL1-transfected Drosophila S2 cells. These observations further confirmed that the signal of Cy3-labeled dsRNA was rapidly attenuated with time in ApSIL1-transfected Drosophila S2 cells. Overall, these findings conclusively establish that ApSIL1 is involved in dsRNA uptake in A.pisum.

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double-stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient-1 (Sid-1) transmembrane channel-mediated uptake and clathrin-mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi-of-RNAi experiments, we demonstrated the contribution of two different sid-1-like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H(+) ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid-1-like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well-devised reporter system in any future experiments on cellular dsRNA uptake.

Clathrin-dependent endocytosis plays a predominant role in cellular uptake of double-stranded RNA in the red flour beetle

Systemic gene silencing from oral uptake of dsRNA in Litopenaeus vannamei requires both clathrin-mediated endocytosis and LvSID-1

RNA interference (RNAi) caused by exogenous double-stranded RNA (dsRNA) has developed into a powerful technique for down-regulating gene expression in a wide range of organisms, following its discovery in the nematode Caenorhabditis elegans. Not only does import of dsRNA into cells to produce small interfering RNAs (siRNAs) take place readily in C. elegans, but also systemic RNAi effects can be shown to occur, which persist over distinct developmental stages. These effects are mediated by amplification of siRNAs and export to other cells in the organism. However, the sensitivity to RNAi in C. elegans, where specific genes can be down-regulated by feeding dsRNA, or even by soaking nematodes in dsRNA solutions, have not been duplicated in other organisms, nor have systemic and persistent RNAi effects been widely observed. Uptake of dsRNA was limited in the model insect species Drosophila melanogaster (fruit fly), and no evidence of systemic and persistent RNAi effects was observed. However, more recent research has shown that full RNAi effects can occur in insects, although they are variable from species to species. Down-regulation of gene expression through delivery of dsRNA to insects can cause mortality, through a range of altered phenotypes, such as interference with developmental processes, or metabolism, or responses to the environment, and systemic and persistent RNAi effects have been reported, most notably in the beetle Tribolium confusum. Whereas injection of dsRNA remains the method of choice for delivery to insects, in some species feeding dsRNA has also been shown to produce RNAi effects. Further, expression of dsRNAs directed against insect genes in transgenic plants has been shown to result in RNAi effects and to afford protection against insect herbivores. This technology has the potential to be the basis of a new generation of pest-resistant GM crops, complementing existing technologies, but further development to improve efficacy of protection, and range of species affected, will be necessary.

In filamentous fungi, gene silencing through RNA interference (RNAi) shapes many biological processes, including pathogenicity. We explored the requirement of key components of fungal RNAi machineries, including DICER-like 1 and 2 (FgDCL1, FgDCL2), ARGONAUTE 1 and 2 (FgAGO1, FgAGO2), AGO-interacting protein FgQIP (QDE2-interacting protein), RecQ helicase (FgQDE3), and four RNA-dependent RNA polymerases (FgRdRP1, FgRdRP2, FgRdRP3, FgRdRP4), in the ascomycete mycotoxin-producing fungal pathogen Fusarium graminearum (Fg) for sexual and asexual multiplication, pathogenicity, and its sensitivity to double-stranded (ds)RNA. We corroborate and extend earlier findings that conidiation, ascosporogenesis, and Fusarium head blight (FHB) symptom development require an operable RNAi machinery. The involvement of RNAi in conidiation is dependent on environmental conditions as it is detectable only under low light (<2 μmol m−2 s−1). Although both DCLs and AGOs partially share their functions, the sexual ascosporogenesis is mediated primarily by FgDCL1 and FgAGO2, while FgDCL2 and FgAGO1 contribute to asexual conidia formation and germination. FgDCL1 and FgAGO2 also account for pathogenesis as their knockout (KO) results in reduced FHB development. Apart from KO mutants Δdcl2 and Δago1, mutants Δrdrp2, Δrdrp3, Δrdrp4, Δqde3, and Δqip are strongly compromised for conidiation, while KO mutations in all RdPRs, QDE3, and QIP strongly affect ascosporogenesis. Analysis of trichothecenes mycotoxins in wheat kernels showed that the relative amount of deoxynivalenol (DON), calculated as [DON] per amount of fungal genomic DNA was reduced in all spikes infected with RNAi mutants, suggesting the possibility that the fungal RNAi pathways affect Fg’s DON production. Moreover, silencing of fungal genes by exogenous target gene-specific double-stranded RNA (dsRNA) (spray-induced gene silencing, SIGS) is dependent on DCLs, AGOs, and QIP, but not on QDE3. Together these data show that in F. graminearum, different key components of the RNAi machinery are crucial in different steps of fungal development and pathogenicity.

Involvement of LvSID-1 in dsRNA uptake in Litopenaeus vannamei

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism whereby target gene messenger RNA (mRNA) is neutralized by double-stranded RNA (dsRNA) homologous to the mRNA sequence. The pathway can be exploited for pest and disease control purposes by delivery of exogenous dsRNA targeting a gene essential for the target organism’s survival. The most likely dsRNA delivery strategy for invertebrate pest control is through oral uptake, but transdermal dsRNA uptake has been reported to lead to gene silencing in some species as well. To combat plant-pathogenic fungi and viruses, methods that efficiently deliver dsRNAs into plant tissues are needed. While transgenic plants allow for efficient production of such dsRNAs in the plants, non-transgenic spray-based applications are being developed as well. Although RNAi is highly effective in some species, for example, certain beetle species, many insects and nematodes show a variable or lower sensitivity to dietary uptake of dsRNA. In the past decade, several factors and barriers affecting RNAi efficiency in insects have been identified, including dsRNA degradation in the insect body, inefficient cellular uptake of dsRNA, and an impaired endosomal escape into the cytoplasm. Nanocarriers could play a role in enhancing the efficacy of sprayable RNAi-based pesticides by helping to overcome some of these barriers. Several proof-of-concept studies have shown that polymers, liposomes, and peptides, among others could be used in this context but further advances are necessary to optimize these delivery systems. Gathering inspiration from the medical field, where RNAi is also being investigated as a potential therapeutics strategy, could drive forward these agricultural applications. In this chapter, we present an overview of the literature on RNAi barriers and the use of these nanoparticles to increase RNAi efficacy in agricultural pests. Finally, we also discuss a number of biosafety considerations regarding RNAi and the use of these nanoparticle formulations.

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.

RNA interference (RNAi) has become a widely used technique of knocking down a gene's expression in insects, but its efficacy in some species is limited by a reduced ability of the cells to take in and disperse the double-stranded RNA (dsRNA) throughout the cytoplasm. While RNA transport proteins such as SID-1 and its orthologues can facilitate dsRNA uptake in some invertebrate species, dsRNA uptake in many insects examined to date appears to be facilitated by clathrin-mediated endocytosis (CME). In this study, we used pharmacological inhibitors and RNAi-mediated knockdown of endocytic genes to provide evidence that CME is the primary means of dsRNA uptake in the mosquito Aedes aegypti. Inhibition of clathrin-mediated endocytosis was sufficient to supress uptake of short (21 nt) interfering RNAs (siRNAs), short (23 nt) hairpin RNAs (shRNAs), and long (>200 nt) dsRNA molecules in Aedes aegypti cultured cells and larvae. In contrast, we observed that short (23 nt) “paperclip” RNAs (pcRNAs), with partially closed ends, efficiently enter cells via a clathrin-independent pathway and effectively facilitate transcript knockdown. This alternative dsRNA structure may prove useful in insects generally considered recalcitrant to RNAi and in insect populations where resistance to RNAi-insecticides may arise through changes in dsRNA uptake mechanisms.

RNA Interference: Systemic RNAi SIDes with Endosomes

As the second largest organ in insects, the insect midgut is the major tissue involved in the digestion of food and detoxification of xenobiotics, such as insecticides, and the first barrier and target for oral RNA interference (RNAi). In this study, we performed a midgut-specific transcriptome analysis in the oriental fruit fly, Bactrocera dorsalis, an economically important worldwide pest, with many populations showing high levels of insecticide resistance. Using high-throughput sequencing, 52 838 060 short reads were generated and assembled to 25 236 unigenes with a mean length of 758 bp. Interestingly, 34 unique sequences encoding digestion enzymes were newly described and these included aminopeptidase and trypsin, genes associated with Bacillus thuringiensis resistance and fitness cost. Second, 41 transcripts were annotated to particular detoxification genes such as glutathione S-transferases, carboxylesterases and cytochrome P450s, and the subsequent phylogenetic analysis indicated homology with tissue-specific and insecticide resistance-related genes of Drosophila melanogaster. Third, we identified the genes involved in the mechanism of RNAi and the uptake of double-stranded RNA. The sequences encoding Dicer-2, R2D2, AGO2, and Eater were confirmed, but SID and SR-CI were absent in the midgut transcriptome. In conclusion, the results provide basic molecular information to better understand the mechanisms of food digestion, insecticide resistance and oral RNAi in this important pest insect in agriculture. Specific genes in these systems can be used in the future as potential targets for pest control, for instance, with RNAi technology.

RNA interference (RNAi) is a post-transcriptional gene silencing process where short interfering RNAs degrade targeted mRNA. Exploration of gene function through reverse genetics is the major achievement of RNAi discovery. Besides, RNAi can be used as a potential strategy for the control of insect pests. This has led totheidea of developing RNAi-based pesticides. Differential RNAi efficiency in the different insect orders is the biggest biological obstacle in developing RNAi-based pesticides. dsRNA stability, the sensitivity of core RNAi machinery, uptake of dsRNA and amplification and spreading of the RNAi signal are the key factors responsible for RNAi efficiency in insects. This review discusses the physiological and adaptive factors responsible for reduced RNAi in insects that pose a major challenge in developing dsRNA- based pesticides.

The vast majority of lepidopterans, about 90%, are moths. Some moths, particularly their caterpillars, are major agricultural and forestry pests in many parts of the world. However, some other members of moths, such as the silkworm Bombyx mori, are famous for their economic value. Fire et al. in 1998 initially found that exogenous double-stranded RNA (dsRNA) can silence the homolog endogenous mRNA in organisms, which is called RNA interference (RNAi). Soon after, the RNAi technique proved to be very promising not only in gene function determination but also in pest control. However, later studies demonstrate that performing RNAi in moths is not as straightforward as shown in other insect taxa. Nevertheless, since 2007, especially after 2010, an increasing number of reports have been published that describe successful RNAi experiments in different moth species either on gene function analysis or on pest management exploration. So far, more than 100 peer-reviewed papers have reported successful RNAi experiments in moths, covering 10 families and 25 species. By using classic and novel dsRNA delivery methods, these studies effectively silence the expression of various target genes and determine their function in larval development, reproduction, immunology, resistance against chemicals, and other biological processes. In addition, a number of laboratory and field trials have demonstrated that RNAi is also a potential strategy for moth pest management. In this review, therefore, we summarize and discuss the mechanisms and applications of the RNAi technique in moths by focusing on recent progresses.