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.
MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that have crucial roles in regulating gene expression. Increasing evidence supports a role for miRNAs in many human diseases, ...including cancer and autoimmune disorders. The function of miRNAs can be efficiently and specifically inhibited by chemically modified antisense oligonucleotides, supporting their potential as targets for the development of novel therapies for several diseases. In this Review we summarize our current knowledge of the design and performance of chemically modified miRNA-targeting antisense oligonucleotides, discuss various in vivo delivery strategies and analyse ongoing challenges to ensure the specificity and efficacy of therapeutic oligonucleotides in vivo. Finally, we review current progress on the clinical development of miRNA-targeting therapeutics.
Glutathione S-transferases (GSTs) are multifunctional enzymes, and insect GSTs play a pivotal role in the metabolism of insecticides. Grapholita molesta is a worldwide pest that causes substantial ...economic losses to the fruit industry. However, it remains unclear how imidacloprid, a commonly used insecticide in orchards, is metabolized by G. molesta. In the present study, the synergist diethyl maleate (DEM), which inhibits the GST activity, exhibited a 22-fold synergistic ratio against imidacloprid. Two new GST genes, GmGSTD2 (OR096251) and GmGSTD3 (OR096252), were identified and successfully cloned, showing the highest expression in the Malpighian tubes. Knockdown of GmGSTD2 and GmGSTD3 by RNA interference, increased the mortality of G. molesta from 28% to 47% following imidacloprid treatment. Both recombinant GmGSTD2 and GmGSTD3 proteins exhibited 1-chloro-2,4-dinitrobenzene (CDNB) activity and could be inhibited by imidacloprid in vitro, with maximum inhibition was 60% for GmGSTD2 and 80% for GmGSTD3. These results suggested that GSTs participate in the metabolism of imidacloprid with GmGSTD2 and GmGSTD3 playing key roles in this process.
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•Glutathione S-transferases play an important role in the metabolism of imidacloprid.•GmGSTD2 and GmGSTD3 are highly expressed in the Malpighian tubes.•Knockdown of GmGSTD2 and GmGSTD3 increases the susceptibility of the Grapholita molesta to imidacloprid.•The activity of GmGSTD2 and GmGSTD3 proteins was inhibited by imidacloprid in vitro.
Protection against microbial infection in eukaryotes is provided by diverse cellular and molecular mechanisms. Here, we present a comparative view of the antiviral activity of virus-derived small ...interfering RNAs in fungi, plants, invertebrates and mammals, detailing the mechanisms for their production, amplification and activity. We also highlight the recent discovery of viral PIWI-interacting RNAs in animals and a new role for mobile host and pathogen small RNAs in plant defence against eukaryotic pathogens. In turn, viruses that infect plants, insects and mammals, as well as eukaryotic pathogens of plants, have evolved specific virulence proteins that suppress RNA interference (RNAi). Together, these advances suggest that an antimicrobial function of the RNAi pathway is conserved across eukaryotic kingdoms.
Summary
Arbuscular mycorrhizal (AM) symbiosis is a mutualistic interaction between fungi and most land plants that is underpinned by a bidirectional exchange of nutrients. AM development is a tightly ...regulated process that encompasses molecular communication for reciprocal recognition, fungal accommodation in root tissues and activation of symbiotic function. As such, a complex network of transcriptional regulation and molecular signaling underlies the cellular and metabolic reprogramming of host cells upon AM fungal colonization. In addition to transcription factors, small RNAs (sRNAs) are emerging as important regulators embedded in the gene network that orchestrates AM development. In addition to controlling cell‐autonomous processes, plant sRNAs also function as mobile signals capable of moving to different organs and even to different plants or organisms that interact with plants. AM fungi also produce sRNAs; however, their function in the AM symbiosis remains largely unknown. Here, we discuss the contribution of host sRNAs in the development of AM symbiosis by considering their role in the transcriptional reprogramming of AM fungal colonized cells. We also describe the characteristics of AM fungal‐derived sRNAs and emerging evidence for the bidirectional transfer of functional sRNAs between the two partners to mutually modulate gene expression and control the symbiosis.
RNA interference (RNAi) on parasitic nematodes has been described as a valuable tool for screening putative targets that could be used as novel drug and/or vaccine candidates. This study aimed to set ...up a pipeline to identify potential targets using RNAi for vaccine/anti-parasite therapy development against Haemonchus contortus, a blood-feeding abomasal nematode parasite. The available H. contortus sequence data was mined for targets, which were tested for essentiality using RNAi electroporation assays. A total of 56 genes were identified and tested for knockdown using electroporation of first-stage larvae (L1) H. contortus with the target double-stranded RNA. Electroporation of L1 proved to be effective overall; 17 targets had a strong phenotype and significant reduction in alive H. contortus, and another 24 had a moderate phenotype with a significant reduction in larvae development. A total of 28 targets showed a significant reduction in the development of H. contortus larvae to the infective stage (L3) following the RNAi assay. Down-regulation of target transcript levels was evaluated in some targets by semi-quantitative PCR. Four out of five genes tested showed complete knockdown of mRNA levels via semi-quantitative PCR, whereas the knockdown was partial for one. In conclusion, the results indicate that the RNAi pathway is confirmed in H. contortus and that several target genes have the potential to be investigated further as possible vaccine candidates.
•41 out of 56 H.contortus genes screened using RNAi gives a significant phenotype•28 of 56 targets significantly reduced larvae-to-infective-stage development•Semi quantitative PCR has confirmed complete knockdown of 4 targets
RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency ...of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects.
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