RNA Interference Pathway Research Articles

Antiviral defense against Cypovirus 1 (Reoviridae) infection in the silkworm, Bombyx mori.

Recent years have shown a large increase in studies of infection of the silkworm (Bombyx mori) with Cypovirus 1 (previously designated as B. mori cytoplasmic polyhedrosis virus), that causes serious damage in sericulture. Cypovirus 1 has a single-layered capsid that encapsulates a segmented double-strand RNA (dsRNA) genome which are attractive features for the establishment of a biotechnological platform for the production of specialized gene silencing agents, either as recombinant viruses or as viral-like particles with nonreplicative dsRNA cargo. For both combatting viral disease and application of Cypovirus-based pest control, however, a better understanding is needed of the innate immune response caused by Cypovirus infection of the midgut of lepidopteran larvae. Studies of deep sequencing of viral small RNAs have indicated the importance of the RNA interference pathway in the control of Cypovirus infection although many functional aspects still need to be elucidated and conclusive evidence is lacking. A considerable number of transcriptome studies were carried out that revealed a complex response that hitherto remains uncharacterized because of a dearth in functional studies. Also, the uptake mechanism of Cypovirus by the midgut cells remains unclarified because of contrasting mechanisms revealed by electron microscopy and functional studies. The field will benefit from an increase in functional studies that will depend on transgenic silkworm technology and reverse genetics systems for Cypovirus 1.

A Novel Method to Silence Genes in Cryptosporidium.

The parasites of the genus Cryptosporidium are important causes of diarrheal diseases, specifically cryptosporidiosis, worldwide. A major bottleneck for developing drugs and vaccines against cryptosporidiosis is the lack of methods to study gene function in this parasite. Silencing of genes by RNA interference (RNAi) is a powerful method to investigate gene function that has been widely used in the identification of targets for several pathogens. Unfortunately, as Cryptosporidium does not possess the enzymes of the RNAi pathway, its genes cannot be silenced by standard siRNA technology. To circumvent that problem, we have developed a novel strategy to knock down Cryptosporidium genes by reconstituting the effector arm of the siRNA pathway. We have induced silencing of several genes in Cryptosporidium by transfecting parasites with hybrid complexes formed between recombinant human Argonaute (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA). This novel methodology provides an effective strategy to study the role of selected genes in host-parasite interactions, and also can be used to identify potential targets for chemotherapy. The standardized methodology based on this strategy is described in this chapter.

Nanocarrier Lipid Composition Modulates the Impact of Pulmonary Surfactant Protein B (SP-B) on Cellular Delivery of siRNA.

Two decades since the discovery of the RNA interference (RNAi) pathway, we are now witnessing the approval of the first RNAi-based treatments with small interfering RNA (siRNA) drugs. Nevertheless, the widespread use of siRNA is limited by various extra- and intracellular barriers, requiring its encapsulation in a suitable (nanosized) delivery system. On the intracellular level, the endosomal membrane is a major barrier following endocytosis of siRNA-loaded nanoparticles in target cells and innovative materials to promote cytosolic siRNA delivery are highly sought after. We previously identified the endogenous lung surfactant protein B (SP-B) as siRNA delivery enhancer when reconstituted in (proteo) lipid-coated nanogels. It is known that the surface-active function of SP-B in the lung is influenced by the lipid composition of the lung surfactant. Here, we investigated the role of the lipid component on the siRNA delivery-promoting activity of SP-B proteolipid-coated nanogels in more detail. Our results clearly indicate that SP-B prefers fluid membranes with cholesterol not exceeding physiological levels. In addition, SP-B retains its activity in the presence of different classes of anionic lipids. In contrast, comparable fractions of SP-B did not promote the siRNA delivery potential of DOTAP:DOPE cationic liposomes. Finally, we demonstrate that the beneficial effect of lung surfactant on siRNA delivery is not limited to lung-related cell types, providing broader therapeutic opportunities in other tissues as well.

Natural Viruses of Caenorhabditis Nematodes.

Caenorhabditis elegans has long been a laboratory model organism with no known natural pathogens. In the past ten years, however, natural viruses have been isolated from wild-caught C. elegans (Orsay virus) and its relative Caenorhabditis briggsae (Santeuil virus, Le Blanc virus, and Melnik virus). All are RNA positive-sense viruses related to Nodaviridae; they infect intestinal cells and are horizontally transmitted. The Orsay virus capsid structure has been determined and the virus can be reconstituted by transgenesis of the host. Recent use of the Orsay virus has enabled researchers to identify evolutionarily conserved proviral and antiviral genes that function in nematodes and mammals. These pathways include endocytosis through SID-3 and WASP; a uridylyltransferase that destabilizes viral RNAs by uridylation of their 3' end; ubiquitin protein modifications and turnover; and the RNA interference pathway, which recognizes and degrades viral RNA.

Different Components of the RNA Interference Machinery Are Required for Conidiation, Ascosporogenesis, Virulence, Deoxynivalenol Production, and Fungal Inhibition by Exogenous Double-Stranded RNA in the Head Blight Pathogen Fusarium graminearum.

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.

Three novel RNA viruses in the spider mite Tetranychus urticae and their possible interactions with the host RNA interference response

As a polyphagous herbivore, the two-spotted spider mite Tetranychus urticae is engaged with various plant hosts and interacts with diverse organisms that share the same ecological niche. Thus, T. urticae faces frequent challenges from viral infections. However, the RNA viruses of T. urticae are still unknown. Here, we constructed two libraries (~8 Gb for RNA and ~10 Mb for small RNA) from a strain of T. urticae using deep sequencing, and identified three novel RNA viruses from the families Kitaviridae, Dicistroviridae, and Chuviridae. Among them, the Kitaviridae and Dicistroviridae viruses presented a possible interaction pattern with the host RNA interference pathway.

Rapid evolution of piRNA pathway and its transposon targets in Japanese flounder (Paralichthys olivaceus)

Piwi-interacting RNA (piRNA) pathway is essential for germline specification, gametogenesis, and genome integrity as defense against transposable elements (TEs). This pathway has been suggested to have undergone rapid adaptive evolution in spite of its conserved role in TE silencing. However, with diverse sexual development patterns, piRNA pathway evolution and its adaptation to transposon activity in teleost lineages remain less known. This study illustrated the evolutionary significance of piRNA pathway via a systematic comparative analysis on diverse teleosts, including flatfish lineages. Molecular evolution of piRNA pathway and microRNA/small interfering RNA pathway genes indicated a faster evolution of piRNA pathway in teleosts than in mammals. Positive selection was detected at the PAZ (Piwi-Argonaute-Zwille) domain involved in Piwi–piRNA interaction, thereby suggesting that the amino acid substitutions were adaptive to their functions in teleost piRNA pathway. Notably, Piwil1 evolved faster in Japanese flounder than in other teleosts, and the piRNA pathway genes expressed higher in testes than in ovaries. In addition, gonadal transcriptomic analysis revealed male under-represented TE families mainly from DNA transposons, which were the potential targets of the complex formed by male-biased Piwi genes and male over-represented piRNAs in Japanese flounder testes. The potential piRNA-TE regulatory relationships suggested that the rapidly evolved piRNA pathway in Japanese flounder was likely involved in the regulation of transposon activity in germlines and could play important roles in Japanese flounder gonadal development and spermatogenesis.

A Family of Nuclear Argonaute Interacting Proteins Gates Nuclear RNAi

Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational inheritance (TEI) of gene silencing. How the nuclear activities of RNAi are physiologically controlled remains poorly understood. Here, we identify and characterize a family of nuclear Argonaute-interacting proteins (NIPs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. NIP-1 and NIP-2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, NIP-1 and NIP-2 are negative regulators of nuclear RNAi triggered from exogenous sources (exo-RNAi). Loss of NIP-3, an unstable protein mostly expressed in the male germline, misdirects the RNAi response to transposable elements and impairs TEI.Our data support a model wherein NIPs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes in both somatic and germline tissues.

The Activation of Antiviral RNA Interference Not Only Exist in Neural Progenitor Cells But Also in Somatic Cells in Mammals

The RNA interference (RNAi) pathway directs an important antiviral immunity mechanism in plants and invertebrates. Recently, we and others have demonstrated that the induction and suppression of antiviral RNAi response are also conserved in mammals, at least to five distinct RNA viruses, including Zika virus (ZIKV). ZIKV preferentially infects neuronal progenitor cells (NPCs) in the developing fetal brain. Xu et al. have revealed that ex vivo ZIKV infection induces RNAi-mediated antiviral response in human NPCs, but not in the more differentiated NPCs or somatic cells. However, less is known about the in vivo property or function of the virus-derived small-interfering RNAs (vsiRNAs) targeting Zika virus. Here we report a surprising observation: viral small RNAs (vsRNAs) targeting ZIKV were produced in vivo upon infection in both central neuron system (CNS) and muscle tissues. In addition, our findings demonstrate the production of canonical vsiRNAs in murine CNS upon antiviral RNAi activation by Sindbis virus, suggesting the possibility of antiviral immune strategy applied by mammals in the CNS.

MiR403a and SA Are Involved in NbAGO2 Mediated Antiviral Defenses Against TMV Infection in Nicotiana benthamiana

RNAi (RNA interference) is an important defense response against virus infection in plants. The core machinery of the RNAi pathway in plants include DCL (Dicer Like), AGO (Argonaute) and RdRp (RNA dependent RNA polymerase). Although involvement of these RNAi components in virus infection responses was demonstrated in Arabidopsis thaliana, their contribution to antiviral immunity in Nicotiana benthamiana, a model plant for plant-pathogen interaction studies, is not well understood. In this study, we investigated the role of N. benthamiana NbAGO2 gene against TMV (Tomato mosaic virus) infection. Silencing of NbAGO2 by transient expression of an hpRNA construct recovered GFP (Green fluorescent protein) expression in GFP-silenced plant, demonstrating that NbAGO2 participated in RNAi process in N. benthamiana. Expression of NbAGO2 was transcriptionally induced by both MeSA (Methylsalicylate acid) treatment and TMV infection. Down-regulation of NbAGO2 gene by amiR-NbAGO2 transient expression compromised plant resistance against TMV infection. Inhibition of endogenous miR403a, a predicted regulatory microRNA of NbAGO2, reduced TMV infection. Our study provides evidence for the antiviral role of NbAGO2 against a Tobamovirus family virus TMV in N. benthamiana, and SA (Salicylic acid) mediates this by induction of NbAGO2 expression upon TMV infection. Our data also highlighted that miR403a was involved in TMV defense by regulation of target NbAGO2 gene in N. Benthamiana.

Dicer prevents genome instability in response to replication stress.

Dicer, an endoribonuclease best-known for its role in microRNA biogenesis and RNA interference pathway, has been shown to play a role in the DNA damage response and repair of double-stranded DNA breaks (DSBs) in mammalian cells. However, it remains unknown whether Dicer is also important to preserve genome integrity upon replication stress. To address this question, we focused our study on common fragile sites (CFSs), which are susceptible to breakage after replication stress. We show that inhibition of the Dicer pathway leads to an increase in CFS expression upon induction of replication stress and to an accumulation of 53BP1 nuclear bodies, indicating transmission of replication-associated damage. We also show that in absence of a functional Dicer or Drosha, the assembly into nuclear foci of the Fanconi anemia (FA) protein FANCD2 and of the replication and checkpoint factor TopBP1 in response to replication stress is impaired, and the activation of the S-phase checkpoint is defective. Based on these results, we propose that Dicer pre-vents genomic instability after replication stress, by allowing the proper recruitment to stalled forks of proteins that are necessary to maintain replication fork stability and activate the S-phase checkpoint, thus limiting cells from proceeding into mitosis with under-replicated DNA.

The South American Fruit Fly: An Important Pest Insect With RNAi-Sensitive Larval Stages.

RNA interference (RNAi) technology has been used in the development of approaches for pest control. The presence of some essential genes, the so-called “core genes,” in the RNAi machinery is crucial for its efficiency and robust response in gene silencing. Thus, our study was designed to examine whether the RNAi machinery is functional in the South American (SA) fruit fly Anastrepha fraterculus (Diptera: Tephritidae) and whether the sensitivity to the uptake of double-stranded RNA (dsRNA) could generate an RNAi response in this fruit fly species. To prepare a transcriptome database of the SA fruit fly, total RNA was extracted from all the life stages for later cDNA synthesis and Illumina sequencing. After the de novo transcriptome assembly and gene annotation, the transcriptome was screened for RNAi pathway genes, as well as the duplication or loss of genes and novel target genes to dsRNA delivery bioassays. The dsRNA delivery assay by soaking was performed in larvae to evaluate the gene-silencing of V-ATPase, and the upregulation of Dicer-2 and Argonaute-2 after dsRNA delivery was analyzed to verify the activation of siRNAi machinery. We tested the stability of dsRNA using dsGFP with an in vitro incubation of larvae body fluid (hemolymph). We identified 55 genes related to the RNAi machinery with duplication and loss for some genes and selected 143 different target genes related to biological processes involved in post-embryonic growth/development and reproduction of A. fraterculus. Larvae soaked in dsRNA (dsV-ATPase) solution showed a strong knockdown of V-ATPase after 48 h, and the expression of Dicer-2 and Argonaute-2 responded with an increase upon the exposure to dsRNA. Our data demonstrated the existence of a functional RNAi machinery in the SA fruit fly, and we present an easy and robust physiological bioassay with the larval stages that can further be used for screening of target genes at in vivo organisms’ level for RNAi-based control of fruit fly pests. This is the first study that provides evidence of a functional siRNA machinery in the SA fruit fly.

Diversity of RNA interference pathways in regulation of endogenous and exogenous sequences expression in ciliates Tetrahymena and Paramecium

RNA interference plays a major role in biology of ciliates. Diverse small RNAs regulate many processes in vegetative cells of ciliates Tetrahymena and Paramecium. Different types of endogenous and exogenous nucleotide sequences induce different RNAi pathways resulting in silencing of the homologous sequences in the macronuclear genome. Likely this way ciliates are able to quickly inactivate heterogeneous sequences and to adapt efficiently to the environmental conditions and external stimuli.

Theranostic Nanoparticles for RNA-Based Cancer Treatment.

Certain genetic mutations lead to the development of cancer through unchecked cell growth and division. Cancer is typically treated through surgical resection, radiotherapy, and small-molecule chemotherapy. A relatively recent approach to cancer therapy involves the use of a natural process wherein small RNA molecules regulate gene expression in a pathway known as RNA interference (RNAi). RNA oligomers pair with a network of proteins to form an RNA-induced silencing complex, which inhibits the translation of mRNA into proteins, thereby controlling the expression of gene products. Synthetically produced RNA oligomers may be designed to target and silence specific oncogenes to provide cancer therapy. The primary challenges facing the use of the RNAi pathway for cancer therapy are the safe and efficacious delivery of RNA payloads and their release at pertinent sites within disease-causing cells. Nucleases are abundant in the bloodstream and intracellular environment, and therapeutic RNA sequences often require a suitable carrier to provide protection from degradation prior to reaching their site of action in the body. The use of metal core nanoparticles (NPs) serving as targeted delivery vehicles able to shield and direct RNA payloads to their intended destinations have recently gained favor. Biological barriers present in the body establish a size prerequisite for drug delivery vehicles; to overcome recognition by the body's immune system and to gain access to intracellular environments, drug carriers must be small (< 100 nm). Iron oxide and gold core NPs can be synthesized with a high degree of control to create uniform ultrasmall drug delivery vehicles capable of bypassing key biological barriers. While progress is being made in size control of liposomal and polymer NPs, such advances still lag in comparison to the exquisite tunability and time stability of size engineering achievable with metal core NPs at bulk scales. Further, unlike lipid- and viral-based transfection agents, the biodistribution of metal core NPs can be traced using noninvasive imaging techniques that capitalize on the interaction of electromagnetic radiation and the inorganic atoms at the core of the NPs. Finally, metal core NPs have been shown to match the transfection efficiency of conventional RNA-delivery vehicles while also providing less immunogenicity and minimal side effects through the addition of tumor-targeting ligands on their surface. This Account reviews recent advances in the use of iron oxide and gold NPs for RNAi therapy. An overview of the different types of RNA-based therapies is provided along with a discussion of the advantages and current limitations of the technique. We highlight design considerations for the use of iron oxide and gold NP carriers in RNAi, including a discussion of the importance of size and its role in traversing biological barriers, NP surface modifications required for targeted delivery and RNA payload release, and auxiliary properties supporting imaging functionality for treatment monitoring. Applications of NPs for combination therapies including the pairing of RNAi with chemotherapy, photothermal therapy, immunotherapy, and radiotherapy are explored through examples. Finally, future perspectives are provided with a focus on the current limitations and the potential for clinical translation of iron oxide and gold NPs in RNAi therapy.

Antiviral RNAi in Insects and Mammals: Parallels and Differences.

The RNA interference (RNAi) pathway is a potent antiviral defense mechanism in plants and invertebrates, in response to which viruses evolved suppressors of RNAi. In mammals, the first line of defense is mediated by the type I interferon system (IFN); however, the degree to which RNAi contributes to antiviral defense is still not completely understood. Recent work suggests that antiviral RNAi is active in undifferentiated stem cells and that antiviral RNAi can be uncovered in differentiated cells in which the IFN system is inactive or in infections with viruses lacking putative viral suppressors of RNAi. In this review, we describe the mechanism of RNAi and its antiviral functions in insects and mammals. We draw parallels and highlight differences between (antiviral) RNAi in these classes of animals and discuss open questions for future research.

Comparative molecular cell biology of phototrophic euglenids and parasitic trypanosomatids sheds light on the ancestor of Euglenozoa.

Parasitic trypanosomatids and phototrophic euglenids are among the most extensively studied euglenozoans. The phototrophic euglenid lineage arose relatively recently through secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga that evolved into the euglenid secondary chloroplast. The parasitic trypanosomatids (i.e. Trypanosoma spp. and Leishmania spp.) and the freshwater phototrophic euglenids (i.e. Euglena gracilis) are the most evolutionary distant lineages in the Euglenozoa phylogenetic tree. The molecular and cell biological traits they share can thus be considered as ancestral traits originating in the common euglenozoan ancestor. These euglenozoan ancestral traits include common mitochondrial presequence motifs, respiratory chain complexes containing various unique subunits, a unique ATP synthase structure, the absence of mitochondria-encoded transfer RNAs (tRNAs), a nucleus with a centrally positioned nucleolus, closed mitosis without dissolution of the nuclear membrane and nucleoli, a nuclear genome containing the unusual 'J' base (β-D-glucosyl-hydroxymethyluracil), processing of nucleus-encoded precursor messenger RNAs (pre-mRNAs) via spliced-leader RNA (SL-RNA) trans-splicing, post-transcriptional gene silencing by the RNA interference (RNAi) pathway and the absence of transcriptional regulation of nuclear gene expression. Mitochondrial uridine insertion/deletion RNA editing directed by guide RNAs (gRNAs) evolved in the ancestor of the kinetoplastid lineage. The evolutionary origin of other molecular features known to be present only in either kinetoplastids (i.e. polycistronic transcripts, compaction of nuclear genomes) or euglenids (i.e. monocistronic transcripts, huge genomes, many nuclear cis-spliced introns, polyproteins) is unclear.

Characterization of a new bunyavirus and its derived small RNAs in the brown citrus aphid, Aphis citricidus.

High-throughput sequencing is widely used for virus discovery, and many RNA viruses have been discovered and identified. A new negative-sense single-stranded RNA virus was identified in the brown citrus aphid and named Aphis citricidus bunyavirus. The genome consists of large (7037nt), medium (3462nt), and small (1163nt) segments. Phylogenetic analysis and amino acid sequences identities of this virus with other bunyaviruses suggest that it is a new species belonging to the family Phenuiviridae. The small interfering RNA pathway could be involved against the infection of this virus in brown citrus aphid as supported by the viral derived small RNAs. The discovery of this virus illustrates the diversity of RNA viruses and contributes to the classification of bunyaviruses.

Widespread gene duplication and adaptive evolution in the RNA interference pathways of the Drosophila obscura group

BackgroundRNA interference (RNAi) related pathways provide defense against viruses and transposable elements, and have been implicated in the suppression of meiotic drive elements. Genes in these pathways often exhibit high levels of adaptive substitution, and over longer timescales show gene duplication and loss—most likely as a consequence of their role in mediating conflict with these parasites. This is particularly striking for Argonaute 2 (Ago2), which is ancestrally the key effector of antiviral RNAi in insects, but has repeatedly formed new testis-specific duplicates in the recent history of the obscura species-group of Drosophila.ResultsHere we take advantage of publicly available genomic and transcriptomic data to identify six further RNAi-pathway genes that have duplicated in this clade of Drosophila, and examine their evolutionary history. As seen for Ago2, we observe high levels of adaptive amino-acid substitution and changes in sex-biased expression in many of the paralogs. However, our phylogenetic analysis suggests that co-duplications of the RNAi machinery were not synchronous, and our expression analysis fails to identify consistent male-specific expression.ConclusionsThese results confirm that RNAi genes, including genes of the antiviral and piRNA pathways, have undergone multiple independent duplications and that their history has been particularly labile within the obscura group. However, they also suggest that the selective pressures driving these changes have not been consistent, implying that more than one selective agent may be responsible.

Genome-wide identification and analysis of genes associated with RNA interference in Bemisia tabaci.

As a method of RNA-mediated gene silencing, RNA interference (RNAi) is a useful reverse genetic tool with which to study gene function, and holds great promise for pest management. Bemisia tabaci is a cosmopolitan pest that causes extensive damage to crops. The mechanism underlying RNAi efficiency in B. tabaci is not well known. We identified and analyzed candidate genes in the RNAi pathway to understand the RNAi mechanism and provide a basis for the application of RNAi in pest management. We identified 33 genes putatively involved in the RNAi pathway from the B. tabaci Q genome. Phylogenetic and structural analyses confirmed the characteristics of these genes. Furthermore, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and transcriptomic analysis profiled gene expression patterns during different developmental stages. Gene expression levels estimated by qRT-PCR and RNA-seq analyses were significantly correlated. Moreover, gene functions were verified by RNAi. When accompanied by knockdown of AGO2, Dicer2 and Sid1, the efficiency of CYP6DB3 RNAi decreased correspondingly. In this study, we annotated and validated genes involved in B. tabaci RNAi. A better understanding of the building blocks of the RNAi process in B. tabaci facilitates integration of this novel biotechnology into the management of this emerging pest, either directly or indirectly. © 2019 Society of Chemical Industry.

Function of heat shock protein 70 in the thermal stress response of Dermatophagoides farinae and establishment of an RNA interference method

Dermatophagoides farinae are an important mite species that cause stored product deterioration and allergic diseases. They widely breed in human habitats because of their strong tolerance to extreme external temperatures. However, mechanisms underlying the stress response and tolerance of D. farinae are unclear. We hypothesized that heat shock protein 70 plays an important role in the heat stress response of D. farinae. In this study, we determined the survival rates of D. farinae at high temperatures (37 °C–45 °C) by performing temperature-gradient experiments in vitro and assessed the expression level of HSP70 by performing RT-qPCR. First, we confirmed that HSP70 regulated the heat stress response of D. farinae, with maximum heat stress regulation observed at 41 °C. Next, we confirmed the presence of a Dicer enzyme-mediated RNA interference (RNAi) pathway in D. farinae by searching the NCBI database and a Dicer site prediction website. Finally, we performed RNAi in D. farinae by using an immersion method with screened dsHSP70 fragments. Moreover, we performed concentration-gradient experiments to determine that 600 ng/μl was the minimal effective concentration of dsHSP70 for silencing HSP70. These results confirm that HSP70 regulates the heat stress response of D. farinae. The present study is the first to report the use of the non-invasive and highly sensitive immersion method for performing RNAi in D. farinae. The results of the present study provide a technical foundation for performing functional gene research and for developing molecular prevention and control strategies against medically important mites.