Identification of leaf chloroplast-specific promoter to efficiently control of Colorado potato beetle with reduced dsRNA accumulation in potato tubers.

BACKGROUND: By expressing double-stranded RNA (dsRNA) in potato plastids targeting the ß-Actin (ACT) gene of the Colorado potato beetle (CPB), transplastomic plants can trigger the beetle's RNA interference response to kill the CPB larvae. High expression of dsACT driven by rrn16 promoter (Prrn) in the leaf chloroplasts of transplastomic plants confers strong resistance to CPB. However, there are still residual amounts of dsRNA in the tubers, which are unnecessary for CPB control and may raise a potential food exposure issue. RESULTS: In order to reduce dsRNA accumulation in the tubers while maintaining stable resistance to CPB, we selected two promoters (PrbcL and PpsbD) from potato plastid-encoded rbcL and psbD genes and compared their activities with Prrn promoter for dsRNA synthesis in the leaf chloroplasts and tuber amyloplasts. We found that the dsACT accumulation levels in leaves of transplastomic plants St-PrbcL-ACT and St-PpsbD-ACT were significantly reduced when compared to St-Prrn-ACT, but they still maintained high resistance to CPB. By contrast, a few amounts of dsACT were still accumulated in the tubers of St-PrbcL-ACT, whereas no dsACT accumulation in tubers was detectable in St-PpsbD-ACT. CONCLUSION: We identified PpsbD as a useful promoter to reduce dsRNA accumulation in potato tubers while maintaining the high resistance of the potato leaves to CPB. © 2023 Society of Chemical Industry.

RNAi efficacy is enhanced by chronic dsRNA feeding in pollen beetle.

Double-stranded RNAs (dsRNAs) represent a promising class of biosafe insecticidal compounds. We examined the ability to induce RNA interference (RNAi) in the pollen beetle Brassicogethes aeneus via anther feeding, and compared short-term (3 d) to chronic (17 d) feeding of various concentrations of dsRNA targeting αCOP (dsαCOP). In short-term dsαCOP feeding, only the highest concentration resulted in significant reductions in B. aeneus survival; whereas in chronic dsαCOP feeding, all three concentrations resulted in significant mortality. Chronic dsαCOP feeding also resulted in significantly greater mortality compared to short-term feeding of equivalent dsαCOP concentrations. Our results have implications for the economics and development of dsRNA spray approaches for managing crop pests, in that multiple lower-concentration dsRNA spray treatments across crop growth stages may result in greater pest management efficacy, compared to single treatments using higher dsRNA concentrations. Furthermore, our results highlight the need for research into the development of RNAi cultivars for oilseed rape protection, given the enhanced RNAi efficacy resulting from chronic, compared to short-term, dsRNA feeding in B. aeneus.

Phosphatidylcholine Extends Lifespan via DAF-16 and Reduces Amyloid-Beta-Induced Toxicity in Caenorhabditis elegans.

Phosphatidylcholine is one of the major phospholipids comprising cellular membrane and is known to have several health-promoting activities, including the improvement of brain function and liver repair. In this paper, we examine the in vivo effect of dietary supplementation with phosphatidylcholine on the response to environmental stressors and aging in C. elegans. Treatment with phosphatidylcholine significantly increased the survival of worms under oxidative stress conditions. However, there was no significant difference in response to stresses caused by heat shock or ultraviolet irradiation. Oxidative stress is believed to be one of the major causal factors of aging. Then, we examined the effect of phosphatidylcholine on lifespan and age-related physiological changes. Phosphatidylcholine showed a lifespan-extending effect and a reduction in fertility, possibly as a tradeoff for long lifespan. Age-related decline of motility was also significantly delayed by supplementation with phosphatidylcholine. Interestingly, the expressions of well-known longevity-assuring genes, hsp-16.2 and sod-3, were significantly upregulated by dietary intervention with phosphatidylcholine. DAF-16, a transcription factor modulating stress response genes, was accumulated in the nucleus by phosphatidylcholine treatment. Increase of the ROS level with phosphatidylcholine suggests that the antioxidant and lifespan-extending effects are due to the hormetic effect of phosphatidylcholine. Phosphatidylcholine also showed a protective effect against amyloid beta-induced toxicity in Alzheimer's disease model animals. Experiments with long-lived mutants revealed that the lifespan-extending effect of phosphatidylcholine specifically overlapped with that of reduced insulin/IGF-1-like signaling and required DAF-16. These findings showed the antioxidant and antiaging activities of phosphatidylcholine for the first time in vivo. Further studies focusing on the identification of underlying cellular mechanisms involved in the antiaging effect will increase the possibility of using phosphatidylcholine for the development of antiaging therapeutics.

Nucleoside analogs in the study of the epitranscriptome.

Over 150 unique RNA modifications are now known including several nonstandard nucleotides present in the body of messenger RNAs. These modifications can alter a transcript's function and are collectively referred to as the epitrancriptome. Chemically modified nucleoside analogs are poised to play an important role in the study of these epitranscriptomic marks. Introduced chemical features on nucleic acid strands provide unique structures or reactivity that can be used for downstream detection or quantification. Three methods are used in the field to synthesize RNA containing chemically modified nucleoside analogs. Nucleoside analogs can be introduced by metabolic labeling, via polymerases with modified nucleotide triphosphates or via phosphoramidite-based chemical synthesis. In this review, these methods for incorporation of nucleoside analogs will be discussed with specific recently published examples pertaining to the study of the epitranscriptome.

Double-Stranded RNAs High-Efficiently Protect Transgenic Potato from Leptinotarsa decemlineata by Disrupting Juvenile Hormone Biosynthesis.

RNA interference (RNAi) has been developed for plant pest control. In this study, hairpin-type double-stranded RNA (dsRNA) targeting the juvenile hormone (JH) acid methyltransferase ( JHAMT) gene ( dsJHAMT) was introduced in potato plants via Agrobacterium-mediated transformation. The results indicated that the transcriptional RNA of dsJHAMT accumulated in the transgenic plants. The transcripts and proteins of the L. decemlineata JHAMT gene were significantly reduced in larvae feeding on dsJHAMT transgenic foliage. The dsJHAMT had a significant negative effect on the growth and development of L. decemlineata, especially resulting in less oviposition. Importantly, in the field trials, transgenic plants are high-efficiently protected from insect damage mainly because surviving insects laid fewer or no eggs. Even full protection from beetle damage can be acquired by continuously lowering insect population size at large scale in the field over the years. Therefore, the transgenic plants expressing dsJHAMT successfully provided an additional option for plant pest control.

Identification of Myricetin as an Ebola Virus VP35-Double-Stranded RNA Interaction Inhibitor through a Novel Fluorescence-Based Assay.

Ebola virus (EBOV) is a filovirus that causes a severe and rapidly progressing hemorrhagic syndrome; a recent epidemic illustrated the urgent need for novel therapeutic agents because no drugs have been approved for treatment of Ebola virus. A key contribution to the high lethality observed during EBOV outbreaks comes from viral evasion of the host antiviral innate immune response in which viral protein VP35 plays a crucial role, blocking interferon type I production, first by masking the viral double-stranded RNA (dsRNA) and preventing its detection by the pattern recognition receptor RIG-I. Aiming to identify inhibitors of the interaction of VP35 with the viral dsRNA, counteracting the VP35 viral innate immune evasion, we established a new methodology for high-yield recombinant VP35 (rVP35) expression and purification and a novel and robust fluorescence-based rVP35-RNA interaction assay ( Z' factor of 0.69). Taking advantage of such newly established methods, we screened a small library of Sardinian natural extracts, identifying Limonium morisianum as the most potent inhibitor extract. A bioguided fractionation led to the identification of myricetin as the component that can inhibit rVP35-dsRNA interaction with an IC50 value of 2.7 µM. Molecular docking studies showed that myricetin interacts with the highly conserved region of the VP35 RNA binding domain, laying the basis for further structural optimization of potent inhibitors of VP35-dsRNA interaction.

An origin of the immunogenicity of in vitro transcribed RNA.

The emergence of RNA-based therapeutics demands robust and economical methods to produce RNA with few byproducts from aberrant activity. While in vitro transcription using the bacteriophage T7 RNA polymerase is one such popular method, its transcripts are known to display an immune-stimulatory activity that is often undesirable and uncontrollable. We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long double-stranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. This promoter-independent transcriptional activity of the T7 RNA polymerase was observed for a wide range of DNA sequences and lengths, but can be suppressed by altering the transcription reaction with modified nucleotides or by reducing the Mg2+ concentration. The current work thus not only offers a previously unappreciated mechanism by which T7 transcripts stimulate the innate immune system, but also shows that the immune-stimulatory activity can be readily regulated.

Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat.

Friedreich's Ataxia (FA) is an inherited neurologic disorder caused by an expanded GAA repeat within intron 1 of the frataxin (FXN) gene that reduces expression of FXN protein. Agents that increase expression of FXN have the potential to alleviate the disease. We previously reported that duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) complementary to the GAA repeat could enhance expression of FXN protein. We now explore the potential of a diverse group of chemically modified dsRNAs and ASOs to define the breadth of repeat-targeted synthetic nucleic acids as a platform for therapeutic development for FA. ASOs and dsRNAs can activate FXN protein expression in FA patient-derived cell lines that possess varied numbers of GAA repeats. Increased FXN protein expression was achieved by ASOs incorporating diverse chemical modifications with low nanomolar potencies, suggesting substantial flexibility in choosing compounds for further chemical optimization and animal studies. Our data encourage further development of ASOs as agents to treat FA.

Biochemical analysis of NSs from different tospoviruses.

Tospoviruses suppress antiviral RNA interference by coding for an RNA silencing suppressor (NSs) protein. Previously, using NSs-containing crude plant and insect cell extracts, the affinity of NSs for double-stranded (ds)RNA molecules was demonstrated by electrophoretic mobility shifts assays (EMSAs). While NSs from tomato spotted wilt virus (TSWV) and groundnut ringspot virus (GRSV) were able to bind small and long dsRNA molecules, the one from tomato yellow ring virus (TYRV), a distinct Asian tospovirus, only bound small dsRNA. Here, using bacterially expressed and purified NSs from GRSV and TYRV, it is shown that they are both able to bind to small and long dsRNA. Binding of siRNAs by NSs revealed two consecutive shifts, i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When NSs of TSWV resistance inducing (RI) and resistance breaking (RB) isolates were analyzed using extracts from infected plants only a major siRNA shift was observed. In contrast, plant extracts containing the respective transiently expressed NSs proteins showed only the lower shift with NSsRI but no shift with NSsRB. The observed affinity for RNA duplexes, as well as the two-stepwise shift pattern, is discussed in light of NSs as a suppressor of silencing and its importance for tospovirus infection.

RIG-I ATPase activity and discrimination of self-RNA versus non-self-RNA.

UNLABELLED: Many RNA viruses are detected by retinoic acid-inducible gene i (RIG-I), a cytoplasmic sensor that triggers an antiviral response upon binding non-self-RNA that contains a stretch of double-stranded RNA (dsRNA) bearing a base-paired 5' ppp nucleotide. To gain insight into how RIG-I discriminates between self-RNA and non-self-RNA, we used duplexes whose complementary bottom strand contained both ribo- and deoxynucleotides. These duplexes were examined for their binding to RIG-I and their relative abilities to stimulate ATPase activity, to induce RIG-I dimerization on the duplex, and to induce beta interferon (IFN-ß) expression. We show that the chemical nature of the bottom strand is not critical for RIG-I binding. However, two key ribonucleotides, at positions 2 and 5 on the bottom strand, are minimally required for the RIG-I ATPase activity, which is necessary but not sufficient for IFN-ß stimulation. We find that duplexes with shorter stretches of dsRNA, as model self-RNAs, bind less stably to RIG-I but nevertheless have an enhanced ability to stimulate the ATPase. Moreover, ATPase activity promotes RIG-I recycling on RIG-I/dsRNA complexes. Since pseudo-self-RNAs bind to RIG-I less stably, they are preferentially recycled by ATP hydrolysis that weakens the helicase domain binding of dsRNA. Our results suggest that one function of the ATPase is to restrict RIG-I signaling to its interaction with non-self-RNA. A model of how this discrimination occurs as a function of dsRNA length is presented. IMPORTANCE: The innate immune response to pathogens is based on the discrimination between self-RNA and non-self-RNA. The main determinants of this detection for RNA viruses are specific pathogen-associated molecular patterns (PAMPs) of RNA, which are detected by dedicated cytoplasmic pattern recognition receptors (PRRs). RIG-I is a PRR that specifically detects short viral dsRNAs amid a sea of cellular RNAs. Here we study the determinants of this discrimination and how RIG-I ATPase activity, the only enzymatic activity of this sensor, contributes to its activation in a manner restricted to its interaction with non-self-RNAs. We also show how the innate immune response evolves during infection via IFN expression, from a state in which discrimination of self-RNA from non-self-RNA is most important to one in which this discrimination is sacrificed for the effectiveness of the antiviral response.

Knocking down a putative Δ(1) -pyrroline-5-carboxylate dehydrogenase gene by RNA interference inhibits flight and causes adult lethality in the Colorado potato beetle Leptinotarsa decemlineata (Say).

BACKGROUND: Leptinotarsa decemlineata is an able disperser by flight. Novel control strategies must be explored to control the damage and inhibit the dispersal efficiently. Proline is a major energy substrate during flight. Δ-Pyrroline-5-carboxylate dehydrogenase (P5CDh) catalyses the second step of proline degradation for the production of ATP. RESULTS: A full-length Ldp5cdh cDNA was cloned. Ldp5cdh was ubiquitously expressed in the eggs, the first through fourth larval instars, wandering larvae, pupae and adults. In the adults, Ldp5cdh mRNA was widely distributed in thorax muscles, midgut, foregut, hindgut, Malpighian tubules, ventral ganglion, fat body and epidermis, with the expression levels from the highest to the lowest. Two double-stranded RNAs (dsRNAs) (dsLdp5cdh1 and dsLdp5cdh2) targeting Ldp5cdh were constructed and bacterially expressed. Ingestion of dsLdp5cdh1 and dsLdp5cdh2 successfully silenced Ldp5cdh, significantly increased the contents of proline, arginine and alanine, but strongly decreased the contents of asparate, asparagine, glutamate and glutamine in the haemolymph. Moreover, knocking down Ldp5cdh significantly reduced ATP content, decreased flight speed, shortened flight distance and increased adult mortality. CONCLUSIONS: It seems that identified Ldp5cdh encodes a functional P5CDh enzyme, and Ldp5cdh may serve as a potential target for dsRNA-based pesticide for controlling the damage and dispersal of L. decemlineata adults. © 2014 Society of Chemical Industry.

Screening for lead compounds and herbal extracts with potential anti-influenza viral activity.

Nonstructural protein 1 (NS1) of the highly pathogenic avian influenza virus (H5N1) contains a conserved RNA binding domain (RBD) that inhibits antiviral functions of host-innate immune response. Dimerization of NS1 forms a central groove and binds to double stranded (ds) RNA. This region might serve as a potential drug target. In this study, three dimensional structure model of NS1 RBD protein was constructed and virtual screening was performed to identify lead compounds that bound within and around the central groove. The virtual screening showed that 5 compounds bound within the central groove with binding energy ranging between -16.05 and -17.36 Kcal/mol. Two commercially available compounds, estradiol and veratridine, were selected for using in an in vitro screening assay. The results showed that neither of the compounds could inhibit the association between dsRNA and NS1 RBD protein. In addition, 34 herbal extracts were examined for their inhibitory effects. Five of them were able to inhibit association between NS1 RBD and dsRNA in electrophoresis mobility shift assay. Four herbs, Terminalia belirica, Salacia chinensis, Zingiber montanum and Peltophorum pterocarpum, could reduce > 50% of infectivity of H5N1 in a cell-based assay, and it is worth further studying their potential use as source of antiviral drugs.

Impact of folic acid supplementation on single- and double-stranded RNA degradation in human colostrum and mature milk.

Sufficient intake of folic acid is necessary for normal embryogenesis, fetal, and neonatal development. Folic acid facilitates nucleic acid internalization, and protects cellular DNA from nuclease degradation. Human milk contains enzymes, antimicrobial proteins, and antibodies, along with macrophages, that protect against infections and allergies. However, little to no information is available on the effects of folic acid supplementation on degradation of nucleic acids in human milk. In the present study, we aimed to determine the RNase activity (free and inhibitor-bound) in colostrum and mature milk, following folic acid supplementation. The study design included a total of 59 women, 27 of whom received 400 µg of folic acid daily periconceptionally and after. Folic acid supplementation increased the free RNase and polyadenylase activity following lactation. However, the increased RNase activity was not due to de novo enzyme synthesis, as the inhibitor-bound (latent) RNase activity was significantly lower and disappeared after one month. Folic acid reduced RNase activity by using double-stranded RNA as substrate. Data suggests that folic acid supplementation may improve viral RNAs degradation and mRNA degradation, but not dsRNA degradation, preserving in this way the antiviral defense.

Template role of double-stranded RNA in tombusvirus replication.

UNLABELLED: Replication of plus-strand RNA [(+)RNA] viruses of plants is a relatively simple process that involves complementary minus-strand RNA [(-)RNA] synthesis and subsequent (+)RNA synthesis. However, the actual replicative form of the (-)RNA template in the case of plant (+)RNA viruses is not yet established unambiguously. In this paper, using a cell-free replication assay supporting a full cycle of viral replication, we show that replication of Tomato bushy stunt virus (TBSV) leads to the formation of double-stranded RNA (dsRNA). Using RNase digestion, DNAzyme, and RNA mobility shift assays, we demonstrate the absence of naked (-)RNA templates during replication. Time course experiments showed the rapid appearance of dsRNA earlier than the bulk production of new (+)RNAs, suggesting an active role for dsRNA in replication. Radioactive nucleotide chase experiments showed that the mechanism of TBSV replication involves the use of dsRNA templates in strand displacement reactions, where the newly synthesized plus strand replaces the original (+)RNA in the dsRNA. We propose that the use of dsRNA as a template for (+)RNA synthesis by the viral replicase is facilitated by recruited host DEAD box helicases and the viral p33 RNA chaperone protein. Altogether, this replication strategy allows TBSV to separate minus- and plus-strand syntheses in time and regulate asymmetrical RNA replication that leads to abundant (+)RNA progeny. IMPORTANCE: Positive-stranded RNA viruses of plants use their RNAs as the templates for replication. First, the minus strand is synthesized by the viral replicase complex (VRC), which then serves as a template for new plus-strand synthesis. To characterize the nature of the (-)RNA in the membrane-bound viral replicase, we performed complete RNA replication of Tomato bushy stunt virus (TBSV) in yeast cell-free extracts and in plant extracts. The experiments demonstrated that the TBSV (-)RNA is present as a double-stranded RNA that serves as the template for TBSV replication. During the production of new plus strands, the viral replicase displaces the old plus strand in the dsRNA template, leading to asymmetrical RNA synthesis. The presented data are in agreement with the model that the dsRNA is present in nuclease-resistant membranous VRCs. This strategy likely allows TBSV to protect the replicating viral RNA from degradation as well as to evade the early detection of viral dsRNAs by the host surveillance system.

A dimeric form of N-methoxycarbonyl-2-amino-1,8-naphthyridine bound to the A-A mismatch in the CAG/CAG base triad in dsRNA.

A dimeric form of N-methoxycarbonyl-2-amino-1,8-naphthyridine (MCND) connected at the C2 position with a three-atom linker was examined for the binding to mismatches in double stranded RNA. Despite the fully complementary hydrogen bonding groups to guanine, MCND did not bind to guanine-guanine mismatch but did to adenine-adenine mismatch. The base pairs flanking the mismatch had weak effect on the binding, with showing the strongest binding to the A-A mismatch in the CAG/CAG sequence. The A-A mismatch in the GAC/GAC sequence was a poor substrate for the MCND binding. A monomeric derivative of MCND and another derivative lacking a methylcarbamate group showed negligilble binding to the A-A mismatch and the sequence selectivity. These results are important clues for the better molecular design of RNA binding small molecules.

The Caenorhabditis elegans RDE-10/RDE-11 complex regulates RNAi by promoting secondary siRNA amplification.

BACKGROUND: In nematodes, plants, and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans (C. elegans), the interaction between the RNA-induced silencing complex (RISC) loaded with primary small interfering RNAs (siRNAs) and the target messenger RNA (mRNA) leads to the recruitment of RdRPs and synthesis of secondary siRNAs using the target mRNA as the template. The mechanism and genetic requirements for secondary siRNA accumulation are not well understood. RESULTS: From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10, and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, and other candidate RNAi factors. The RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10, rde-11, and other dosage-sensitive RNAi-defective genes rsd-2, rsd-6, and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways. CONCLUSIONS: The RDE-10/RDE-11 complex is essential for the amplification of RNAi in C. elegans by promoting secondary siRNA accumulation.

Characterization of a novel bipartite double-stranded RNA mycovirus conferring hypovirulence in the phytopathogenic fungus Botrytis porri.

The ascomycete Botrytis porri causes clove rot and leaf blight of garlic worldwide. We report here the biological and molecular features of a novel bipartite double-stranded RNA (dsRNA) mycovirus named Botrytis porri RNA virus 1 (BpRV1) from the hypovirulent strain GarlicBc-72 of B. porri. The BpRV1 genome comprises two dsRNAs, dsRNA-1 (6,215 bp) and dsRNA-2 (5,879 bp), which share sequence identities of 62 and 95% at the 3'- and 5'-terminal regions, respectively. Two open reading frames (ORFs), ORF I (dsRNA-1) and ORF II (dsRNA-2), were detected. The protein encoded by the 3'-proximal coding region of ORF I shows sequence identities of 19 to 23% with RNA-dependent RNA polymerases encoded by viruses in the families Totiviridae, Chrysoviridae, and Megabirnaviridae. However, the proteins encoded by the 5'-proximal coding region of ORF I and by the entire ORF II lack sequence similarities to any reported virus proteins. Phylogenetic analysis showed that BpRV1 belongs to a separate clade distinct from those of other known RNA mycoviruses. Purified virions of ~35 nm in diameter encompass dsRNA-1 and dsRNA-2, and three structural proteins (SPs) of 70, 80, and 85 kDa, respectively. Peptide mass fingerprinting analysis revealed that the 80- and 85-kDa SPs are encoded by ORF I, while the 70-kDa SP is encoded by ORF II. Introducing BpRV1 purified virions into the virulent strain GarlicBc-38 of B. porri caused derivative 38T reduced mycelial growth and hypovirulence. These combined results suggest that BpRV1 is a novel bipartite dsRNA virus that possibly belongs to a new virus family.

Old World hantaviruses do not produce detectable amounts of dsRNA in infected cells and the 5' termini of their genomic RNAs are monophosphorylated.

dsRNA and 5'-triphosphate RNA are considered critical activators of the innate immune response because of their interaction with pattern recognition receptors. It has been reported that no dsRNA is detected in negative-sense RNA virus-infected cells and that Hantaan virus (HTNV) genomic RNA bears a 5' monophosphate group. In this paper we examine the 5' termini of genomic RNAs of and dsRNA production by two major groups of Old World hantaviruses. No detectable amounts of dsRNA were found in infected cells. Also, the genomic RNAs of these hantaviruses bear a 5' monophosphate group and therefore are unable to trigger interferon induction. Taken together with the earlier data on HTNV, these results suggest that in addition to the dsRNA and genomic RNA, which may be only minimally involved in the induction of innate immunity, other cellular signalling pathways may also be involved and that these await further investigation.

Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata.

BACKGROUND: RNA interference (RNAi) is a breakthrough technology for conducting functional genomics studies and also as a potential tool for crop protection against insect pests. The major challenge for efficient pest control using RNAi in the field is the development of efficient and reliable methods for production and delivery of double-stranded RNA (dsRNA). In this paper, the potential of feeding dsRNA expressed in bacteria or synthesized in vitro to manage populations of Colorado potato beetle, Leptinotarsa decemlineata (Say) (CPB), was investigated. RESULTS: Feeding RNAi successfully triggered the silencing of all five target genes tested and caused significant mortality and reduced body weight gain in the treated beetles. This study provides the first example of an effective RNAi response in insects after feeding dsRNA produced in bacteria. CONCLUSION: These results suggest that the efficient induction of RNAi using bacteria to deliver dsRNA is a possible method for management of CPB. This could be also a promising bioassay approach for genome-wide screens to identify effective target genes for use as novel RNAi-based insecticides.

Narrowband ultraviolet B inhibits innate cytosolic double-stranded RNA receptors in psoriatic skin and keratinocytes.

BACKGROUND: The mode of action of narrowband ultraviolet B (NB-UVB) therapy in clearing psoriasis is incompletely understood, and in vivo studies at the molecular level in patients undergoing NB-UVB therapy are limited. We previously demonstrated increased expression and activity of double-stranded RNA (dsRNA) receptors in psoriasis lesions, and suggested that this enhanced innate signalling contributed to the maintenance of psoriatic inflammation. OBJECTIVES: We investigated whether NB-UVB affects dsRNA receptor expression and function in vivo as well as in vitro. METHODS: Skin samples of patients with psoriasis undergoing NB-UVB treatment were analysed for epidermal messenger RNA (mRNA) expression of the various dsRNA receptors by microarray and quantitative reverse transcription-polymerase chain reaction. Primary human keratinocytes were irradiated with NB-UVB and stimulated with interferon (IFN)-α or IFN-γ, critical cytokines in psoriasis. The dsRNA analogue polyriboinosinic-polyribocytidylic acid was used to assess the functional responsiveness of the cells to dsRNA. RESULTS: NB-UVB therapy of patients with psoriasis resulted in a significantly reduced mRNA expression of the activating dsRNA receptors MDA5 (IFIH1) and RIG-I (DDX58). On the other hand, expression of LGP2 (DHX58), toll-like receptor 3 (TLR3) and PKR (EIF2AK2) was not affected. In vitro, NB-UVB irradiation completely blocked the upregulation of four of the dsRNA receptors in primary human keratinocytes stimulated with IFN-α or IFN-γ, resulting in an attenuated inflammatory response to dsRNA. CONCLUSIONS: Our results show that NB-UVB irradiation inhibits the local innate inflammatory response to dsRNA, and suggest a novel mechanism of action of NB-UVB phototherapy in psoriasis.