dsRNA Expression Research Articles

Utilization of recombinant Chlorella expressing dsRNA for protection against grouper nervous necrosis virus

In mammals and fish, dsRNA can induce the type I interferon (IFN-I), leading to the expression of antiviral and immune-related genes. The use of microalgae expressing dsRNA as an oral delivery system to activate innate immunity in fish has not been documented. In this study, wild Chlorella sorokiniana was transformed with NNV (nervous necrosis virus) RdRp and GIV (grouper iridovirus) ORF086R dsRNA expression vectors. The recombinant Chlorella strains effectively protected pearl gentian groupers (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂) against NNV and reduced viral titers. The survival rates of the NNV RdRp and GIV ORF086R dsRNA treatment groups increased by 32 % and 38 %, respectively, compared with that of the control group. These findings indicate that recombinant Chlorella has the potential to be used as an oral delivery strategy for protection against NNV disease in groupers.

Functional canonical RNAi in mice expressing a truncated Dicer isoform and long dsRNA

Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer’s adaptation to produce another small RNA class—microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in DicerΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. DicerΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo.

Control of Sclerotinia sclerotiorum via an RNA interference (RNAi)-mediated targeting of SsPac1 and SsSmk1.

The study evaluates the potential of Spray-Induced Gene Silencing and Host-Induced Gene Silencing for sustainable crop protection against thebroad-spectrum necrotrophic fungus Sclerotinia sclerotiorum. Sclerotinia sclerotiorum (Lib.) de Bary, an aggressive ascomycete fungus causes white rot or cottony rot on a broad range of crops including Brassica juncea. The lack of sustainable control measures has necessitated biotechnological interventions such as RNA interference (RNAi) for effective pathogen control. Here we adopted two RNAi-based strategies-Spray-Induced Gene Silencing (SIGS) and Host-Induced Gene Silencing (HIGS) to control S. sclerotiorum. SIGS was successful in controlling white rot on Nicotiana benthamiana and B. juncea by targeting SsPac1, a pH-responsive transcription factor and SsSmk1, a MAP kinase involved in fungal development and pathogenesis. Topical application of dsRNA targeting SsPac1 and SsSmk1 delayed infection initiation and progression on B. juncea. Further, altered hyphal morphology and reduced radial growth were also observed following dsRNA application. We also explored the impact of stable dsRNA expression in A. thaliana against S. sclerotiorum. In this report, we highlight the utility of RNAi as a biofungicide and a tool for preliminary functional genomics.

Construction of an infectious clone for enterovirus A89 and mutagenesis analysis of viral infection and cell binding.

Enterovirus A89 (EV-A89) is an unconventional strain belonging to the Enterovirus A species. Limited research has been conducted on EV-A89, leaving its biological and pathogenic properties unclear. Developing reverse genetic tools for EV-A89 would help to unravel its infection mechanisms and aid in the development of vaccines and anti-viral drugs. In this study, an infectious clone for EV-A89 was successfully constructed and recombinant enterovirus A89 (rEV-A89) was generated. The rEV-A89 exhibited similar characteristics such as growth curve, plaque morphology, and dsRNA expression with parental strain. Four amino acid substitutions were identified in the EV-A89 capsid, which were found to enhance viral infection. Mechanistic studies revealed that these substitutions increased the virus's cell-binding ability. Establishing reverse genetic tools for EV-A89 will significantly contribute to understanding viral infection and developing anti-viral strategies.IMPORTANCEEnterovirus A species contain many human pathogens and have been classified into conventional cluster and unconventional cluster. Most of the research focuses on various conventional members, while understanding of the life cycle and infection characteristics of unconventional viruses is still very limited. In our study, we constructed the infectious cDNA clone and single-round infectious particles for the unconventional EV-A89, allowing us to investigate the biological properties of recombinant viruses. Moreover, we identified key amino acids residues that facilitate EV-A89 infection and elucidate their roles in enhancing viral binding to host cells. The establishment of the reverse genetics system will greatly facilitate future study on the life cycle of EV-A89 and contribute to the development of prophylactic vaccines and anti-viral drugs.

RNA surveillance by ADAR1 is required for vascular endothelial resilience

Abstract Introduction Long dsRNAs are recognised as danger-associated molecular patterns by the cytosolic innate immune sensors inducing thus innate immune responses. RNA editing is a post-transcriptional modification induced by the binding of Adenosine Deaminases Acting on RNA (ADARs) to double-stranded RNA structures (dsRNAs). We have recently reported that ADAR1 is the main RNA editor in endothelial cells (ECs) but the role of ADAR1 in vascular homeostatic processes remains yet elusive. Methods Genetic deletion strategies were employed by crossing mice carrying a conditional (floxed) Adar1 , Ifh1 or Tlr3 allele with either a Tie2-Cre or with a tamoxifen-inducible VE-Cadherin-CreERT2 mouse line. Primary human and murine vascular EC culture assays, dsRNA metabolism studies, gene-silencing and expression techniques, immunohistochemistry and confocal microscopy were used to assess the EC-specific ADAR1 effects. Results EC-specific ADAR1 deletion resulted in prenatal embryonic lethality evidencing an absolute requirement of EC-ADAR1 in life. Inducible EC-ADAR1 ablation in adult mice elicited premature sudden death due to a widespread lung vascular leakage and pleural effusion, reflecting a compromised EC barrier function. Mechanistically, silencing of endothelial ADAR1 disrupted long-to-short dsRNA metabolism witnessed by the striking accumulation of cytoplasmic long dsRNAs. Subsequently, the cytoplasmic accumulation of endogenous long dsRNAs induced aberrant levels of interferon-β and activation of innate immune system. Finally, activation of the cytosolic dsRNA sensors inflicted the dissociation of β-catenin from VE-cadherin in EC junctions, while, co-silencing of the dsRNA sensor, MDA5, was sufficient to restore the integrity of the ADAR1-deficient endothelium. While double EC-specific ADAR1 and TLR3 did not fully rescue the lethality of the EC-ADAR1 animals, in vivo experiments using double EC-specific ADAR1 and MDA5 KO mice fully rescued the phenotype. Validation experiments using murine vascular ECs isolated from the mouse strains of our studies confirmed our mechanistic and phenotypic findings. Conclusion Endogenous suppression of double-stranded RNA-induced MDA5 autoinflammatory signaling by the RNA editor ADAR1 is essential for the maintenance of vascular integrity in adult mice.

RNA interference‐mediated silencing of NOA and V‐ATPase subunit D genes results in deformities and mortality in Bactrocera dorsalis (Hendel, 1912)

AbstractThe Oriental fruit fly, Bactrocera dorsalis (Hendel) is one of the five disastrous pests in the world with an economical significance. The guava germplasm resilient to fruit fly is not reported worldwide to date. The utilization of RNA interference (RNAi) approach can offer a promising alternative for fruit fly management in guava. The goal of our investigation is to evaluate the efficiency of dsRNA expression based on Escherichia coli used in B. dorsalis management by addressing its two indispensable genes, fatty acid elongase (NOA) and Vacuolar‐type H+‐ATPases D (v‐ATPase D). An artificial diet having E. coli strain HT115 (DE3) containing BdNOA, Bdv‐ATPase D dsRNA fed to maggots resulted in deformities with drastic mortality in treated adults, emerged pupae and maggots. The deformity together with total mortality in B. dorsalis was recorded at maximum as 74.66% and 64.20% in bacteria expressing dsRNAs of BdNOA and Bdv‐ATPase D, respectively, at 700 μL as compared to 350, 200 μL treatments. Severe deformities were reported such as underdeveloped abdomen, absence of wings, legs and inadequate eclosion along with suppressed target gene expression in B. dorsalis. This investigation thus provides a potential RNAi‐based pest management by targeting two key genes through feeding dsRNA‐expressing bacteria in B. dorsalis.

Novel bacterial plasmid produces small interfering RNAs (siRNAs) that induce effective gene silencing in the Asian citrus psyllid Diaphorina citri

BackgroundThe Asian citrus psyllid, Diaphorina citri, is a vector of the plant pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing disease. HLB represents the main threat to the citrus industry around the world due to its fast spreading, high infectivity, and incurability. An alternative for the control of this plague in Rutaceae is the implementation of RNA interference (RNAi). Here, we propose a novel method to produce small interfering RNA (siRNA) in Escherichia coli. The method includes a reporter system to visually demonstrate dsRNA expression. We produced and tested siRNAs against three D. citri genes: Abnormal Wing Disk (AWD), Superoxide dismutase 1 (SOD), and Wingless (WNT). These genes play key roles in psyllid development and maturity, and thus, represent promising targets for potential vector control. ResultsThe blue coloration in bacterial cultures was easily observable, and it corroborated the generation of the dsRNA that is in the same transcript. We evaluated the dosage efficiency of siRNA using the AWD siRNA. The RNAi treatment was evaluated, and all the siRNAs tested were able to induce silencing (-3.05 for AWD, -2.60 for SOD and -2.57 for WNT). ConclusionsThe novel bacterial plasmid effectively produces siRNAs, and the blue color reporter is visually facilitated to check the expression of each dsRNA. Treating D. citri with the produced siRNAs resulted in a decrease in gene expression. In addition, psyllid mortality was observed, being the highest when treated with WNT-siRNA. Our results suggest the potential of siRNA treatment as a method for controlling Huanglongbing.How to cite: Rueda-Silva JC, González-Campos LI, Durán-Armenta LF, et al. Novel bacterial plasmid produces small interfering RNAs (siRNAs) that induce effective gene silencing in the Asian citrus psyllid Diaphorina citri. Electron J Biotechnol 2023;64. https://doi.org/10.1016/j.ejbt.2023.03.006.

Functional characterization of five developmental signaling network genes in the white-backed planthopper: Potential application for pest management.

The white-backed planthopper (WBPH, Sogatella furcifera) is a major rice pest that exhibits condition dependent wing dimorphisms - a macropterous (long wing) form and a brachypterous (short wing) form. Although, the gene cascade that regulates wing development and dimorphic differentiation has been largely defined, the utility of these genes as targets for pest control has yet to be fully explored. Five genes typically associated with the developmental signaling network, armadillo (arm), apterous A (apA), scalloped (sd), dachs (d), and yorkie (yki) were identified from the WBPH genome and their roles in wing development assessed following RNA interference (RNAi)-mediated knockdown. At 5 days-post injection, transcript levels for all five targets were substantially decreased compared with the dsGFP control group. Among the treatment groups, those injected with dsSfarm had the most pronounced effects on transcript reduction, mortality (95 ± 3%), and incidence (45 ± 3%) of wing deformities, whereas those injected with dsSfyki had the lowest incidence (6.7 ± 4%). To assess the utility of topical RNAi for Sfarm, we used a spray-based approach that complexed a large-scale, bacteria-based double-stranded RNA (dsRNA) expression pipeline with star polycation (SPc) nanoparticles. Rice seedlings infested with third and fourth instar nymphs were sprayed with SPc-dsRNA formulations and RNAi phenotypic effects were assessed over time. At 2 days post-spray, Sfarm transcript levels decreased by 86 ± 9.5% compared with dsGFP groups, and the subsequent incidences of mortality and wing defects were elevated in the treatment group. This study characterized five genes in the WBPH developmental signaling cascade, assessed their impact on survival and wing development via RNAi, and developed a nanoparticle-dsRNA spray approach for potential field control of WBPH. © 2023 Society of Chemical Industry.

Honey bee functional genomics using symbiont-mediated RNAi.

Honey bees are indispensable pollinators and model organisms for studying social behavior, development and cognition. However, their eusociality makes it difficult to use standard forward genetic approaches to study gene function. Most functional genomics studies in bees currently utilize double-stranded RNA (dsRNA) injection or feeding to induce RNAi-mediated knockdown of a gene of interest. However, dsRNA injection is laborious and harmful, and dsRNA feeding is difficult to scale cheaply. Further, both methods require repeated dsRNA administration to ensure a continued RNAi response. To fill this gap, we engineered the bee gut bacterium Snodgrassella alvi to induce a sustained host RNA interference response that reduces expression of a targeted gene. To employ this functional genomics using engineered symbionts (FUGUES) procedure, a dsRNA expression plasmid is cloned in Escherichia coli using Golden Gate assembly and then transferred to S. alvi. Adult worker bees are then colonized with engineered S. alvi. Finally, gene knockdown is verified through qRT-PCR, and bee phenotypes of interest can be further assessed. Expression of targeted genes is reduced by as much as 50-75% throughout the entire bee body by 5 d after colonization. This protocol can be accomplished in 4 weeks by bee researchers with microbiology and molecular cloning skills. FUGUES currently offers a streamlined and scalable approach for studying the biology of honey bees. Engineering other microbial symbionts to influence their hosts in ways that are similar to those described in this protocol may prove useful for studying additional insect and animal species in the future.

TDP-43 knockdown in mouse model of ALS leads to dsRNA deposition, gliosis, and neurodegeneration in the spinal cord.

Transactive response DNA binding protein 43 kilodaltons (TDP-43) is a DNA and RNA binding protein associated with severe neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), primarily affecting motor neurons in the brain and spinal cord. Partial knockdown of TDP-43 expression in a mouse model (the amiR-TDP-43 mice) leads to progressive, age-related motor dysfunction, as observed in ALS patients. Work in Caenorhabditis elegans suggests that TDP-43 dysfunction can lead to deficits in chromatin processing and double-stranded RNA (dsRNA) accumulation, potentially activating the innate immune system and promoting neuroinflammation. To test this hypothesis, we used immunostaining to investigate dsRNA accumulation and other signs of CNS pathology in the spinal cords of amiR-TDP-43 mice. Compared with wild-type controls, TDP-43 knockdown animals show increases in dsRNA deposition in the dorsal and ventral horns of the spinal cord. Additionally, animals with heavy dsRNA expression show markedly increased levels of astrogliosis and microgliosis. Interestingly, areas of high dsRNA expression and microgliosis overlap with regions of heavy neurodegeneration, indicating that activated microglia could contribute to the degeneration of spinal cord neurons. This study suggests that loss of TDP-43 function could contribute to neuropathology by increasing dsRNA deposition and subsequent innate immune system activation.

The Metarhizium anisopliae Strains Expressing dsRNA of the NlCHSA Enhance Virulence to the Brown Planthopper Nilaparvata lugens

Metarhizium anisopliae is a broad-spectrum entomopathogenic fungus with large-scale applications in pest control; however, its slow-killing action is a limiting factor. To enhance fungal virulence, we constructed transgenic M. anisopliae strains that expressed dsRNA targeting the chitin synthase gene A in the brown planthopper Nilaparvata lugens (NlCHSA). The transcription of NlCHSA was suppressed in N. lugens infected by transgenic M. anisopliae strains. Consequently, the cumulative mortality of N. lugens caused by those transgenic strains was increased by 34.7–40.0% on day 6 after inoculation, and their median lethal times (LT50) were about 50% compared to the wild-type strain. Our results demonstrate that dsRNA expression in M. anisopliae caused RNA silencing in the host insect after infection and hence greatly enhanced the fungal virulence, highlighting a new strategy for augmentation of fungal virulence against sucking pests.

Mitochondrial double-stranded RNAs govern the stress response in chondrocytes to promote osteoarthritis development.

Protein kinase R (PKR) is an immune response protein that becomes activated by double-stranded RNAs (dsRNAs). PKR overactivation is associated with degenerative diseases with inflammation, including osteoarthritis (OA), but the dsRNA activator remains largely unknown. Here, we find that mitochondrial dsRNA (mt-dsRNA) expression and its cytosolic efflux are facilitated in chondrocytes under OA-eliciting conditions, leading to innate immune activation. Moreover, mt-dsRNAs are released to the extracellular space and activate Toll-like receptor 3 at the plasma membrane. Elevated levels of mt-dsRNAs in the synovial fluids and damaged cartilage of OA patients and in the cartilage of surgery-induced OA mice further support our data. Importantly, autophagy prevents PKR activation and protects chondrocytes from mitochondrial stress partly by removing cytosolic mtRNAs. Our study provides a comprehensive understanding of innate immune activation by mt-dsRNAs during stress responses that underlie the development of OA and suggests mt-dsRNAs as a potential target for chondroprotective intervention.

Resistance to RNA interference by plant‐derived double‐stranded RNAs but not plant‐derived short interfering RNAs in Helicoverpa armigera

Plant-mediated RNA interference (RNAi) has emerged as a promising technology for pest control through expression of double-stranded RNAs (dsRNAs) targeted against essential insect genes. However, little is known about the underlying molecular mechanisms and whether long dsRNA or short interfering RNAs (siRNAs) are the effective triggers of the RNAi response. Here we generated transplastomic and nuclear transgenic tobacco plants expressing dsRNA against the Helicoverpa armigera ATPaseH gene. We showed that expression of long dsRNA of HaATPaseH was at least three orders of magnitude higher in transplastomic plants than in transgenic plants. HaATPaseH-derived siRNAs are absent from transplastomic plants, while they are abundant in transgenic plants. Feeding transgenic plants to H. armigera larvae reduced gene expression of HaATPaseH and delayed growth. Surprisingly, no effect of transplastomic plants on insect growth was observed, despite efficient dsRNA expression in plastids. Furthermore, we found that dsRNA ingested by H. armigera feeding on transplastomic plants was rapidly degraded in the intestinal fluid. In contrast, siRNAs are relatively stable in the digestive system. These results suggest that plant-derived siRNAs may be more effective triggers of RNAi in Lepidoptera than dsRNAs, which will aid the optimization of the strategies for plant-mediated RNAi to pest control.

Development of a Viral RdRp-Assisted Gene Silencing System and Its Application in the Identification of Host Factors of Plant (+)RNA Virus.

Gene silencing induced by hairpin RNA or virus infection expression is one of the major tools in genetics studies in plants. However, when dealing with essential genes, virus-induced gene silencing (VIGS) and transgenic expression of hairpin RNA could lead to plant death, while transient expression of hairpin RNA in leaves is often less competent in downregulating target gene mRNA levels. Here, we developed a transient double-stranded RNA (dsRNA) expression system assisted by a modified viral RNA-dependent RNA polymerase (RdRp) in plant leaves. We show that this system is more effective in inducing gene silencing than the intron-spliced hairpin RNA expression. Furthermore, by using this system, we tested the role of the early secretory pathway during infection of Soybean mosaic potyvirus (SMV). We found that key components of the coat protein complex II vesicles are required for the multiplication of SMV. Overall, this dsRNA-based gene silencing system is effective in downregulating plant gene expression and can be used to identify host genes involved in plant-virus interactions.

Efficient production of long double-stranded RNAs applicable to agricultural pest control by Corynebacterium glutamicum equipped with coliphage T7-expression system

RNA-based pesticides exert their function by suppressing the expression of an essential gene in the target pest through RNA interference caused by double-stranded RNA (dsRNA). Here, we selected target genes for growth suppression of the solanaceous crop pests ladybird beetle (Henosepilachna vigintioctopunctata) and Colorado potato beetle (Leptinotarsa decemlineata)-the death-associated inhibitor of apoptosis protein 1 gene (diap1), and an orthologous gene of the COPI coatomer protein complex (copI), respectively. We constructed a cost-competitive overproduction system for dsRNA using Corynebacterium glutamicum as a host bacterium. The dsRNA expression unit was equipped with two sets of promoters and terminators derived from coliphage T7, and the convergent expression system was designed to be selectively transcribed by T7 RNA polymerase. This expression system efficiently overproduced both target dsRNAs. On culture in a jar fermentor, the yield of diap1-targeting dsRNA (approximately 360 bp) was > 1 g per liter of culture. Long-chain diap1-targeting dsRNAs (up to around 1 kbp) could be produced without a substantial loss of efficiency. dsRNA accumulated in C. glutamicum significantly suppressed larval growth of H. vigintioctopunctata. The dsRNA expression technology developed here is expected to substantially reduce dsRNA production costs. Our method can be applied for a wide range of industrial uses, including agricultural pest control.Key points• Overexpression of dsRNA was achieved in C. glutamicum using a coliphage T7 system.• The best strain produced > 1 g/L of the target dsRNA species, for use as an insecticide.• The developed system efficiently produced long dsRNA species, up to ~ 1 kbp.

ERVs-TLR3-IRF axis is linked to myelodysplastic syndrome pathogenesis

Toll-like receptors are mutated or overexpressed in up to 50% of patients with myelodysplastic syndrome (MDS). Endogenous retroviruses (ERV) trigger TLR3 leading to interferon regulatory genes (IRFs) activation. We evaluated if the ERVs-TLR3-IRF axis activation would be linked to MDS pathogenesis and we also conducted a detailed cancer analysis of the ERVs, TLR3 and IRFs gene expression in 30 cancer types using GEPIA database. Seventy-nine bone marrow samples from patients with MDS were evaluated for cytogenetics and quantitative real‑time PCR of TLR3, ERVK6, ERVW-1, ERV3-1, IRF3 and IRF7. Patients with dyserythropoiesis showed higher TLR3 (p = 0.035), ERVK6 (p = 0.001), ERVW1 (p = 0.045) and ERV3-1 (p = 0.016) expression than patients without dyserythropoiesis. Upregulation of Interferon Regulatory Factors, IRF3 and IRF7, was associated with poor prognostic markers in MDS such as > 10% of blasts (p = 0.003-IRF3; p = 0.009-IRF7), low platelets count (< 50.000/mm3) (p = 0.001-IRF3; p = 0.021-IRF7), transfusion dependence (p = 0.014-IRF3) and chromosomal abnormalities (p = 0.036-IRF7). We found strong correlations between ERVK6-ERVW1 (r = 0.800; r2 = 0.640; p = 0.000), ERVW1-ERV3-1 (r = 0.715; r2 = 0.511; p = 0.000), and IRF7-IRF3 (r = 0.567; r2 = 0.321; p = 0.000) and moderate correlation between ERVK6-ERV3-1(r = 0.485; r2 = 0.235; p = 0.000), ERVW1-IRF7 (r = 0.389; r2 = 0.151; p = 0.001), ERVW1-IRF3 (r = 0.357; r2 = 0.127; p = 0.004), ERV3-1-IRF7 (r = 0.314; r2 = 0.098; p = 0.009), and ERV3-1-IRF3 (r = 0.324; r2 = 0.104; p = 0.007). Using GEPIA Database in 30 cancer types, we detected a typical pattern of upregulation as here presented in MDS. We suggest TLR3 activation by ERVs is linked to MDS pathogenesis leading to bone marrow failure. Abnormal double-stranded RNA (dsRNA) expression of Endogenous Retroviruses (ERV) triggers TLR3 hyperactivation. This induces IRF3, IRF7, and NF-kB to translocate to the nucleus and activate transcription of IFNα/β which binds to the type I-IFN receptor promoting interferon response. Thus, just as TLR4 induces a crucial myeloid shift, the ERVs-TLR3 axis may play an important role in establishing one of the most striking characteristics in MDS, dyserythropoiesis.

Ingestion of bacteria expressing dsRNA to maggots produces severe mortality and deformities in fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

BackgroundThe oriental fruit fly, Bactrocera dorsalis (Hendel), is the world’s most damaging (30–100%) pest infesting important fruits and vegetables. Its control is highly challenging due to its polyphagous, multivoltine nature, and unexposed developmental stages. No known fruit fly-resistant guava germplasm is reported so far worldwide. RNAi approach in guava against fruit fly can provide an attractive alternative to overcome this problem.Main bodyEscherichia coli-based dsRNA expression strategy was used to investigate its potential in control of B. dorsalis via targeting its two vital genes, ecr (ecdysone receptor) and rpl19 (a ribosomal protein L19). Effects of feeding E. coli, HT115 (DE3) expressing dsRNA of Bdecr and Bdrpl19 with artificial diet to maggots of B. dorsalis resulted in severe mortality and deformities in treated maggots, emerged pupae, and adults. Total mortality (including deformity) of maggots, pupae, and adult fruit flies was 86.3% and 87.9% and was highest in 700 μl (× 200 of 3.5 × 108 cells) dsRNAs of Bdecr and Bdrpl19 bacterial treatment respectively, compared to 350 and 200 μl bacterial treatments. Severe defects in terms of developmental changes like melanisation and deformities of maggots and pupae, absence of wings, underdeveloped abdomen/absence of complete abdomen, absence of legs, severely curled wings, malformed legs, and incomplete eclosion, and suppression of these target genes expression were observed in emerged adults.Short conclusionThe study provides a proof of concept of feasibility to silence two potential genes by feeding bacteria expressing dsRNA in all developmental stages of B. dorsalis to step further to perform RNAi-based pest control.

Negligible transcriptome and metabolome alterations in RNAi insecticidal maize against Monolepta hieroglyphica

Key messageRNAi-based genetically modified maize resistant to Monolepta hieroglyphica (Motschulsky) was demonstrated with negligible transcriptome and metabolome alterations compared to its unmodified equivalent.As one of the most prevalent insect pests afflicting various crops, Monolepta hieroglyphica (Motschulsky) causes severe loss of agricultural and economic productivity for many years in China. In an effort to reduce damages, in this study, an RNA interference (RNAi)-based genetically modified (GM) maize was developed. It was engineered to produce MhSnf7 double-stranded RNAs (dsRNAs), which can suppress the Snf7 gene expression and then lead M. hieroglyphica to death. Field trail analysis confirmed the robustly insecticidal ability of the MhSnf7 GM maize to resist damages by M. hieroglyphica. RNA sequencing analysis identified that only one gene was differentially expressed in the MhSnf7 GM maize compared to non-GM maize, indicating that the transcriptome in MhSnf7 GM maize is principally unaffected by the introduction of the MhSnf7 dsRNA expression vector. Likewise, metabolomics analysis identified that only 8 out of 5787 metabolites were significantly changed. Hence, the integration of transcriptomics and metabolomics demonstrates that there are negligible differences between MhSnf7 GM maize and its unmodified equivalent. This study not only presents a comprehensive assessment of cellular alteration in terms of gene transcription and metabolite abundance in RNAi-based GM maize, but also could be used as a reference for evaluating the unintended effect of GM crops.

Effective suppression of yellow head virus replication in Penaeus monodon hemocytes using constitutive expression vector for long-hairpin RNA (lhRNA)

Double-stranded RNA (dsRNA) is employed to down-regulate the expression of specific genes of shrimp viral pathogens through the RNA interference (RNAi) pathway. The administration of dsRNA into shrimp has been shown to be an effective strategy to block yellow head virus (YHV) progression. In this study, a vector (pLVX-AcGFP1-N1) was developed to introduce a long-hairpin RNA (lhRNA) silencing cassette under a CMV promoter, so-called “pLVX-lhRdRp”, against the RNA-dependent RNA polymerase (RdRp) gene of YHV. A primary culture of hemocytes isolated from Penaeus monodon was transfected with the pLVX-lhRdRp vector, generating transcripts of lhRNAs as early as 12 h post transfection. Twelve hours prior to YHV challenge, the primary hemocyte cell culture was transfected with pLVX-lhRdRp, whereas control groups were transfected with pLVX-AcGFP1-N1 or no transfection. The group treated with pLVX-lhRdRp significantly suppressed YHV replication at 24–72 h after YHV challenge. The results from RT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of YHV were effectively inhibited by the pLVX-lhRdRp vector. Thus, our hemocyte culture and dsRNA expression plasmid with constitutive promoter have potential as a platform to test DNA constructs expressing long-hairpin RNA against pathogenic viral infection and as a RNAi-based DNA vaccine in shrimp.

Biosafety of GM Crop Plants Expressing dsRNA: Data Requirements and EU Regulatory Considerations.

The use of RNA interference (RNAi) enables the silencing of target genes in plants or plant-dwelling organisms, through the production of double stranded RNA (dsRNA) resulting in altered plant characteristics. Expression of properly synthesized dsRNAs in plants can lead to improved crop quality characteristics or exploit new mechanisms with activity against plant pests and pathogens. Genetically modified (GM) crops exhibiting resistance to viruses or insects via expression of dsRNA have received authorization for cultivation outside Europe. Some products derived from RNAi plants have received a favourable opinion from the European Food Safety Authority (EFSA) for import and processing in the European Union (EU). The authorization process in the EU requires applicants to produce a risk assessment considering food/feed and environmental safety aspects of living organisms or their derived food and feed products. The present paper discusses the main aspects of the safety assessment (comparative assessment, molecular characterization, toxicological assessment, nutritional assessment, gene transfer, interaction with target and non-target organisms) for GM plants expressing dsRNA, according to the guidelines of EFSA. Food/feed safety assessment of products from RNAi plants is expected to be simplified, in the light of the consideration that no novel proteins are produced. Therefore, some of the data requirements for risk assessment do not apply to these cases, and the comparative compositional analysis becomes the main source of evidence for food/feed safety of RNAi plants. During environmental risk assessment, the analysis of dsRNA expression levels of the GM trait, and the data concerning the observable effects on non-target organisms (NTO) will provide the necessary evidence for ensuring safety of species exposed to RNAi plants. Bioinformatics may provide support to risk assessment by selecting target gene sequences with low similarity to the genome of NTOs possibly exposed to dsRNA. The analysis of these topics in risk assessment indicates that the science-based regulatory process in Europe is considered to be applicable to GM RNAi plants, therefore the evaluation of their safety can be effectively conducted without further modifications. Outcomes from the present paper offer suggestions for consideration in future updates of the EFSA Guidance documents on risk assessment of GM organisms.