Effects of Nanoparticles on Double-Stranded RNA Stability in Corn Soil

Double-stranded RNA (dsRNA) can potentially be used as a pesticide because these molecules trigger an immune response called RNA interference (RNAi). If the expression of essential genes matching the dsRNA sequence are silenced, then the pest dies. New classes of pesticides, including RNAi-based pesticides, are needed to overcome pesticide resistance and reduce the environmental impacts of pesticides. Unfortunately, dsRNA is easily degraded by enzymes in the environment, particularly those produced by microbes in the soil (Dubelmanet al., 2014),severely limiting delivery of dsRNA to cryptic (soil dwelling) species unless transgenic plants are used. Here we investigate dsRNA stability when incubated in corn soil supernatant ex situ to determine if encapsulation of dsRNA in chitosan-basednanoparticles (CB-NPs) enhances stability in corn soil. Interestingly, dsRNA stability was not affected by soil supernatant, possibly due to the time of year when sampling was performed (Icozet al., 2008). Nonetheless, these findings provide insight into dsRNA stability in soil, and in the future may lead to a method for protecting dsRNA from environmental degradation using CB-NPs

Effects of Nanoparticles on Double-Stranded RNA Stability in Corn Soil

Double-stranded RNA (dsRNA) can potentially be used as a pesticide because these molecules trigger an immune response called RNA interference (RNAi). If the expression of essential genes matching the dsRNA sequence are silenced, then the pest dies. New classes of pesticides, including RNAi-based pesticides, are needed to overcome pesticide resistance and reduce the environmental impacts of pesticides. Unfortunately, dsRNA is easily degraded by enzymes in the environment, particularly those produced by microbes in the soil (Dubelmanet al., 2014),severely limiting delivery of dsRNA to cryptic (soil dwelling) species unless transgenic plants are used. Here we investigate dsRNA stability when incubated in corn soil supernatant ex situ to determine if encapsulation of dsRNA in chitosan-basednanoparticles (CB-NPs) enhances stability in corn soil. Interestingly, dsRNA stability was not affected by soil supernatant, possibly due to the time of year when sampling was performed (Icozet al., 2008). Nonetheless, these findings provide insight into dsRNA stability in soil, and in the future may lead to a method for protecting dsRNA from environmental degradation using CB-NPs

Double-Stranded RNA Attenuates the Barrier Function of Human Pulmonary Artery Endothelial Cells

Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs). The effect of natural and synthetic double-stranded RNA (dsRNA) on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca2+) homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca2+ signaling in hPAECs by inhibiting the sarco-endoplasmic Ca2+-ATPase (SERCA) which is involved in the regulation of the intracellular Ca2+ homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes

Next-generation sequencing of dsRNA is greatly improved by treatment with the inexpensive denaturing reagent DMSO.

dsRNA is the genetic material of important viruses and a key component of RNA interference-based immunity in eukaryotes. Previous studies have noted difficulties in determining the sequence of dsRNA molecules that have affected studies of immune function and estimates of viral diversity in nature. DMSO has been used to denature dsRNA prior to the reverse-transcription stage to improve reverse transcriptase PCR and Sanger sequencing. We systematically tested the utility of DMSO to improve the sequencing yield of a dsRNA virus (Φ6) in a short-read next-generation sequencing platform. DMSO treatment improved sequencing read recovery by over two orders of magnitude, even when RNA and cDNA concentrations were below the limit of detection. We also tested the effects of DMSO on a mock eukaryotic viral community and found that dsRNA virus reads increased with DMSO treatment. Furthermore, we provide evidence that DMSO treatment does not adversely affect recovery of reads from a ssRNA viral genome (influenza A/California/07/2009). We suggest that up to 50 % DMSO treatment be used prior to cDNA synthesis when samples of interest are composed of or may contain dsRNA

Effects of pH on Double Stranded RNA Stability in European Corn Borer Nucleases

RNA interference (RNAI) is an immune response that can be exploited to make greener pesticides. It works by inciting suppression of a specific target gene using fed or injected dsRNA. Targeting a specific gene sequence also means RNAi can be used to target a specific organism. However, some insects, such as lepidopterans, have nucleases, called dsRNases, in their gut and hemolymph that sever dsRNA and lower RNAi efficiency (1). Ostrinia nubilalis, the European corn borer, (ECB), is a prime example of a lepidopteran pest which decimates corn supplies across the Midwest and does not respond to RNAi. Comparison of dsRNA stability in dsRNase genes in ECB and western corn rootworm (WCR), a coleopteran pest that has very high RNAi efficiency, indicates that dsRNA is rapidly degraded in ECB tissues, but not WCR tissues, despite similar expression of dsRNase genes in both species. These findings suggest that another variable, such as pH may be influencing dsRNA stability in insects (2)

RNAi efficiency, systemic properties, and novel delivery methods for pest insect control : what we know so far

In recent years, the research on the potential of using RNA interference (RNAi) to suppress crop pests has made an outstanding growth. However, given the variability of RNAi efficiency that is observed in many insects, the development of novel approaches toward insect pest management using RNAi requires first to unravel factors behind the efficiency of dsRNA-mediated gene silencing. In this review, we explore essential implications and possibilities to increase RNAi efficiency by delivery of dsRNA through non-transformative methods. We discuss factors influencing the RNAi mechanism in insects and systemic properties of dsRNA. Finally, novel strategies to deliver dsRNA are discussed, including delivery by symbionts, plant viruses, trunk injections, root soaking, and transplastomic plants

Suppression of Argonaute 2 Transcript Levels in Du182A Cells

RNA interference (RNAi) uses double-stranded RNA (dsRNA) molecules to degrade and suppress the transcript level of a complementary mRNA target1. The RNAi pathway is complex and includes many different proteins, like argonautes, in the core machinery. Argonautes are dsRNA binding proteins which help recognize and cleave target mRNA molecules. In our experiments, we attempted to suppress the transcript level of argonaute 2 (Ago2) in a Diabrotica undecimpunctata cell line (Du182A) using dsRNA, with the idea of disrupting the RNAi pathway using an RNAi of RNAi technique. Ago2 transcript levels were suppressed following treatment with dsRNA. Future experiments can now use this technique, with some modification to better understand the RNAi pathway

An RNAi-Based Control of Fusarium graminearum Infections Through Spraying of Long dsRNAs Involves a Plant Passage and Is Controlled by the Fungal Silencing Machinery

Meeting the increasing food and energy demands of a growing population will require the development of ground-breaking strategies that promote sustainable plant production. Host-induced gene silencing has shown great potential for controlling pest and diseases in crop plants. However, while delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a promising concept, it requires the generation of transgenic crop plants which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species. Using the agronomically important barley—Fusarium graminearum pathosystem, we alternatively demonstrate that a spray application of a long noncoding dsRNA (791 nt CYP3-dsRNA), which targets the three fungal cytochrome P450 lanosterol C-14a-demethylases, required for biosynthesis of fungal ergosterol, inhibits fungal growth in the directly sprayed (local) as well as the non-sprayed (distal) parts of detached leaves. Unexpectedly, efficient spray-induced control of fungal infections in the distal tissue involved passage of CYP3-dsRNA via the plant vascular system and processing into small interfering (si)RNAs by fungal DICER-LIKE 1 (FgDCL-1) after uptake by the pathogen. We discuss important consequences of this new finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential as a new plant protection strategy

Drosophila RecQ4 Is Directly Involved in Both DNA Replication and the Response to UV Damage in S2 Cells.

The RecQ4 protein shows homology to both the S.cerevisiae DNA replication protein Sld2 and the DNA repair related RecQ helicases. Experimental data also suggest replication and repair functions for RecQ4, but the precise details of its involvement remain to be clarified.Here we show that depletion of DmRecQ4 by dsRNA interference in S2 cells causes defects consistent with a replication function for the protein. The cells show reduced proliferation associated with an S phase block, reduced BrdU incorporation, and an increase in cells with a subG1 DNA content. At the molecular level we observe reduced chromatin association of DNA polymerase-alpha and PCNA. We also observe increased chromatin association of phosphorylated H2AvD--consistent with the presence of DNA damage and increased apoptosis.Analysis of DmRecQ4 repair function suggests a direct role in NER, as the protein shows rapid but transient nuclear localisation after UV treatment. Re-localisation is not observed after etoposide or H₂O₂ treatment, indicating that the involvement of DmRecQ4 in repair is likely to be pathway specific.Deletion analysis of DmRecQ4 suggests that the SLD2 domain was essential, but not sufficient, for replication function. In addition a DmRecQ4 N-terminal deletion could efficiently re-localise on UV treatment, suggesting that the determinants for this response are contained in the C terminus of the protein. Finally several deletions show differential rescue of dsRNA generated replication and proliferation phenotypes. These will be useful for a molecular analysis of the specific role of DmRecQ4 in different cellular pathways

ICTV Virus Taxonomy Profile: Chrysoviridae

Members of the family Chrysoviridae are isometric, non-enveloped viruses with segmented, linear, dsRNA genomes. There are 3–7 genomic segments, each of which is individually encapsidated. Chrysoviruses infect fungi, plants and possibly insects, and may cause hypovirulence in their fungal hosts. Chrysoviruses have no known vectors and lack an extracellular phase to their replication cycle; they are transmitted via intracellular routes within an individual during hyphal growth, in asexual or sexual spores, or between individuals via hyphal anastomosis. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the family Chrysoviridae, which is available at ictv.global/report/chrysoviridae.Peer reviewe