dsRNA Sequences Research Articles

Characterization of Cronartium ribicola dsRNAs reveals novel members of the family Totiviridae and viral association with fungal virulence

BackgroundMycoviruses were recently discovered in the white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.). Detection and characterization of their double stranded RNA (dsRNA) would facilitate understanding of pathogen virulence and disease pathogenesis in WPBR systems.MethodsFull-length cDNAs were cloned from the dsRNAs purified from viral-infected C. ribicola, and their cDNA sequences were determined by DNA sequencing. Evolutionary relationships of the dsRNAs with related mycoviruses were determined by phylogenetic analysis. Dynamic distributions of the viral RNAs within samples of their fungal host C. ribicola were investigated by measurement of viral genome prevalence and viral gene expression.ResultsIn this study we identified and characterized five novel dsRNAs from C. ribicola, designated as Cronartium ribicola totivirus 1–5 (CrTV1 to CrTV5). These dsRNA sequences encode capsid protein and RNA-dependent RNA polymerase with significant homologies to dsRNA viruses of the family Totiviridae. Phylogenetic analysis showed that the CrTVs were grouped into two distinct clades. CrTV2 through CrTV5 clustered within the genus Totivirus. CrTV1 along with a few un-assigned dsRNAs constituted a distinct phyletic clade that is genetically distant from presently known genera in the Totiviridae family, indicating that CrTV1 represents a novel genus in the Totiviridae family. The CrTVs were prevalent in fungal samples obtained from infected western white pine, whitebark pine, and limber pines. Viral RNAs were generally expressed at higher levels during in planta mycelium growth than in aeciospores and urediniospores. CrTV4 was significantly associated with C. ribicola virulent pathotype and specific C. ribicola host tree species, suggesting dsRNAs as potential tools for dissection of pathogenic mechanisms of C. ribicola and diagnosis of C. ribicola pathotypes.ConclusionPhylogenetic and expression analyses of viruses in the WPBR pathogen, C. ribicola, have enchanced our understanding of virus diversity in the family Totiviridae, and provided a potential strategy to utilize pathotype-associated mycoviruses to control fungal forest diseases.

DsRNA-Seq: Identification of Viral Infection by Purifying and Sequencing dsRNA.

RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that RNA viruses produce double-stranded RNA (dsRNA) while replicating. Purifying and sequencing dsRNA from the total RNA isolated from infected tissue allowed us to recover dsRNA virus sequences and replicated sequences from single-stranded RNA (ssRNA) viruses. We refer to this approach as dsRNA-Seq. By assembling dsRNA sequences into contigs we identified full length or partial RNA viral genomes of varying genome types infecting mammalian culture samples, identified a known viral disease agent in laboratory infected mice, and successfully detected naturally occurring RNA viral infections in reptiles. Here, we show that dsRNA-Seq is a preferable method for identifying viruses in organisms that don’t have sequenced genomes and/or commercially available rRNA depletion reagents. In addition, a significant advantage of this method is the ability to identify replicated viral sequences of ssRNA viruses, which is useful for distinguishing infectious viral agents from potential noninfectious viral particles or contaminants.

High resolution fingerprinting of single and double-stranded RNA using ion-pair reverse-phase chromatography

The emergence of new sustainable approaches for insect management using RNA interference (RNAi) based insecticides has created the demand for high throughput analytical techniques to fully characterise and accurately quantify double stranded RNA (dsRNA) prior to downstream RNAi applications. In this study we have developed a method for the rapid characterisation of single stranded and double stranded RNA using high resolution RNase mapping in conjunction with ion-pair reverse-phase chromatography utilising a column with superficially porous particles. The high resolution oligoribonucleotide map provides an important ‘fingerprint’ for identity testing and bioprocess monitoring. Reproducible RNA mapping chromatograms were generated from replicate analyses. Moreover, this approach was used to provide a method to rapidly distinguish different RNA sequences of the same size, based on differences in the resulting chromatograms. Principal components analysis of the high resolution RNA mapping data enabled us to rapidly compare multiple HPLC chromatograms and distinguish two dsRNA sequences of different size which share 72% sequence homology. We used the high resolution RNase mapping method to rapidly fingerprint biomanufactured dsRNA across a number of different batches. The resulting chromatograms in conjunction with principal components analysis demonstrated high similarity in the dsRNA produced across the different batches highlighting the potential ability of this method to provide information for batch release in a high throughput manner.

The crystal structure of Staufen1 in complex with a physiological RNA sheds light on substrate selectivity.

During mRNA localization, RNA-binding proteins interact with specific structured mRNA localization motifs. Although several such motifs have been identified, we have limited structural information on how these interact with RNA-binding proteins. Staufen proteins bind structured mRNA motifs through dsRNA-binding domains (dsRBD) and are involved in mRNA localization in Drosophila and mammals. We solved the structure of two dsRBDs of human Staufen1 in complex with a physiological dsRNA sequence. We identified interactions between the dsRBDs and the RNA sugar-phosphate backbone and direct contacts of conserved Staufen residues to RNA bases. Mutating residues mediating nonspecific backbone interactions only affected Staufen function in Drosophila when in vitro binding was severely reduced. Conversely, residues involved in base-directed interactions were required in vivo even when they minimally affected in vitro binding. Our work revealed that Staufen can read sequence features in the minor groove of dsRNA and suggests that these influence target selection in vivo.

Double-stranded RNA injected into female Black Tiger shrimp (Penaeus monodon) prior to spawning does not transfer to progeny

Injecting double-stranded (ds)RNA into abdominal muscle of female shrimp may provide a relatively simple means of reducing pre-existing virus infection loads and thus the propensity for infection to be transmitted vertically to seedstock used for aquaculture. However, to obtain regulatory approvals to use such RNA interference (RNAi) technology in hatcheries producing seedstock to be farmed for human consumption, there is a need to demonstrate that injected dsRNA will not transfer from broodstock or be maintained/amplified in their farmed progeny. To investigate this, at the time they were eyestalk ablated, female Black Tiger shrimp (Penaeus monodon) broodstock naturally impregnated with male spermatophores were injected with a 1:1 mix of dsRNAs synthesised to a 954 bp region of the gill-associated virus (GAV) genome and a 464 bp region of the firefly luciferase (Luc) gene. Eggs spawned 6 to 14 days later from each of 3 non-injected control females and 3 females injected at a dosage of 0.5 μg each dsRNA/g shrimp weight were reared separately and sampled at key developmental stages until they reached post-larval (PL) age. To detect residual dsRNA, cDNA synthesised to both native and heat-denatured (HD) total nucleic acid (TNA = RNA + DNA) extracted from the larval samples and pleopod, lymphoid organ and ovary tissues sampled from each female immediately after spawning was amplified using highly-sensitive TaqMan quantitative real-time (q)PCR tests targeting the dsGAV1 and dsLuc dsRNA sequences. Using HD TNA to strand-separate dsRNA and thus improve the detection sensitivity of the RT-qPCR tests, both dsRNAs were detected in all 3 tissues examined from injected females. Direct qPCR testing of the DNA component of each TNA confirmed that the detections were not due to residual template DNA in the purified dsRNA preparations. Similar RT-qPCR testing of both native and HD TNA extracted from pools of 50 embryos, nauplii, protozoea, and mysis and 32 individual PL reared separately for each female generated no unequivocal or consistent detections of dsGAV1 or dsLuc dsRNA. These findings provide confidence that purified dsRNA injected into female broodstock at the time an eyestalk is ablated and at the dosage tested was not effectively transferred to/amplified in progeny, and thus that the human health risks of using this RNAi strategy to reduce virus disease risks during shrimp grow-out will be negligible.

Enhancing RNAi by using concatemerized double-stranded RNA.

RNA interference (RNAi) is a potential tool for functional characterization of genes and also in the management of insect pests. Accumulated literature reveals that the RNAi efficiency varies among insect species and is reported to be less efficient in lepidopteran insects. We attempted to enhance RNAi efficiency by concatemerizing short double-stranded RNA (dsRNA) sequence. Then the effectiveness of concatemerized dsRNAs (C-dsRNAs) was compared with non-concatemerized long dsRNA (NCL-dsRNA) in silencing of acetylcholinesterase (AChE) in the diamondback moth, Plutella xylostella (Lepidoptera), a major pest on cruciferous vegetables. Results revealed that the C-dsRNAs enhanced the RNAi efficiency in terms of higher target gene silencing and consequently resulted in lower larval weight gain and higher mortality compared to the NCL-dsRNA treatment. Even the lower concentration (0.5µg) of C-dsRNAs had a relatively similar effect to that of higher concentrations (2µg) of NCL-dsRNA. The enhanced RNAi efficiency with C-dsRNAs was plausibly due to the higher expression of core RNAi pathway genes, Dicer-2 and Argonaute-2 (Ago-2), which are known to govern the efficiency of RNAi. Overall, C-dsRNA enhanced the RNAi activity over routinely used long dsRNA in P. xylostella and this strategy may be applied for effective management of insect pests. © 2018 Society of Chemical Industry.

A myosin5 dsRNA that reduces the fungicide resistance and pathogenicity of Fusarium asiaticum

Fungal resistance to fungicides is a serious challenge in crop protection. Although strategies have been found to prevent the development of fungicide resistance, rare strategy has been found to quickly reduce such resistance once it has occurred. We demonstrate that the application of dsRNAs, which inhibit the expression of the phenamacril (fungicide JS399-19) target gene−Myosin 5 (Myo5) in Fusarium, decreased F. asiaticum resistance to phenamacril and infection. RNAi molecules derived from different regions of Myo5 gene had different effects on phenamacril-resistance. Myo5-8 (one of Myo5 segments) exhibited great and stable effect on phenamacril-resistant reduction both in vivo and in vitro. Myo5 mRNA and protein were both reduced when mycelium was treated with Myo5-8 dsRNA. After a mixture of Myo5-8 dsRNA and phenamacril treatment, plants can highly control the infection of phenamacril-resistant strain. The antifungal activity of Myo5-8 dsRNA plus phenamacril effected longer than a single Myo5-8 dsRNA. In addition, no off-target sequences were found in wheat and/or other plant and animal species for Myo5-8 dsRNA sequence. Our findings suggest a new strategy for fungicide resistant reduction and for designing new fungicides to control pathogens which easily develop fungicide resistance.

Synthetic biology approach for plant protection using dsRNA.

SummaryPathogens induce severe damages on cultivated plants and represent a serious threat to global food security. Emerging strategies for crop protection involve the external treatment of plants with double‐stranded (ds)RNA to trigger RNA interference. However, applying this technology in greenhouses and fields depends on dsRNA quality, stability and efficient large‐scale production. Using components of the bacteriophage phi6, we engineered a stable and accurate in vivo dsRNA production system in Pseudomonas syringae bacteria. Unlike other in vitro or in vivo dsRNA production systems that rely on DNA transcription and postsynthetic alignment of single‐stranded RNA molecules, the phi6 system is based on the replication of dsRNA by an RNA‐dependent RNA polymerase, thus allowing production of high‐quality, long dsRNA molecules. The phi6 replication complex was reprogrammed to multiply dsRNA sequences homologous to tobacco mosaic virus (TMV) by replacing the coding regions within two of the three phi6 genome segments with TMV sequences and introduction of these constructs into P. syringae together with the third phi6 segment, which encodes the components of the phi6 replication complex. The stable production of TMV dsRNA was achieved by combining all the three phi6 genome segments and by maintaining the natural dsRNA sizes and sequence elements required for efficient replication and packaging of the segments. The produced TMV‐derived dsRNAs inhibited TMV propagation when applied to infected Nicotiana benthamiana plants. The established dsRNA production system enables the broad application of dsRNA molecules as an efficient, highly flexible, nontransgenic and environmentally friendly approach for protecting crops against viruses and other pathogens.

Kinetic and thermodynamic analysis of triplex formation between peptide nucleic acid and double-stranded RNA.

Kinetics and thermodynamics of triplex formation between 9-mer homopyrimidine PNA (H2N-Lys-TCTCCTCCC-CONH2) and double-stranded RNA (dsRNA, 5'-AGAGGAGGG-3'/3'-UCUCCUCCC-5') at acidic pH were studied by means of a stopped-flow technique and isothermal titration calorimetry (ITC). These results revealed the following main findings: (i) the stable PNA-dsRNA triplex formation mostly originated from the large association rate constant (kon), which was dominated by both the charge neutral PNA backbone and the protonation level of the PNA cytosine. (ii) The temperature dependence of the enthalpy change (ΔH) and kon suggested that the association phase of the PNA-dsRNA triplex formation comprised a non-directional nucleation-zipping mechanism that was coupled with the conformational transition of the unbound PNA. (iii) The destabilization by a mismatch in the dsRNA sequence mainly resulted from the decreased magnitude of both kon and ΔH. (iv) There was sequence and position dependence of the mismatch on ΔH and the activation energy (Eon), which illustrated the importance of base pairing in the middle of the sequence. Our results for the first time revealed an association mechanism for the PNA-dsRNA triplex formation. A set of the kinetic and thermodynamic data we reported here will also expand the scope of understanding for nucleic acid recognition by PNA.

Triplex-Forming Peptide Nucleic Acid Probe Having Thiazole Orange as a Base Surrogate for Fluorescence Sensing of Double-stranded RNA.

We have developed a new fluorescent sensing probe for double-stranded RNA (dsRNA) by integrating thiazole orange (TO) as a base surrogate into triplex-forming PNA. Our probe forms the thermally stable triplex with the target dsRNA at acidic pH; and the triplex formation is accompanied by the remarkable light-up response of the TO unit. The binding of our probe to the target dsRNA proceeds very rapidly, allowing real-time monitoring of the triplex formation. Importantly, we found the TO base surrogate in our probe functions as a universal base for the base pair opposite the TO unit in the triplex formation. Furthermore, the TO unit is significantly more responsive for the fully matched dsRNA sequence compared to the mismatch-containing sequences, which enables the analysis of the target dsRNA sequence at the single-base pair resolution. The binding and sensing functions of our probe are described for the development of fluorescent probes applicable to sensing biologically relevant dsRNA.

RNA intereference (ENAi): a novel technology with potential for pest insect management

RNA Interference (RNAi) is a biological mechanism widely distributed in eukaryotes that allows silencing of genes in the presence of double-stranded RNA (dsRNA) molecules. This mechanism was discovered more than 15 years ago, since then a lot of research projects have been performed in order to obtain a better understanding of how this mechanism works, its function in different organisms, utility for specific gene function description and its potential in the technologic development of other areas. Gene silencing occurs by the interaction between enzymatic complexes in the cytoplasm, with small interference RNA molecules (siRNA), which act on the endogenous messenger RNA (mRNA), therefore preventing their translation to proteins. It is a fact that RNAi could be used as an alternative technology for pest control, through silencing essential genes for the target species. The implementation of this technology involve many necessary studies, ranging from the identification of target genes including dsRNA sequence design, development of bioassays and field tests that show the real effects of dsRNA on gene silencing, and the evaluation of associated factors that could produce variability during the process. Due to this, is necessary more research studies for a marketing establishing of this technology. This paper presents the theory behind the RNAi mechanism, current achievements of this technology, as well as the potential of this technology for insect pest control.

Temporal Gene Expression Analysis and RNA Silencing of Single and Multiple Members of Gene Family in the Lone Star Tick Amblyomma americanum.

Saliva is an integral factor in the feeding success of veterinary and medically important ticks. Therefore, the characterization of the proteins present in tick saliva is an important area of tick research. Here, we confirmed previously generated sialotranscriptome data using quantitative real-time PCR. The information obtained in this in-depth study of gene expression was used to measure the effects of metalloprotease gene silencing on tick feeding. We analyzed the temporal expression of seven housekeeping genes and 44 differentially expressed salivary molecules selected from a previously published Amblyomma americanum sialotranscriptome. Separate reference genes were selected for the salivary glands and midgut from among the seven housekeeping genes, to normalize the transcriptional expression of differentially expressed genes. The salivary gland reference gene, ubiquitin, was used to normalize the expression of 44 salivary genes. Unsurprisingly, each gene family was expressed throughout the blood meal, but the expression of specific genes differed at each time point. To further clarify the complex nature of the many proteins found in the saliva, we disrupted the translation of several members of the metalloprotease family. Intriguingly, the nucleotide sequence similarity of the reprolysin metalloprotease gene family is so homologous that a single synthesized dsRNA sequence knocked down multiple members of the family. The use of multigene knockdown yielded a more significant picture of the role of metalloproteases in tick feeding success, and changes were observed in the female engorgement weight and larval hatching success. Interestingly, the depletion of metalloprotease transcripts also reduced the total number of bacteria present in the salivary glands. These data provide insight into the expression and functions of tick salivary proteins expressed while feeding on its host.

Molecular epidemiological analyses of Cryptosporidium parvum virus 1 (CSpV1), a symbiotic virus of Cryptosporidium parvum, in Japan

We show that Cryptosporidium parvum virus 1 (CSpV1), a member of the family Partitiviridae, genus Cryspovirus that can infect Cryptosporidium parvum, is a new candidate for high-resolution tool for tracing C. parvum. CSpV1 was detected in all C. parvum-positive samples tested. Phylogenetic analysis of dsRNA1 sequence from CSpV1 can distinguish infected areas of C. parvum on the national level. Sequences detected in samples from Iwate prefecture and other islands (Tanegashima, and Okinawa) belonged to a single clade. This system can differentiate the samples from Hokkaido and south part of Japan as well as from other countries. Samples from Iwate, Tanegashima, and Okinawa belonged to a single subclade, respectively. Therefore, the CSpV1 dsRNA sequences reflect the regional distribution of their host and have potential as a high-resolution tool to trace C. parvum IIaA15G2R1 subtype.

Non-Invasive Delivery of dsRNA into De-Waxed Tick Eggs by Electroporation.

RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT) / Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis.

Sequence-specific cleavage of dsRNA by Mini-III RNase.

Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner. Here, we present evidence for a sequence-dependent cleavage of long dsRNA by RNase Mini-III from Bacillus subtilis (BsMiniIII). Analysis of the sites cleaved by this enzyme in limited digest of bacteriophage Φ6 dsRNA led to the identification of a consensus target sequence. We defined nucleotide residues within the preferred cleavage site that affected the efficiency of the cleavage and were essential for the discrimination of cleavable versus non-cleavable dsRNA sequences. We have also determined that the loop α5b-α6, a distinctive structural element in Mini-III RNases, is crucial for the specific cleavage, but not for dsRNA binding. Our results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be used for targeted cleavage of dsRNA.

Complete dsRNA sequence of a novel victorivirus isolated from the pear stem wart fungus Botryosphaeria dothidea.

Botryosphaeria dothidea is a pathogen that causes many important diseases of deciduous fruit trees worldwide. In this study, a double-stranded (ds) RNA of 5322 base pairs (bp) in length from strain GY25 of B. dothidea was isolated and sequenced. The dsRNA potentially contained two overlapping open reading frames (ORFs). The predicted gene product of ORF1 had the highest amino acid sequence similarity, 37% (E-value, 8e(-101)), to the capsid protein of Rosellinia necatrix victorivirus 1 (RnV1), and ORF2 had the highest similarity, 44% (E-value, 0.0), to the RNA-directed RNA polymerase of Beauveria bassiana victorivirus NZL/1980(BbV-NZL/1980). Both of these viruses are members of the genus Victorivirus in the family Totiviridae. There was a small ORF at the 5' terminus whose predicted gene product did not show homology to any known proteins. The dsRNA from B. dothidea strain GY25 appears to be a genomic component of a victorivirus, which we have tentatively named "Botryosphaeria dothidea victorivirus 1" (BdV1). This is the first full-length genome sequence of a putative victorivirus infecting B. dothidea.

A 28-day oral toxicity evaluation of small interfering RNAs and a long double-stranded RNA targeting vacuolar ATPase in mice

New biotechnology-derived crop traits have been developed utilizing the natural process of RNA interference (RNAi). However, plant-produced double stranded RNAs (dsRNAs) are not known to present a hazard to mammals because numerous biological barriers limit uptake and potential for activity. To evaluate this experimentally, dsRNA sequences matching the mouse vATPase gene (an established target for control of corn rootworms) were evaluated in a 28-day toxicity study with mice. Test groups were orally gavaged with escalating doses of either a pool of four 21-mer vATPase small interfering RNAs (siRNAs) or a 218-base pair vATPase dsRNA. There were no treatment-related effects on body weight, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. The highest dose levels tested were considered to be the no observed adverse effect levels (NOAELs) for the 21-mer siRNAs (48mg/kg/day) and the 218bp dsRNA (64mg/kg/day). As an additional exploratory endpoint, vATPase gene expression, was evaluated in selected gastrointestinal tract and systemic tissues. The results of this assay did not indicate treatment-related suppression of vATPase. The results of this study indicate that orally ingested dsRNAs, even those targeting a gene in the test species, do not produce adverse health effects in mammals.

Analysis of Double-Stranded RNA from Microbial Communities Identifies Double-Stranded RNA Virus-like Elements

Double-stranded RNA (dsRNA) can function as genetic information and may have served as genomic material before the existence of DNA-based life. By developing a method to purify dsRNA, we have investigated the diversity of dsRNA in microbial populations. We detect large dsRNAs in multiple microbial populations. Analysis of an aquatic microbial population reveals that some dsRNA sequences match metagenomic DNA, suggesting that microbes contain pools of sense-antisense transcripts. In addition, ∼30% of the dsRNA sequences are not present in the corresponding DNA pool and are strongly biased toward encoding novel proteins. Of these "dsRNA unique" sequences, only a small percentage share similarity to known viruses, a large fraction assemble into RNA virus-like contigs, and the remaining fraction has an unexplained origin. These results have uncovered dsRNA virus-like elements and underscore that dsRNA potentially represents an additional reservoir of genetic information in microbial populations.

Reporters Transiently Transfected into Mammalian Cells Are Highly Sensitive to Translational Repression Induced by dsRNA Expression

In mammals, double-stranded RNA (dsRNA) can mediate sequence-specific RNA interference, activate sequence-independent interferon response, or undergo RNA editing by adenosine deaminases. We showed that long hairpin dsRNA expression had negligible effects on mammalian somatic cells—expressed dsRNA was slightly edited, poorly processed into siRNAs, and it did not activate the interferon response. At the same time, we noticed reduced reporter expression in transient co-transfections, which was presumably induced by expressed dsRNA. Since transient co-transfections are frequently used for studying gene function, we systematically explored the role of expressed dsRNA in this silencing phenomenon. We demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits the expression of co-transfected reporter plasmids but not the expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent, it is found in different cell types, and it is independent of transfection method and dsRNA sequence. The inhibition occurs at the level of translation and involves protein kinase R, which binds the expressed dsRNA. Thus, dsRNA expression represents a hidden danger in transient transfection experiments and must be taken into account during interpretation of experimental results.

Isolation and amplification of cDNA from the conserved region of the nematode Heterodera schachtii 8H07 gene with a close similarity to its homolog in rape plants

An original method was developed for isolation of small regulatory RNAs (si/miRNAs) from plant cells. PCR-amplification was carried out for the cDNA fragment of the nematode Heterodera schachtii 8H07 gene. Northern-blot hybridization of plant si/miRNAs with the cDNA fragment of the conserved region from the nematode’s 8H07 gene confirmed the high degree of their homology. In the future, the amplified cDNA fragment of the nematode 8H07 gene will be used for creating a recombinant gene with an antisense dsRNA sequence for increasing the resistance of rape plants to parasitic nematodes.