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1
Han, Zhenhao, Jiwen Liu, Linghong Kong, Yunqiang He, Hongqu Wu, and Wenxing Xu. "A special satellite-like RNA of a novel hypovirus from Pestalotiopsis fici broadens the definition of fungal satellite." PLOS Pathogens 19, no. 6 (June 7, 2023): e1010889. http://dx.doi.org/10.1371/journal.ppat.1010889.
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Satellites associated with plant or animal viruses have been largely detected and characterized, while those from mycoviruses together with their roles remain far less determined. Three dsRNA segments (dsRNA 1 to 3 termed according to their decreasing sizes) were identified in a strain of phytopathogenic fungus Pestalotiopsis fici AH1-1 isolated from a tea leaf. The complete sequences of dsRNAs 1 to 3, with the sizes of 10316, 5511, and 631 bp, were determined by random cloning together with a RACE protocol. Sequence analyses support that dsRNA1 is a genome of a novel hypovirus belonging to genus Alphahypovirus of the family Hypoviridae, tentatively named Pestalotiopsis fici hypovirus 1 (PfHV1); dsRNA2 is a defective RNA (D-RNA) generating from dsRNA1 with septal deletions; and dsRNA3 is the satellite component of PfHV1 since it could be co-precipitated with other dsRNA components in the same sucrose fraction by ultra-centrifuge, suggesting that it is encapsulated together with PfHV1 genomic dsRNAs. Moreover, dsRNA3 shares an identical stretch (170 bp) with dsRNAs 1 and 2 at their 5′ termini and the remaining are heterogenous, which is distinct from a typical satellite that generally has very little or no sequence similarity with helper viruses. More importantly, dsRNA3 lacks a substantial open reading frame (ORF) and a poly (A) tail, which is unlike the known satellite RNAs of hypoviruses, as well as unlike those in association with Totiviridae and Partitiviridae since the latters are encapsidated in coat proteins. As up-regulated expression of RNA3, dsRNA1 was significantly down-regulated, suggesting that dsRNA3 negatively regulates the expression of dsRNA1, whereas dsRNAs 1 to 3 have no obvious impact on the biological traits of the host fungus including morphologies and virulence. This study indicates that PfHV1 dsRNA3 is a special type of satellite-like nucleic acid that has substantial sequence homology with the host viral genome without encapsidation in a coat protein, which broadens the definition of fungal satellite.
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Wen, Caiyi, Xinru Wan, Yuanyuan Zhang, Hongyan Du, Chenxing Wei, Rongrong Zhong, Han Zhang, et al. "Molecular Characterization of the First Alternavirus Identified in Fusarium oxysporum." Viruses 13, no. 10 (October 8, 2021): 2026. http://dx.doi.org/10.3390/v13102026.
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A novel mycovirus named Fusarium oxysporum alternavirus 1(FoAV1) was identified as infecting Fusarium oxysporum strain BH19, which was isolated from a fusarium wilt diseased stem of Lilium brownii. The genome of FoAV1 contains four double-stranded RNA (dsRNA) segments (dsRNA1, dsRNA 2, dsRNA 3 and dsRNA 4, with lengths of 3.3, 2.6, 2.3 and 1.8 kbp, respectively). Additionally, dsRNA1 encodes RNA-dependent RNA polymerase (RdRp), and dsRNA2- dsRNA3- and dsRNA4-encoded hypothetical proteins (ORF2, ORF3 and ORF4), respectively. A homology BLAST search, along with multiple alignments based on RdRp, ORF2 and ORF3 sequences, identified FoAV1 as a novel member of the proposed family “Alternaviridae”. Evolutionary relation analyses indicated that FoAV1 may be related to alternaviruses, thus dividing the family “Alternaviridae” members into four clades. In addition, we determined that dsRNA4 was dispensable for replication and may be a satellite-like RNA of FoAV1—and could perhaps play a role in the evolution of alternaviruses. Our results provided evidence for potential genera establishment within the proposed family “Alternaviridae”. Additionally, FoAV1 exhibited biological control of Fusarium wilt. Our results also laid the foundations for the further study of mycoviruses within the family “Alternaviridae”, and provide a potential agent for the biocontrol of diseases caused by F. oxysporum.
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Jiang, Daohong, and Said A. Ghabrial. "Molecular characterization of Penicillium chrysogenum virus: reconsideration of the taxonomy of the genus Chrysovirus." Journal of General Virology 85, no. 7 (July 1, 2004): 2111–21. http://dx.doi.org/10.1099/vir.0.79842-0.
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Molecular cloning and complete nucleotide sequencing of Penicillium chrysogenum virus (PcV) dsRNAs indicated that PcV virions contained four dsRNA segments with sizes of 3562, 3200, 2976 and 2902 bp. Each dsRNA segment had unique sequences and contained a single large open reading frame (ORF). In vitro translation of transcripts derived from full-length cDNA clones of PcV dsRNAs yielded single products of sizes similar to those predicted from the deduced amino acid sequences of the individual ORFs. Sequence similarity searches revealed that dsRNA1 encodes a putative RNA-dependent RNA polymerase. In this study, it was determined that dsRNA2 encodes the major capsid protein and that p4, encoded by dsRNA4, is virion-associated as a minor component. All four dsRNAs of PcV, like the genomic segments of viruses with multipartite genomes, were found to have extended regions of highly conserved terminal sequences at both ends. In addition to the strictly conserved 5′-terminal 10 nt, a second region consisting of reiteration of the sequence CAA was found immediately upstream of the AUG initiator codon. These (CAA) n repeats are reminiscent of the translational enhancer elements of tobamoviruses. The 3′-terminal 14 nt were also strictly conserved. As PcV and related viruses with four dsRNA segments (genus Chrysovirus) have not been previously characterized at the molecular level, they were provisionally classified in the family Partitiviridae, comprising viruses with bipartite genomes. This study represents the first report on molecular characterization of a chrysovirus and the results suggest the creation of a new family of mycoviruses with multipartite dsRNA genomes to accommodate PcV and related viruses.
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Wang, Yanfen, Hang Zhao, Jiayuan Cao, Xinming Yin, Yashuang Guo, Lihua Guo, Haiyan Wu, and Meng Zhang. "Characterization of a Novel Mycovirus from the Phytopathogenic Fungus Botryosphaeria dothidea." Viruses 14, no. 2 (February 6, 2022): 331. http://dx.doi.org/10.3390/v14020331.
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Botryosphaeria dothidea is, globally, one of the most economically important phytopathogenic fungi worldwide, causing the canker and dieback of fruit trees. An increasing number of viruses infecting B. dothidea have lately been reported, several of which could confer hypovirulence. In this study, isolated from strain ZM170285-1 of B. dothidea, a novel double-stranded RNA (dsRNA) mycovirus, tentatively named Botryosphaeria dothidea partitivirus 2 (BdPV2), was identified well. The BdPV2 harbored three dsRNA segments (1–3) with lengths of 1751, 1568, and 1198 bp, which encoded an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), and a hypothetical protein of unknown function, respectively. BLASTp searches revealed that the predicted protein sequences of dsRNA1 and dsRNA2 had the highest identities (74.95% and 61.01%) with the corresponding dsRNAs of Penicillium stoloniferum virus S (PsV-S), whereas dsRNA3 shared the highest identity (32.95%) with the dsRNA3 of Aspergillus ochraceous virus 1 (AoV1). Phylogenetic analysis indicated that BdPV2 belonged to the Gammapartitivirus genus and Partitiviridae family. To our knowledge, this is the first report of a Gammapartitivirus in B. dothidea.
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Nameth, S. T., and S. L. Cheng. "Identification and Partial Characterization of Endogenous Double-stranded Ribonucleic Acid in Mulberry." Journal of the American Society for Horticultural Science 119, no. 4 (July 1994): 859–61. http://dx.doi.org/10.21273/jashs.119.4.859.
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Double-stranded ribonucleic acid (dsRNA) analysis of apparently healthy red mulberry (Morus rubra L.) yielded four distinct dsRNA banding profiles. dsRNA type 1 contained three dsRNA bands with approximate molecular weights (MWs) of 12.0, 1.0, and 0.9 × 106, respectively. dsRNA type 2 contained two dsRNA bands with MWs of 1.0 and 0.9 × 106. dsRNA type 3 contained four dsRNA bands with MWs of 1.0, 0.9, 0.89, and 0.88 × 106. dsRNA type 4 contained three dsRNA bands with MWs of 1.0, 0.88, and 0.87 × 106. No virus particles were associated with any of the samples analyzed. All four types of dsRNA were resistant to DNase I and RNase A in high salt and susceptible to RNase A in low salt. Mulberry dsRNAs were somewhat similar to endogenous dsRNAs (edsRNA) associated with other hosts. This is the first report of edsRNA associated with a deciduous tree.
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Zhai, Lifeng, Mengmeng Yang, Meixin Zhang, Ni Hong, and Guoping Wang. "Characterization of a Botybirnavirus Conferring Hypovirulence in the Phytopathogenic Fungus Botryosphaeria dothidea." Viruses 11, no. 3 (March 17, 2019): 266. http://dx.doi.org/10.3390/v11030266.
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A double-stranded RNA (dsRNA) virus was isolated and characterized from strain EW220 of the phytopathogenic fungus Botryosphaeria dothidea. The full-length cDNAs of the dsRNAs were 6434 bp and 5986 bp in size, respectively. The largest dsRNA encodes a cap-pol fusion protein that contains a coat protein gene and an RNA-dependent RNA polymerase (RdRp) domain, and the second dsRNA encodes a hypothetical protein. Genome sequence analysis revealed that the sequences of the dsRNA virus shared 99% identity with Bipolaris maydis botybirnavirus 1(BmBRV1) isolated from the causal agent of corn southern leaf blight, Bipolaris maydis. Hence, the dsRNA virus constitutes a new strain of BmBRV1 and was named Bipolaris maydis botybirnavirus 1 strain BdEW220 (BmBRV1-BdEW220). BmBRV1-BdEW220 contains spherical virions that are 37 nm in diameter and consist of two dsRNA segments. The structural proteins of the BmBRV1-BdEW220 virus particles were 110 kDa, 90 kDa, and 80 kDa and were encoded by dsRNA1 and 2-ORFs. Phylogenetic reconstruction indicated that BmBRV1 and BmBRV1-BdEW220 are phylogenetically related to the genus Botybirnavirus. Importantly, BmBRV1-BdEW220 influences the growth of B. dothidea and confers hypovirulence to the fungal host. To our knowledge, this is the first report of a botybirnavirus in B. dothidea.
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Peever, Tobin L., Yir-Chung Liu, Kerong Wang, Bradley I. Hillman, Robert Foglia, and Michael G. Milgroom. "Incidence and Diversity of Double-Stranded RNAs Occurring in the Chestnut Blight Fungus, Cryphonectria parasitica, in China and Japan." Phytopathology® 88, no. 8 (August 1998): 811–17. http://dx.doi.org/10.1094/phyto.1998.88.8.811.
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Isolates of the chestnut blight fungus, Cryphonectria parasitica, were randomly sampled from 10 subpopulations in China and 8 subpopulations in Japan and screened for the presence of double-stranded (ds) RNA using an immunoblot procedure with a monoclonal antibody specific for dsRNA. The overall incidence of dsRNA in C. parasitica was 2 and 6% in China and Japan, respectively, much lower than the 28% found previously in North American populations. Genetic relatedness of dsRNAs within and among populations in China and Japan was examined using RNA-RNA hybridizations with labeled-dsRNA probes. The majority of Chinese and Japanese dsRNAs were members of a single hybridization group, related to Cryphonectria hypovirus 1 (CHV1) from Europe, and are referred to as CHV1-type dsRNAs. No evidence was obtained for genetic differentiation between CHV1-type dsRNAs sampled in China and Japan. Five Japanese isolates contained two genetically distinct dsRNAs. The larger segments (approximately 12 kilobases [kb]) were members of the CHV1 hybridization group, while the smaller segments (approximately 3 kb) did not hybridize with any known dsRNA from C. parasitica including the 2.7-kb dsRNA from isolate NB631 from New Jersey or dsRNA from isolate RC1 from Michigan. Two small dsRNA segments (approximately 1.8 and 2 kb) from one isolate sampled from Liaoning Province in northeastern China did not hybridize with any of the dsRNA probes tested including several described dsRNAs of similar size from C. parasitica in North America. Three dsRNAs from Anhui Province, China, hybridized to Cryphonectria hypovirus 2 (CHV2)-specific probes and are thus referred to as CHV2-type dsRNAs. Sequence analysis of 1,627 base pairs of these three CHV2-type dsRNAs from Anhui revealed that they were identical to each other in the region sequenced and very closely related to CHV2-NB58, isolated from New Jersey. We speculate that CHV2-NB58 may have been introduced into North America from this part of China. This is the first record of a North American C. parasitica dsRNA that is genetically related to a dsRNA from Asia.
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Valverde, Rodrigo A., and James F. Fontenot. "Variation in Double-stranded Ribonucleic Acid among Pepper Cultivars." Journal of the American Society for Horticultural Science 116, no. 5 (September 1991): 903–5. http://dx.doi.org/10.21273/jashs.116.5.903.
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Double-stranded ribonucleic acid (dsRNA) was found associated with 51 of 80 healthy pepper (Capsicum annuum L., C. frutescent L., C. chinense Jacq.) cultivars analyzed. In general, dsRNAs were consistent within particular cultivars. Twelve distinct dsRNA profiles that varied in the number and size of the dsRNA segments were obtained. All bell and pimento pepper cultivars analyzed had a similar dsRNA profile. Furthermore, all six cherry pepper cultivars tested were free of dsRNAs. However, an association between the dsRNA profile and the pepper group was not obtained with other cultivars. Selected dsRNAs were transmitted at a high rate through the seed of self-pollinated plants but were not transmitted through grafts to plants that lacked them.
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He, Wanwan, Wenbo Xu, Letian Xu, Kaiyun Fu, Wenchao Guo, Ralph Bock, and Jiang Zhang. "Length-dependent accumulation of double-stranded RNAs in plastids affects RNA interference efficiency in the Colorado potato beetle." Journal of Experimental Botany 71, no. 9 (January 6, 2020): 2670–77. http://dx.doi.org/10.1093/jxb/eraa001.
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Abstract Transplastomic potato plants expressing double-stranded RNA (dsRNA) targeted against essential genes of the Colorado potato beetle (CPB) can be lethal to larvae by triggering an RNA interference (RNAi) response. High accumulation levels of dsRNAs in plastids are crucial to confer an efficient RNAi response in the insects. However, whether length and sequence of the dsRNA determine the efficacy of RNAi and/or influence the level of dsRNA accumulation in plastids is not known. We compared the RNAi efficacy of different lengths of dsRNA targeted against the CPB β-Actin gene (ACT) by feeding in vitro-synthesized dsRNAs to larvae. We showed that, while the 60 bp dsRNA induced only a relatively low RNAi response in CPB, dsRNAs of 200 bp and longer caused high mortality and similar larval growth retardation. When the dsRNAs were expressed from the plastid (chloroplast) genome of potato plants, we found that their accumulation were negatively correlated with length. The level of dsRNA accumulation was positively associated with the observed mortality, suppression of larval growth, and suppression of target gene expression. Importantly, transplastomic potato plants expressing the 200 bp dsRNA were better protected from CPB than plants expressing the 297 bp dsRNA, the best-performing line in our previous study. Our results suggest that the length of dsRNAs is an important factor that influences their accumulation in plastids and thus determines the strength of the insecticidal RNAi effect. Our findings will aid the design of optimized dsRNA expression constructs for plant protection by plastid-mediated RNAi.
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Ahn, II-Pyung, and Yong-Hwan Lee. "A Viral Double-Stranded RNA Up Regulates the Fungal Virulence of Nectria radicicola." Molecular Plant-Microbe Interactions® 14, no. 4 (April 2001): 496–507. http://dx.doi.org/10.1094/mpmi.2001.14.4.496.
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Double-stranded RNAs (dsRNAs) are widespread in plant pathogenic fungi, but their functions in fungal hosts remain mostly unclear, with a few exceptions. We analyzed dsRNAs from Nectria radicicola, the causal fungus of ginseng root rot. Four distinct sizes of dsRNAs, 6.0, 5.0, 2.5, and 1.5 kbp, were detected in 24 out of the 81 strains tested. Curing tests of individual dsRNAs suggested that the presence of 6.0-kbp dsRNA was associated with high levels of virulence, sporulation, laccase activity, and pigmentation in this fungus. The 6.0-kbp dsRNA-cured strains completely lost virulence-related phenotypes. This 6.0-kbp dsRNA was reintroduced by hyphal anastomosis to a dsRNA-cured strain marked with hygromycin resistance, which resulted in the restoration of virulence-related phenotypes. These results strongly suggest that 6.0-kbp dsRNA up regulates fungal virulence in N. radicicola. Sequencing of several cDNA clones derived from 6.0-kbp dsRNA revealed the presence of a RNA-dependent RNA polymerase (RDRP) gene. Phylogenetic analysis showed that this gene is closely related to those of plant cryptic viruses. Biochemical analyses suggested that the 6.0-kbp dsRNA may regulate fungal virulence through signal-transduction pathways involving cyclic AMP-dependent protein kinase and protein kinase C.
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Peever, Tobin L., Yir-Chung Liu, and Michael G. Milgroom. "Diversity of Hypoviruses and Other Double-Stranded RNAs in Cryphonectria parasitica in North America." Phytopathology® 87, no. 10 (October 1997): 1026–33. http://dx.doi.org/10.1094/phyto.1997.87.10.1026.
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Double-stranded (ds) RNAs in Cryphonectria parasitica were randomly sampled from nine subpopulations in North America using an antibody-based detection system for dsRNA. dsRNA was detected in 166 (28%) of a total of 595 C. parasitica isolates sampled by immunoblotting. Incidence of dsRNA infection within subpopulations ranged from 0% in samples from New Hampshire and Ontario to 100% in County Line, MI. Most of the dsRNAs sampled were approximately 9 to 13 kb in size. dsRNAs from 72 isolates analyzed by probing Northern blots with 32P-labeled dsRNAs were in one of three hybridization groups. One hybridization group was widespread throughout eastern North America, being found in New York, New Jersey, Maryland, West Virginia, Kentucky, and Michigan. These dsRNAs hybridized to dsRNA from the previously described C. parasitica isolate SR2 from Maryland and are referred to as SR2-type dsRNAs. The second hybridization group was found almost exclusively in Michigan. The Michigan dsRNAs cross-hybridized to Cryphonectria hypovirus 3-GH2 (CHV3-GH2) and are referred to as CHV3-type dsRNAs.One dsRNA sampled from Kentucky hybridized to CHV3-type dsRNAs from Michigan. This dsRNA was probably derived from a fungal isolate that had been intentionally released for biological control at this same site 10 years previously and had become established in Kentucky. The third hybridization group was found only in New Jersey. These dsRNAs were much smaller than all other dsRNAs (3 and 5 kb) and were found in all 11 isolates that were probed; two of these isolates also had SR2-type dsRNA in mixed infections. None of the North American dsRNAs hybridized to CHV1 from Europe, even though CHV1 has been released in numerous locations in eastern North America for biological control of chestnut blight. Similarly, no dsRNAs hybridized to CHV2-NB58, a hypovirus found previously in New Jersey. Mixed infections of SR2-type and CHV3-type dsRNAs were found in 13 of 15 isolates from Frankfort, MI, while another nearby subpopulation (County Line) was infected with only CHV3-type dsRNAs. The distribution of dsRNA hybridization groups in C. parasitica thus presents a mixed picture, since one hybridization group is widespread, whereas two others are primarily restricted to smaller geographic areas.
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Varga, János, Edit Rinyu, Éva Kevei, Beáta Tóth, and Zofia Kozakiewicz. "Double-stranded RNA mycoviruses in species ofAspergillussectionsCircumdatiandFumigati." Canadian Journal of Microbiology 44, no. 6 (June 1, 1998): 569–74. http://dx.doi.org/10.1139/w98-038.
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Isolates (178) belonging to Aspergillus sections Fumigati, Candidi, Clavati, and Circumdati were tested for the presence of double-stranded RNA (dsRNA) genomes. Altogether, 5.6% of the Aspergillus strains examined were infected with dsRNAs. dsRNA segments indicative of mycovirus infection were observed for the first time in Neosartorya hiratsukae, Neosartorya quadricincta, Petromyces alliaceus, and Aspergillus clavatus strains. Correlation was not observed between ochratoxin production and dsRNA content of the strains. This is the first report on the detection of naturally occurring dsRNAs in Aspergillus species that are able to reproduce sexually. The detection of dsRNA in sexual aspergilli gave us a chance to examine the transmission of these segments through ascospores. A Neosartorya hiratsukae strain transmitted the dsRNAs efficiently through sexual spores, while the stromata embedding the asci in Petromyces alliaceus did not transmit one of the dsRNA segments. The 0.6-kb dsRNA segment that was present in the single-stromatal cultures was found to be located in the mitochondrial fraction of this strain. This observation indicates that some mechanisms exist in aspergilli to exclude cytoplasmically located dsRNA molecules from stromatal structures.Key words: Aspergillus, double-stranded RNA, mycovirus, Petromyces, Neosartorya.
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Mérai, Zsuzsanna, Zoltán Kerényi, Sándor Kertész, Melinda Magna, Lóránt Lakatos, and Dániel Silhavy. "Double-Stranded RNA Binding May Be a General Plant RNA Viral Strategy To Suppress RNA Silencing." Journal of Virology 80, no. 12 (June 15, 2006): 5747–56. http://dx.doi.org/10.1128/jvi.01963-05.
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ABSTRACT In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3′ overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus γB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.
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Delgado-Martín, Josemaría, Alejo Delgado-Olidén, and Leonardo Velasco. "Carbon Dots Boost dsRNA Delivery in Plants and Increase Local and Systemic siRNA Production." International Journal of Molecular Sciences 23, no. 10 (May 10, 2022): 5338. http://dx.doi.org/10.3390/ijms23105338.
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In this work, we obtained carbon dots from glucose or saccharose as the nucleation source and passivated them with branched polyethylenimines for developing dsRNA nanocomposites. The CDs were fully characterized using hydrodynamic analyses, transmission electron microscopy, X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The ζ potential determined that the CDs had positive charges, good electrophoretic mobility and conductivity, and were suitable for obtaining dsRNA nanocomposites. DsRNA naked or coated with the CDs were delivered to leaves of cucumber plants by spraying. Quantitation of the dsRNA that entered the leaves showed that when coated with the CDs, 50-fold more dsRNA was detected than when naked dsRNA. Moreover, specific siRNAs derived from the sprayed dsRNAs were 13 times more abundant when the dsRNA was coated with the CDs. Systemic dsRNAs were determined in distal leaves and showed a dramatic increase in concentration when delivered as a nanocomposite. Similarly, systemic siRNAs were significantly more abundant in distal leaves when spraying with the CD-dsRNA nanocomposite. Furthermore, FITC-labeled dsRNA was shown to accumulate in the apoplast and increase its entry into the plant when coated with CDs. These results indicate that CDs obtained by hydrothermal synthesis are suitable for dsRNA foliar delivery in RNAi plant applications.
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Marin-Gonzalez, Alberto, J. G. Vilhena, Ruben Perez, and Fernando Moreno-Herrero. "Understanding the mechanical response of double-stranded DNA and RNA under constant stretching forces using all-atom molecular dynamics." Proceedings of the National Academy of Sciences 114, no. 27 (June 20, 2017): 7049–54. http://dx.doi.org/10.1073/pnas.1705642114.
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Multiple biological processes involve the stretching of nucleic acids (NAs). Stretching forces induce local changes in the molecule structure, inhibiting or promoting the binding of proteins, which ultimately affects their functionality. Understanding how a force induces changes in the structure of NAs at the atomic level is a challenge. Here, we use all-atom, microsecond-long molecular dynamics to simulate the structure of dsDNA and dsRNA subjected to stretching forces up to 20 pN. We determine all of the elastic constants of dsDNA and dsRNA and provide an explanation for three striking differences in the mechanical response of these two molecules: the threefold softer stretching constant obtained for dsRNA, the opposite twist-stretch coupling, and its nontrivial force dependence. The lower dsRNA stretching resistance is linked to its more open structure, whereas the opposite twist-stretch coupling of both molecules is due to the very different evolution of molecules’ interstrand distance with the stretching force. A reduction of this distance leads to overwinding in dsDNA. In contrast, dsRNA is not able to reduce its interstrand distance and can only elongate by unwinding. Interstrand distance is directly correlated with the slide base-pair parameter and its different behavior in dsDNA and dsRNA traced down to changes in the sugar pucker angle of these NAs.
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Deng, F., and G. J. Boland. "A Satellite RNA of Ophiostoma novo-ulmi Mitovirus 3a in Hypovirulent Isolates of Sclerotinia homoeocarpa." Phytopathology® 94, no. 9 (September 2004): 917–23. http://dx.doi.org/10.1094/phyto.2004.94.9.917.
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Two genetically distinct double-stranded RNA (dsRNA) elements were identified in hypovirulent isolates of Sclerotinia homoeocarpa, the causal agent of dollar spot of turfgrass. The large dsRNA (L-dsRNA) was consistently present in all hypovirulent isolates, whereas the small dsRNA (S-dsRNA) was found only in some hypovirulent isolates. Virulence comparisons revealed that there was no significant difference between isolates containing one or both dsRNAs. Therefore, the L-dsRNA appears to be the genetic determinant of hypovirulence, while the S-dsRNA is not essential for hypovirulence in S. homoeocarpa. The L-dsRNA in hypovirulent isolate Sh12B of S. homoeocarpa was previously characterized as a fungal mitochondrial virus and designated Ophiostoma novo-ulmi mitovirus 3a-Sh12B (OnuMV3a-Sh12B) because it was conspecific with O. novo-ulmi mitovirus 3a-Ld from O. novo-ulmi, the causal agent of Dutch elm disease. In the present study, the nucleotide sequences of the S-dsRNAs (738 to 767 nucleotides) in hypovirulent isolates Sh12B, Sh279B, and Sh286B were determined. Nucleotide sequence analysis indicated that the S-dsRNA was not derived from the OnuMV3a dsRNA and it could not encode an RNA-dependent RNA polymerase. These results are consistent with biological data that the S-dsRNA was always associated with the L-dsRNA and was never found independently. Therefore, the S-dsRNA can be regarded as a satellite RNA of OnuMV3a in S. homoeocarpa. Northern blotting analysis indicated that nucleic acid extracts from isolate Sh12B of S. homoeocarpa contained more single (+) stranded RNA than dsRNA for this satellite RNA. The 5′- and 3′-terminal sequences of the positive strand of the S-dsRNA each could be folded into a stem-loop structure and the terminal 21 nucleotides were complementary to each other, potentially forming a panhandle structure.
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Jian, JianHua, Dilip K. Lakshman, and Stellos M. Tavantzis. "Association of Distinct Double-Stranded RNAs with Enhanced or Diminished Virulence in Rhizoctonia solani Infecting Potato." Molecular Plant-Microbe Interactions® 10, no. 8 (November 1997): 1002–9. http://dx.doi.org/10.1094/mpmi.1997.10.8.1002.
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A virulent field isolate of Rhizoctonia solani AG 3, Rhs 1AP, has given rise to three sectors in a period of several years. Subculturing of these sectors resulted in three hypovirulent isolates—Rhs 1A1, Rhs 1A2, and Rhs 1A3. We reported previously that five genetically different double-stranded (ds) RNAs occur in these four isolates, with each isolate containing a unique combination of two or more dsRNAs. We report here that all five dsRNA elements occur in the cytoplasm, and none in the nucleus. The mitochondria contains low to moderate concentrations of the four larger dsRNAs. The four isolates were paired in selected combinations in an attempt to transmit specific dsRNAs to cultures lacking these dsRNAs. This approach generated groups of near-isogenic lines possessing the same dsRNA elements. As many as six cultures possessing the same dsRNA genotype were found to have the same degree of pathogenicity. Consistently, acquisition of a 6.4-kb dsRNA brought about increased virulence, whereas the presence or acquisition of a 3.6-kb dsRNA resulted in diminished virulence in the recipient culture. All cultures had the same mitochondrial DNA haplotype as that of the original field isolate, Rhs 1AP. This, in conjunction with the fact that all cultures in this study had a single nuclear DNA origin (Rhs 1AP), indicates a strong correlation between certain phenotypic characters or changes thereof and dsRNA profiles or acquisition of particular dsRNAs, respectively.
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Holeva, Maria C., Athanasios Sklavounos, Rajendran Rajeswaran, Mikhail M. Pooggin, and Andreas E. Voloudakis. "Topical Application of Double-Stranded RNA Targeting 2b and CP Genes of Cucumber mosaic virus Protects Plants against Local and Systemic Viral Infection." Plants 10, no. 5 (May 12, 2021): 963. http://dx.doi.org/10.3390/plants10050963.
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Cucumber mosaic virus (CMV) is a destructive plant virus with worldwide distribution and the broadest host range of any known plant virus, as well as a model plant virus for understanding plant–virus interactions. Since the discovery of RNA interference (RNAi) as a major antiviral defense, RNAi-based technologies have been developed for plant protection against viral diseases. In plants and animals, a key trigger of RNAi is double-stranded RNA (dsRNA) processed by Dicer and Dicer-like (DCL) family proteins in small interfering RNAs (siRNAs). In the present study, dsRNAs for coat protein (CP) and 2b genes of CMV were produced in vitro and in vivo and applied onto tobacco plants representing a systemic solanaceous host as well as on a local host plant Chenopodium quinoa. Both dsRNA treatments protected plants from local and systemic infection with CMV, but not against infection with unrelated viruses, confirming sequence specificity of antiviral RNAi. Antiviral RNAi was effective when dsRNAs were applied simultaneously with or four days prior to CMV inoculation, but not four days post inoculation. In vivo-produced dsRNAs were more effective than the in vitro-produced; in treatments with in vivo dsRNAs, dsRNA-CP was more effective than dsRNA-2b, while the effects were opposite with in vitro dsRNAs. Illumina sequencing of small RNAs from in vivo dsRNA-CP treated and non-treated tobacco plants revealed that interference with CMV infection in systemic leaves coincides with strongly reduced accumulation of virus-derived 21- and 22-nucleotide (nt) siRNAs, likely generated by tobacco DCL4 and DCL2, respectively. While the 21-nt class of viral siRNAs was predominant in non-treated plants, 21-nt and 22-nt classes accumulated at almost equal (but low) levels in dsRNA treated plants, suggesting that dsRNA treatment may boost DCL2 activity. Taken together, our findings confirm the efficacy of topical application of dsRNA for plant protection against viruses and shed more light on the mechanism of antiviral RNAi.
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Deng, F., R. Xu, and G. J. Boland. "Hypovirulence-Associated Double-Stranded RNA from Sclerotinia homoeocarpa Is Conspecific with Ophiostoma novo-ulmi Mitovirus 3a-Ld." Phytopathology® 93, no. 11 (November 2003): 1407–14. http://dx.doi.org/10.1094/phyto.2003.93.11.1407.
Full textAbstract:
The nucleotide sequence of the hypovirulence-associated double-stranded RNA (dsRNA) in hypovirulent isolate Sh12B of Sclerotinia homoeocarpa, the causal agent of dollar spot of turf grass, was determined. This large dsRNA (L-dsRNA) is 2,632 bp long and is A and U rich (61.0% A+U residues). One strand of this dsRNA contains an open reading frame (ORF) with the potential to encode a protein of 720 amino acids. This ORF contains 12 UGA codons, predicted to encode tryptophan in ascomycete mitochondria, and has a codon bias typical of mitochondrial genes, which is consistent with a mitochondrial localization of this dsRNA. The amino acid sequence contains conserved motifs typical of RNA-dependent RNA polymerases (RdRps). Sequence analyses of the nucleotide and RdRp-like protein revealed that the L-dsRNA is homologous with previously characterized mitochondrial viruses and dsRNAs from other phytopathogenic fungi, and shares 92.4% nucleotide and 95.1% amino acid sequence identities with the Ophiostoma novo-ulmi mitovirus 3a-Ld from Ophiostoma novo-ulmi, the causal agent of Dutch elm disease. The results indicate that these two dsRNAs are conspecific. This is the first report that a hypovirulence-associated dsRNA virus naturally occurs in two taxonomically distinct fungi, and indicates that horizontal transmission of this dsRNA virus may have occurred between these fungi.
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Tayler, Alison, Daniel Heschuk, David Giesbrecht, Jae Yeon Park, and Steve Whyard. "Efficiency of RNA interference is improved by knockdown of dsRNA nucleases in tephritid fruit flies." Open Biology 9, no. 12 (December 2019): 190198. http://dx.doi.org/10.1098/rsob.190198.
Full textAbstract:
RNA interference (RNAi) in insects is routinely used to ascertain gene function, but also has potential as a technology to control pest species. For some insects, such as beetles, ingestion of small quantities of double-stranded RNA (dsRNA) is able to knock down a targeted gene's expression. However, in other species, ingestion of dsRNA can be ineffective owing to the presence of nucleases within the gut, which degrade dsRNA before it reaches target cells. In this study, we observed that nucleases within the gut of the Queensland fruit fly ( Bactrocera tryoni ) rapidly degrade dsRNA and reduce RNAi efficacy. By complexing dsRNA with liposomes within the adult insect's diet, RNAi-mediated knockdown of a melanin synthesis gene, yellow , was improved significantly, resulting in strong RNAi phenotypes. RNAi efficiency was also enhanced by feeding both larvae and adults for several days on dsRNAs that targeted two different dsRNase gene transcripts. Co-delivery of both dsRNase-specific dsRNAs and yellow dsRNA resulted in almost complete knockdown of the yellow transcripts. These findings show that the use of liposomes or co-feeding of nuclease-specific dsRNAs significantly improves RNAi inhibition of gene expression in B. tryoni and could be a useful strategy to improve RNAi-based control in other insect species.
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Chen, Xijun, Tong Shi, Tao Tang, Chen Chen, You Liang, and Shimin Zuo. "Nanosheet-Facilitated Spray Delivery of dsRNAs Represents a Potential Tool to Control Rhizoctonia solani Infection." International Journal of Molecular Sciences 23, no. 21 (October 26, 2022): 12922. http://dx.doi.org/10.3390/ijms232112922.
Full textAbstract:
Rhizoctonia solani is one of the important pathogenic fungi causing several serious crop diseases, such as maize and rice sheath blight. Current methods used to control the disease mainly depend on spraying fungicides because there is no immunity or high resistance available in crops. Spraying double-strand RNA (dsRNA) for induced-gene silencing (SIGS) is a new potentially sustainable and environmentally friendly tool to control plant diseases. Here, we found that fluorescein-labelled EGFP-dsRNA could be absorbed by R. solani in co-incubation. Furthermore, three dsRNAs, each targeting one of pathogenicity-related genes, RsPG1, RsCATA, and RsCRZ1, significantly downregulated the transcript levels of the target genes after co-incubation, leading to a significant reduction in the pathogenicity of the fungus. Only the spray of RsCRZ1 dsRNA, but not RsPG1 or RsCATA dsRNA, affected fungal sclerotium formation. dsRNA stability on leaf surfaces and its efficiency in entering leaf cells were significantly improved when dsRNAs were loaded on layered double hydroxide (LDH) nanosheets. Notably, the RsCRZ1-dsRNA-LDH approach showed stronger and more lasting effects than using RsCRZ1-dsRNA alone in controlling pathogen development. Together, this study provides a new potential method to control crop diseases caused by R. solani.
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Shadle, Sean C., Sean R. Bennett, Chao-Jen Wong, Nancy A. Karreman, Amy E. Campbell, Silvère M. van der Maarel, Brenda L. Bass, and Stephen J. Tapscott. "DUX4-induced bidirectional HSATII satellite repeat transcripts form intranuclear double-stranded RNA foci in human cell models of FSHD." Human Molecular Genetics 28, no. 23 (October 19, 2019): 3997–4011. http://dx.doi.org/10.1093/hmg/ddz242.
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Abstract The DUX4 transcription factor is normally expressed in the cleavage-stage embryo and regulates genes involved in embryonic genome activation. Misexpression of DUX4 in skeletal muscle, however, is toxic and causes facioscapulohumeral muscular dystrophy (FSHD). We recently showed DUX4-induced toxicity is due, in part, to the activation of the double-stranded RNA (dsRNA) response pathway and the accumulation of intranuclear dsRNA foci. Here, we determined the composition of DUX4-induced dsRNAs. We found that a subset of DUX4-induced dsRNAs originate from inverted Alu repeats embedded within the introns of DUX4-induced transcripts and from DUX4-induced dsRNA-forming intergenic transcripts enriched for endogenous retroviruses, Alu and LINE-1 elements. However, these repeat classes were also represented in dsRNAs from cells not expressing DUX4. In contrast, pericentric human satellite II (HSATII) repeats formed a class of dsRNA specific to the DUX4 expressing cells. Further investigation revealed that DUX4 can initiate the bidirectional transcription of normally heterochromatin-silenced HSATII repeats. DUX4-induced HSATII RNAs co-localized with DUX4-induced nuclear dsRNA foci and with intranuclear aggregation of EIF4A3 and ADAR1. Finally, gapmer-mediated knockdown of HSATII transcripts depleted DUX4-induced intranuclear ribonucleoprotein aggregates and decreased DUX4-induced cell death, suggesting that HSATII-formed dsRNAs contribute to DUX4 toxicity.
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Yoon, Jimin, Doyeong Ku, Minseok Lee, Namseok Lee, Sung Gap Im, and Yoosik Kim. "Resveratrol Attenuates the Mitochondrial RNA-Mediated Cellular Response to Immunogenic Stress." International Journal of Molecular Sciences 24, no. 8 (April 17, 2023): 7403. http://dx.doi.org/10.3390/ijms24087403.
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Human mitochondria contain a circular genome that encodes 13 subunits of the oxidative phosphorylation system. In addition to their role as powerhouses of the cells, mitochondria are also involved in innate immunity as the mitochondrial genome generates long double-stranded RNAs (dsRNAs) that can activate the dsRNA-sensing pattern recognition receptors. Recent evidence shows that these mitochondrial dsRNAs (mt-dsRNAs) are closely associated with the pathogenesis of human diseases that accompany inflammation and aberrant immune activation, such as Huntington’s disease, osteoarthritis, and autoimmune Sjögren’s syndrome. Yet, small chemicals that can protect cells from a mt-dsRNA-mediated immune response remain largely unexplored. Here, we investigate the potential of resveratrol (RES), a plant-derived polyphenol with antioxidant properties, on suppressing mt-dsRNA-mediated immune activation. We show that RES can revert the downstream response to immunogenic stressors that elevate mitochondrial RNA expressions, such as stimulation by exogenous dsRNAs or inhibition of ATP synthase. Through high-throughput sequencing, we find that RES can regulate mt-dsRNA expression, interferon response, and other cellular responses induced by these stressors. Notably, RES treatment fails to counter the effect of an endoplasmic reticulum stressor that does not affect the expression of mitochondrial RNAs. Overall, our study demonstrates the potential usage of RES to alleviate the mt-dsRNA-mediated immunogenic stress response.
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Hashiro, Shuhei, Yasuhiko Chikami, Haruka Kawaguchi, Alexander A. Krylov, Teruyuki Niimi, and Hisashi Yasueda. "Efficient production of long double-stranded RNAs applicable to agricultural pest control by Corynebacterium glutamicum equipped with coliphage T7-expression system." Applied Microbiology and Biotechnology 105, no. 12 (June 2021): 4987–5000. http://dx.doi.org/10.1007/s00253-021-11324-9.
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Abstract 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.
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Mat Jalaluddin, Nurzatil Sharleeza, Maimunah Asem, Jennifer Ann Harikrishna, and Abdullah Al Hadi Ahmad Fuaad. "Recent Progress on Nanocarriers for Topical-Mediated RNAi Strategies for Crop Protection—A Review." Molecules 28, no. 6 (March 16, 2023): 2700. http://dx.doi.org/10.3390/molecules28062700.
Full textAbstract:
To fulfil the growing needs of the global population, sustainability in food production must be ensured. Insect pests and pathogens are primarily responsible for one-third of food losses and harmful synthetic pesticides have been applied to protect crops from these pests and other pathogens such as viruses and fungi. An alternative pathogen control mechanism that is more “friendly” to the environment can be developed by externally applying double-stranded RNAs (dsRNAs) to suppress gene expression. However, the use of dsRNA sprays in open fields is complicated with respect to variable efficiencies in the dsRNA delivery, and the stability of the dsRNA on and in the plants, and because the mechanisms of gene silencing may differ between plants and between different pathogen targets. Thus, nanocarrier delivery systems have been especially used with the goal of improving the efficacy of dsRNAs. Here, we highlight recent developments in nanoparticle-mediated nanocarriers to deliver dsRNA, including layered double hydroxide, carbon dots, carbon nanotubes, gold nanoparticles, chitosan nanoparticles, silica nanoparticles, liposomes, and cell-penetrating peptides, by review of the literature and patent landscape. The effects of nanoparticle size and surface modification on the dsRNA uptake efficiency in plants are also discussed. Finally, we emphasize the overall limitation of dsRNA sprays, the risks associated, and the potential safety concerns for spraying dsRNAs on crops.
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Punja, Zamir K. "Influence of double-stranded RNAs on growth, sporulation, pathogenicity, and survival of Chalara elegans." Canadian Journal of Botany 73, no. 7 (July 1, 1995): 1001–9. http://dx.doi.org/10.1139/b95-109.
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Three strains of Chalara elegans from diverse geographical areas that contained multiple (4 or 5) double-stranded RNA fragments were compared with spontaneously derived cultures from these strains that were either partially cured or completely free of dsRNA. In the wild-type strains, presence of the dsRNAs was found to significantly enhance phialospore production and pigmentation of colonies, whereas radial growth and mycelial dry weight accumulation were reduced. The rate and overall percentage of phialospore germination on 1% Noble water agar were also significantly reduced by the presence of the dsRNAs. In two partially cured strains (only one 2.8-kb fragment remaining), pathogenicity to various plant tissues was significantly enhanced when compared with the wild-type strains containing multiple dsRNA. However, survival in field soil was enhanced in one strain and reduced in the other. In the completely cured strain, the loss of multiple dsRNA fragments was associated with enhanced growth, reduced phialospore production, and a complete loss of pathogenicity and capability for survival in soil. These results indicate that the effects of dsRNAs in C. elegans vary with the strain. In general, the presence of multiple dsRNAs in this fungus enhanced sporulation, altered colony morphology, and reduced growth and pathogenicity. However, since the complete loss of dsRNA was found to eliminate pathogenicity and reduce survival, it suggests that some dsRNA fragments in C. elegans may confer an advantage to this soil-borne facultative plant pathogen. Key words: black root rot, soil-borne plant pathogen, Thielaviopsis basicola.
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Marin-Gonzalez, Alberto, Clara Aicart-Ramos, Mikel Marin-Baquero, Alejandro Martín-González, Maarit Suomalainen, Abhilash Kannan, J. G. Vilhena, Urs F. Greber, Fernando Moreno-Herrero, and Rubén Pérez. "Double-stranded RNA bending by AU-tract sequences." Nucleic Acids Research 48, no. 22 (November 27, 2020): 12917–28. http://dx.doi.org/10.1093/nar/gkaa1128.
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Abstract Sequence-dependent structural deformations of the DNA double helix (dsDNA) have been extensively studied, where adenine tracts (A-tracts) provide a striking example for global bending in the molecule. However, in contrast to dsDNA, sequence-dependent structural features of dsRNA have received little attention. In this work, we demonstrate that the nucleotide sequence can induce a bend in a canonical Watson-Crick base-paired dsRNA helix. Using all-atom molecular dynamics simulations, we identified a sequence motif consisting of alternating adenines and uracils, or AU-tracts, that strongly bend the RNA double-helix. This finding was experimentally validated using atomic force microscopy imaging of dsRNA molecules designed to display macroscopic curvature via repetitions of phased AU-tract motifs. At the atomic level, this novel phenomenon originates from a localized compression of the dsRNA major groove and a large propeller twist at the position of the AU-tract. Moreover, the magnitude of the bending can be modulated by changing the length of the AU-tract. Altogether, our results demonstrate the possibility of modifying the dsRNA curvature by means of its nucleotide sequence, which may be exploited in the emerging field of RNA nanotechnology and might also constitute a natural mechanism for proteins to achieve recognition of specific dsRNA sequences.
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Nityagovsky, Nikolay N., Konstantin V. Kiselev, Andrey R. Suprun, and Alexandra S. Dubrovina. "Exogenous dsRNA Induces RNA Interference of a Chalcone Synthase Gene in Arabidopsis thaliana." International Journal of Molecular Sciences 23, no. 10 (May 10, 2022): 5325. http://dx.doi.org/10.3390/ijms23105325.
Full textAbstract:
Recent investigations have shown the possibility of artificial induction of RNA interference (RNAi) via plant foliar treatments with naked double-stranded RNA (dsRNA) to silence essential genes in plant fungal pathogens or to target viral RNAs. Furthermore, several studies have documented the downregulation of plant endogenous genes via external application of naked gene-specific dsRNAs and siRNAs to the plant surfaces. However, there are limited studies on the dsRNA processing and gene silencing mechanisms after external dsRNA application. Such studies would assist in the development of innovative tools for crop improvement and plant functional studies. In this study, we used exogenous gene-specific dsRNA to downregulate the gene of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, in Arabidopsis. The nonspecific NPTII-dsRNA encoding the nonrelated neomycin phosphotransferase II bacterial gene was used to treat plants in order to verify that any observed effects and processing of AtCHS mRNA were sequence specific. Using high-throughput small RNA (sRNA) sequencing, we obtained six sRNA-seq libraries for plants treated with water, AtCHS-dsRNA, or NPTII-dsRNA. After plant foliar treatments, we detected the emergence of a large number of AtCHS- and NPTII-encoding sRNAs, while there were no such sRNAs after control water treatment. Thus, the exogenous AtCHS-dsRNAs were processed into siRNAs and induced RNAi-mediated AtCHS gene silencing. The analysis showed that gene-specific sRNAs mapped to the AtCHS and NPTII genes unevenly with peak read counts at particular positions, involving primarily the sense strand, and documented a gradual decrease in read counts from 17-nt to 30-nt sRNAs. Results of the present study highlight a significant potential of exogenous dsRNAs as a promising strategy to induce RNAi-based downregulation of plant gene targets for plant management and gene functional studies.
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Dickinson, M. J., C. R. Wellings, and A. Pryor. "Variation in the double-stranded RNA phenotype between and within different rust species." Canadian Journal of Botany 68, no. 3 (March 1, 1990): 599–604. http://dx.doi.org/10.1139/b90-079.
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The dsRNA phenotype of 34 isolates representing 10 Australian pathotypes of Puccinia striiformis, 2 of P. recondita, and 1 each of P. graminis, P. sorghi, P. menthae, and Melampsora lini were examined. Each rust species had its own distinctive dsRNA pattern. Variation occurred in the dsRNA phenotype between the two isolates of P. recondita and among the 34 different isolates of P. striiformis but did not correlate with variation in pathotype. Single pustule isolates of P. graminis and P. recondita were examined to establish that all the dsRNAs were probably present in individual uredospores and were consistently transmitted asexually. The potential use of dsRNA phenotype as a cytoplasmic marker in rust population studies is discussed. Key words: dsRNA, Puccinia, rust.
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Bragg, Zachary, and Lynne K. Rieske. "Spatial Distribution and Retention in Loblolly Pine Seedlings of Exogenous dsRNAs Applied through Roots." International Journal of Molecular Sciences 23, no. 16 (August 15, 2022): 9167. http://dx.doi.org/10.3390/ijms23169167.
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Exogenously applied double-stranded RNA (dsRNA) can induce potent host specific gene knockdown and mortality in insects. The deployment of RNA-interference (RNAi) technologies for pest suppression is gaining traction in both agriculture and horticulture, but its implementation in forest systems is lagging. While numerous forest pests have demonstrated susceptibility to RNAi mediated gene silencing, including the southern pine beetle (SPB), Dendroctonus frontalis, multiple barriers stand between laboratory screening and real-world deployment. One such barrier is dsRNA delivery. One possible delivery method is through host plants, but an understanding of exogenous dsRNA movement through plant tissues is essential. Therefore, we sought to understand the translocation and persistence of dsRNAs designed for SPB throughout woody plant tissues after hydroponic exposure. Loblolly pine, Pinus taeda, seedlings were exposed to dsRNAs as a root soak, followed by destructive sampling. Total RNA was extracted from different tissue types including root, stem, crown, needle, and meristem, after which gel electrophoresis confirmed the recovery of the exogenous dsRNAs, which were further verified using Sanger sequencing. Both techniques confirmed the presence of the exogenously applied target dsRNAs in each tissue type after 1, 3, 5, and 7 d of dsRNA exposure. These findings suggest that root drench applications of exogenous dsRNAs could provide a viable delivery route for RNAi technology designed to combat tree feeding pests.
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Esteban, R., and R. B. Wickner. "Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis." Molecular and Cellular Biology 6, no. 5 (May 1986): 1552–61. http://dx.doi.org/10.1128/mcb.6.5.1552-1561.1986.
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Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).
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Esteban, R., and R. B. Wickner. "Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis." Molecular and Cellular Biology 6, no. 5 (May 1986): 1552–61. http://dx.doi.org/10.1128/mcb.6.5.1552.
Full textAbstract:
Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).
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Strauss, Ethan E., Dilip K. Lakshman, and Stellos M. Tavantzis. "Molecular characterization of the genome of a partitivirus from the basidiomycete Rhizoctonia solani." Microbiology 81, no. 2 (February 1, 2000): 549–55. http://dx.doi.org/10.1099/0022-1317-81-2-549.
Full textAbstract:
The bisegmented genome of a double-stranded (ds) RNA virus from the fungus Rhizoctonia solani isolate Rhs 717 was characterized. The larger segment, dsRNA 1, is 2363 bases long whereas the smaller segment, dsRNA 2, has 2206 bases. The 5′ ends of the coding strands of dsRNA 1 and dsRNA 2 are highly conserved (100% identity over 47 bases), and contain inverted repeats capable of forming stable stem–loop structures. Analysis of the coding potential of each of the two segments showed that dsRNAs 1 and 2 could code for polypeptides of 730 aa (bases 86–2275; molecular mass 86 kDa) and 683 aa (bases 79–2130; molecular mass 76 kDa), respectively. The 86 kDa polypeptide has all the motifs of dsRNA RNA-dependent RNA polymerases (RDRP), and has significant homology with putative RDRPs of partitiviruses from Fusarium poae and Atkinsonella hypoxylon. The 76 kDa protein shows homology with the putative capsid proteins (CP) of the same viruses. Northern blot analysis revealed no subgenomic RNA species, consistent with the fact that the long open reading frames encoding the putative RDRP and CP cover the entire length of the respective dsRNAs.
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Kumar, Madhur, and Gordon G. Carmichael. "Antisense RNA: Function and Fate of Duplex RNA in Cells of Higher Eukaryotes." Microbiology and Molecular Biology Reviews 62, no. 4 (December 1, 1998): 1415–34. http://dx.doi.org/10.1128/mmbr.62.4.1415-1434.1998.
Full textAbstract:
SUMMARY There is ample evidence that cells of higher eukaryotes express double-stranded RNA molecules (dsRNAs) either naturally or as the result of viral infection or aberrant, bidirectional transcriptional readthrough. These duplex molecules can exist in either the cytoplasmic or nuclear compartments. Cells have evolved distinct ways of responding to dsRNAs, depending on the nature and location of the duplexes. Since dsRNA molecules are not thought to exist naturally within the cytoplasm, dsRNA in this compartment is most often associated with viral infections. Cells have evolved defensive strategies against such molecules, primarily involving the interferon response pathway. Nuclear dsRNA, however, does not induce interferons and may play an important posttranscriptional regulatory role. Nuclear dsRNA appears to be the substrate for enzymes which deaminate adenosine residues to inosine residues within the polynucleotide structure, resulting in partial or full unwinding. Extensively modified RNAs are either rapidly degraded or retained within the nucleus, whereas transcripts with few modifications may be transported to the cytoplasm, where they serve to produce altered proteins. This review summarizes our current knowledge about the function and fate of dsRNA in cells of higher eukaryotes and its potential manipulation as a research and therapeutic tool.
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Adeyinka, Olawale Samuel. "A Protective dsRNA is Crucial for Optimum RNAi Gene Silencing in Chilo partellus." International Journal of Agriculture and Biology 25, no. 06 (June 1, 2021): 1238–48. http://dx.doi.org/10.17957/ijab/15.1785.
Full textAbstract:
RNAi technology is currently employed as an alternate control measure for agricultural pests. However, the variability of RNAi efficiency in insect pests limits the extensive usage of this technology and demands identifying the best target gene for effective RNAi. Four different bacterially-expressed dsRNA and purified dsRNAs coated on artificial diet were fed to the larvae. The transcripts expression was analyzed at 5 days and 15 days post-exposure to various dsRNAs. In the larvae fed on bacterially-expressed dsRNA, knockdown percentages were 80 and 57% knockdown in Acetylcholinesterase transcript, 40 and 60% gene knockdown in Arginine kinase, 74 and 73% knockdown in Chymotrypsin, and 80 and 20% reduction in V-ATPase transcript expression. Overall, the mRNA knockdown percentages in the targeted genes were more pronounced at 5 days of exposure to bacterially-expressed crude dsRNA than 15 days of exposure. However, most purified dsRNAs rarely induce any significant knockdown except dsARG, which reduced the arginine kinase transcript by 40%. Our findings suggest that for optimum RNAi in C. partellus, the dsRNA must be protected from direct access with nucleases. © 2021 Friends Science Publishers
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Hur, Sun. "Double-Stranded RNA Sensors and Modulators in Innate Immunity." Annual Review of Immunology 37, no. 1 (April 26, 2019): 349–75. http://dx.doi.org/10.1146/annurev-immunol-042718-041356.
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Detection of double-stranded RNAs (dsRNAs) is a central mechanism of innate immune defense in many organisms. We here discuss several families of dsRNA-binding proteins involved in mammalian antiviral innate immunity. These include RIG-I-like receptors, protein kinase R, oligoadenylate synthases, adenosine deaminases acting on RNA, RNA interference systems, and other proteins containing dsRNA-binding domains and helicase domains. Studies suggest that their functions are highly interdependent and that their interdependence could offer keys to understanding the complex regulatory mechanisms for cellular dsRNA homeostasis and antiviral immunity. This review aims to highlight their interconnectivity, as well as their commonalities and differences in their dsRNA recognition mechanisms.
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Kato, Hiroki, Osamu Takeuchi, Eriko Mikamo-Satoh, Reiko Hirai, Tomoji Kawai, Kazufumi Matsushita, Akane Hiiragi, Terence S. Dermody, Takashi Fujita, and Shizuo Akira. "Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid–inducible gene-I and melanoma differentiation–associated gene 5." Journal of Experimental Medicine 205, no. 7 (June 30, 2008): 1601–10. http://dx.doi.org/10.1084/jem.20080091.
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The ribonucleic acid (RNA) helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation–associated gene 5 (MDA5) recognize distinct viral and synthetic RNAs, leading to the production of interferons. Although 5′-triphosphate single-stranded RNA is a RIG-I ligand, the role of RIG-I and MDA5 in double-stranded (ds) RNA recognition remains to be characterized. In this study, we show that the length of dsRNA is important for differential recognition by RIG-I and MDA5. The MDA5 ligand, polyinosinic-polycytidylic acid, was converted to a RIG-I ligand after shortening of the dsRNA length. In addition, viral dsRNAs differentially activated RIG-I and MDA5, depending on their length. Vesicular stomatitis virus infection generated dsRNA, which is responsible for RIG-I–mediated recognition. Collectively, RIG-I detects dsRNAs without a 5′-triphosphate end, and RIG-I and MDA5 selectively recognize short and long dsRNAs, respectively.
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Leelesh, Ramya Shanivarsanthe, and Lynne K. Rieske. "Oral Ingestion of Bacterially Expressed dsRNA Can Silence Genes and Cause Mortality in a Highly Invasive, Tree-Killing Pest, the Emerald Ash Borer." Insects 11, no. 7 (July 14, 2020): 440. http://dx.doi.org/10.3390/insects11070440.
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RNA interference (RNAi) is a naturally occurring process inhibiting gene expression, and recent advances in our understanding of the mechanism have allowed its development as a tool against insect pests. A major challenge for deployment in the field is the development of convenient and efficient methods for production of double stranded RNA (dsRNA). We assessed the potential for deploying bacterially produced dsRNA as a bio-pesticide against an invasive forest pest, the emerald ash borer (EAB). EAB feeds on the cambial tissue of ash trees (Fraxinus spp.), causing rapid death. EAB has killed millions of trees in North America since its discovery in 2002, prompting the need for innovative management strategies. In our study, bacterial expression and synthesis of dsRNA were performed with E. coli strain HT115 using the L4440 expression vector. EAB-specific dsRNAs (shi and hsp) over-expressed in E. coli were toxic to neonate EAB after oral administration, successfully triggering gene silencing and subsequent mortality; however, a non-specific dsRNA control was not included. Our results suggest that ingestion of transformed E. coli expressing dsRNAs can induce an RNAi response in EAB. To our knowledge, this is the first example of an effective RNAi response induced by feeding dsRNA-expressing bacteria in a forest pest.
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Kaplanoglu, Emine, Igor Kolotilin, Rima Menassa, and Cam Donly. "Plastid Transformation of Micro-Tom Tomato with a Hemipteran Double-Stranded RNA Results in RNA Interference in Multiple Insect Species." International Journal of Molecular Sciences 23, no. 7 (April 1, 2022): 3918. http://dx.doi.org/10.3390/ijms23073918.
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Plant-mediated RNA interference (RNAi) holds great promise for insect pest control, as plants can be transformed to produce double-stranded RNA (dsRNA) to selectively down-regulate insect genes essential for survival. For optimum potency, dsRNA can be produced in plant plastids, enabling the accumulation of unprocessed dsRNAs. However, the relative effectiveness of this strategy in inducing an RNAi response in insects using different feeding mechanisms is understudied. To investigate this, we first tested an in vitro-synthesized 189 bp dsRNA matching a highly conserved region of the v-ATPaseA gene from cotton mealybug (Phenacoccus solenopsis) on three insect species from two different orders that use leaf-chewing, lacerate-and-flush, or sap-sucking mechanisms to feed, and showed that the dsRNA significantly down-regulated the target gene. We then developed transplastomic Micro-tom tomato plants to produce the dsRNA in plant plastids and showed that the dsRNA is produced in leaf, flower, green fruit, red fruit, and roots, with the highest dsRNA levels found in the leaf. The plastid-produced dsRNA induced a significant gene down-regulation in insects using leaf-chewing and lacerate-and-flush feeding mechanisms, while sap-sucking insects were unaffected. Our results suggest that plastid-produced dsRNA can be used to control leaf-chewing and lacerate-and-flush feeding insects, but may not be useful for sap-sucking insects.
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Kiselev, Konstantin V., Andrey R. Suprun, Olga A. Aleynova, Zlata V. Ogneva, Alexander V. Kalachev, and Alexandra S. Dubrovina. "External dsRNA Downregulates Anthocyanin Biosynthesis-Related Genes and Affects Anthocyanin Accumulation in Arabidopsis thaliana." International Journal of Molecular Sciences 22, no. 13 (June 23, 2021): 6749. http://dx.doi.org/10.3390/ijms22136749.
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Exogenous application of double-stranded RNAs (dsRNAs) and small-interfering RNAs (siRNAs) to plant surfaces has emerged as a promising method for regulation of essential genes in plant pathogens and for plant disease protection. Yet, regulation of plant endogenous genes via external RNA treatments has not been sufficiently investigated. In this study, we targeted the genes of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, and two transcriptional factors, MYBL2 and ANAC032, negatively regulating anthocyanin biosynthesis in Arabidopsis. Direct foliar application of AtCHS-specific dsRNAs and siRNAs resulted in an efficient downregulation of the AtCHS gene and suppressed anthocyanin accumulation in A. thaliana under anthocyanin biosynthesis-modulating conditions. Targeting the AtMYBL2 and AtANAC032 genes by foliar dsRNA treatments markedly reduced their mRNA levels and led to a pronounced upregulation of the AtCHS gene. The content of anthocyanins was increased after treatment with AtMYBL2-dsRNA. Laser scanning microscopy showed a passage of Cy3-labeled AtCHS-dsRNA into the A. thaliana leaf vessels, leaf parenchyma cells, and stomata, indicating the dsRNA uptake and spreading into leaf tissues and plant individual cells. Together, these data show that exogenous dsRNAs were capable of downregulating Arabidopsis genes and induced relevant biochemical changes, which may have applications in plant biotechnology and gene functional studies.
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Wang, Qiaoqiao, and Gordon G. Carmichael. "Effects of Length and Location on the Cellular Response to Double-Stranded RNA." Microbiology and Molecular Biology Reviews 68, no. 3 (September 1, 2004): 432–52. http://dx.doi.org/10.1128/mmbr.68.3.432-452.2004.
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SUMMARY Since double-stranded RNA (dsRNA) has not until recently generally been thought to be deliberately expressed in cells, it has commonly been assumed that the major source of cellular dsRNA is viral infections. In this view, the cellular responses to dsRNA would be natural and perhaps ancient antiviral responses. While the cell may certainly react to some dsRNAs as an antiviral response, this does not represent the only response or even, perhaps, the major one. A number of recent observations have pointed to the possibility that dsRNA molecules are not seen only as evidence of viral infection or recognized for degradation because they cannot be translated. In some instances they may also play important roles in normal cell growth and function. The purpose of this review is to outline our current understanding of the fate of dsRNA in cells, with a focus on the apparent fact that their fates and functions appear to depend critically not only on where in the cell dsRNA molecules are found, but also on how long they are and perhaps on how abundant they are.
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Fenner, Beau J., Winnie Goh, and Jimmy Kwang. "Sequestration and Protection of Double-Stranded RNA by the Betanodavirus B2 Protein." Journal of Virology 80, no. 14 (July 15, 2006): 6822–33. http://dx.doi.org/10.1128/jvi.00079-06.
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ABSTRACT Betanodavirus B2 belongs to a group of functionally related proteins from the sense-strand RNA virus family Nodaviridae that suppress cellular RNA interference. The B2 proteins of insect alphanodaviruses block RNA interference by binding to double-stranded RNA (dsRNA), thus preventing Dicer-mediated cleavage and the subsequent generation of short interfering RNAs. We show here that the fish betanodavirus B2 protein also binds dsRNA. Binding is sequence independent, and maximal binding occurs with dsRNA substrates greater than 20 bp in length. The binding of B2 to long dsRNA is sufficient to completely block Dicer cleavage of dsRNA in vitro. Protein-protein interaction studies indicated that B2 interacts with itself and with other dsRNA binding proteins, the interaction occurring through binding to shared dsRNA substrates. Induction of the dsRNA-dependent interferon response was not antagonized by B2, as the interferon-responsive Mx gene of permissive fish cells was induced by wild-type viral RNA1 but not by a B2 mutant. The induction of Mx instead relied solely on viral RNA1 accumulation, which is impaired in the B2 mutant. Hyperediting of virus dsRNA and site-specific editing of 5-HT2C mRNA were both antagonized by B2. RNA editing was not, however, observed in transfected wild-type or B2 mutant RNA1, suggesting that this pathway does not contribute to the RNA1 accumulation defect of the B2 mutant. We thus conclude that betanodavirus B2 is a dsRNA binding protein that sequesters and protects both long and short dsRNAs to protect betanodavirus from cellular RNA interference.
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Wytinck, Nick, Christopher L. Manchur, Vivian H. Li, Steve Whyard, and Mark F. Belmonte. "dsRNA Uptake in Plant Pests and Pathogens: Insights into RNAi-Based Insect and Fungal Control Technology." Plants 9, no. 12 (December 16, 2020): 1780. http://dx.doi.org/10.3390/plants9121780.
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Efforts to develop more environmentally friendly alternatives to traditional broad-spectrum pesticides in agriculture have recently turned to RNA interference (RNAi) technology. With the built-in, sequence-specific knockdown of gene targets following delivery of double-stranded RNA (dsRNA), RNAi offers the promise of controlling pests and pathogens without adversely affecting non-target species. Significant advances in the efficacy of this technology have been observed in a wide range of species, including many insect pests and fungal pathogens. Two different dsRNA application methods are being developed. First, host induced gene silencing (HIGS) harnesses dsRNA production through the thoughtful and precise engineering of transgenic plants and second, spray induced gene silencing (SIGS) that uses surface applications of a topically applied dsRNA molecule. Regardless of the dsRNA delivery method, one aspect that is critical to the success of RNAi is the ability of the target organism to internalize the dsRNA and take advantage of the host RNAi cellular machinery. The efficiency of dsRNA uptake mechanisms varies across species, and in some uptake is negligible, rendering them effectively resistant to this new generation of control technologies. If RNAi-based methods of control are to be used widely, it is critically important to understand the mechanisms underpinning dsRNA uptake. Understanding dsRNA uptake mechanisms will also provide insight into the design and formulation of dsRNAs for improved delivery and provide clues into the development of potential host resistance to these technologies.
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Liu, Shaoshuai, Maria Jose Ladera-Carmona, Minna M. Poranen, Aart J. E. van Bel, Karl-Heinz Kogel, and Jafargholi Imani. "Evaluation of dsRNA delivery methods for targeting macrophage migration inhibitory factor MIF in RNAi-based aphid control." Journal of Plant Diseases and Protection 128, no. 5 (May 3, 2021): 1201–12. http://dx.doi.org/10.1007/s41348-021-00464-9.
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AbstractMacrophage migration inhibitory factors (MIFs) are multifunctional proteins regulating major processes in mammals, including activation of innate immune responses. In invertebrates, MIF proteins participate in the modulation of host immune responses when secreted by parasitic organisms, such as aphids. In this study, we assessed the possibility to use MIF genes as targets for RNA interference (RNAi)-based control of the grain aphid Sitobion avenae (Sa) on barley (Hordeum vulgare). When nymphs were fed on artificial diet containing double-stranded (ds)RNAs (SaMIF-dsRNAs) that target sequences of the three MIF genes SaMIF1, SaMIF2 and SaMIF3, they showed higher mortality rates and these rates correlated with reduced MIF transcript levels as compared to the aphids feeding on artificial diet containing a control dsRNA (GFP-dsRNA). Comparison of different feeding strategies showed that nymphs’ survival was not altered when they fed from barley seedlings sprayed with naked SaMIF-dsRNAs, suggesting they did not effectively take up dsRNA from the sieve tubes of these plants. Furthermore, aphids’ survival was also not affected when the nymphs fed on leaves supplied with dsRNA via basal cut ends of barley leaves. Consistent with this finding, the use of sieve tube-specific YFP-labeled Arabidopsis reporter lines confirmed that fluorescent 21 nt dsRNACy3, when supplied via petioles or spraying, co-localized with xylem structures, but not with phloem tissue. Our results suggest that MIF genes are a potential target for insect control and also imply that application of naked dsRNA to plants for aphid control is inefficient. More efforts should be put into the development of effective dsRNA formulations.
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Arora, Arinder K., Seung Ho Chung, and Angela E. Douglas. "Non-Target Effects of dsRNA Molecules in Hemipteran Insects." Genes 12, no. 3 (March 12, 2021): 407. http://dx.doi.org/10.3390/genes12030407.
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Insect pest control by RNA interference (RNAi)-mediated gene expression knockdown can be undermined by many factors, including small sequence differences between double-stranded RNA (dsRNA) and the target gene. It can also be compromised by effects that are independent of the dsRNA sequence on non-target organisms (known as sequence-non-specific effects). This study investigated the species-specificity of RNAi in plant sap-feeding hemipteran pests. We first demonstrated sequence-non-specific suppression of aphid feeding by dsRNA at dietary concentrations ≥0.5 µg µL−1. Then we quantified the expression of NUC (nuclease) genes in insects administered homologous dsRNA (with perfect sequence identity to the target species) or heterologous dsRNA (generated against a related gene of non-identical sequence in a different insect species). For the aphids Acyrthosiphon pisum and Myzus persicae, significantly reduced NUC expression was obtained with the homologous but not heterologous dsRNA at 0.2 µg µL−1, despite high dsNUC sequence identity. Follow-up experiments demonstrated significantly reduced expression of NUC genes in the whitefly Bemisia tabaci and mealybug Planococcus maritimus administered homologous dsNUCs, but not heterologous aphid dsNUCs. Our demonstration of inefficient expression knockdown by heterologous dsRNA in these insects suggests that maximal dsRNA sequence identity is required for RNAi targeting of related pest species, and that heterologous dsRNAs at appropriate concentrations may not be a major risk to non-target sap-feeding hemipterans.
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Necira, Khouloud, Mongia Makki, Eugenio Sanz-García, Tomás Canto, Fattouma Djilani-Khouadja, and Francisco Tenllado. "Topical Application of Escherichia coli-Encapsulated dsRNA Induces Resistance in Nicotiana benthamiana to Potato Viruses and Involves RDR6 and Combined Activities of DCL2 and DCL4." Plants 10, no. 4 (March 29, 2021): 644. http://dx.doi.org/10.3390/plants10040644.
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Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants represents an attractive alternative to transgene-based silencing approaches. However, improvement of dsRNA stability in natural conditions is required in order to provide long-term protection against the targeted virus. Here, we tested the protective effect of topical application of Escherichia coli-encapsulated dsRNA compared to naked dsRNA against single and dual infection by Potato virus X expressing the green fluorescent protein (PVX-GFP) and Potato virus Y (PVY) in Nicotiana benthamiana. We found that, in our conditions, the effectiveness of E. coli-encapsulated dsRNA in providing RNAi-mediated protection did not differ from that of naked dsRNA. dsRNA vaccination was partly effective against a dual infection by PVX-GFP and PVY, manifested by a delay in the expression of the synergistic symptoms at early times after inoculation. Using PVX-GFP as a reporter virus together with a suite of RNAi knockdown transgenic lines, we have also shown that RNA-directed RNA polymerase 6 and the combined activities of DICER-like 2 (DCL2) and DCL4 act to promote efficient resistance to virus infection conferred by topical application of dsRNA in N. benthamiana. Our results provide evidence that exogenous dsRNA molecules are processed by the RNA silencing pathways commonly used by the host in response to virus infection.
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Guo, Peixuan, and Tae Jin Lee. "Viral nanomotors for packaging of dsDNA and dsRNA." Molecular Microbiology 64, no. 4 (May 2007): 886–903. http://dx.doi.org/10.1111/j.1365-2958.2007.05706.x.
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Brown, B. A., and A. Rich. "The left-handed double helical nucleic acids." Acta Biochimica Polonica 48, no. 2 (June 30, 2001): 295–312. http://dx.doi.org/10.18388/abp.2001_3916.
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The conversion of right-handed dsDNA and dsRNA to the left-handed Z-conformation involves a reorganization of the nucleotides relative to each other. This conversion can be facilitated by the tight binding of a Z-conformation-specific protein domain from the editing enzyme dsRNA adenosine deaminase. This may influence the modification of both pre-mRNAs as well as some replicating RNA viruses.
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Tretiakova, Polina Ya, and Aleksandr A. Soloviev. "Application of small RNAs for plant protection." Ecological genetics 18, no. 4 (December 12, 2020): 467–82. http://dx.doi.org/10.17816/ecogen35203.
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Double-stranded small RNAs (dsRNA) perform various regulatory functions via RNA-interference. Additionally, they can be transported between various plant species and their pathogens and pests via extracellular vesicles, protecting RNA from nucleases. Plants secrete short dsRNA molecules to defend themselves against pathogens. The latter also use small RNAs when infecting crops. Some dsRNAs of pathogens are known as ribonucleic effectors. Host-induced gene silencing (HIGS) was shown to be effective when breeding resistant varieties and analyzing plant-pathogen interactions. However, complexity of transgenesis and society fear of genetically modified products make HIGS application difficult. The appearance of a new strategy based on plant spraying with dsRNA gave a new perspective of plant protection. Currently such a strategy requires accurate studying as well as the development of efficient systems stably producing high-quality dsRNA.
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Chu, Yeon-Mee, Jae-Jin Jeon, Sang-Jin Yea, Yong-Ho Kim, Sung-Hwan Yun, Yin-Won Lee, and Kook-Hyung Kim. "Double-Stranded RNA Mycovirus from Fusarium graminearum." Applied and Environmental Microbiology 68, no. 5 (May 2002): 2529–34. http://dx.doi.org/10.1128/aem.68.5.2529-2534.2002.
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ABSTRACT Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.