Relevant bibliographies by topics / DsRNase

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  1. Journal articles
  2. Dissertations / Theses
  3. Books
  4. Book chapters
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Academic literature on the topic 'DsRNase'

Author: Grafiati
Published: 11 March 2023

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Journal articles on the topic "DsRNase"

1

Giesbrecht, David, Daniel Heschuk, Ian Wiens, David Boguski, Parker LaChance, and Steve Whyard. "RNA Interference Is Enhanced by Knockdown of Double-Stranded RNases in the Yellow Fever Mosquito Aedes aegypti." Insects 11, no. 6 (May 27, 2020): 327. http://dx.doi.org/10.3390/insects11060327.

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RNA interference (RNAi) techniques are being developed for a range of pest insect control technologies, including the sterile insect technique (SIT) and double-stranded RNA (dsRNA)-based insecticides. In SIT applications, where >99% of the released males should be sterile to meet industry standards, the efficiency of RNAi will need to be improved for many insect species if this technology is to be adopted. Endogenous dsRNases can impede dsRNA delivery in some insects, and, here, we investigated whether dsRNases in the midgut could limit RNAi efficacy in the mosquito Aedes aegypti. Ten putative dsRNases were identified in the Ae. aegypti genome, with two highly expressed in the midguts of larvae. Using an ex vivo assay, we observed that dsRNA was rapidly degraded within the mosquito larva’s gut. Double-stranded RNA targeting these two dsRNases, when fed to the larvae, effectively reduced gut dsRNase activity. When these dsRNase-specific dsRNAs were co-delivered with dsRNA targeting a cyan fluorescent protein (CFP) reporter gene, greater knockdown of CFP fluorescence was observed. These results suggest that inhibiting dsRNase activity could enable the implementation of RNAi-based mosquito control methods.
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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.

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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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Li, Jiajing, Juan Du, Shangwei Li, and Xin Wang. "Identification and Characterization of a Double-Stranded RNA Degrading Nuclease Influencing RNAi Efficiency in the Rice Leaf Folder Cnaphalocrocis medinalis." International Journal of Molecular Sciences 23, no. 7 (April 2, 2022): 3961. http://dx.doi.org/10.3390/ijms23073961.

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Rice leaf folder Cnaphalocrocis medinalis is one of the most serious pests of rice in rice-planting regions worldwide. DsRNA-degrading nucleases (dsRNases) are important factors in reducing the efficiency of RNA interference (RNAi) in different insects. In this study, a dsRNase gene from C. medinalis (CmdsRNase) was cloned and characterized. The CmdsRNase cDNA was 1395 bp in length, encoding 464 amino acids. The CmdsRNase zymoprotein contains a signal peptide and an endonuclease NS domain that comprises six active sites, three substrate-binding sites, and one Mg2+-binding site. The mature CmdsRNase forms a homodimer with a total of 16 α-helices and 20 β-pleated sheets. Homology and phylogenetic analyses revealed that CmdsRNase is closely related to dsRNase2 in Ostrinia nubilalis. Expression pattern analysis by droplet digital PCR indicated that the expression levels of CmdsRNase varied throughout the developmental stages of C. medinalis and in different adult tissues, with the highest expression levels in the fourth-instar larvae and the hemolymph. CmdsRNase can degrade dsRNA to reduce the efficiency of RNAi in C. medinalis. Co-silencing of CmCHS (chitin synthase from C. medinalis) and CmdsRNase affected significantly the growth and development of C. medinalis and thus improved RNAi efficacy, which increased by 27.17%. These findings will be helpful for green control of C. medinalis and other lepidopteran pests by RNAi.
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Sharma, Rohit, Clauvis Nji Tizi Taning, Guy Smagghe, and Olivier Christiaens. "Silencing of Double-Stranded Ribonuclease Improves Oral RNAi Efficacy in Southern Green Stinkbug Nezara viridula." Insects 12, no. 2 (January 28, 2021): 115. http://dx.doi.org/10.3390/insects12020115.

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Variability in RNA-interference (RNAi) efficacy among different insect orders poses a big hurdle in the development of RNAi-based pest control strategies. The activity of double-stranded ribonucleases (dsRNases) in the digestive canal of insects can be one of the critical factors affecting oral RNAi efficacy. Here, the involvement of these dsRNases in the southern green stinkbug Nezara viridula was investigated. First, the full sequence of the only dsRNase (NvdsRNase) in the transcriptome of N. viridula was obtained, followed by an oral feeding bioassay to evaluate the effect of NvdsRNase-silencing on oral RNAi efficacy. The NvdsRNase was first silenced in nymphs by NvdsRNase-dsRNA injections, followed by exposure to an artificial diet containing a lethal αCop-specific dsRNA. A significantly higher mortality was observed in the NvdsRNase-silenced nymphs when placed on the dsαCop-containing diet (65%) than in the dsGFP injected and dsαCop fed control (46.67%). Additionally, an ex vivo dsRNA degradation assay showed a higher stability of dsRNA in the saliva and midgut juice of NvdsRNase-silenced adults. These results provide evidence for the involvement of NvdsRNase in the reduction of oral RNAi efficacy in N. viridula. This information will be useful in further improving potential RNAi-based strategies to control this pest.
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Zhang, Xun, Zhizhi Fan, Qinghua Wang, Xiangbo Kong, Fu Liu, Jiaxing Fang, Sufang Zhang, and Zhen Zhang. "RNAi Efficiency through dsRNA Injection Is Enhanced by Knockdown of dsRNA Nucleases in the Fall Webworm, Hyphantria cunea (Lepidoptera: Arctiidae)." International Journal of Molecular Sciences 23, no. 11 (May 31, 2022): 6182. http://dx.doi.org/10.3390/ijms23116182.

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RNA interference (RNAi) technology is a promising approach used in pest control. The efficiency of RNAi varies considerably among different insect species, and growing evidence suggests that degradation of double-stranded RNA (dsRNA) prior to uptake is an important factor that limits RNAi efficiency in insects. Our recent work on fall webworm (Hyphantria cunea), an important invasive pest in China, showed a relatively low silencing efficiency of RNAi through dsRNA injection, which is considered the most feasible dsRNA delivery method for inducing RNAi, and the factors involved in the mechanism remain unknown. Herein, we first detected the dsRNA-degrading activity in the hemolymph and gut content of H. cunea in ex vivo assays and observed rapid degradation of dsRNA, especially in the hemolymph, which was complete within only 10 min. To determine whether dsRNA degradation could contribute to the low effectiveness of RNAi in H. cunea, four dsRNA nuclease (dsRNase) genes, HcdsRNase1, HcdsRNase2, HcdsRNase3, and HcdsRNase4, were identified by homology searching against the H. cunea transcriptome database, and their transcript levels were subsequently investigated in different tissues, developmental stages, and after dsRNA injection. Our results show that HcdsRNases are highly expressed mainly in gut tissues and hemolymph, and the expression of HcdsRNase3 and HcdsRNase4 were significantly upregulated by dsGFP induction. RNAi-of-RNAi studies, using HcCht5 as a reporter gene, demonstrated that silencing HcdsRNase3 and HcdsRNase4 significantly increases RNAi efficacy via dsHcCht5 injection, and co-silencing these two HcdsRNase genes results in a more significant improvement in efficacy. These results confirm that the RNAi efficacy in H. cunea through dsRNA injection is certainly impaired by dsRNase activity, and that blocking HcdsRNases could potentially improve RNAi, providing a reference for related studies on insects where RNAi has low efficiency.
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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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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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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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Krishnan, Niranjana, Maura J. Hall, Richard L. Hellmich, Joel R. Coats, and Steven P. Bradbury. "Evaluating toxicity of Varroa mite (Varroa destructor)-active dsRNA to monarch butterfly (Danaus plexippus) larvae." PLOS ONE 16, no. 6 (June 2, 2021): e0251884. http://dx.doi.org/10.1371/journal.pone.0251884.

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Varroa mites (Varroa destructor) are parasitic mites that, combined with other factors, are contributing to high levels of honey bee (Apis mellifera) colony losses. A Varroa-active dsRNA was recently developed to control Varroa mites within honey bee brood cells. This dsRNA has 372 base pairs that are homologous to a sequence region within the Varroa mite calmodulin gene (cam). The Varroa-active dsRNA also shares a 21-base pair match with monarch butterfly (Danaus plexippus) calmodulin mRNA, raising the possibility of non-target effects if there is environmental exposure. We chronically exposed the entire monarch larval stage to common (Asclepias syriaca) and tropical (Asclepias curassavica) milkweed leaves treated with concentrations of Varroa-active dsRNA that are one- and ten-fold higher than those used to treat honey bee hives. This corresponded to concentrations of 0.025–0.041 and 0.211–0.282 mg/g leaf, respectively. Potassium arsenate and a previously designed monarch-active dsRNA with a 100% base pair match to the monarch v-ATPase A mRNA (leaf concentration was 0.020–0.034 mg/g) were used as positive controls. The Varroa mite and monarch-active dsRNA’s did not cause significant differences in larval mortality, larval or pupal development, pupal weights, or adult eclosion rates when compared to negative controls. Irrespective of control or dsRNA treatment, larvae that consumed approximately 7500 to 10,500-mg milkweed leaf within 10 to 12 days had the highest pupal weights. The lack of mortality and sublethal effects following dietary exposure to dsRNA with 21-base pair and 100% base pair match to mRNAs that correspond to regulatory genes suggest monarch mRNA may be refractory to silencing by dsRNA or monarch dsRNase may degrade dsRNA to a concentration that is insufficient to silence mRNA signaling.
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Li, Danhua, Fangfang Guo, Hongfang Yue, Yaqi Huang, Chenchen Lu, Yubai Guo, Qinghua Liu, and Yanqiang Li. "An Artificial Small RNA Editor by Chimeric dsRNase with RNA Binding Protein." Journal of Biomedical Nanotechnology 18, no. 5 (May 1, 2022): 1349–61. http://dx.doi.org/10.1166/jbn.2022.3333.

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RNA plays a vital role in cell functions, but tools to manipulate it is limited. RNA interference (RNAi) is an important approach for biological and clinical applications, but the prone of non-target knockdown effects limited the usage. CRISPR-Cas13 systems recently have been identified for RNA-guided RNA-interfering activity, and can be used in therapeutics, but the large size of Cas13 proteins and the off-targets effect also limit their further usage. Here we report that the chimeric protein containing a double strand nuclease/domain and a structure RNA binding domain (dsRNase-stRBD) with structure guided RNA (sgRNA) can be engineered for mammalian RNA silencing effectively. The RNA knockdown mediated by this method was durable, efficient and stringent without off-target interfering by the sense strand of shRNA base method. Moreover, at size of only 307 aa, allowing dsRNase-stRBD fitting for the versatile scAAV, while the most recent report displays that the smallest Cas13 protein is 775 aa. These results establish sgRNA-dsRBD-RNase as an excellent method for studying RNA function of cells and further clinical application.
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Dissertations / Theses on the topic "DsRNase"

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Souza, Sandra Maria de. "Avaliação do silenciamento gênico em plantas utilizando estratégias baseadas em dsRNAs." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2006. http://hdl.handle.net/10183/165640.

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O silenciamento gênico tem sido utilizado extensivamente para facilitar a investigação de eventos da biologia vegetal. Ele pode ser induzido de diferentes modos, sendo que o passo chave para sua ocorrência é a presença de RNA fita dupla (dsRNA). A fim de avaliar um método rápido de obtenção de silenciamento para análise funcional em larga escala, foi inoculado dsRNA ou siRNA diretamente em folhas de plantas de arroz e de Nicotiana benthamiana. Os dsRNAs de seqüências do gene pds e do gene codificante da ferritina foram obtidos através da síntese in vivo e in vitro e o siRNA de PDS de arroz, derivado somente de síntese in vitro. O gene pds codifica a enzima fitoeno desaturase (PDS), uma enzima chave na biossíntese de carotenóides, que protege a clorofila das plantas contra o foto-branqueamento. O silenciamento deste gene mostra um fenótipo visível, claramente demonstrado através do uso de um inibidor químico da síntese desta enzima. No presente estudo, plantas de arroz e de N. benthamiana inoculadas com dsRNAs derivados do gene pds não apresentaram mudanças fenotípicas. Para avaliação de um outro método foi adaptado um vetor de silenciamento estável, no qual foram inseridos fragmentos de cDNA nas orientações senso e antisenso do gene de ferritina de arroz. O papel da ferritina, segundo experimentação in vitro, pode estar ligada à tolerância de certos cultivares de arroz às altas concentrações de ferro. O aprimoramento das metodologias de silenciamento e de transformação poderá resultar em uma ferramenta mais útil para a análise funcional em larga escala de genes de arroz.
Gene silencing has been used extensively to facilitate the investigation of different events in plant biology. It can be induced through different ways. The key step for its occurrence is the presence of double strand RNA (dsRNA). In order to evaluate a fast method to obtain silencing for functional analysis in a wide scale, either dsRNAs or siRNAs were directly inoculated in the leaves of rice and N. benthamiana plants. The dsRNAs the sequences of the pds gene and of the coding region of the ferritin gene were obtained through in vivo or in vitro synthesis, the siRNA PDS of rice were derived from synthesis in vitro. The pds gene codifies for the enzyme phytoene desaturase (PDS), a key enzyme in the biosynthesis of carotenoids, which protects the clorophil from the plants against photo-bleaching. The silencing of this gene shows a visible phenotype, clearly demonstrated through a chemical inhibition of PDS synthesis. However, no phenotypical changes were observed in either rice or N. benthamiana plants inoculated with dsRNA of the pds gene. In the present study, it was also adapted a vector for stable transformation, in which were inserted cDNA fragments of both, sense and antisense, orientations of the rice ferritin gene. In vitro experiments show that ferritin could be connected to the tolerance to high concentrations of iron of certain cultivars of rice. The improvement of silencing and transformation methodologies could result in a more useful tool for the functional analysis of rice genes in a wide scale.
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Cole, Thomas Edwin. "Molecular studies of virus-like dsRNAs in a diseased isolate of Ophiostoma novo-ulmi." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327031.

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Vyas, Meenal, Amir Raza, Muhammad Yousaf Ali, Muhammad Aleem Ashraf, Shahid Mansoor, Ahmad Ali Shahid, and Judith K. Brown. "Knock down of Whitefly Gut Gene Expression and Mortality by Orally Delivered Gut Gene-Specific dsRNAs." PUBLIC LIBRARY SCIENCE, 2017. http://hdl.handle.net/10150/622632.

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Control of the whitefly Bemisia tabaci (Genn.) agricultural pest and plant virus vector relies on the use of chemical insecticides. RNA-interference (RNAi) is a homology-dependent innate immune response in eukaryotes, including insects, which results in degradation of the corresponding transcript following its recognition by a double-stranded RNA (dsRNA) that shares 100% sequence homology. In this study, six whitefly `gut' genes were selected from an in silico-annotated transcriptome library constructed from the whitefly alimentary canal or 'gut' of the B biotype of B. tabaci, and tested for knock down efficacy, post-ingestion of dsRNAs that share 100% sequence homology to each respective gene target. Candidate genes were: Acetylcholine receptor subunit a, Alpha glucosidase 1, Aquaporin 1, Heat shock protein 70, Trehalasel, and Trehalose transported. The efficacy of RNAi knock down was further tested in a gene-specific functional bioassay, and mortality was recorded in 24 hr intervals, six days, post-treatment. Based on qPCR analysis, all six genes tested showed significantly reduced gene expression. Moderate-to-high whitefly mortality was associated with the down-regulation of osmoregulation, sugar metabolism and sugar transport -associated genes, demonstrating that whitefly survivability was linked with RNAi results. Silenced Acetylcholine receptor subunit a and Heat shock protein 70 genes showed an initial low whitefly mortality, however, following insecticide or high temperature treatments, respectively, significantly increased knockdown efficacy and death was observed, indicating enhanced post-knockdown sensitivity perhaps related to systemic silencing. The oral delivery of gut-specific dsRNAs, when combined with qPCR analysis of gene expression and a corresponding gene-specific bioassay that relates knockdown and mortality, offers a viable approach for functional genomics analysis and the discovery of prospective dsRNA biopesticide targets. The approach can be applied to functional genomics analyses to facilitate, species-specific dsRNA-mediated control of other non-model hemipterans.
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Reddy, Vidita. "Role of dsRNA-induced DRAK1 in Apoptosis." University of Toledo / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1513349817056948.

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Lu, Lenette L. "dsRNA Signaling in Innate Immunity and Viral Inhibition." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1220030971.

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Muccioli, Maria. "Characterizing dsRNA-induced inflammation in ovarian cancer cells." Ohio University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1405707670.

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Kilic, Ozlem III. "Effect of dsRNA-containing and dsRNA-free hypovirulent isolates of Fusarium oxysporum on severity of Fusarium seedling disease of Essex soybean." Thesis, Virginia Tech, 1997. http://hdl.handle.net/10919/36965.

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Sixty-six isolates of F. oxysporum and F. solani were recovered from healthy and necrotic Essex soybean seedlings grown in naturally infested soil. These were tested for pathogenicity at 20 C and -0.01 MPa water potential in artificially infested, autoclaved field soil. Highly pathogenic, moderately pathogenic, and hypovirulent isolates of both species were identified. Fifty-seven F. oxysporum and nine F. solani isolates were tested for the presence of dsRNA. The presence of dsRNA was not associated with hypovirulence in F. oxysporum since some hypovirulent isolates contained dsRNA while other hypovirulent isolates did not. Furthermore, of six dsRNA-containing F. oxysporum isolates, three were hypovirulent, two were moderately pathogenic, and one isolate was highly pathogenic. Four segments of dsRNA, with sizes of 4.0, 3.1, 2.7, and 2.2 kb, were detected in extracts of all six F. oxysporum isolates. No morphological differences were found between dsRNA-containing and dsRNA-free F. oxysporum isolates. Attempts to cure dsRNA-containing hypovirulent F. oxysporum isolates, either by single-sporing of isolates or by using a range of concentrations of cycloheximide, were not successful. No dsRNA was found in any of the F. solani isolates tested. Pythium ultimum, an associate in Essex seedling disease, was isolated from water-soaked lesions and interfered with evaluations of disease caused by the Fusarium spp. Metalaxyl was used to control P. ultimum and had no apparent effect on symptoms associated with F. oxysporum and F. solani in field soil. Prior inoculation of Essex soybean seeds with conidia of dsRNA-free hypovirulent F. oxysporum isolates, plus metalaxyl seed treatment, significantly (p<0.05) reduced disease severity on both cotyledons and hypocotyls and increased the rate of seedling emergence in field soil, compared to the control plants treated with metalaxyl alone or not treated with metalaxyl. No significant (p>0.05) differences were found between dsRNA-containing and dsRNA-free hypovirulent F. oxysporum isolates in their effects on the reduction of disease severity. A mixture of two hypovirulent F. oxysporum isolates was significantly (p<0.05) more effective than single hypovirulent F. oxysporum isolates in increasing the rate of seedling emergence. Symptoms associated with P. ultimum were not affected by the prior inoculation of seeds with individual hypovirulent F. oxysporum isolates.
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Rodrigues, Paula. "Produção e caracterização de um antissoro policlonal para detecção de ds-RNA." Bachelor's thesis, UTAD, 1998. http://hdl.handle.net/10198/997.

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Os fitopatologistas têm feito um uso cada vez maior dos métodos serológicos na detecção e caracterização de fitopatogénios, por se tratar de técnicas rápidas, práticas e de elevada sensibilidade, que se podem adaptar às necessidades. De entre estes testes, as várias adaptações do ELISA (enzyme-linked immunosorbent assay) são, actualmente, os métodos mais divulgados, uma vez que permitem testar um elevado número de amostras num curto espaço de tempo e a preço moderado. A maioria dos vírus fitopatogénicos tem genoma de ss-RNA (ácido ribonucleico monocatenário) que, durante o processo replicativo, no interior das células do hospedeiro, dá origem a uma forma replicativa de ds-RNA (ácido ribonucleico bicatenário). Considerando que as plantas não infectadas não contêm quantidades detectáveis de ds-RNA, a sua presença em extractos vegetais é uma forte indicação de infecção viral. O presente trabalho desenvolveu-se no sentido de produzir um antissoro policlonal para um polinucleótido sintético bicatenário [poli(I):poli(C)] para detecção de ds-RNA. O antissoro foi caracterizado através de várias técnicas serológicas (ELISA-indirecto em placa de poliestireno, ELISA-indirecto em membrana de nitrocelulose e teste de difusão dupla em agar). O teste ELISA-indirecto em placa revelou ser mais sensível e prático do que o respectivo teste em membrana de nitrocelulose, tanto na detecção de poli(I):poli(C) como de ds-RNA purificado. Ambos se mostraram, no entanto, incapazes de detectar ds-RNA a partir de extractos aquosos de videira, o que dificulta o processo de detecção, uma vez que a extracção de ds-RNA de material vegetal é morosa e de baixo rendimento.
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Potgieter, Abraham Christiaan. "Cloning viral dsRNA genomes : analysis and application / A.C. Potgieter." Thesis, North-West University, 2004. http://hdl.handle.net/10394/334.

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Double-stranded RNA viruses occur in a large number of hosts in nature ranging from bacteria to mammals. Molecular studies of the double-stranded RNA viruses have greatly enhanced man's understanding of this large group of viruses as far as structure and function of their genes and epidemiology is concerned. However, one of the major prerequisites of obtaining this information is the ability to clone the genomes of these viruses for nucleotide sequencing and recombinant protein expression studies. In the dsRNA field, cloning viral genomes has historically been difficult and time consuming and created a bottleneck that hampered molecular studies. The main aim of this investigation was to optimise a method for cloning viral dsRNA genomes to the extent that it would be easy and fast as well as applicable to most dsRNA viruses. In this study a sequence-independent, oligo-ligation mediated dsRNA cloning procedure for large genes (up to 6.8 kb) was perfected and tailored for routine use to amplify and clone complete genome sets or individual genes. Complete genome sets could be amplified and cloned from as little as 1 ng dsRNA. The method was shown to be simple and efficient compared to other methods and is currently the only method that allows the amplification of complete genomes in a single PCR reaction. Complete gene sets of seven genomes from the Reovirus family, one from the Cystovirus family and one mycovirus, have been amplified and cloned. The full-length VP2 genes of all 9 AHSV and 24 BTV serotypes were also cloned. Phylogenetic analysis of VP2-genes revealed the same grouping of AHSVs and BTVs as serology. Several cloned genes of AHSV, rotavirus and EEV have been utilised for recombinant protein production establishing that the cloned cDNAs have full open reading frames. The nine AHSV VP2 genes have been developed as serotype-specific probes which allowed serotyping of AHSV isolates within 4 days compared to 2-4 weeks needed with the traditional serological serotyping. The new cloning procedure finally opens the bottleneck that hamstrung the development of complete repertoires of recombinant vaccines, molecular diagnostics and epidemiology to combat dsRNA viral diseases. It should now be possible to deliver on many of the expectations that were envisaged for dsRNA virus research and biotechnology since the advent of recombinant DNA technology.
Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2004.
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Robinson, Helen Lynne. "Characterization of double-stranded RNA (dsRNA) from Rhizoctonia solani." Thesis, University of Edinburgh, 1999. http://webex.lib.ed.ac.uk/abstracts/robins01.pdf.

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Books on the topic "DsRNase"

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Michael, Tavantzis Stylianos, ed. dsRNA genetic elements: Concepts and applications in agriculture, forestry, and medicine. Boca Raton, FL: CRC Press, 2002.

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Tavantzis, Stellos M. Dsrna Genetic Elements: Concepts and Applications in Agriculture, Forestry, and Medicine. Taylor & Francis Group, 2002.

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Tavantzis, Stellos M. DsRNA Genetic Elements: Concepts and Applications in Agriculture, Forestry, and Medicine. Taylor & Francis Group, 2001.

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McEwan, Deborah Lyn. Mechanisms of intercellular dsRNA transport by SID-1 and SID-2. 2010.

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Tavantzis, Stellos M. dsRNA Genetic Elements: Concepts and Applications in Agriculture, Forestry, and Medicine. CRC, 2001.

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Tavantzis, Stellos M. DsRNA Genetic Elements: Concepts and Applications in Agriculture, Forestry, and Medicine. Taylor & Francis Group, 2001.

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Chong, Karen Lynn. Characterization of the human, interferon inducible, dsRNA-activated protein kinase (p68 kinase). 1993.

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Book chapters on the topic "DsRNase"

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de Schutter, Kristof, Olivier Christiaens, Clauvis Nji Tizi Taning, and Guy Smagghe. "Boosting dsRNA delivery in plant and insect cells with peptide- and polymer-based carriers: case-based current status and future perspectives." In RNAi for plant improvement and protection, 102–16. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781789248890.0011.

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Abstract Since the discovery of this naturally occurring endogenous regulatory and defence mechanism, RNA interference (RNAi) has been exploited as a powerful tool for functional genomic research. In addition, it has evolved as a promising candidate for a sustainable, specific and ecofriendly strategy for pest management and plant improvement. A key element in this technology is the efficient delivery of dsRNAs into the pest or plant tissues. While several examples using transgenic plants expressing the dsRNAs have proved the potential of this technology, nontransgenic approaches are investigated as alternatives, allowing flexibility and circumventing technical limitations of the transgenic approach. However, the efficacy of environmental RNAi is affected by several barriers, such as extracellular degradation of the dsRNA, inefficient internalization of the dsRNA in the cell and low endosomal escape into the cytoplasm, resulting in variable or low RNAi responses. In the medical field, carrier systems are commonly used to enhance RNA delivery and these systems are being rapidly adopted by the agricultural industry. Using four case studies, this chapter demonstrates the potential of carriers to improve the RNAi response in pest control for aquatic-living mosquito larvae and RNAi-resilient Lepidoptera and to cross the plant cell wall, allowing efficient environmental RNAi in plants.
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de Schutter, Kristof, Olivier Christiaens, Clauvis Nji Tizi Taning, and Guy Smagghe. "Boosting dsRNA delivery in plant and insect cells with peptide- and polymer-based carriers: case-based current status and future perspectives." In RNAi for plant improvement and protection, 102–16. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781789248890.0102.

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Abstract Since the discovery of this naturally occurring endogenous regulatory and defence mechanism, RNA interference (RNAi) has been exploited as a powerful tool for functional genomic research. In addition, it has evolved as a promising candidate for a sustainable, specific and ecofriendly strategy for pest management and plant improvement. A key element in this technology is the efficient delivery of dsRNAs into the pest or plant tissues. While several examples using transgenic plants expressing the dsRNAs have proved the potential of this technology, nontransgenic approaches are investigated as alternatives, allowing flexibility and circumventing technical limitations of the transgenic approach. However, the efficacy of environmental RNAi is affected by several barriers, such as extracellular degradation of the dsRNA, inefficient internalization of the dsRNA in the cell and low endosomal escape into the cytoplasm, resulting in variable or low RNAi responses. In the medical field, carrier systems are commonly used to enhance RNA delivery and these systems are being rapidly adopted by the agricultural industry. Using four case studies, this chapter demonstrates the potential of carriers to improve the RNAi response in pest control for aquatic-living mosquito larvae and RNAi-resilient Lepidoptera and to cross the plant cell wall, allowing efficient environmental RNAi in plants.
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Kontogiannatos, Dimitrios, Anna Kolliopoulou, and Luc Swevers. "The 'Trojan horse' approach for successful RNA interference in insects." In RNAi for plant improvement and protection, 25–39. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781789248890.0004a.

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Abstract Since the discovery of RNA interference in 1998 as a potent molecular tool for the selective downregulation of gene expression in almost all eukaryotes, increasing research is being performed in order to discover applications that are useful for the pharmaceutical and chemical industry. The ease of use of double-stranded RNA for targeted in vivo gene silencing in animal cells and tissues gave birth to a massive interest from industry in order to discover biotechnological applications for human health and plant protection. For insects, RNAi became the 'Holy Grail' of pesticide manufacturing, because this technology is a promising species-specific environmentally friendly approach to killing natural enemies of cultured plants and farmed animals. The general idea to use RNAi as a pest-control agent originated with the realization that dsRNAs that target developmentally or physiologically important insect genes can cause lethal phenotypes as a result of the specific gene downregulation. Most importantly to achieve this, dsRNA is not required to be constitutively expressed via a transgene in the targeted insect but it can be administrated orally after direct spraying on the infested plants. Similarly, dsRNAs can be administered to pests after constitutive expression as a hairpin in plants or bacteria via stable transgenesis. Ideally, this technology could have already been applied in integrated pest management (IPM) if improvements were not essential in order to achieve higher insecticidal effects. There are many limitations that decrease RNAi efficiency in insects, which arise from the biochemical nature of the insect gut as well as from deficiencies in the RNAi core machinery, a common phenomenon mostly observed in lepidopteran species. To overcome these obstacles, new technologies should be assessed to ascertain that the dsRNA will be transferred intact, stable and in high amounts to the targeted insect cells. In this chapter we will review a wide range of recent discoveries that address the delivery issues of dsRNAs in insect cells, with a focus on the most prominent and efficient technologies. We will also review the upcoming and novel use of viral molecular components for the successful and efficient delivery of dsRNA to the insect cell.
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Kontogiannatos, Dimitrios, Anna Kolliopoulou, and Luc Swevers. "The 'Trojan horse' approach for successful RNA interference in insects." In RNAi for plant improvement and protection, 25–39. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781789248890.0025.

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Abstract Since the discovery of RNA interference in 1998 as a potent molecular tool for the selective downregulation of gene expression in almost all eukaryotes, increasing research is being performed in order to discover applications that are useful for the pharmaceutical and chemical industry. The ease of use of double-stranded RNA for targeted in vivo gene silencing in animal cells and tissues gave birth to a massive interest from industry in order to discover biotechnological applications for human health and plant protection. For insects, RNAi became the 'Holy Grail' of pesticide manufacturing, because this technology is a promising species-specific environmentally friendly approach to killing natural enemies of cultured plants and farmed animals. The general idea to use RNAi as a pest-control agent originated with the realization that dsRNAs that target developmentally or physiologically important insect genes can cause lethal phenotypes as a result of the specific gene downregulation. Most importantly to achieve this, dsRNA is not required to be constitutively expressed via a transgene in the targeted insect but it can be administrated orally after direct spraying on the infested plants. Similarly, dsRNAs can be administered to pests after constitutive expression as a hairpin in plants or bacteria via stable transgenesis. Ideally, this technology could have already been applied in integrated pest management (IPM) if improvements were not essential in order to achieve higher insecticidal effects. There are many limitations that decrease RNAi efficiency in insects, which arise from the biochemical nature of the insect gut as well as from deficiencies in the RNAi core machinery, a common phenomenon mostly observed in lepidopteran species. To overcome these obstacles, new technologies should be assessed to ascertain that the dsRNA will be transferred intact, stable and in high amounts to the targeted insect cells. In this chapter we will review a wide range of recent discoveries that address the delivery issues of dsRNAs in insect cells, with a focus on the most prominent and efficient technologies. We will also review the upcoming and novel use of viral molecular components for the successful and efficient delivery of dsRNA to the insect cell.
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Gries, Oliver, and Thomas Ly. "Reoviridae [dsRNA]." In Infektologie - Kompendium humanpathogener Infektionskrankheiten und Erreger, 191–93. Berlin, Heidelberg: Springer Berlin Heidelberg, 2019. http://dx.doi.org/10.1007/978-3-662-58219-0_24.

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Pantchev, Ivelin, Goritsa Rakleova, and Atanas Atanassov. "The stability of dsRNA during external applications - an overview." In RNAi for plant improvement and protection, 94–101. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781789248890.0010.

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Abstract The research community is deeply convinced that RNA is unstable in the environment. Its roots rise from numerous failed attempts to isolate functional cellular RNA molecules. Further support had originated from the fast turnover of RNA in the cells. The situation changed recently with the discovery that externally applied dsRNA can produce targeted gene silencing in plant-feeding insects. First results have demonstrated that external dsRNA can successfully pass the insect gastrointestinal tract and reach its final destination within the body cells. This was somewhat unexpected and sparked new interest in RNA stability in the environment and its fate in the insect organism. In this brief review we make an attempt to summarize current knowledge and to propose a model of how dsRNA can perform its function under these settings.
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Pantchev, Ivelin, Goritsa Rakleova, and Atanas Atanassov. "The stability of dsRNA during external applications - an overview." In RNAi for plant improvement and protection, 94–101. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781789248890.0094.

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Abstract The research community is deeply convinced that RNA is unstable in the environment. Its roots rise from numerous failed attempts to isolate functional cellular RNA molecules. Further support had originated from the fast turnover of RNA in the cells. The situation changed recently with the discovery that externally applied dsRNA can produce targeted gene silencing in plant-feeding insects. First results have demonstrated that external dsRNA can successfully pass the insect gastrointestinal tract and reach its final destination within the body cells. This was somewhat unexpected and sparked new interest in RNA stability in the environment and its fate in the insect organism. In this brief review we make an attempt to summarize current knowledge and to propose a model of how dsRNA can perform its function under these settings.
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Mindich, Leonard. "Packaging in dsRNA Viruses." In Viral Molecular Machines, 601–8. Boston, MA: Springer US, 2011. http://dx.doi.org/10.1007/978-1-4614-0980-9_26.

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Obadia, Benjamin, and Maria-Carla Saleh. "dsRNA Uptake in Adult Drosophila." In Antiviral RNAi, 253–63. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-037-9_16.

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Mitchell, Diane J., and E. Alan Bevan. "dsRNA killer systems in yeast." In Yeast Biotechnology, 104–55. Dordrecht: Springer Netherlands, 1987. http://dx.doi.org/10.1007/978-94-009-3119-0_5.

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Conference papers on the topic "DsRNase"

1

Zhang, Jianzhen. "Effect of dsRNase on the efficiency of RNA interference." In 2016 International Congress of Entomology. Entomological Society of America, 2016. http://dx.doi.org/10.1603/ice.2016.94702.

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Suprunova, T. P., N. V. Markin, A. N. Ignatov, A. G. Solovyov, N. O. Kalinina, and M. E. Talyansky. "Use of dsRNA-based antiviral compounds to protect potato plants." In Растениеводство и луговодство. Тимирязевская сельскохозяйственная академия, 2020. http://dx.doi.org/10.26897/978-5-9675-1762-4-2020-132.

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One of the most important food crops in the world, the potato (Solanum tuberosum L.) is infected with many viruses, of which the y virus (Potato virus Y, PVY) is the most important economically, causing significant crop losses. Several alternative methods of dsRNA delivery have been tested, with the most promising being spray - induced gene silencing (SIGS). The results showed a high effect of preventive use of dsRNA. Treatment with the initial working concentration of dsRNA protected 100% and 65% of plants from virus propagation for 14 and 21 days, respectively, and 65% of plants were protected by the minimum tested concentration (10 ng/MCL) for 14 days. Therapeutic use of dsRNA 3 days after inoculation did not significantly affect the dynamics of virus accumulation in the plant. Thus, in the course of the experiment, a high biological antiviral effectiveness of dsRNA was demonstrated in the preventive treatment of potato plants against the background of artificial infection of plants with the PVY virus.
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Lee, How-Jing. "Oral delivery of dsRNA lipoplexes to German cockroach,Blattella germanica, protects dsRNA from degradation." In 2016 International Congress of Entomology. Entomological Society of America, 2016. http://dx.doi.org/10.1603/ice.2016.113415.

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Hosseini, Ramtin, Xingyi Yang, and Pengtao Xie. "DSRNA: Differentiable Search of Robust Neural Architectures." In 2021 IEEE/CVF Conference on Computer Vision and Pattern Recognition (CVPR). IEEE, 2021. http://dx.doi.org/10.1109/cvpr46437.2021.00613.

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Wong, Colin R. "Environmental fate of dsRNA in an aqueous system." In 2016 International Congress of Entomology. Entomological Society of America, 2016. http://dx.doi.org/10.1603/ice.2016.113822.

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Davis, Ian C. "Double-stranded RNA (dsRNA) Impairs Alveolar Fluid Clearance In Mice." In American Thoracic Society 2010 International Conference, May 14-19, 2010 • New Orleans. American Thoracic Society, 2010. http://dx.doi.org/10.1164/ajrccm-conference.2010.181.1_meetingabstracts.a6458.

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Mahmutovic Persson, Irma, Angelica Brandelius, and Lena Uller. "DsRNA-Induced TSLP Expression And Neutrophilia In Mouse Asthma Exacerbation Model." In American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California. American Thoracic Society, 2012. http://dx.doi.org/10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a5385.

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Liu, Jisheng. "Transcriptional inhibition of BmToll9-1 by dsRNA in the silkworm larval midgut." In 2016 International Congress of Entomology. Entomological Society of America, 2016. http://dx.doi.org/10.1603/ice.2016.108954.

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Gil, M. Maqueda, and M. Ramírez Fernández. "A simple method for simultaneously isolating mitDNA and virus dsRNA from wine yeasts." In Proceedings of the II International Conference on Environmental, Industrial and Applied Microbiology (BioMicroWorld2007). WORLD SCIENTIFIC, 2009. http://dx.doi.org/10.1142/9789812837554_0075.

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Asokan, R. "Differential expression of miRNAs in response to dsRNA of various target genes in Noctuidae." In 2016 International Congress of Entomology. Entomological Society of America, 2016. http://dx.doi.org/10.1603/ice.2016.94537.

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Reports on the topic "DsRNase"

1

Gal-On, Amit, Shou-Wei Ding, Victor P. Gaba, and Harry S. Paris. role of RNA-dependent RNA polymerase 1 in plant virus defense. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597919.bard.

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Objectives: Our BARD proposal on the impact of RNA-dependent RNA polymerase 1 (RDR1) in plant defense against viruses was divided into four original objectives. 1. To examine whether a high level of dsRNA expression can stimulate RDR1 transcription independent of salicylic acid (SA) concentration. 2. To determine whether the high or low level of RDR1 transcript accumulation observed in virus resistant and susceptible cultivars is associated with viral resistance and susceptibility. 3. To define the biogenesis and function of RDR1-dependent endogenous siRNAs. 4. To understand why Cucumber mosaic virus (CMV) can overcome RDR1-dependent resistance. The objectives were slightly changed due to the unique finding that cucumber has four different RDR1 genes. Background to the topic: RDR1 is a key plant defense against viruses. RDR1 is induced by virus infection and produces viral and plant dsRNAs which are processed by DICERs to siRNAs. siRNAs guide specific viral and plant RNA cleavage or serve as primers for secondary amplification of viral-dsRNA by RDR. The proposal is based on our preliminary results that a. the association of siRNA and RDR1 accumulation with multiple virus resistance, and b. that virus infection induced the RDR1-dependent production of a new class of endogenous siRNAs. However, the precise mechanisms underlying RDR1 induction and siRNA biogenesis due to virus infection remain to be discovered in plants. Major conclusions, solutions and achievements: We found that in the cucurbit family (cucumber, melon, squash, watermelon) there are 3-4 RDR1 genes not documented in other plant families. This important finding required a change in the emphasis of our objectives. We characterized 4 RDR1s in cucumber and 3 in melon. We demonstrated that in cucumber RDR1b is apparently a new broad spectrum virus resistance gene, independent of SA. In melon RDR1b is truncated, and therefore is assumed to be the reason that melon is highly susceptible to many viruses. RDR1c is dramatically induced due to DNA and RNA virus infection, and inhibition of RDR1c expression led to increased virus accumulation which suggested its important on gene silencing/defense mechanism. We show that induction of antiviral RNAi in Arabidopsis is associated with production of a genetically distinct class of virus-activated siRNAs (vasiRNAs) by RNA dependent RNA polymerase-1 targeting hundreds of host genes for RNA silencing by Argonaute-2. Production of vasiRNAs is induced by viruses from two different super groups of RNA virus families, targeted for inhibition by CMV, and correlated with virus resistance independently of viral siRNAs. We propose that antiviral RNAi activate broad-spectrum antiviral activity via widespread silencing of host genes directed by vasiRNAs, in addition to specific antiviral defense Implications both scientific and agricultural: The RDR1b (resistance) gene can now be used as a transcription marker for broad virus resistance. The discovery of vasiRNAs expands the repertoire of siRNAs and suggests that the siRNA-processing activity of Dicer proteins may play a more important role in the regulation of plant and animal gene expression than is currently known. We assume that precise screening of the vasiRNA host targets will lead in the near future for identification of plant genes associate with virus diseases and perhaps other pathogens.
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Bartholomay, Lyric, and D. L. Hank Harris. Evaluation of Highly Targeted dsRNA for the Treatment of Infectious Myonecrosis Virus (IMNV) in Litopenaeus vannamei. Ames (Iowa): Iowa State University, January 2012. http://dx.doi.org/10.31274/ans_air-180814-1050.

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Gonsalves, Dennis, Edna Tanne, and Deborah Golino. Isolation, Serological Detection of Closteroviruses Assoc. with Grape Corky Bark, and ELISA-dsRNA Analysis to Monitor Grape Leafroll Elimination Procedures. United States Department of Agriculture, August 1994. http://dx.doi.org/10.32747/1994.7604311.bard.

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Aly, Radi, and John I. Yoder. Development of resistant crop plants to parasitic weeds based on trans-specific gene silencing. United States Department of Agriculture, January 2013. http://dx.doi.org/10.32747/2013.7598146.bard.

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Broomrapes (Orobanche/Phelipanchespp.) are holo parasitic plants that subsist on the roots of a variety of agricultural crops and cause severe losses to the yield quality and quantity. Effective methods for controlling parasitic weeds are scarce, with only a few known cases of genetic resistance. In the current study, we proposed an improved strategy for the control of parasitic weeds based on trans-specific gene-silencing of three parasite genes at once. We used two strategies to express dsRNA containing selected sequences of three Phelipancheaegyptiacagenes PaACS, PaM6PR and PaPrx1 (pma): transient expression using Tobacco rattle virus (TRV:pma) as a virus-induced gene-silencing (VIGS) vector and stable expression in transgenic tomato Solanumlycopersicum(Mill.) plants harboring a hairpin construct (pBINPLUS35:pma). siRNA-mediated transgene-silencing (20–24 nt) was detected in the host plants. Our results demonstrate that the quantities of PaACSand PaM6PR transcripts from P. aegyptiacatubercles grown on transgenic tomato or on Tobacco rattle virus-infected Nicotianabenthamianaplants were significantly reduced. However, only partial reductions in the quantity of PaPrx1 transcripts were observed in the parasite tubercles grown on tomato and on N. benthamianaplants. Concomitant with the suppression of the target genes, there were significant decreases in the number and weight of the parasite tubercles that grew on the host plants, in both the transient and the stable experimental systems. The results of the work carried out using both strategies point to the movement of mobile exogenous siRNA from the host to the parasite, leading to the impaired expression of essential parasite target genes. In light of the importance of parasitic weeds to world agriculture and the difficulty of obtaining resistance by conventional methods, we assume that genetic resistance based on the silencing of key metabolic genes in the parasite is now feasible. BARD Report - Project4622 Page 2 of 60
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Mevarech, Moshe, Jeremy Bruenn, and Yigal Koltin. Virus Encoded Toxin of the Corn Smut Ustilago Maydis - Isolation of Receptors and Mapping Functional Domains. United States Department of Agriculture, September 1995. http://dx.doi.org/10.32747/1995.7613022.bard.

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Ustilago maydis is a fungal pathogen of maize. Some strains of U. maydis encode secreted polypeptide toxins capable of killing other susceptible strains of U. maydis. Resistance to the toxins is conferred by recessive nuclear genes. The toxins are encoded by genomic segments of resident double-strande RNA viruses. The best characterized toxin, KP6, is composed of two polypeptides, a and b, which are not covalently linked. It is encoded by P6M2 dsRNA, which has been cloned, sequenced and expressed in a variety of systems. In this study we have shown that the toxin acts on the membranes of sensitive cells and that both polypeptides are required for toxin activity. The toxin has been shown to function by creating new pores in the cell membrane and disrupting ion fluxes. The experiments performed on artificial phospholipid bilayers indicated that KP6 forms large voltage-independent, cation-selective channels. Experiments leading to the resolution of structure-function relationship of the toxin by in vitro analysis have been initiated. During the course of this research the collaboration also yielded X-ray diffracion data of the crystallized a polypeptide. The effect of the toxin on the pathogen has been shown to be receptor-mediated. A potential receptor protein, identified in membrane fractions of sensitive cells, was subjected to tryptic hydrolysis followed by amino-acid analysis. The peptides obtained were used to isolate a cDNA fragment by reverse PCR, which showed 30% sequence homology to the human HLA protein. Analysis of other toxins secreted by U. maydis, KP1 and KP4, have demonstrated that, unlike KP6, they are composed of a single polypeptide. Finally, KP6 has been expressed in transgenic tobacco plants, indicating that accurate processing by Kex2p-like activity occurs in plants as well. Using tobacco as a model system, we determined that active antifungal toxins can be synthesized and targeted to the outside of transgenic plant cells. If this methodology can be applied to other agronomically crop species, then U. maydis toxins may provide a novel means for biological control of pathogenic fungi.
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Davis, Eric L., Yuji Oka, Amit Gal-On, Todd Wehner, and Aaron Zelcer. Broad-spectrum Resistance to Root-Knot Nematodes in Transgenic Cucurbits. United States Department of Agriculture, June 2013. http://dx.doi.org/10.32747/2013.7593389.bard.

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Root-knot nematodes (RKN), Meloidogyne spp., are extremely destructive pathogens of cucurbit crops grown in the United States and Israel. The safety and environmental concerns of toxic nematicides, and limited sources of natural cucurbit resistance to the four major species of Meloidogyne that threaten these crops in Israel and the U.S., have emphasized the use of biotechnology to develop cucurbits with novel RKN resistance. The U.S. scientists have identified over 40 unique RKN parasitism genes that encode nematode secretions involved in successful plant root infection by RKN, and they have demonstrated that expression of a double-stranded RNA (dsRNA) complementary to a RKN parasitism gene (called 16DIO) in Arabidopsis thaliana induced RNA interference (RNAi)-mediated silencing of the RKN16DlO gene and produced transgenic plants with strong resistance to all four major RKN species. The expression 8D05 parasitism gene was found to coincide with the timing of upregulation of NtCel7 promoter (identified to be upregulated in giantcells by US scientists). NtCel7 promoter was used to express the genes at the right time (early stages of infection) and in the right place (giant-cells) in transgenic plants. US partners produced NtCel7 (nematode-induced promoter)-driven 16DlO-RNAi and 8DOS-RNAi constructs, pHANNIBAL 4D03-RNAi construct and modified 16DlO-RNAi construct (for increased RNAi expression and efficacy) for cucurbit transformation in Israel. In Arabidopsis, some 16DlO-RNAi plant lines show greater levels of resistance to M. incognita than others, and within these lines resistance of greater than 90% reduction in infection is observed among almost all replicates in US. The level of observed nematode resistance is likely to be directly correlated with the level of RNAi expression in individual plants. In Israel, all the RKN parasitism genes-RNAi constructs were successfully transformed into cucumber and melon. The transgenic lines were evaluated for expression of the transgene siRNA in leaves and roots. Those displaying transgene siRNA accumulation were passed on for nematode resistance analysis. Rl seedlings from different lines were subjected to evaluation for resistance to M. javanica. None of the lines was resistant to the nematode in contrast with US partner's results in Arabidopsis. This could be for the following reasons: a) The level of transgene siRNA was insufficient in cucumber and tomato to cause resislance. b) 111e nemalode species on cucwnber IIlay be different ur act in a different manner. c) The assay was performed in soil with a high level of nematode inoculation, and not in petri dish, which may not permit the observation of a low level of resistance.
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Elizur, Abigail, Amir Sagi, Gideon Hulata, Clive Jones, and Wayne Knibb. Improving Crustacean Aquaculture Production Efficiencies through Development of Monosex Populations Using Endocrine and Molecular Manipulations. United States Department of Agriculture, June 2010. http://dx.doi.org/10.32747/2010.7613890.bard.

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Background Most of Australian prawn aquaculture production is based on P. monodon. However, the Australian industry is under intense competition from lower priced overseas imports. The availability of all-female monosex populations, by virtue of their large size and associated premium prize, will offer competitive advantage to the industry which desperately needs to counteract competitors within this market. As for the redclaw production in Israel, although it is at its infancy, the growers realized that the production of males is extremely advantageous and that such management strategy will change the economic assumptions and performances of this aquaculture to attract many more growers. Original objectives (as in original proposal) Investigating the sex inheritance mechanism in the tiger prawn. Identification of genes expressed uniquely in the androgenic gland (AG) of prawns and crayfish. The above genes and/or their products will be used to localize the AG in the prawn and manipulate the AG activity in both species. Production of monosex populations through AG manipulation. In the prawn, production of all-female populations and in the crayfish, all-male populations. Achievements In the crayfish, the AG cDNA library was further screened and a third AG specific transcript, designated Cq-AG3, had been identified. Simultaneously the two AG specific genes, which were previously identified, were further characterized. Tissue specificity of one of those genes, termed Cq-AG2, was demonstrated by northern blot hybridization and RNA in-situ hybridization. Bioinformatics prediction, which suggested a 42 amino acid long signal anchor at the N-terminus of the deduced Cq-AG2, was confirmed by immunolocalization of a recombinant protein. Cq-IAG's functionality was demonstrated by dsRNA in-vivo injections to intersex crayfish. Cq-IAGsilencing induced dramatic sex-related alterations, including male feature feminization, reduced sperm production, extensive testicular apoptosis, induction of the vitellogeningene expression and accumulation of yolk proteins in the ovaries. In the prawn, the AG was identified and a cDNA library was created. The putative P. monodonAG hormone encoding gene (Pm-IAG) was identified, isolated and characterized for time of expression and histological localization. Implantation of the AG into prawn post larvae (PL) and juveniles resulted in phenotypic transformation which included the appearance of appendix masculina and enlarged petasma. The transformation however did not result in sex change or the creation of neo males thus the population genetics stage to be executed with Prof. Hulata did not materialized. Repeated AG implantation is currently being trialed. Major conclusions and Implications, both scientific and agricultural Cq-IAG's involvement in male sexual differentiation had been demonstrated and it is strongly suggested that this gene encodes an AG hormone in this crayfish. A thorough screening of the AG cDNA library shows Cq-IAG is the prominent transcript within the library. However, the identification of two additional transcripts hints that Cq-IAG is not the only gene mediating the AG effects. The successful gene silencing of Cq-IAG, if performed at earlier developmental stages, might accomplish full and functional sex reversal which will enable the production of all-male crayfish populations. Pm-IAG is likely to play a similar role in prawns. It is possible that repeated administration of the AG into prawn will lead to the desired full sex reversal, so that WZ neo males, crossed with WZ females can result in WW females, which will form the basis for monosex all-female population.
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