Canonical RNAi, one of the so-called RNA-silencing mechanisms, is defined as sequence-specific RNA degradation induced by long double-stranded RNA (dsRNA). RNAi occurs in four basic steps: (i) processing of long dsRNA by RNase III Dicer into small interfering RNA (siRNA) duplexes, (ii) loading of one of the siRNA strands on an Argonaute protein possessing endonucleolytic activity, (iii) target recognition through siRNA basepairing, and (iv) cleavage of the target by the Argonaute's endonucleolytic activity. This basic pathway diversified and blended with other RNA silencing pathways employing small RNAs. In some organisms, RNAi is extended by an amplification loop employing an RNA-dependent RNA polymerase, which generates secondary siRNAs from targets of primary siRNAs. Given the high specificity of RNAi and its presence in invertebrates, it offers an opportunity for highly selective pest control. The aim of this text is to provide an introductory overview of key mechanistic aspects of RNA interference for understanding its potential and constraints for its use in pest control.

Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

See more in PubMed

Ameres S. L., Martinez J., Schroeder R. (2007). Molecular basis for target RNA recognition and cleavage by human RISC. Cell 130, 101–112. 10.1016/j.cell.2007.04.037 PubMed DOI

Aoki K., Moriguchi H., Yoshioka T., Okawa K., Tabara H. (2007). In vitro analyses of the production and activity of secondary small interfering RNAs in C. elegans. EMBO J. 26, 5007–5019. 10.1038/sj.emboj.7601910 PubMed DOI PMC

Bachman P. M., Bolognesi R., Moar W. J., Mueller G. M., Paradise M. S., Ramaseshadri P., et al. (2013). Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte). Transgenic Res. 22, 1207–1222. 10.1007/s11248-013-9716-5 PubMed DOI PMC

Baum J. A., Bogaert T., Clinton W., Heck G. R., Feldmann P., Ilagan O., et al. (2007). Control of coleopteran insect pests through RNA interference. Nat. Biotechnol. 25, 1322–1326. 10.1038/nbt1359 PubMed DOI

Bhatia V., Bhattacharya R., Uniyal P. L., Singh R., Niranjan R. S. (2012). Host Generated siRNAs Attenuate Expression of Serine Protease Gene in Myzus persicae. PloS One 7, e46343–e46343. 10.1371/journal.pone.0046343 PubMed DOI PMC

Billi A. C., Fischer S. E., Kim J. K. (2014). Endogenous RNAi pathways in C. elegans. WormBook, ed. The C. elegans Research Community, WormBook; 1–49. 10.1895/wormbook.1.170.1 PubMed DOI PMC

Bologna N. G., Voinnet O. (2014). The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu. Rev. Plant Biol. 65, 473–503. 10.1146/annurev-arplant-050213-035728 PubMed DOI

Borges F., Martienssen R. A. (2015). The expanding world of small RNAs in plants. Nat. Rev. Mol. Cell Biol. 16, 727–741. 10.1038/nrm4085 PubMed DOI PMC

Brodersen P., Sakvarelidze-Achard L., Bruun-Rasmussen M., Dunoyer P., Yamamoto Y. Y., Sieburth L., et al. (2008). Widespread translational inhibition by plant miRNAs and siRNAs. Science 320, 1185–1190. 10.1126/science.1159151 PubMed DOI

Buck A. H., Blaxter M. (2013). Functional diversification of Argonautes in nematodes: an expanding universe. Biochem. Soc. Trans. 41, 881–886. 10.1042/BST20130086 PubMed DOI PMC

Burroughs A. M., Ando Y., De Hoon M. J. L., Tomaru Y., Suzuki H., Hayashizaki Y., et al. (2011). Deep-sequencing of human argonaute-associated small RNAs provides insight into miRNA sorting and reveals argonaute association with RNA fragments of diverse origin. RNA Biol. 8, 158–177. 10.4161/rna.8.1.14300 PubMed DOI PMC

Cai Q., He B., Kogel K. H., Jin H. (2018). Cross-kingdom RNA trafficking and environmental RNAi-nature’s blueprint for modern crop protection strategies. Curr. Opin. Microbiol. 46, 58–64. 10.1016/j.mib.2018.02.003 PubMed DOI PMC

Cappelle K., De Oliveira C. F., Van Eynde B., Christiaens O., Smagghe G. (2016). The involvement of clathrin-mediated endocytosis and two Sid-1-like transmembrane proteins in double-stranded RNA uptake in the Colorado potato beetle midgut. Insect Mol. Biol. 25, 315–323. 10.1111/imb.12222 PubMed DOI

Cerutti H., Casas-Mollano J. A. (2006). On the origin and functions of RNA-mediated silencing: from protists to man. Curr. Genet. 50, 81–99. 10.1007/s00294-006-0078-x PubMed DOI PMC

Chandradoss S. D., Schirle N. T., Szczepaniak M., Macrae I. J., Joo C. (2015). A Dynamic Search Process Underlies MicroRNA Targeting. Cell 162, 96–107. 10.1016/j.cell.2015.06.032 PubMed DOI PMC

Chen P. Y., Weinmann L., Gaidatzis D., Pei Y., Zavolan M., Tuschl T., et al. (2008). Strand-specific 5 ‘-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity. Rna-a Publ. RNA Soc. 14, 263–274. 10.1261/rna.789808 PubMed DOI PMC

Chen Y. H., Jia X. T., Zhao L., Li C. Z., Zhang S. A., Chen Y. G., et al. (2011). Identification and functional characterization of Dicer2 and five single VWC domain proteins of Litopenaeus vannamei. Dev. Comp. Immunol. 35, 661–671. 10.1016/j.dci.2011.01.010 PubMed DOI

Chera S., De Rosa R., Miljkovic-Licina M., Dobretz K., Ghila L., Kaloulis K., et al. (2006). Silencing of the hydra serine protease inhibitor Kazal1 gene mimics the human SPINK1 pancreatic phenotype. J. Cell Sci. 119, 846–857. 10.1242/jcs.02807 PubMed DOI

Correa R. L., Steiner F. A., Berezikov E., Ketting R. F. (2010). MicroRNA-directed siRNA biogenesis in Caenorhabditis elegans. PloS Genet. 6, e1000903. 10.1371/journal.pgen.1000903 PubMed DOI PMC

Czech B., Zhou R., Erlich Y., Brennecke J., Binari R., Villalta C., et al. (2009). Hierarchical Rules for Argonaute Loading in Drosophila. Mol. Cell 36, 445–456. 10.1016/j.molcel.2009.09.028 PubMed DOI PMC

Dalzell J. J., Mcveigh P., Warnock N. D., Mitreva M., Bird D. M., Abad P., et al. (2011). RNAi effector diversity in nematodes. PloS Negl. Trop. Dis. 5, e1176. 10.1371/journal.pntd.0001176 PubMed DOI PMC

Demeter T., Vaskovicova M., Malik R., Horvat F., Pasulka J., Svobodova E., et al. (2019). Main constraints for RNAi induced by expressed long dsRNA in mouse cells. Life Sci. Alliance 2, 1–13. 10.26508/lsa.201800289 PubMed DOI PMC

Deng P., Muhammad S., Wu L. (2018). Biogenesis and regulatory hierarchy of phased small interfering RNAs in plants. Plant Biotechnol. J. 16 (5), 965–975. 10.1111/pbi.12882 PubMed DOI PMC

Ding S. W., Voinnet O. (2007). Antiviral immunity directed by small RNAs. Cell 130, 413–426. 10.1016/j.cell.2007.07.039 PubMed DOI PMC

Dowling D., Pauli T., Donath A., Meusemann K., Podsiadlowski L., Petersen M., et al. (2016). Phylogenetic Origin and Diversification of RNAi Pathway Genes in Insects. Genome Biol. Evol. 8, 3784–3793. 10.1093/gbe/evw281 PubMed DOI PMC

Du Q., Thonberg H., Wang J., Wahlestedt C., Liang Z. (2005). A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 33, 1671–1677. 10.1093/nar/gki312 PubMed DOI PMC

Dueck A., Ziegler C., Eichner A., Berezikov E., Meister G. (2012). microRNAs associated with the different human Argonaute proteins. Nucleic Acids Res. 40, 9850–9862. 10.1093/nar/gks705 PubMed DOI PMC

Echeverri C. J., Beachy P. A., Baum B., Boutros M., Buchholz F., Chanda S. K., et al. (2006). Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat. Methods 3, 777–779. 10.1038/nmeth1006-777 PubMed DOI

Elkayam E., Kuhn C. D., Tocilj A., Haase A. D., Greene E. M., Hannon G. J., et al. (2012). The Structure of Human Argonaute-2 in Complex with miR-20a. Cell 150, 100–110. 10.1016/j.cell.2012.05.017 PubMed DOI PMC

Elvin M., Snoek L. B., Frejno M., Klemstein U., Kammenga J. E., Poulin G. B. (2011). A fitness assay for comparing RNAi effects across multiple C. elegans genotypes. BMC Genomics 12, 510. 10.1186/1471-2164-12-510 PubMed DOI PMC

Feinberg E. H., Hunter C. P. (2003). Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301, 1545–1547. 10.1126/science.1087117 PubMed DOI

Felix M. A., Ashe A., Piffaretti J., Wu G., Nuez I., Belicard T., et al. (2011). Natural and experimental infection of Caenorhabditis nematodes by novel viruses related to nodaviruses. PloS Biol. 9, e1000586. 10.1371/journal.pbio.1000586 PubMed DOI PMC

Fire A., Xu S., Montgomery M. K., Kostas S. A., Driver S. E., Mello C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811. 10.1038/35888 PubMed DOI

Fluiter K., Mook O. R., Baas F. (2009). The therapeutic potential of LNA-modified siRNAs: reduction of off-target effects by chemical modification of the siRNA sequence. Methods Mol. Biol. 487, 189–203. 10.1007/978-1-60327-547-7_9 PubMed DOI

Forstemann K., Horwich M. D., Wee L., Tomari Y., Zamore P. D. (2007). Drosophila microRNAs are sorted into functionally distinct argonaute complexes after production by dicer-1. Cell 130, 287–297. 10.1016/j.cell.2007.05.056 PubMed DOI PMC

Gantier M. P., Williams B. R. (2007). The response of mammalian cells to double-stranded RNA. Cytokine Growth Factor Rev. 18, 363–371. 10.1016/j.cytogfr.2007.06.016 PubMed DOI PMC

Geldhof P., Visser A., Clark D., Saunders G., Britton C., Gilleard J., et al. (2007). RNA interference in parasitic helminths: current situation, potential pitfalls and future prospects. Parasitology 134, 609–619. 10.1017/S0031182006002071 PubMed DOI

Ghildiyal M., Xu J., Seitz H., Weng Z. P., Zamore P. D. (2010). Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway. Rna-a Publ. RNA Soc. 16, 43–56. 10.1261/rna.1972910 PubMed DOI PMC

Haasnoot J., De Vries W., Geutjes E. J., Prins M., De Haan P., Berkhout B. (2007). The Ebola virus VP35 protein is a suppressor of RNA silencing. PloS Pathog. 3, e86. 10.1371/journal.ppat.0030086 PubMed DOI PMC

Haley B., Zamore P. D. (2004). Kinetic analysis of the RNAi enzyme complex. Nat. Struct. Mol. Biol. 11, 599–606. 10.1038/nsmb780 PubMed DOI

Haley B., Foys B., Levine M. (2010). Vectors and parameters that enhance the efficacy of RNAi-mediated gene disruption in transgenic Drosophila. Proc. Natl. Acad. Sci. U.S.A. 107, 11435–11440. 10.1073/pnas.1006689107 PubMed DOI PMC

Hannus M., Beitzinger M., Engelmann J. C., Weickert M. T., Spang R., Hannus S., et al. (2014). siPools: highly complex but accurately defined siRNA pools eliminate off-target effects. Nucleic Acids Res. 42, 8049–8061. 10.1093/nar/gku480 PubMed DOI PMC

Himber C., Dunoyer P., Moissiard G., Ritzenthaler C., Voinnet O. (2003). Transitivity-dependent and -independent cell-to-cell movement of RNA silencing. EMBO J. 22, 4523–4533. 10.1093/emboj/cdg431 PubMed DOI PMC

Holen T., Moe S. E., Sorbo J. G., Meza T. J., Ottersen O. P., Klungland A. (2005). Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo . Nucleic Acids Res. 33, 4704–4710. 10.1093/nar/gki785 PubMed DOI PMC

Hoy M. A., Waterhouse R. M., Wu K., Estep A. S., Ioannidis P., Palmer W. J., et al. (2016). Genome sequencing of the phytoseiid predatory mite Metaseiulus occidentalis reveals completely atomised Hox genes and super-dynamic intron evolution. Genome Biol. Evol. 8 (6), 1762–1775. 10.1093/gbe/evw048 PubMed DOI PMC

Huang T. Z., Zhang X. B. (2013). Host defense against DNA virus infection in shrimp is mediated by the siRNA pathway. Eur. J. Immunol. 43, 137–146. 10.1002/eji.201242806 PubMed DOI

Hunter W., Ellis J., Vanengelsdorp D., Hayes J., Westervelt D., Glick E., et al. (2010). Large-scale field application of RNAi technology reducing Israeli acute paralysis virus disease in honey bees (Apis mellifera, Hymenoptera: Apidae). PloS Pathog. 6, e1001160. 10.1371/journal.ppat.1001160 PubMed DOI PMC

Huvenne H., Smagghe G. (2010). Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review. J. Insect Physiol. 56, 227–235. 10.1016/j.jinsphys.2009.10.004 PubMed DOI

Ivashuta S., Zhang Y., Wiggins B. E., Ramaseshadri P., Segers G. C., Johnson S., et al. (2015). Environmental RNAi in herbivorous insects. RNA 21, 840–850. 10.1261/rna.048116.114 PubMed DOI PMC

Jackson A. L., Bartz S. R., Schelter J., Kobayashi S. V., Burchard J., Mao M., et al. (2003). Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637. 10.1038/nbt831 PubMed DOI

Jackson A. L., Burchard J., Schelter J., Chau B. N., Cleary M., Lim L., et al. (2006). Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. Rna 12, 1179–1187. 10.1261/rna.25706 PubMed DOI PMC

Jaskiewicz L., Filipowicz W. (2008). Role of Dicer in posttranscriptional RNA silencing. Curr. Top. Microbiol. Immunol. 320, 77–97. 10.1007/978-3-540-75157-1_4 PubMed DOI

Jo M. H., Shin S., Jung S. R., Kim E., Song J. J., Hohng S. (2015. a). Human Argonaute 2 Has Diverse Reaction Pathways on Target RNAs. Mol. Cell 59, 117–124. 10.1016/j.molcel.2015.04.027 PubMed DOI

Jo M. H., Song J.-J., Hohng S. (2015. b). Single-molecule fluorescence measurements reveal the reaction mechanisms of the core-RISC, composed of human Argonaute 2 and a guide RNA. Bmb Rep. 48, 643–644. 10.5483/BMBRep.2015.48.12.235 PubMed DOI PMC

Jones-Rhoades M. W., Bartel D. P., Bartel B. (2006). “MicroRNAs and their regulatory roles in plants,”. Annu. Rev. Plant Biol.) 19–53. 10.1146/annurev.arplant.57.032905.105218 PubMed DOI

Kang W., Bang-Berthelsen C. H., Holm A., Houben A. J., Muller A. H., Thymann T., et al. (2017). Survey of 800+ data sets from human tissue and body fluid reveals xenomiRs are likely artifacts. RNA 23, 433–445. 10.1261/rna.059725.116 PubMed DOI PMC

Kennedy E. M., Whisnant A. W., Kornepati A. V., Marshall J. B., Bogerd H. P., Cullen B. R. (2015). Production of functional small interfering RNAs by an amino-terminal deletion mutant of human Dicer. Proc. Natl. Acad. Sci. U.S.A. 112, E6945–E6954. 10.1073/pnas.1513421112 PubMed DOI PMC

Kennedy E. M., Kornepati A. V., Bogerd H. P., Cullen B. R. (2017). Partial reconstitution of the RNAi response in human cells using Drosophila gene products. RNA 23, 153–160. 10.1261/rna.059345.116 PubMed DOI PMC

Ketting R. F. (2011). The many faces of RNAi. Dev. Cell 20, 148–161. 10.1016/j.devcel.2011.01.012 PubMed DOI

Khajuria C., Ivashuta S., Wiggins E., Flagel L., Moar W., Pleau M., et al. (2018). Development and characterization of the first dsRNA-resistant insect population from western corn rootworm, Diabrotica virgifera virgifera LeConte. PloS One 13, e0197059. 10.1371/journal.pone.0197059 PubMed DOI PMC

Khvorova A., Reynolds A., Jayasena S. D. (2003). Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216. 10.1016/S0092-8674(03)00801-8 PubMed DOI

Kim V. N., Han J., Siomi M. C. (2009). Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell Biol. 10, 126–139. 10.1038/nrm2632 PubMed DOI

Koch A., Biedenkopf D., Furch A., Weber L., Rossbach O., Abdellatef E., et al. (2016). An RNAi-Based Control of Fusarium graminearum Infections Through Spraying of Long dsRNAs Involves a Plant Passage and Is Controlled by the Fungal Silencing Machinery. PloS Pathog. 12, e1005901. 10.1371/journal.ppat.1005901 PubMed DOI PMC

Kola V. S., Renuka P., Padmakumari A. P., Mangrauthia S. K., Balachandran S. M., Ravindra Babu V., et al. (2016). Silencing of CYP6 and APN Genes Affects the Growth and Development of Rice Yellow Stem Borer, Scirpophaga incertulas. Front. Physiol. 7:20. 10.3389/fphys.2016.00020 PubMed DOI PMC

Komiya R. (2017). Biogenesis of diverse plant phasiRNAs involves an miRNA-trigger and Dicer-processing. J. Plant Res. 130, 17–23. 10.1007/s10265-016-0878-0 PubMed DOI PMC

Konakalla N. C., Kaldis A., Masarapu H., Voloudakis A. E. (2019). Topical application of double stranded RNA molecules deriving from Sesbania mosaic virus (SeMV) CP and MP genes protects Sesbania plants against SeMV. Eur. J. Plant Pathol. 155, 1345–1352. 10.1007/s10658-019-01821-z DOI

Kunte N., Mcgraw E., Bell S., Held D., Avila L. A. (2020). Prospects, challenges and current status of RNAi through insect feeding. Pest Manag. Sci. 76, 26–41. 10.1002/ps.5588 PubMed DOI

Kurscheid S., Lew-Tabor A. E., Valle M. R., Bruyeres A. G., Doogan V. J., Munderloh U. G., et al. (2009). Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila. BMC Mol. Biol. 10, 26–26. 10.1186/1471-2199-10-26 PubMed DOI PMC

Lanet E., Delannoy E., Sormani R., Floris M., Brodersen P., Crete P., et al. (2009). Biochemical Evidence for Translational Repression by Arabidopsis MicroRNAs. Plant Cell 21, 1762–1768. 10.1105/tpc.108.063412 PubMed DOI PMC

Lau P. W., Guiley K. Z., De N., Potter C. S., Carragher B., Macrae I. J. (2012). The molecular architecture of human Dicer. Nat. Struct. Mol. Biol. 19, 436–440. 10.1038/nsmb.2268 PubMed DOI PMC

Li F., Li P., Yang L. M., Tang B. (2012). Simple and sensitive fluorescence detection of the RNA endonuclease activity of mammalian argonaute2 protein based on an RNA molecular beacon. Chem. Commun. 48, 12192–12194. 10.1039/c2cc36404b PubMed DOI

Lingel A., Simon B., Izaurralde E., Sattler M. (2003). Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain. Nature 426, 465–469. 10.1038/nature02123 PubMed DOI

Lu R., Maduro M., Li F., Li H. W., Broitman-Maduro G., Li W. X., et al. (2005). Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans. Nature 436, 1040–1043. 10.1038/nature03870 PubMed DOI PMC

Ma J. B., Ye K., Patel D. J. (2004). Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429, 318–322. 10.1038/nature02519 PubMed DOI PMC

Ma Y., Creanga A., Lum L., Beachy P. A. (2006). Prevalence of off-target effects in Drosophila RNA interference screens. Nature 443, 359–363. 10.1038/nature05179 PubMed DOI

Macrae I. J., Zhou K., Li F., Repic A., Brooks A. N., Cande W. Z., et al. (2006). Structural basis for double-stranded RNA processing by Dicer. Science 311, 195–198. 10.1126/science.1121638 PubMed DOI

Macrae I. J., Zhou K., Doudna J. A. (2007). Structural determinants of RNA recognition and cleavage by Dicer. Nat. Struct. Mol. Biol. 14, 934–940. 10.1038/nsmb1293 PubMed DOI

Maida Y., Masutomi K. (2011). RNA-dependent RNA polymerases in RNA silencing. Biol. Chem. 392, 299–304. 10.1515/bc.2011.035 PubMed DOI

Maillard P. V., Van Der Veen A. G., Deddouche-Grass S., Rogers N. C., Merits A., Reis E Sousa C. (2016). Inactivation of the type I interferon pathway reveals long double-stranded RNA-mediated RNA interference in mammalian cells. EMBO J. 35, 2505–2518. 10.15252/embj.201695086 PubMed DOI PMC

Malik R., Svoboda P. (2012). “Nuclear RNA silencing and related phenomena in animals,” in Toxicology and Epigenetics. Ed. Sahu S. C. (Chichester, West Sussex, United Kingdom: John Wiley & Sons; ).

Mao Y. B., Cai W. J., Wang J. W., Hong G. J., Tao X. Y., Wang L. J., et al. (2007). Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat. Biotechnol. 25, 1307–1313. 10.1038/nbt1352 PubMed DOI

Matveyev A. V., Alves J. M., Serrano M. G., Lee V., Lara A. M., Barton W. A., et al. (2017). The Evolutionary Loss of RNAi Key Determinants in Kinetoplastids as a Multiple Sporadic Phenomenon. J. Mol. Evol. 84, 104–115. 10.1007/s00239-017-9780-1 PubMed DOI PMC

Mcfarlane L., Svingen T., Braasch I., Koopman P., Schartl M., Wilhelm D. (2011). Expansion of the Ago gene family in the teleost clade. Dev. Genes Evol. 221, 95–104. 10.1007/s00427-011-0363-7 PubMed DOI

Meister G., Landthaler M., Patkaniowska A., Dorsett Y., Teng G., Tuschl T. (2004). Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197. 10.1016/j.molcel.2004.07.007 PubMed DOI

Meister G. (2013). Argonaute proteins: functional insights and emerging roles. Nat. Rev. Genet. 14, 447–459. 10.1038/nrg3462 PubMed DOI

Minkina O., Hunter C. P. (2017). Stable Heritable Germline Silencing Directs Somatic Silencing at an Endogenous Locus. Mol. Cell 65 659-670, e655. 10.1016/j.molcel.2017.01.034 PubMed DOI PMC

Moissiard G., Parizotto E. A., Himber C., Voinnet O. (2007). Transitivity in Arabidopsis can be primed, requires the redundant action of the antiviral Dicer-like 4 and Dicer-like 2, and is compromised by viral-encoded suppressor proteins. RNA 13, 1268–1278. 10.1261/rna.541307 PubMed DOI PMC

Murphy D., Dancis B., Brown J. R. (2008). The evolution of core proteins involved in microRNA biogenesis. BMC Evol. Biol. 8, 92. 10.1186/1471-2148-8-92 PubMed DOI PMC

Nakayashiki H., Kadotani N., Mayama S. (2006). Evolution and diversification of RNA silencing proteins in fungi. J. Mol. Evol. 63, 127–135. 10.1007/s00239-005-0257-2 PubMed DOI

Namgial T., Kaldis A., Chakraborty S., Voloudakis A. (2019). Topical application of double-stranded RNA molecules containing sequences of Tomato leaf curl virus and Cucumber mosaic virus confers protection against the cognate viruses. Physiol. Mol. Plant Pathol. 108, 1–9. 10.1016/j.pmpp.2019.101432 DOI

Nayak A., Berry B., Tassetto M., Kunitomi M., Acevedo A., Deng C., et al. (2010). Cricket paralysis virus antagonizes Argonaute 2 to modulate antiviral defense in Drosophila. Nat. Struct. Mol. Biol. 17, 547–554. 10.1038/nsmb.1810 PubMed DOI PMC

Newmark P. A., Reddien P. W., Cebria F., Sanchez Alvarado A. (2003). Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians. Proc. Natl. Acad. Sci. U.S.A. 100 Suppl 1, 11861–11865. 10.1073/pnas.1834205100 PubMed DOI PMC

Obbard D. J., Gordon K. H., Buck A. H., Jiggins F. M. (2009). The evolution of RNAi as a defence against viruses and transposable elements. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 364, 99–115. 10.1098/rstb.2008.0168 PubMed DOI PMC

Okamura K., Chung W. J., Ruby J. G., Guo H. L., Bartel D. P., Lai E. C. (2008). The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453, 803–U808. 10.1038/nature07015 PubMed DOI PMC

Okamura K., Liu N., Lai E. C. (2009). Distinct Mechanisms for MicroRNA Strand Selection by Drosophila Argonautes. Mol. Cell 36, 431–444. 10.1016/j.molcel.2009.09.027 PubMed DOI PMC

Orii H., Mochii M., Watanabe K. (2003). A simple “soaking method” for RNA interference in the planarian Dugesia japonica. Dev. Genes Evol. 213, 138–141. 10.1007/s00427-003-0310-3 PubMed DOI

Paces J., Nic M., Novotny T., Svoboda P. (2017). Literature review of baseline information to support the risk assessment of RNAi-based GM plants. EFSA Support. Publ. 14, 1246E. 10.2903/sp.efsa.2017.EN-1246 DOI

Palmer W. J., Jiggins F. M. (2015). Comparative Genomics Reveals the Origins and Diversity of Arthropod Immune Systems. Mol. Biol. Evol. 32, 2111–2129. 10.1093/molbev/msv093 PubMed DOI PMC

Parrish S., Fire A. (2001). Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans. Rna 7, 1397–1402. PubMed PMC

Provost P., Dishart D., Doucet J., Frendewey D., Samuelsson B., Radmark O. (2002). Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J. 21, 5864–5874. 10.1093/emboj/cdf578 PubMed DOI PMC

Rechavi O., Lev I. (2017). Principles of Transgenerational Small RNA Inheritance in Caenorhabditis elegans. Curr. Biol. 27, R720–R730. 10.1016/j.cub.2017.05.043 PubMed DOI

Roignant J. Y., Carre C., Mugat B., Szymczak D., Lepesant J. A., Antoniewski C. (2003). Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila. RNA 9, 299–308. 10.1261/rna.2154103 PubMed DOI PMC

Roth B. M., Pruss G. J., Vance V. B. (2004). Plant viral suppressors of RNA silencing. Virus Res. 102, 97–108. 10.1016/j.virusres.2004.01.020 PubMed DOI

Salomon W. E., Jolly S. M., Moore M. J., Zamore P. D., Serebrov V. (2015). Single-Molecule Imaging Reveals that Argonaute Reshapes the Binding Properties of Its Nucleic Acid Guides. Cell 162, 84–95. 10.1016/j.cell.2015.06.029 PubMed DOI PMC

Santos D., Mingels L., Vogel E., Wang L., Christiaens O., Cappelle K., et al. (2019). Generation of Virus- and dsRNA-Derived siRNAs with Species-Dependent Length in Insects. Viruses 11, 1–15. 10.3390/v11080738 PubMed DOI PMC

Schirle N. T., Macrae I. J. (2012). The Crystal Structure of Human Argonaute2. Science 336, 1037–1040. 10.1126/science.1221551 PubMed DOI PMC

Schirle N. T., Sheu-Gruttadauria J., Macrae I. J. (2014). Structural basis for microRNA targeting. Science 346, 608–613. 10.1126/science.1258040 PubMed DOI PMC

Schirle N. T., Sheu-Gruttadauria J., Chandradoss S. D., Joo C., Macrae I. J. (2015). Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets. Elife 4, 1–16. 10.7554/eLife.07646 PubMed DOI PMC

Schmitter D., Filkowski J., Sewer A., Pillai R. S., Oakeley E. J., Zavolan M., et al. (2006). Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells. Nucleic Acids Res. 34, 4801–4815. 10.1093/nar/gkl646 PubMed DOI PMC

Schnettler E., Tykalova H., Watson M., Sharma M., Sterken M. G., Obbard D. J., et al. (2014). Induction and suppression of tick cell antiviral RNAi responses by tick-borne flaviviruses. Nucleic Acids Res. 42, 9436–9446. 10.1093/nar/gku657 PubMed DOI PMC

Schwarz D. S., Hutvagner G., Haley B., Zamore P. D. (2002). Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol. Cell 10, 537–548. 10.1016/S1097-2765(02)00651-2 PubMed DOI

Schwarz D. S., Hutvagner G., Du T., Xu Z., Aronin N., Zamore P. D. (2003). Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208. 10.1016/S0092-8674(03)00759-1 PubMed DOI

Seok H., Jang E.-S., Chi S. W. (2016). Rationally designed siRNAs without miRNA-like off-target repression. Bmb Rep. 49, 135–136. 10.5483/BMBRep.2016.49.3.019 PubMed DOI PMC

Shih J. D., Hunter C. P. (2011). SID-1 is a dsRNA-selective dsRNA-gated channel. RNA 17, 1057–1065. 10.1261/rna.2596511 PubMed DOI PMC

Shih J. D., Fitzgerald M. C., Sutherlin M., Hunter C. P. (2009). The SID-1 double-stranded RNA transporter is not selective for dsRNA length. RNA 15, 384–390. 10.1261/rna.1286409 PubMed DOI PMC

Sijen T., Fleenor J., Simmer F., Thijssen K. L., Parrish S., Timmons L., et al. (2001). On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107, 465–476. 10.1016/S0092-8674(01)00576-1 PubMed DOI

Snead N. M., Escamilla-Powers J. R., Rossi J. J., Mccaffrey A. P. (2013). 5 ‘ Unlocked Nucleic Acid Modification Improves siRNA Targeting. Mol. Therapy-Nucleic Acids 2, e103–e103. 10.1038/mtna.2013.36 PubMed DOI PMC

Song J. J., Liu J., Tolia N. H., Schneiderman J., Smith S. K., Martienssen R. A., et al. (2003). The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nat. Struct. Biol. 10, 1026–1032. 10.1038/nsb1016 PubMed DOI

Song J. J., Smith S. K., Hannon G. J., Joshua-Tor L. (2004). Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437. 10.1126/science.1102514 PubMed DOI

Stein P., Svoboda P., Anger M., Schultz R. M. (2003). RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase. Rna 9, 187–192. 10.1261/rna.2860603 PubMed DOI PMC

Stein P., Zeng F., Pan H., Schultz R. M. (2005). Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes. Dev. Biol. 286, 464–471. 10.1016/j.ydbio.2005.08.015 PubMed DOI

Su H., Trombly M. I., Chen J., Wang X. Z. (2009). Essential and overlapping functions for mammalian Argonautes in microRNA silencing. Genes Dev. 23, 304–317. 10.1101/gad.1749809 PubMed DOI PMC

Svoboda P. (2007). Off-targeting and other non-specific effects of RNAi experiments in mammalian cells. Curr. Opin. Mol. Ther. 9, 248–257. PubMed

Svoboda P. (2014). Renaissance of mammalian endogenous RNAi. FEBS Lett. 588, 2550–2556. 10.1016/j.febslet.2014.05.030 PubMed DOI

Svobodova E., Kubikova J., Svoboda P. (2016). Production of small RNAs by mammalian Dicer. Pflugers Arch. 468, 1089–1102. 10.1007/s00424-016-1817-6 PubMed DOI PMC

Tabara H., Grishok A., Mello C. C. (1998). RNAi in C. elegans: soaking in the genome sequence. Science 282, 430–431. 10.1126/science.282.5388.430 PubMed DOI

Tabara H., Sarkissian M., Kelly W. G., Fleenor J., Grishok A., Timmons L., et al. (1999). The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123–132. 10.1016/S0092-8674(00)81644-X PubMed DOI

Tabara H., Yigit E., Siomi H., Mello C. C. (2002). The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109, 861–871. 10.1016/S0092-8674(02)00793-6 PubMed DOI

Tam O. H., Aravin A. A., Stein P., Girard A., Murchison E. P., Cheloufi S., et al. (2008). Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453, 534–538. 10.1038/nature06904 PubMed DOI PMC

Tijsterman M., Okihara K. L., Thijssen K., Plasterk R. H. (2002). PPW-1, a PAZ/PIWI protein required for efficient germline RNAi, is defective in a natural isolate of C. elegans. Curr. Biol. 12, 1535–1540. 10.1016/S0960-9822(02)01110-7 PubMed DOI

Timmons L., Fire A. (1998). Specific interference by ingested dsRNA. Nature 395, 854. 10.1038/27579 PubMed DOI

Tomari Y., Zamore P. D. (2005). Perspective: machines for RNAi. Genes Dev. 19, 517–529. 10.1101/gad.1284105 PubMed DOI

Tomari Y., Du T., Zamore P. D. (2007). Sorting of Drosophila small silencing RNAs. Cell 130, 299–308. 10.1016/j.cell.2007.05.057 PubMed DOI PMC

Tomoyasu Y., Miller S. C., Tomita S., Schoppmeier M., Grossmann D., Bucher G. (2008). Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium. Genome Biol. 9, R10. 10.1186/gb-2008-9-1-r10 PubMed DOI PMC

Vaistij F. E., Jones L., Baulcombe D. C. (2002). Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14, 857–867. 10.1105/tpc.010480 PubMed DOI PMC

Van Der Veen A. G., Maillard P. V., Schmidt J. M., Lee S. A., Deddouche-Grass S., Borg A., et al. (2018). The RIG-I-like receptor LGP2 inhibits Dicer-dependent processing of long double-stranded RNA and blocks RNA interference in mammalian cells. EMBO J. 37, 1–14. 10.15252/embj.201797479 PubMed DOI PMC

Wang F., Polydore S., Axtell M. J. (2015). More than meets the eye? Factors that affect target selection by plant miRNAs and heterochromatic siRNAs. Curr. Opin. Plant Biol. 27, 118–124. 10.1016/j.pbi.2015.06.012 PubMed DOI PMC

Wassenegger M. (2005). The role of the RNAi machinery in heterochromatin formation. Cell 122, 13–16. 10.1016/j.cell.2005.06.034 PubMed DOI

Watanabe T., Totoki Y., Toyoda A., Kaneda M., Kuramochi-Miyagawa S., Obata Y., et al. (2008). Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539–543. 10.1038/nature06908 PubMed DOI

Wee L. M., Flores-Jasso C. F., Salomon W. E., Zamore P. D. (2012). Argonaute Divides Its RNA Guide into Domains with Distinct Functions and RNA-Binding Properties. Cell 151, 1055–1067. 10.1016/j.cell.2012.10.036 PubMed DOI PMC

Whangbo J. S., Hunter C. P. (2008). Environmental RNA interference. Trends Genet. 24, 297–305. 10.1016/j.tig.2008.03.007 PubMed DOI

Wilkins C., Dishongh R., Moore S. C., Whitt M. A., Chow M., Machaca K. (2005). RNA interference is an antiviral defence mechanism in Caenorhabditis elegans. Nature 436, 1044–1047. 10.1038/nature03957 PubMed DOI

Winston W. M., Molodowitch C., Hunter C. P. (2002). Systemic RNAi in C-elegans requires the putative transmembrane protein SID-1. Science 295, 2456–2459. 10.1126/science.1068836 PubMed DOI

Winston W. M., Sutherlin M., Wright A. J., Feinberg E. H., Hunter C. P. (2007). Caenorhabditis elegans SID-2 is required for environmental RNA interference. Proc. Natl. Acad. Sci. U.S.A. 104, 10565–10570. 10.1073/pnas.0611282104 PubMed DOI PMC

Worrall E. A., Bravo-Cazar A., Nilon A. T., Fletcher S. J., Robinson K. E., Carr J. P., et al. (2019). Exogenous Application of RNAi-Inducing Double-Stranded RNA Inhibits Aphid-Mediated Transmission of a Plant Virus. Front. Plant Sci. 10:265. 10.3389/fpls.2019.00265 PubMed DOI PMC

Xu W., Han Z. (2008). Cloning and phylogenetic analysis of sid-1-like genes from aphids. J. Insect Sci. 8, 1–6. 10.1673/031.008.3001 PubMed DOI PMC

Yan K. S., Yan S., Farooq A., Han A., Zeng L., Zhou M. M. (2003). Structure and conserved RNA binding of the PAZ domain. Nature 426, 468–474. 10.1038/nature02129 PubMed DOI

Yang L. S., Li X. L., Jiang S., Qiu L. H., Zhou F. L., Liu W. J., et al. (2014). Characterization of Argonaute2 gene from black tiger shrimp (Penaeus monodon) and its responses to immune challenges. Fish Shellfish Immunol. 36, 261–269. 10.1016/j.fsi.2013.11.010 PubMed DOI

Yekta S., Shih I. H., Bartel D. P. (2004). MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596. 10.1126/science.1097434 PubMed DOI

Yigit E., Batista P. J., Bei Y., Pang K. M., Chen C. C., Tolia N. H., et al. (2006). Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell 127, 747–757. 10.1016/j.cell.2006.09.033 PubMed DOI

Yuan Y. R., Pei Y., Ma J. B., Kuryavyi V., Zhadina M., Meister G., et al. (2005). Crystal structure of A-aeolicus Argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol. Cell 19, 405–419. 10.1016/j.molcel.2005.07.011 PubMed DOI PMC

Zander A., Holzmeister P., Klose D., Tinnefeld P., Grohmann D. (2014). Single-molecule FRET supports the two-state model of Argonaute action. RNA Biol. 11, 45–56. 10.4161/rna.27446 PubMed DOI PMC

Zhang H., Kolb F. A., Brondani V., Billy E., Filipowicz W. (2002). Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21, 5875–5885. 10.1093/emboj/cdf582 PubMed DOI PMC

Zhang H., Kolb F. A., Jaskiewicz L., Westhof E., Filipowicz W. (2004). Single processing center models for human Dicer and bacterial RNase III. Cell 118, 57–68. 10.1016/j.cell.2004.06.017 PubMed DOI

Zhang L., Hou D. X., Chen X., Li D. H., Zhu L. Y., Zhang Y. J., et al. (2012). Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res. 22, 107–126. 10.1038/cr.2011.158 PubMed DOI PMC

Zhang J., Khan S. A., Hasse C., Ruf S., Heckel D. G., Bock R. (2015). Pest control. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids. Science 347, 991–994. 10.1126/science.1261680 PubMed DOI

RNAi-mediated gene silencing of Phlebotomus papatasi defensins favors Leishmania major infection

High Diversity of Novel Viruses in the Tree Pathogen Phytophthora castaneae Revealed by High-Throughput Sequencing of Total and Small RNA