Ecological geneticsEcological geneticsЭкологическая генетика1811-09322411-9202Eco-Vector3520310.17816/ecogen35203Genetic basis of ecosystems evolutionГенетические основы эволюции экосистемResearch ArticleApplication of small RNAs for plant protectionМалые РНК в защите растений от болезней8930-8251TretiakovaPolina Ya.ТретьяковаПолина Яковлевна
Russian Federation

PhD student, department of Genetics, Plant Breeding and Seed Production

аспирант, кафедра генетики, селекции и семеноводства

polina.tretiakova@yandex.ru35732425900Q-1589-20153431-5168SolovievAleksandr A.СоловьевАлександр Александрович
Russian Federation

Doctor of Science, Head of the Laboratory of Marker-Assisted and Genomic Selection of Plants

-р биол. наук, заведующий лабораторией маркерной и геномной селекции растений

a.soloviev70@gmail.comRussian State Agrarian University – Moscow Timiryazev Agricultural AcademyФедеральное государственное бюджетное образовательное учреждение высшего образования «Российский государственный аграрный университет — МСХА им. К.А. Тимирязева»All-Russia Research Institute of Agricultural BiotechnologyФедеральное государственное бюджетное научное учреждение «Всероссийский научно-исследовательский институт сельскохозяйственной биотехнологии»04112020121220201844674821607202004112020Copyright ©; 2021, Tretiakova P.Y., Soloviev A.A.Copyright ©; 2021, ООО "Эко-вектор"2021Tretiakova P.Y., Soloviev A.A.ООО "Эко-вектор"http://creativecommons.org/licenses/by/4.0https://journals.eco-vector.com/ecolgenet/article/view/35203

Double-stranded small RNAs (dsRNA) perform various regulatory functions via RNA-interference. Additionally, they can be transported between various plant species and their pathogens and pests via extracellular vesicles, protecting RNA from nucleases. Plants secrete short dsRNA molecules to defend themselves against pathogens. The latter also use small RNAs when infecting crops. Some dsRNAs of pathogens are known as “ribonucleic effectors”. Host-induced gene silencing (HIGS) was shown to be effective when breeding resistant varieties and analyzing plant-pathogen interactions. However, complexity of transgenesis and society fear of genetically modified products make HIGS application difficult. The appearance of a new strategy based on plant spraying with dsRNA gave a new perspective of plant protection. Currently such a strategy requires accurate studying as well as the development of efficient systems stably producing high-quality dsRNA.

Двухцепочечные малые РНК (дцРНК), включаясь в процесс РНК-интерференции, выполняют различные регуляторные функции во многих организмах. Были описаны случаи обмена малыми РНК между разными видами растений и поражающими их патогенами и вредителями посредством внеклеточных везикул (крошечных пузырьков), которые защищают их от действия нуклеаз. Растения секретируют двухцепочечные короткие молекулы РНК для борьбы с возбудителями заболеваний, а те, в свою очередь, используют малые РНК как инструмент, позволяющий им ослабить иммунитет хозяев. Некоторые дцРНК патогенов получили название «рибонуклеиновых эффекторов». Технология HIGS (host induced gene silencing — сайленсинг, индуцируемый растением-хозяином) показала свою эффективность в создании устойчивых сортов сельскохозяйственных культур и изучении различных патосистем. Однако использование ее ограничено из-за сложности создания трансгенных линий и запрета на их выращивание во многих странах. Распыление дцРНК по поверхности листьев, стеблей и соцветий растений может стать новым приемом контроля заболеваний, способным заменить использование пестицидов. На данный момент такая стратегия требует тщательного анализа и доработки, а также создания дешевых систем, стабильно синтезирующих дцРНК хорошего качества.

small RNAsilencingHIGSSIGSRNA-interferencehostpathogenvesicleeffectordsRNAмалые РНКсайленсингHIGSSIGSРНК-интерференцияхозяинпатогенвезикулыэффектордцРНКKim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol. 2009;10(2):126-139. https://doi.org/10.1038/nrm2632.Baulcombe D. RNA silencing in plants. Nature. 2004;431(7006):356-363. https://doi.org/ 10.1038/nature02874.Vaucheret H, Vazquez F, Crété P, et al. The action of Argonaute1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev. 2004;18(10): 1187-1197. https://doi.org/10.1101/gad. 1201404.Brodersen P, Voinnet O. The diversity of RNA silencing pathways in plants. Trends Genet. 2006;22(5):268-280. https://doi.org/10.1016/j.tig.2006.03.003.Koch A, Kumar N, Weber L, et al. Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase-encoding genes confers strong resistance to Fusarium species. Proc Natl Acad Sci USA. 2013;110(48):19324-19329. https://doi.org/10.1073/pnas.1306373110.Lee HC, Li L, Gu W, et al. Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi. Mol Cell. 2010;38(6):803-814. https://doi.org/10.1016/j.molcel.2010.04.005.Weiberg A, Wang M, Bellinger M, et al. Small RNAs: a new paradigm in plant-microbe interactions. Annu Rev Phytopathol. 2014;52: 495-516. https://doi.org/10.1146/annurev-phyto-102313-045933.Wang B, Sun Y, Song N, et al. Puccinia striiformis f. sp. tritici microRNA-like RNA 1 (Pst-milR1), an important pathogenicity factor of Pst, impairs wheat resistance to Pst by suppressing the wheat pathogenesis-related 2 gene. New Phytol. 2017;215(1): 338-350. https://doi.org/10.1111/nph.14577.Cerutti H, Casas-Mollano JA. On the origin and functions of RNA-mediated silencing: from protists to man. Curr Genet. 2006;50(2):81-99. https://doi.org/10.1007/s00294-006-0078-x.Laurie JD, Linning R, Bakkeren G. Hallmarks of RNA silencing are found in the smut fungus Ustilago hordei but not in its close relative Ustilago maydis. Curr Genet. 2008;53(1):49-58. https://doi.org/10.1007/s00294-007-0165-7.Kusch S, Frantzeskakis L, Thieron H, et al. Small RNAs from cereal powdery mildew pathogens may target host plant genes. Fungal Biol. 2018;122(11):1050-1063. https://doi.org/ 10.1016/j.funbio.2018.08.008.Nicolás FE, Garre V. RNA interference in fungi: retention and loss. Microbiol Spectr. 2016;4(6). https://doi.org/10.1128/microbiolspec.funk-0008-2016.Canto-Pastor A, Santos BA, Valli AA, et al. Enhanced resistance to bacterial and oomycete pathogens by short tandem target mimic RNAs in tomato. Proc Natl Acad Sci USA. 2019;116(7):2755-2760. https://doi.org/10. 1073/pnas.1814380116.Pak J, Fire A. Distinct populations of primary and secondary effectors during RNAi in C. elegans. Science. 2007;315(5809):241-244. https://doi.org/10.1126/science.1132839.Molnar A, Melnyk CW, Bassett A, et al. Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science. 2010;328(5980):872-875. https://doi.org/10.1126/science.1187959.Lewsey MG, Hardcastle TJ, Melnyk CW, et al. Mobile small RNAs regulate genome-wide DNA methylation. Proc Natl Acad Sci USA. 2016;113(6):E801-E810. https://doi.org/10. 1073/pnas.1515072113.Koch A, Biedenkopf D, Furch A, et al. 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. 2016;12(10): e1005901. https://doi.org/10.1371/journal.ppat.1005901.Ding B. The biology of viroid-host interactions. Annu Rev Phytopathol. 2009;47:105-131. https://doi.org/10.1146/annurev-phyto-080508- 081927.Buhtz A, Springer F, Chappell L, et al. Identification and characterization of small RNAs from the phloem of Brassica napus. Plant J. 2008;53(5):739-749. https://doi.org/10.1111/j.1365-313x.2007.03368.x.Weiberg A, Jin H. Small RNAs – the secret agents in the plant-pathogen interactions. Curr Opin Plant Biol. 2015;26:87-94. https://doi.org/10.1016/j.pbi.2015.05.033.Wang M, Thomas N, Jin H. Cross-kingdom RNA trafficking and environmental RNAi for powerful innovative pre- and post-harvest plant protection. Curr Opin Plant Biol. 2017;38:133-141. https://doi.org/10.1016/j.pbi.2017.05.003.Weiberg A, Bellinger M, Jin H. Conversations between kingdoms: small RNAs. Curr Opin Biotechnol. 2015;32:207-215. https://doi.org/10.1016/j.copbio.2014.12.025.Wang M, Weiberg A, Dellota E, et al. Botrytis small RNA Bc-siR37 suppresses plant defense genes by cross-kingdom RNAi. RNA Biol. 2017;14(4):421-428. https://doi.org/10.1080/15476286.2017.1291112.Weiberg A, Wang M, Lin FM, et al. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science. 2013;342(6154):118-123. https://doi.org/10.1126/science.1239705.Buck AH, Coakley G, Simbari F, et al. Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity. Nat Commun. 2014;5(1):5488. https://doi.org/10.1038/ncomms6488.Wang M, Weiberg A, Lin FM. Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nat Plants. 2016;2:16151. https://doi.org/10.1038/nplants.2016.151.Shahid S, Kim G, Johnson NR et al. MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs. Nature. 2018;553(7686):82-85. https://doi.org/ 10.1038/nature25027.Zhang T, Zhao YL, Zhao JH, et al. Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen. Nat Plants. 2016;2:16153. https://doi.org/10.1038/nplants.2016.153.Dutta S, Kumar D, Jha S, et al. Identification and molecular characterization of a trans-acting small interfering RNA producing locus regulating leaf rust responsive gene expression in wheat (Triticum aestivum L.) Planta. 2017;246(5):939-957. https://doi.org/10.1007/s00425-017-2744-2.Ghildiyal M, Xu J, Seitz H, et al. Sorting of Drosophila small silencing RNAs partitions microRNA strands into the RNA interference pathway. RNA. 2010;16(1):43-56. https://doi.org/10.1261/rna.1972910.Mi S, Cai T, Hu Y, et al. Sorting of small RNAs into Arabidopsis Argonaute Complexes is directed by the 5'-terminal nucleotide. Cell. 2008;133(1):116-127. https://doi.org/10.1016/j.cell.2008.02.034.Mueth NA, Ramachandran SR, Hulbert SH. Small RNAs from the wheat stripe rust fungus (Puccinia striiformis f. sp. tritici). BMC Genomics. 2015;16(1):718. https://doi.org/10.1186/s12864-015-1895-4.Shapulatov UM, Buriev ZT, Ulloa M, et al. Characterization of small RNAs and their targets from Fusarium oxysporum infected and noninfected cotton root tissues. Plant Mol Biol Report. 2016;34(3):698-706. https://doi.org/10.1007/s11105-015-0945-z.Yang F. Genome-wide analysis of small RNAs in the wheat pathogenic fungus Zymoseptoria tritici. Fungal Biol. 2015;119(7):631-640. https://doi.org/10.1016/j.funbio.2015.03.008.Vetukuri RR, Asman AKM, Tellgren-Roth C, et al. Evidence for small RNAs homologous to effector-encoding genes and transposable elements in the oomycete Phytophthora infestans. PLoS One. 2012;7(12):e51399. https://doi.org/ 10.1371/journal.pone.0051399.Derbyshire M, Mbengue M, Barascud M, et al. Small RNAs from the plant pathogenic fungus Sclerotinia sclerotiorum highlight host candidate genes associated with quantitative disease resistance. Mol Plant Pathol. 2019;20(9): 1279-1297. https://doi.org/10.1111/mpp. 12841.Baldrich P, Rutter BD, Karimi HZ, et al. Plant extracellular vesicles contain diverse small RNA species and are enriched in 10- to 17-nucleotide “tiny” RNAs. Plant Cell. 2019;31(2):315-324. https://doi.org/10.1105/tpc.18.00872.Li LC, Okino ST, Zhao H, et al. Small dsRNAs induce transcriptional activation in human cells. Proc Natl Acad Sci USA. 2006;103(46):17337-17342. https://doi.org/10.1073/pnas.0607015103.Valadi H, Ekström K, Bossios A, et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007:9(6):654-659. https://doi.org/10.1038/ncb1596.Mittelbrunn M, Gutiérrez-Vázquez C, Villarroya-Beltri C, et al. Unidirectional transfer of microRNA- loaded exosomes from T-cells to antigen-presenting cells. Nat Commun. 2011;2(1):282. https://doi.org/10.1038/ncomms1285.Colombo M, Raposo G, Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014:30(1):255-289. https://doi.org/10.1146/annurev-cellbio-101512-122326.Mittelbrunn M, Sánchez-Madrid F. Intercellular communication: diverse structures for exchange of genetic information. Nat Rev Mol Cell Biol. 2012;13(5):328-335. https://doi.org/10.1038/nrm3335.Cai Q, Qiao L, Wang M, et al. Plants send small RNAs in extracellular vesicles to fungal pathogen to silence virulence genes. Science. 2018;360(6393):1126-1129. https://doi.org/10.1126/science.aar4142.Feng H, Wang B, Zhang Q, et al. Exploration of microRNAs and their targets engaging in the resistance interaction between wheat and stripe rust. Front Plant Sci. 2015;6:469. https://doi.org/10.3389/fpls.2015.00469.LaMonte G, Philip N, Reardon J, et al. Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance. Cell Host Microbe. 2012;12(2):187-199. https://doi.org/10.1016/j.chom.2012.06.007.Ashida H, Ogawa M, Kim M, et al. Shigella deploy multiple countermeasures against host innate immune responses. Curr Opin Microbiol. 2011;14(1):16-23. https://doi.org/10.1016/j.mib.2010.08.014.Rafiqi M, Ellis JG, Ludowici VA, et al. Challenges and progress towards understanding the role of effectors in plant-fungal interactions. Curr Opin Plant Biol. 2012;15(4):477-482. https://doi.org/10.1016/j.pbi.2012.05.003.Bozkurt TO, Schornack S, Banfield MJ, et al. Oomycetes, effectors, and all that jazz. Curr Opin Plant Biol. 2012:15(4):483-492. https://doi.org/10.1016/j.pbi.2012.03.008.Muthukrishnan S, Liang GH, Trick HN, et al. Pathogenesis-related proteins and their genes in cereals. Plant Cell Tissue Organ Cult. 2001; 64:93-114.Fudal I, Collemare J, Böhnert HU, et al. Expression of Magnaporthe grisea avirulence gene ACE1 is connected to the initiation of appressorium-mediated penetration. Eukaryot Cell. 2007;6(3):546-554. https://doi.org/10.1128/ec.00330-05.Raman V, Simon SA, Romag A, et al. Physiological stressors and invasive plant infections alter the small RNA transcriptome of the rice blast fungus, Magnaporthe oryzae. BMC Genomics. 2013;14(1):326. https://doi.org/10.1186/1471-2164-14-326.Qutob D, Chapman BP, Gijzen M. Transgenerational gene silencing causes gain of virulence in a plant pathogen. Nat Commun. 2013;4(1):1349. https://doi.org/10.1038/ncomms2354.Alptekin B, Budak H. Wheat miRNA ancestors: evident by transcriptome analysis of A, B, and D genome donors. Funct Integr Genomics. 2017;17(2-3):171-187. https://doi.org/10.1007/s10142-016-0487-y.Van Herpen TW, Riley M, Sparks C, et al. Detailed analysis of the expression of an alpha-gliadin promoter and the deposition of alpha-gliadin protein during wheat grain development. Ann Bot. 2008;102(3):331-342. https://doi.org/10.1093/aob/mcn114.Hohmann U, Lau K, Hothorn M. The structural basis of ligand perception and signal activation by receptor kinases. Annu Rev Plant Biol. 2017;68(1):109-137. https://doi.org/10.1146/annurev-arplant-042916-040957.Zheng Z, Appiano M, Pavan S, et al. Genome-wide study of the tomato SlMLO gene family and its functional characterization in response to the powdery mildew fungus Oidium neolycopersici. Front Plant Sci. 2016;7:380. https://doi.org/10.3389/fpls.2016.00380.Chojak-Koźniewska J, Linkiewicz A, Sowa S, et al. Interactive effects of salt stress and Pseudomonas syringae pv. lachrymans infection in cucumber: involvement of antioxidant enzymes, abscisic acid and salicylic acid. Environ Exp Bot. 2017;136:9-20. https://doi.org/10.1016/j.envexpbot.2017.01.004.Mittler R. ROS are good. Trends Plant Sci. 2017;22(1):11-19. https://doi.org/10.1016/j.tplants.2016.08.002.Dalton DA, Boniface C, Turner Z, et al. Physiological roles of glutathione-S-transferases in soybean root nodules. Plant Physiol. 2009;150(1):521-530. https://doi.org/10.1104/pp.109.136630.Menard GN, Moreno JM, Bryant FM, et al. Genome wide analysis of fatty acid desaturation and its response to temperature. Plant Physiol. 2017;173(3):1594-1605. https://doi.org/10.1104/pp.16.01907.Huang G, Allen R, Davis EL, et al. Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. Proc Natl Acad Sci USA. 2006;103(39):14302-14306. https://doi.org/10.1073/pnas.0604698103.Mao YB, Cai WJ, Wang JW, et al. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol. 2007;25(11):1307-1313. https://doi.org/10.1038/nbt1352.Baum JA, Bogaert T, Clinton W, et al. Control of coleopteran insect pests through RNA interference. Nat Biotechnol. 2007;25(11):1322-1326. https://doi.org/10.1038/nbt1359.Nowara D, Gay A, Lacomme C, et al. HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell. 2010;22(9):3130-3141. https://doi.org/10.1105/tpc.110.077040.Vega-Arreguin JC, Jalloh A, Bos JI, et al. Recognition of an Avr3a homologue plays a major role in mediating nonhost resistance to Phytophthora capsici in Nicotiana species. Mol Plant Microbe Interact. 2014;27(8):770-780. https://doi.org/10.1094/mpmi-01-14-0014-r.Jahan SN, Asman AK, Corcoran P, et al. Plant-mediated gene silencing restricts growth of the potato late blight pathogen Phytophthora infestans. J Exp Bot. 2015;66(9):2785-2794. https://doi.org/10.1093/jxb/erv094.Nunes CC, Dean RA. Host-induced gene silencing: a tool for understanding fungal host interaction and for developing novel disease control strategies. Mol Plant Pathol. 2012;13(5): 519-529. https://doi.org/10.1111/j.1364-3703. 2011.00766.x.Lee WS, Hammond-Kosack KE, Kanyuka K. Barley stripe mosaic virus-mediated tools for investigating gene function in cereal plants and their pathogens: virus-induced gene silencing, host-mediated gene silencing, and virus-mediated overexpression of heterologous protein. Plant Physiol. 2012;160(2):582-590. https://doi.org/10.1104/pp.112.203489.Zhu X, Guo J, He F, et al. Silencing PsKPP4, a MAP kinase kinase kinase gene, reduces pathogenicity of the stripe rust fungus. Mol Plant Pathol. 2018;19(12):2590-2602. https://doi.org/10.1111/mpp.12731.Cheng W, Song XS, Li HP, et al. Host-induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat. Plant Biotechnol J. 2015;13(9):1335-1345. https://doi.org/10.1111/pbi.12352.Song XS, Gu KX, Duan XX, et al. Secondary amplification of siRNA machinery limits the application of spray-induced gene silencing. Mol Plant Pathol. 2018;19(12):2543-2560. https://doi.org/10.1111/mpp.12728.Cerutti H, Ibrahim F. Turnover of Mature miRNAs and siRNAs in Plants and Algae. Adv Exp Med Biol. 2011;700:124-139. https://doi.org/10.1007/978-1-4419-7823-3_11.Carbonell A, Martínez de Alba Á-E, Flores R, et al. Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families. Virology. 2008;371(1):44-53. https://doi.org/10.1016/j.virol.2007.09.031.Konakalla NC, Kaldis A, Berbati M, et al. Exogenous application of double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco. Planta. 2016;244(4):961-969. https://doi.org/10.1007/s00425-016-2567-6.Tenllado F, Díaz-Ruíz JR. Double-stranded RNA-mediated interference with plant virus infection. J Virol. 2001;75(24):12288-12297. https://doi.org/10.1128/jvi.75.24.12288-12297.2001.Gan D, Zhang J, Jiang H, et al. Bacterially expressed dsRNA protects maize against SCMV infection. Plant Cell Rep. 2010;29(11):1261-1268. https://doi.org/10.1007/s00299-010-0911-z.Tenllado F, Barajas D, Vargas M, et al. Transient expression of homologous hairpin RNA causes interference with plant virus infection and is overcome by a virus encoded suppressor of gene silencing. Mol Plant Microbe Interact. 2003;16(2):149-158. https://doi.org/10.1094/mpmi.2003.16.2.149.Yin G, Sun Z, Liu N, et al. Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system. Appl Microbiol Biotechnol. 2009;84(2):323-333. https://doi.org/10.1007/s00253-009-1967-y.Niehl A, Soininen M, Poranen MM, et al. Synthetic biology approach for plant protection using dsRNA. Plant Biotechnol J. 2018;16(9): 1679-1687. https://doi.org/10.1111/pbi.12904.Mitter N, Worrall EA, Robinson KE, et al. Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nat Plants. 2017;3:16207. https://doi.org/10.1038/nplants.2016.207.Dubelman S, Fischer J, Zapata F, et al. Environmental fate of double-stranded RNA in agricultural soils. PLoS One. 2014;9(3):e93155. https://doi.org/10.1371/journal.pone.0093155.Singh IK, Singh S, Mogilicherla K, et al. Comparative analysis of double-stranded RNA degradation and processing in insects. Sci Rep. 2017;7(1):17059. https://doi.org/10.1038/s41598-017-17134-2.Schussler MD, Alexandersson E, Bienert GP, et al. The effects of the loss of TIP1;1 and TIP1;2 aquaporins in Arabidopsis thaliana. Plant J. 2008;56(5):756-767. https://doi.org/10.1111/j.1365-313X.2008.03632.x.De Souza N. Off-targets in RNAi screens. Nat Methods. 2014;11(5):480. https://doi.org/ 10.1038/nmeth.2958.Tang G, Reinhart BJ, Bartel DP, et al. A biochemical framework for RNA silencing in plants. Genes Dev. 2003;17(1):49-63. https://doi.org/10.1101/gad.1048103.Zhang C, Ruvkun G. New insights into siRNA amplification and RNAi. RNA Biol. 2012;9(8): 1045-1049. https://doi.org/10.4161/rna. 21246.Pridgeon JW, Zhao LM, Becnel JJ, et al. Topically applied AaeIAP1 double-stranded RNA kills female adults of Aedes aegypti. J Med Entomol. 2008;45(3):414-420. https://doi.org/10.1603/ 0022-2585(2008)45[414: taadrk]2.0.co;2.Wang YB, Zhang H, Li H, et al. Second-generation sequencing supplies an effective way to screen RNAi targets in large scale for potential application in pest insect control. PLoS One. 2011;6(4): e18644. https://doi.org/10.1371/journal.pone.0018644.Killiny N, Hajeri S, Tiwari S, et al. Double-stranded RNA uptake through topical application, mediates silencing of five CYP4 genes and suppresses insecticide resistance in diaphorina citri. PLoS One. 2014;9(10): e110536. https://doi.org/10.1371/journal.pone.0110536.Miguel SK, Scott JG. The next generation of insecticides: dsRNA is stable as a foliar-applied insecticide. Pest Manag Sci. 2016;72(4):801-809. https://doi.org/10.1002/ps.4056.