Abstract
Post-transcriptional gene silencing (PTGS)-mediated gene silencing exploits the cellular mechanism wherein transcripts having sequence similarity to the double-stranded RNA (dsRNA) molecules present in the cell will be subjected to degradation. PTGS is closely related to natural processes such as RNA-mediated virus resistance and cross-protection in plants. Gene silencing and the cellular machinery for affecting this phenomenon might have evolved as a natural protective measure against viral infection in plants. In PTGS, small interfering RNA (siRNA) molecules of 21–23 nucleotides length act as homology guides for triggering the systemic degradation of transcripts homologous to the siRNA molecules. PTGS phenomenon, first discovered in transgenic petunia plants harbouring chalcone synthase gene and termed co-suppression, has been subsequently exploited to target specific gene transcripts for degradation leading to manifestation of desirable traits in crop plants. Targeted gene silencing has been achieved either through the introduction of DNA constructs encoding dsRNA or antisense RNA or by deploying co-suppression constructs producing siRNAs against the transcript of interest. Understanding the mechanism of gene silencing has led to the development of several alternative strategies for inducing gene silencing in a precise and controlled way. This has paved the way for using PTGS as one of the chief functional genomics tools in plants and has helped in unraveling the mechanism of many cellular processes and identifying the focal points in pathways, besides, opening new vistas in genetic engineering of plants for human benefits. PTGS has shown great potential in silencing the deleterious genes efficiently so that value-added plant products could be obtained. Thus, PTGS has ushered in a new era in the genetic manipulation of plants for both applied and basic studies. In this review, we have outlined the basics of RNAi-mediated gene silencing and summarized the work carried out at our institute using this approach, as case studies. In particular, adopting RNAi-mediated gene silencing (a) as a method to restore fertility in transgenic male sterile lines developed based on orfH522 gene from sunflower PET1-CMS source, (b) as a tool to suppress the production of toxic proteins, ricin and RCA, in castor, and (c) as an approach to induce bud necrosis virus resistance in sunflower has been discussed. Examples from other plant systems also have been mentioned to exemplify the concept and utility of gene silencing in crop plants.
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References
Agrawal N, Dasaradhi PVN, Asif M, Malhotra P, Bhatnagar RK and Mukherjee SK 2003 RNA interference: biology, mechanism, and applications. Microbiol. Mol. Biol. Rev. 67 657–685
Arenz C and Schepers U 2003 RNA interference: From an ancient mechanism to a state of the art therapeutic application? Die Naturwissenschaften. 90 345–359
Ashfaq, MA, Reddy PS, Anil-Kumar C, Selvaraj VM and Kumar VD 2018 Ricin and RCA- The enemies within castor (Ricinus communis L.): A perspective on their biogenesis, mechanism of action, detection methods and detoxification strategies. In: C. Kole and P. Rabinowicz (Eds.), The castor bean genome Springer Nature, Switzerland, pp. 215–236
Ashfaq MA, Kirti PB and Dinesh Kumar V 2007 Development of generic PTGS and PTGS constructs for silencing ricin and RCA genes in castor (Ricinus communis L.). Extended Summaries: National Seminar on Changing Global Vegetable Oils Scenario: Issues and Challenges Before India. January 29–31 2007. Indian Society of Oilseeds Research, Hyderabad. pp 12–14
Ashfaq MA, Reddy PS, Kumar CA, Kirti PB and Kumar VD 2010 Towards functional analysis of ricin promoters. J. Oilseeds Res. 27 12–14
Ashraf MA, Rao NN, Kirti PB and Kumar VD 2009 Isolation of ricin promoters from castor, Ricinus communis L. J. Oilseeds Res. 26 208–210
Brummell DA, Balint-Kurti PJ, Harpster MH, Palys JM, Oeller PW and Gutterson N 2003 Inverted repeat of a heterologous 3′-untranslated region for high-efficiency, high-throughput gene silencing. Plant J. 33 793–800
Chan A, Crabtree J and Zhao Q 2010 Draft genome sequence of the oilseed species Ricinus communis. Nat. Biotechnol. 28 951–956
Chen W, Zhang X, Fan Y, Li B, Ryabov E, Shi N, Zhao M, Yu Z, Qin C, Zheng Q, Zhang P, Wang H, Jackson S, Cheng Q, Liu Y, Gallusci P and Hong Y 2018 A genetic network for systemic RNA silencing in plants. Plant Physiol. 176 2700–2719
Chicas A and Macino G 2001 Characteristics of post-transcriptional gene silencing. EMBO Rep. 2 992–996
Cogoni C and Macino G 1997 Isolation of quelling-defective (qde) mutants impaired in post transcriptional transgene-induced gene silencing in Neurospora crassa. Proc. Natl. Acad. Sci. USA 94 10233–10238
Dalakouras A, Wassenegger, M, Dadami E, Ganopoulos I, Pappas M and Papadopouloua K 2020 Genetically modified organism-free RNA interference: exogenous application of RNA molecules in plants. Plant Physiol. 182 38–50
de Alba AEM, Elvira-Matelot E and Vaucheret H 2013 Gene silencing in plants: a diversity of pathways. Biochim. Biophys. Acta 1829 1300–1308
Fagard M and Vaucheret H 2000 (Trans) Gene silencing in plants: How many mechanisms? Ann. Rev. Plant Physiol. Plant Mol. Biol. 51 167–194
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE and Mello CC 1998 Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391 806–811
Fletcher SJ, Reeves PT, Hoang BT and Mitter N 2020 A perspective on RNAi-based biopesticides. Front. Plant Sci. 11 51
Gray J, Picton S, Shabbeer J, Schuch W and Grierson D 1992 Molecular biology of fruit ripening and its manipulation with antisense genes. Plant Mol. Biol. 9 69–87
Ibrahim AB and Aragão FJL 2015 RNAi-mediated resistance to viruses in genetically engineered plants. In: Mysore K, Senthil-Kumar M (eds) Plant Gene Silencing Methods in Molecular Biology, Vol. 1287. Humana Press, New York, NY
Islam W, Noman A, Qasim M and Wang L 2018 Plant responses to pathogen attack: Small RNAs in focus. Int. J. Mol. Sci. 19 515
Kamthan A, Chaudhuri A, Kamthan M and Datta A 2015 Small RNAs in plants: recent development and application for crop improvement. Front. Plant Sci. 6 208
Khalid A, Zhang Q, Yasir M and Li F 2017 Small RNA based genetic engineering for plant viral resistance: application in crop protection. Front. Microbiol. 8 43
Kinney A J 1996 Development of genetically engineered soybean oils for food applications. J. Food Lipids 3 273–292
Knutzen DS, Thomson GA, Radke SE, Johnson WB, Knauf VC and Kridl JC 1992 Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase. Proc. Natl. Acad. Sci. USA 89 2624–2628
Koch A and Kogel KH 2014 New wind in the sails: improving the agronomic value of crop plants through RNAi-mediated gene silencing. Plant Biotechnol. J. 12 821–831
Kuznetsov V 2003 RNA Interference. An approach to produce knockout organisms and cell lines. Biochemistry 68 1063–76
Lee CH and Carroll BJ 2018 Evolution and diversification of small RNA pathways in flowering plants. Plant Cell Physiol. 59 2169–2187
Lindbo JA 2012 A historical overview of RNAi in plants. Methods Mol. Biol. 894 1–16
Lindbo JA and Dougherty 2005 Plant pathology and RNAi: a brief history. Ann. Rev. Phytopathol. 43 191–204
Lindbo JA and Falk BW 2017 The impact of “coat-protein mediated virus resistance” in applied plant pathology and basic research. Phytopathology 107 624–634
Lindbo JA, Silva-Rosales L, Proebsting WM and Dougherty 2003 Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. Plant Cell 5 1749–1759
Majumdar R, Rajasekaran K and Cary JW 2017 RNA interference (RNAi) as a potential tool for control of mycotoxin contamination in crop plants: concepts and considerations. Front. Plant Sci. 8 200
Mariani C, de Beuckeleer M, Truettner J, Leemans J and Goldberg RB 1990 Induction of male sterility in plant by a chimaeric ribonuclease gene. Nature 347 737–741
Mariani C, Gossele V, de Beuckeleer M, de Block M, Goldberg RB, de Greef W and Leemans J 1992 A chimeric ribonuclease inhibitor gene restores fertility to male sterile plants. Nature 357 384–387
Meena AK, Verma LK and Kumhar BL 2017 RNAi: Its mechanism and potential use in crop improvement: a review. Int. J. Pure App. Biosci. 5 294–311
Mlotshwa S, Voinnet O, Mette MF, Matzke M, Vaucheret H, Ding SW, Pruss G, and Vance VB 2002 RNA silencing and the mobile silencing signal. Plant Cell 14 S289–S301
Napoli C, Lemieux C and Jorgensen R 1990 Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2 279–289
Narayanan U, Kang B and Lee WS 2004 Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells. FEBS Lett. 566 307–10
Nizampatnam NR and Kumar VD 2011 Intron hairpin and transitive RNAi mediated silencing of orfH522 transcripts restores male fertility in transgenic male sterile tobacco plants expressing orfH522. Plant Mol. Biol. 76 557–573
Nizampatnam NR, Harinath D, Yamini KN, Sujatha M and Kumar VD 2009 Expression of sunflower cytoplasmic male sterility-associated open reading frame, orfH522 induces male sterility in transgenic tobacco plants. Planta 229 987–1001
Qi Y and Hannon GJ 2005 Uncovering RNAi mechanisms in plants: biochemistry enters the foray. FEBS Lett. 579 5899–5903
Ramesh JP, Chandrakant ES and Sahebrao WY 2020 RNAi induced gene silencing journey from simple dsRNA to high-throughput intron hairpin RNA construct in crop improvement. DOI: http://dx.doi.org/10.5772/intechopen.93012
Ranjan T, Kumari N, Sahni S and Prasad B 2019 RNA Interference: a versatile tool for functional genomics and unraveling the genes required for viral disease resistance in Plants. Curr. J. Appl. Sci. Technol. 35 1–20
Ray K, Bisht N, Pental D and Burma P 2007 Development of barnase/barstar transgenics for hybrid seed production in Indian oilseed mustard (Brassica juncea L. Czern & Coss) using a mutant acetolactate synthase gene conferring resistance to imidazolinone-based herbicide ‘Pursuit. Curr. Sci. 93 1390–1396
Reddy PS, Ashfaq MA, Kumar CA, Kirti PB and Kumar VD 2009 Development of transgenic tobacco model system as a prelude to identify the efficient PTGS technology for silencing ricin and RCA in castor, Ricinus communis L. J. Oilseeds Res. 26 199–202
Reddy PS, Sharan ST and Kumar VD 2010 Transgenic tobacco - as a model for identifying the most efficient PTGS technology at silencing ricin and RCA in castor, Ricinus communis L. J. Oilseeds Res. 27 7–11
Roberts LM, Lamb FI, Pappin DJC and Lord JM 1985 The primary sequence of Ricinus communis agglutinin. Comparison with ricin. J. Biol. Chem. 260 15682–15688
Romano N and Macino G 1992 Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol. Microbiol. 6 3343–3353
Runo S 2011 Engineering host-derived resistance against plant parasites through RNA interference: challenges and opportunities. Bioeng. Bugs 2 208–213
Saikumar K and Kumar VD 2014 Plant microRNAs: an overview. In: Kavikishore PB, Bandopadhyay R, Suravajhala P (eds) Agricultural Bioinformatics. Springer, New Delhi
Saikumar K, Swamy HHK and Kumar VD 2009 Enemy of enemy: In silico designed amiRNAs to silence ricin genes in castor, Ricinus communis L. J. Oilseeds Res. 26 188–190
Singh A, Gautam V, Singh S, Das SS, Verma S, Mishra V, Mukherjee S and Sarkar AK 2018 Plant small RNAs: advancement in the understanding of biogenesis and role in plant development. Planta 248 545–558
Smith NA, Singh SP and Wang M 2000 Total silencing by intron spliced hairpin RNAs. Nature 407 319–320
Vasavi S, Vijay SR, Tarakeswari M, Revathi T, Jain RK, Rao CS, Varaprasad KS and Sujatha M 2018 Genetic engineering of sunflower (Helianthus annuus L.) for resistance to necrosis disease through deployment of the TSV coat protein gene. Plant Cell Tissue Organ Cult. 135 263–277
Vaucheret H, Béclin C and Fagard M 2001 Post-transcriptional gene silencing in plants. J. Cell Sci. 114 3083–3091
Watanabe Y 2011 Overview of Plant RNAi. In: Kodama H, Komamine A (eds) RNAi and Plant Gene Function Analysis. Methods in Molecular Biology (Methods and Protocols), Vol 744. Humana Press
Waterhouse PM and Helliwell CA 2003 Exploring plant genomes by RNA-induced gene silencing. Nat. Rev. Genet. 4 29–38
Waterhouse PM and Helliwell CA 2003 Exploring plant genomes by RNA-induced gene silencing. Nat. Rev. Genet. 4 29–38
Watson JM, Fusaro AF, Wang M and Waterhouse PM 2005 RNA silencing platforms in plants. FEBS Lett. 579 5982–5987
Weiberg A, Bellinger M and Jin H 2015 Conversations between kingdoms: small RNAs. Curr. Opin. Biotechnol. 32 207–215
Wesley SV, Helliwell CA, Smith NA, Wang M, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG and Waterhouse PM 2001 Construct design for effective and high-throughput gene silencing in plants. Plant J. 27 581–590
Wesley SV, Liu Q, Wielopolska A, Ellacott G, Smith N, Singh S and Helliwell C 2003 Custom knock-outs with hairpin RNA-mediated gene silencing. Methods Mol. Biol. 236 273–286
Yang Z and Li Y 2018 Dissection of RNAi-based antiviral immunity in plants. Curr. Opin. Virol. 32 88–99
Yu XD, Liu ZC, Huang SL, Chen ZQ, Sun YW, Duan PF, Ma YZ and Xia LQ 2016 RNAi-mediated plant protection against aphids. Pest Manag. Sci. 72 1090–1098
Yu Y, Jia T and Chen X 2017 The ‘how’ and ‘where’ of plant microRNAs. New Phytol. 216 1002–1017
Zhang X, Zhu Y, Wu H and Guo H 2016 Post-transcriptional gene silencing in plants: a double-edged sword. Sci. China Life Sci. 59 271–276
Zhang N, Zhang D, Chen SL, Gong BQ, Guo Y, Xu L, Zhang XN and Li JF 2018 Engineering artificial microRNAs for multiplex gene silencing and simplified transgenic screen. Plant Physiol. 178 989–1001
Acknowledgements
The financial help by Indian Council of Agricultural Research, New Delhi, through a research grant (Code No. 030558005), Department of Biotechnology, New Delhi, through research grants (Code No. BT/PR9589/AGR/02/441/07 and BT/PR/9836/AGR/36/17/2007) to VDK for carrying out the research work on developing cell ablation system using orfH522 work; research Grant (Code SR/SO/BB-58/2005) by Department of Science Technology, New Delhi, to VDK to carry out research work on reducing ricin and RCA in castor through PTGS approaches; and the financial assistance from the Department of Biotechnology, New Delhi, through a research Grant (BT/PR9572/ARG/02/432/2007) to MS for work on developing transgenic sunflower lines are gratefully acknowledged. Research fellowship provided by CSIR to MAA is also acknowledged. The authors are also grateful to the Director, ICAR-IIOR, Hyderabad, for providing all the logistics support to carry out the research work presented in this article.
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This article is part of the Topical Collection: Genetic Intervention in Plants: Mechanisms and Benefits.
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Ashfaq, M.A., Dinesh Kumar, V., Soma Sekhar Reddy, P. et al. Post-transcriptional gene silencing: Basic concepts and applications. J Biosci 45, 128 (2020). https://doi.org/10.1007/s12038-020-00098-3
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DOI: https://doi.org/10.1007/s12038-020-00098-3