Strategy for Generic Resistance Against Begomoviruses Through RNAi

  • Nikita Shukla
  • Saurabh Verma
  • G Sunil Babu
  • Sangeeta Saxena


RNA interference (RNAi) is a natural gene regulatory mechanism that limits gene expression either by suppressing transcription (transcriptional gene silencing) or by promoting the sequence-specific mRNA degradation (posttranscriptional gene silencing). RNAi utilizes dsRNA molecule along with a group of proteins consisting of Argonaute (AGO), Dicer, and few RISC-associated proteins for generation of small noncoding RNAs (ncRNAs), i.e., microRNA (miRNA) and small interfering RNA (siRNA) of 21–23 nt in length which actually bind with target mRNA and regulate their gene expression. However, there is a slight difference in their mechanism of action; for instance, miRNA partially binds to target mRNA and mainly results in translational suppression, while siRNA shows complete complementarity to putative mRNA and cleaves it resulting in gene silencing. With growing evidence every day, one of the important functions of RNAi in molecular biology seems to be protection of host genome against viruses. In case of plant viruses, begomoviruses impose a serious threat to mankind as they infect several crops like tomato, cotton, papaya, etc. leading to huge economic losses. Though several physical, chemical, and transgenic strategies are in practice to provide resistance against begomoviruses, none of them have proved out to be successful. Here we propose a strategy to develop generic resistance against begomoviruses by generating small siRNAs using various in silico strategies.


  1. Abel PP, Nelson RS et al (1986) Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232(4751):738–743CrossRefPubMedGoogle Scholar
  2. Ahlquist P (2002) RNA dependent RNA polymerases, viruses and RNA silencing. Science 296:1270–1274CrossRefPubMedGoogle Scholar
  3. Ali I, Amin I et al (2013) Artificial microRNA-mediated resistance against the monopartite begomovirus cotton leaf curl Burewala virus. Virol J 10(1):231CrossRefPubMedPubMedCentralGoogle Scholar
  4. Alvarez JP, Pekker I et al (2006) Endogenous and synthetic microRNAs stimulate simultaneous, efficient and localized regulation of multiple targets in diverse species. Plant Cell 18:1134–1151CrossRefPubMedPubMedCentralGoogle Scholar
  5. Asad S, Haris WAA et al (2003) Transgenic tobacco expressing geminiviral RNAs are resistant to the serious viral pathogen causing cotton leaf curl disease. Arch Virol 148:2341–2352CrossRefPubMedGoogle Scholar
  6. Begomovirus V (2017) Accessed 13 Feb 2017
  7. Bendahmane M, Gronenborn B (1997) Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA. Plant Mol Biol 33(2):351–357CrossRefPubMedGoogle Scholar
  8. Bisaro DM (2006) Silencing suppression by geminivirus proteins. Virology 344(1):158–168CrossRefPubMedGoogle Scholar
  9. Brennecke J, Aravin AA et al (2007) Discrete small RNA-generating loci as master regulators of transposon activity in drosophila. Cell 128(6):1089–1103CrossRefPubMedGoogle Scholar
  10. Briddon RW, Bull SE, Amin I, Idris AM et al (2003) Diversity of DNA beta; a satellite molecule associated with some monopartite begomoviruses. Virology 312(1):106–121CrossRefPubMedGoogle Scholar
  11. Burgess DJ (2013) Small RNAs: defining piRNA expression. Nat Rev Genet 14:301CrossRefPubMedGoogle Scholar
  12. Bucher E, Lohuis D et al (2006) Multiple virus resistance at a high frequency using a single transgene construct. J Gen Virol 87:3697–3701CrossRefPubMedGoogle Scholar
  13. Carbonell A, Takeda A et al (2014) New generation of artificial MicroRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Physiol 165:15–29CrossRefPubMedPubMedCentralGoogle Scholar
  14. Carmell M, Xuan Z, Zhang M, Hannon G (2002) The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev 16(21):2733–2742CrossRefPubMedGoogle Scholar
  15. Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs. Cell 136(4):642–655CrossRefPubMedPubMedCentralGoogle Scholar
  16. Chen J, Li WX et al (2004) Viral virulence protein suppresses RNA silencing-mediated defense but upregulates the role of microRNA in host gene expression. Plant Cell 16:1302–1131CrossRefPubMedPubMedCentralGoogle Scholar
  17. Chung WJ, Okamura K et al (2008) Endogenous RNA interference provides a somatic defense against Drosophila transposons. Curr Biol 18(11):795–802CrossRefPubMedPubMedCentralGoogle Scholar
  18. Cogoni C, Irelan JT et al (1996) Transgene silencing of the AL-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation. EMBO J 15(12):3153–3163PubMedPubMedCentralGoogle Scholar
  19. Cui XF, Tao XR et al (2004) A DNA b associated with tomato yellow leaf curl China virus is required for symptom induction. J Virol 78(24):13966–139744CrossRefPubMedPubMedCentralGoogle Scholar
  20. Cui XF, Li YQ, Hu DW, Zhou XP (2005) Expression of a begomoviral DNA b gene in transgenic Nicotiana plants induced abnormal cell division. J Zhejiang Univ Sci B 6(2):83–86CrossRefPubMedPubMedCentralGoogle Scholar
  21. Czech B, Malone CD et al (2008) An endogenous small interfering RNA pathway in Drosophila. Nature 453(7196):798–802CrossRefPubMedPubMedCentralGoogle Scholar
  22. Duan CG, Wang CH et al (2008) Artificial microRNAs highly accessible to targets confer efficient virus resistance in plants. J Virol 82(22):11084–11095CrossRefPubMedPubMedCentralGoogle Scholar
  23. Elbashir SM, Harborth J et al (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26:199–213CrossRefPubMedGoogle Scholar
  24. Fagoaga C, Lopez C et al (2006) Post-transcriptional gene silencing of the p23 silencing suppressor of citrus tristeza virus confers resistance to the transgenic Mexican lime. Plant Mol Biol 60(2):153–165CrossRefPubMedGoogle Scholar
  25. Fahim M, Larkin PJ (2013) Designing effective amiRNA and multimeric amiRNA against plant viruses. Methods Mol Biol 942:357–377CrossRefPubMedGoogle Scholar
  26. Fahim M, Millar AA et al (2012) Resistance to wheat streak mosaic virus generated by expression of an artificial polycistronic microRNA in wheat. Plant Biotechnol J 10(2):150–163CrossRefPubMedGoogle Scholar
  27. Ferreira SA, Pitz KY et al (2002) Virus coat protein transgenic papaya provides practical control of papaya ringspot virus in Hawaii. Plant Dis 86:101–105CrossRefGoogle Scholar
  28. Fitch MMM, Manshardt RM et al (1992) Virus resistant papaya derived from tissues bombarded with the coat protein gene of papaya ringspot virus. BioTechnol 10:1466–1472Google Scholar
  29. Fire A, Xu S et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811CrossRefPubMedGoogle Scholar
  30. Freier SM, Kierzek R, Jaeger JA et al (1986) Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci 83:9373–9377CrossRefPubMedPubMedCentralGoogle Scholar
  31. Ghoshal B, Sanfacon H (2015) Symptom recovery in virus-infected plants: revisiting the role of RNA silencing complex. Virology 479–480:167–179CrossRefPubMedGoogle Scholar
  32. Gonsalves C, Lee DR, Gonsalves D (2004) Transgenic virus resistant papaya: from hope to reality for controlling papaya ring spot virus in Hawaii. APSnet Features. doi: 10.1094/APSnetFeature-2004-0804.
  33. Hamilton A, Baulcombe D (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286(5441):950–952CrossRefPubMedGoogle Scholar
  34. Hanley-Bowdoin L, Settlage SB et al (1999) Geminiviruses: models for plant DNA replication transcription and cell cycle regulation. CRC Crit Rev Plant Sci 18:71–106CrossRefGoogle Scholar
  35. Hanley-Bowdoin L, Bejarano ER et al (2013) Geminiviruses: masters at redirecting and reprogramming plant processes. Nat Rev Microbiol 11(11):777–788CrossRefPubMedGoogle Scholar
  36. Harborth J, Elbashir SM, Vandenburgh K et al (2003) Sequence, chemical and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Anitisense Nucleic Acid Drug Dev 13(2):83–105CrossRefGoogle Scholar
  37. Hirai S, Kodama H (2008) RNAi vectors for manipulation of gene expression in higher plants. Open Plant Sci J 2:21–30CrossRefGoogle Scholar
  38. Hong Y, Levay K et al (1995) A potyvirus polymerase interacts with the viral coat protein and VPg in yeast cells. Virology 214(1):159–166CrossRefPubMedGoogle Scholar
  39. Jackson AL, Burchard J, Leake D et al (2006) Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 12:1197–1205CrossRefPubMedPubMedCentralGoogle Scholar
  40. Kasschau KD, Xie Z et al (2003) P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Dev Cell 4:205–217CrossRefPubMedGoogle Scholar
  41. Khatoon S, Kumar A et al (2016) RNAi- mediated resistance against cotton leaf curl disease in elite Indian cotton (Gossypium hirsutum) cultivar ‘Narasimha’. Virus Genes 52(4):530–553CrossRefPubMedGoogle Scholar
  42. Khvorova A, Reynolds A, Jayasena SD (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209–216CrossRefPubMedGoogle Scholar
  43. Khraiwesh B, Ossowski S et al (2008) Specific gene silencing by artificial MicroRNAs in Physcomitrella patens: an alternative to targeted gene knockouts. Plant Physiol 148(2):684–693CrossRefPubMedPubMedCentralGoogle Scholar
  44. Kis A, Tholt G et al (2016) Polycistronic artificial miRNA-mediated resistance to wheat dwarf virus in barley is highly efficient at low temperature. Mol Plant Pathol 17(3):427–437CrossRefPubMedGoogle Scholar
  45. Klingelhoefer JW, Moutsianas L, Holmes C (2009) Approximate Bayesian feature selection on a large meta-dataset offers novel insights on factors that effect siRNA potency. Bioinformatics 25:1594–1601CrossRefPubMedPubMedCentralGoogle Scholar
  46. Liang G, He H et al (2012) A new strategy for construction of artificial miRNA vectors in Arabidopsis. Planta 235:1421–1429CrossRefPubMedGoogle Scholar
  47. Liu Q, Zhou H et al (2012) Reconsideration of in-silico siRNA design based on feature selection: a cross-platform data integration perspective. PLoS One 7(5):e37879CrossRefPubMedPubMedCentralGoogle Scholar
  48. Lozano G, Trenado HP et al (2016) Characterization of non-coding DNA satellites associated with Sweepoviruses (genus Begomovirus, Geminiviridae)- definition of a distinct class of Begomovirus-associated satellites. Front Microbiol 7:162CrossRefPubMedPubMedCentralGoogle Scholar
  49. Lu C, Tej SS et al (2005) Elucidation of the small RNA component of the transcriptome. Science 309(5740):1567–1569CrossRefPubMedGoogle Scholar
  50. MacRae IJ, Zhou K et al (2006) Structural basis for double-stranded RNA processing by Dicer. Science 311(5758):195–198CrossRefPubMedGoogle Scholar
  51. Matranga C, Tomari Y et al (2005) Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123(4):607–620CrossRefPubMedGoogle Scholar
  52. Medina-Hernandez D, Rivera-Bustamante R et al (2013) Effects and effectiveness of two RNAi constructs for resistance to pepper golden mosaic virus in Nicotiana benthamiana plants. Virus 5:2931–2945CrossRefGoogle Scholar
  53. Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431(7006):343–349CrossRefPubMedGoogle Scholar
  54. Molecular biology select (2006) Cell 126(2): 223–225. Accessed 12 Dec 2016Google Scholar
  55. Molnar A, Bassett A et al (2009) Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J 58(1):165–174CrossRefPubMedGoogle Scholar
  56. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric Chalcone synthase gene into Petunia results in co-suppression of homologous genes in trans. Plant Cell 2(4):279–289CrossRefPubMedPubMedCentralGoogle Scholar
  57. Niu QW, Lin SS, Reyes JL et al (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 24:1420–1428CrossRefPubMedGoogle Scholar
  58. Padidam M, Beachy RN, Fauquet CM (1996) The role of AV2 (‘precoat’) and coat protein in viral replication and movement in tomato leaf curl geminivirus. Virology 224(2):390–404CrossRefPubMedGoogle Scholar
  59. Patil BL, Ogwok E et al (2011) RNAi-mediated resistance to diverse isolates belonging to two virus species involved in cassava brown streak disease. Mol Plant Pathol 12(1):31–41CrossRefPubMedGoogle Scholar
  60. Pooggin M, Shivprasad PV et al (2003) RNAi targeting of DNA virus in plants. Nat Biotechnol 21:131–132CrossRefPubMedGoogle Scholar
  61. Pratt AJ, MacRae IJ (2009) The RNA-induced silencing complex: a versatile gene-silencing machine. J Biol Chem 284(27):17897–17901CrossRefPubMedPubMedCentralGoogle Scholar
  62. Qu J, Ye J, Fang R (2007) Artificial MicroRNA-mediated virus resistance in plants. J Virol 81(12):6690–6699CrossRefPubMedPubMedCentralGoogle Scholar
  63. Reynolds A, Leake D, Boese Q et al (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22:326–330CrossRefPubMedGoogle Scholar
  64. Sano T, Matsuura Y (2004) Accumulation of small interfering RNAs characteristic of RNA silencing precedes recovery of tomato plants from severe symptoms of potato spindle tuber viroid infection. J Gen Plant Pathol 70(1):50–53CrossRefGoogle Scholar
  65. Saxena S, Kesharwani RK, Singh V (2013) Designing of putative siRNA against geminiviral suppressors of RNAi to develop geminivirus-resistant papaya crop. IJBRA 9(1):3–12CrossRefPubMedGoogle Scholar
  66. Saxena S, Singh N et al (2011) Strategy for a generic resistance to geminiviruses infecting tomato and papaya through in silico siRNA search. Virus Genes 43:409–434CrossRefPubMedGoogle Scholar
  67. Schultz N, Marenstein DR et al (2011) Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-β pathway and reveal microRNA regulation of TGFBR2. Silence 2:3CrossRefPubMedPubMedCentralGoogle Scholar
  68. Schwab R, Ossowski et al (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18(5):1121–1133CrossRefPubMedPubMedCentralGoogle Scholar
  69. Seto AG, Kingston RE, Lau NC (2007) The coming age for Piwi proteins. Mol Cell 26(5):603–609CrossRefPubMedGoogle Scholar
  70. Sharma VK, Kushwaha N et al (2015) Identification of siRNA generating hot spots in multiple viral suppressors to generate broad-spectrum antiviral resistance in plants. Physiol Mol Biol Plants 21(1):9–18CrossRefPubMedGoogle Scholar
  71. Shekhawat UKS, Ganapathi TR, Hadapad AB (2012) Transgenic banana plants expressing small interfering RNAs targeted against viral replication initiation gene display high-level resistance to banana bunchy top virus infection. J Gen Virol 93(8):1804–1813CrossRefPubMedGoogle Scholar
  72. Siomi H, Siomi MC (2009) On the road to reading the RNA-interference code. Nature 457(7228):396–404CrossRefPubMedGoogle Scholar
  73. Siomi MC, Sato K, Pezic D, Aravin AA (2011) PIWI-interacting small RNAs: the vanguard of genome defense. Nat Rev Mol Cell Biol 12:246–258CrossRefPubMedGoogle Scholar
  74. Stein DA, Perry ST, Buck MD (2011) Inhibition of dengue virus infections in cell cultures and in AG129 mice by a small interfering RNA targeting a highly conserved sequence. J Virol 85(19):10154–10166CrossRefPubMedPubMedCentralGoogle Scholar
  75. Sunter G, Hartitz MD et al (1990) Genetic analysis of tomato golden mosaic virus: ORF AL2 is required for coat protein accumulation while ORF AL3 is necessary for efficient DNA replication. Virology 179(1):69–77CrossRefPubMedGoogle Scholar
  76. Sunter G, Bisaro DM (1992) Transactivation of geminivirus AR1 and BR1 gene expression by the AL2 gene product occurs at the level of transcription. Plant Cell 4(10):1321–1331CrossRefPubMedPubMedCentralGoogle Scholar
  77. Tafer H, Ameres SL et al (2008) The impact of target site accessibility on the design of effective siRNAs. Nat Biotechnol 26(5):578–583CrossRefPubMedGoogle Scholar
  78. Tiwari M, Sharma D, Trivedi PK (2014) Artificial microRNA mediated gene silencing in plants: progress and perspectives. Plant Mol Biol 86(1):1–18CrossRefPubMedGoogle Scholar
  79. Tuschl T, Zamore PD, Lehmann R et al (1999) Targeted mRNA degradation by double-stranded RNA invitro. Genes Dev 13:3191–3197CrossRefPubMedPubMedCentralGoogle Scholar
  80. Ui-Tei K, Naito Y, Takahashi F et al (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 32:936–948CrossRefPubMedPubMedCentralGoogle Scholar
  81. Vanitharani R, Chellappan P, Fauquet CM (2003) Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells. Proc Natl Acad Sci U S A 100(16):9632–9636CrossRefPubMedPubMedCentralGoogle Scholar
  82. Vu TV, Do VN (2016) Customization of artificial microRNA design. In: Springer protocol. Methods in molecular biology 1509. pp 235–243Google Scholar
  83. Wang L, Huang C, Yang JY (2010) Predicting siRNA potency with random forests and support vector machines. BMC Genomics 11(Suppl.3):S2CrossRefPubMedPubMedCentralGoogle Scholar
  84. Warthmann N, Chen H et al (2008) Highly specific gene silencing by artificial miRNAs in rice. PLoS One 3(3):e1829CrossRefPubMedPubMedCentralGoogle Scholar
  85. Watanabe T, Totoki Y et al (2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453(7194):539–543CrossRefPubMedGoogle Scholar
  86. Wesley SV, Helliwell CA et al (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27(6):581–590CrossRefPubMedGoogle Scholar
  87. Xuan N, Zhao C et al (2015) Development of transgenic maize with anti-rough dwarf virus artificial miRNA vector and their disease resistance. Shen Wu Gong Cheng Xue Bao 31(9):1375–1386Google Scholar
  88. Yan F, Lu Y et al (2012) A simplified method for constructing artificial microRNAs based on the osa-MIR528 precursor. J Biotechnol 60:146–150CrossRefGoogle Scholar
  89. Ye J, Qu J et al (2014) Engineering Geminivirus resistance in Jatropha curcas. Biotechnol Biofuels 7:149. doi: 10.1186/s13068-014-0149-z CrossRefPubMedPubMedCentralGoogle Scholar
  90. Yu JY, DeRuiter SL, Turner DL (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci 99:6047–6052CrossRefPubMedPubMedCentralGoogle Scholar
  91. Zecherle GN, Whelen S, Hall BD (1996) Purines are required at the 5′ends of newly initiated RNAs for optimal RNA polymerase III gene expression. Mol Cell Biol 16:5801–5810CrossRefPubMedPubMedCentralGoogle Scholar
  92. Zhang P, Vanderschuren H et al (2005) Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes. Plant Biotechnol J 3:385–397CrossRefPubMedGoogle Scholar
  93. Zhou X (2013) Advances in understanding Begomovirus satellites. Annu Rev Phytopathol 51:357–381CrossRefPubMedGoogle Scholar
  94. Zhou M, Luo H (2013) MicroRNA mediated gene regulation: potential applications for plant genetic engineering. Plant Mol Biol 83:59–75CrossRefPubMedGoogle Scholar
  95. Zrachya A, Kumar PP et al (2007) Production of siRNA targeted against TYLCV coat protein transcripts leads to silencing of its expression and resistance to the virus. Transgenic Res 16(3):385–398CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2017

Authors and Affiliations

  • Nikita Shukla
    • 1
  • Saurabh Verma
    • 1
  • G Sunil Babu
    • 1
  • Sangeeta Saxena
    • 1
  1. 1.Department of BiotechnologyBabasaheb Bhimrao Ambedkar UniversityLucknowIndia

Personalised recommendations