Abstract
Viruses are one of the most devastating plant pathogens causing severe economic losses worldwide. RNA silencing is a robust technology to knock down the expression of specific genes. This mechanism can be exploited to generate virus resistant plants through expression of the viral derived sequences. Viruses in turn have evolved to encode suppressors of RNA silencing to combat host defense. Mixed infection of plants is of common occurrence in nature and simultaneous targeting of suppressor(s) of multiple viruses offers an effective strategy. In this study, we have in silico designed siRNAs against suppressors of the two most devastating viruses of tomato, leaf curl causing tomato begomoviruses and Cucumber mosaic virus. Three different siRNA prediction programs were used to evaluate siRNAs generating capability of each sequence and common putative candidate siRNAs were selected fulfilling the stringent parameters. Our results indicated that in the case of each suppressor a particular region of 100–150 base pairs could be source of potent siRNAs referred as hotspots. Expression of these viral hot spots as a single construct in the plants would facilitate development of transgenic plants with a high degree of broad spectrum resistance against multiple viruses.
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References
Aza-Blanc P, Cooper CL, Wagner K, Batalov S, Deveraux QL, Cooke MP (2003) Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol Cell 12:627–637
Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363–366
Bisaro DM (2006) Silencing suppression by geminivirus proteins. Virology 344:158–168
Bohula EA, Salisbury AJ, Sohail M, Playford MP, Riedemann J, Southern EM, Macaulay VM (2003) The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. J Biol Chem 278:15991–15997
Bonfim K, Faria JC, Nogueira EPL, Mendes ÉA, Aragão FJL (2007) RNAi-mediated resistance to bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol Plant Microbe 20:717–726
Boulton MI (2003) Geminiviruses: major threats to world agriculture. Ann App Biol 142:143
Brigneti G, Voinnet O, Li WX, Ji LH, Ding SW, Baulcombe DC (1998) Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J 17:6739–6746
Carr RJ, Kim KS (1983) Evidence that bean golden mosaic virus invades non-phloem tissue in double infections with tobacco mosaic virus. J Gen Virol 64:2489–2492
Chakraborty S, Vanitharani R, Chattopadhyay B, Fauquet CM (2008) Supervirulent pseudorecombination and asymmetric synergism between genomic components of two distinct species of begomovirus associated with severe tomato leaf curl disease in India. J Gen Virol 89:818–828
Chellappan P, Vanitharani R, Fauquet CM (2005) MicroRNA-binding viral protein interferes with Arabidopsis development. Proc Natl Acad Sci U S A 102:10381–10386
Chen Y-K, Lohuis D, Goldbach R, Prins M (2004) High frequency induction of RNA-mediated resistance against Cucumber mosaic virus using inverted repeat constructs. Mol Breed 14:15–226
Di Nicola-Negri E, Brunetti A, Tavazza M, Ilardi V (2005) Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus. Transgenic Res 14:989–994
García-Cano E, Resende RO, Fernández-Muñoz R, Moriones E (2006) Synergistic interaction between tomato chlorosis virus and tomato spotted wilt virus results in breakdown of resistance in tomato. Phytopathology 96:1263–1269
Glick E, Zrachya A, Levy Y, Mett A, Gidoni D, Belausov E, Citovsky V, Gafni Y (2008) Interaction with host SGS3 is required for suppression of RNA silencing by tomato yellow leaf curl virus V2 protein. Proc Natl Acad Sci U S A 105:157–161
Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293–296
Hao L, Wang H, Sunter G, Bisaro DM (2003) Geminivirus AL2 and L2 proteins interact with and inactivate SNF1 kinase. Plant Cell 15:1034–1048
Heale BSE (2005) siRNA target site secondary structure predictions using local stable substructures. Nucleic Acids Res 33:e30
Holen T (2005) Mechanisms of RNAi: mRNA cleavage fragments may indicate stalled RISC. J RNAi Gene Silenc 1:21–25
Jackson AL (2006) Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 12:1197–1205
Khvorova A, Reynolds A, Jayasena SD (2003) Functional siRNAs and miRNAs Exhibit Strand Bias. Cell 115:209–216
Lin X, Ruan X, Anderson MG, McDowell JA, Kroeger PE, Fesik SW, Shen Y (2005) siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res 33:4527–4535
Mansoor S, Amrao L, Amin I, Briddon RW, Malik KA, Zafar Y (2006) First report of cotton leaf curl disease in central and southern Sindh province in Pakistan. Plant Dis 90:826
Moffat AS (1999) Geminiviruses emerge as serious crop threat. Science 286:1835
Muckstein U, Tafer H, Hackermuller J, Bernhart SH, Stadler PF, Hofacker IL (2006) Thermodynamics of RNA-RNA binding. Bioinformatics 22:1177–1182
Palukaitis P, Roossinck MJ, Dietzgen RG, Francki RI (1992) Cucumber mosaic virus. Adv Virus Res 41:281–348
Patil BL, Ogwok E, Wagaba H, Mohammed IU, Yadav JS, Bagewadi B, Taylor NJ, Kreuze JF, Maruthi MN, Alicai T et al (2011) RNAi-mediated resistance to diverse isolates belonging to two virus species involved in cassava brown streak disease. Mol Plant Pathol 12:31–41
Persengiev SP, Zhu X, Green MR (2004) Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10:12–18
Ramesh SV, Mishra AK, Praveen S (2007) Hairpin RNA-mediated strategies for silencing of tomato leaf curl virus AC1 and AC4 genes for effective resistance in plants. Oligonucleotides 17:251–257
Rentería-Canett I, Xoconostle-Cázares B, Ruiz-Medrano R, Rivera-Bustamante RF (2011) Geminivirus mixed infection on pepper plants: Synergistic interaction between PHYVV and PepGMV. Virol J 8:104
Sanford JC, Johnston SA (1985) The concept of parasite-derived resistance-Deriving resistance genes from the parasite’s own genome. J Theo Biol 113:395–405
Saxena S, Jonsson ZO, Dutta A (2003) Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells. J Biol Chem 278:44312–44319
Saxena S, Kesharwani RK, Singh V, Singh S (2013) Designing of putative siRNA against geminiviral suppressors of RNAi to develop geminivirus-resistant papaya crop. IJBRA 9:3
Saxena S, Singh N, Ranade SA, Babu SG (2011) Strategy for a generic resistance to geminiviruses infecting tomato and papaya through in silico siRNA search. Virus Genes 43:409–434
Scacheri PC (2004) Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc Natl Acad Sci U S A 101:1892–1897
Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199–208
Semizarov D, Frost L, Sarthy A, Kroeger P, Halbert DN, Fesik SW (2003) Specificity of short interfering RNA determined through gene expression signatures. Proc Natl Acad Sci U S A 100:6347–6352
Singh AK, Chattopadhyay B, Chakraborty S (2012) Biology and interactions of two distinct monopartite begomoviruses and betasatellites associated with radish leaf curl disease in India. Virol J 9:43
Syller J (2012) Facilitative and antagonistic interactions between plant viruses in mixed infections. Mol Plant Pathol 13:204–216
Tafer H, Ameres SL, Obernosterer G, Gebeshuber CA, Schroeder R, Martinez J, Hofacker IL (2008) The impact of target site accessibility on the design of effective siRNAs. Nat Biotechnol 26:578–583
Tomari Y (2004) A protein sensor for siRNA asymmetry. Science 306:1377–1380
Vanderschuren H, Alder A, Zhang P, Gruissem W (2009) Dose-dependent RNAi-mediated geminivirus resistance in the tropical root crop cassava. Plant Mol Biol 70:265–272
Vert JP, Foveau N, Lajaunie C, Vandenbrouck Y (2006) An accurate and interpretable model for siRNA efficacy prediction. BMC Bioinforma 7:520
Vickers TA, Koo S, Bennett CF, Crooke ST, Dean NM, Baker BF (2003) Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. J Biol Chem 278:7108–7118
Wege C (2009) Mixed infections of geminiviruses and unrelated RNA viruses or viroids in tomato: a multitude of effects with a highly probable impact on epidemiology and agriculture. In: Crop Plant Resistance to Biotic and Abiotic Factors: Current Potential and Future Demands, pp. 233–241
Wege C, Siegmund D (2007) Synergism of a DNA and an RNA virus: Enhanced tissue infiltration of the begomovirus Abutilon mosaic virus (AbMV) mediated by Cucumber mosaic virus (CMV). Virology 357:10–28
Xu P, Zhang Y, Kang L, Roossinck MJ, Mysore KS (2006) Computational estimation and experimental verification of off-target silencing during posttranscriptional gene silencing in plants. Plant Physiol 142:429–440
Yuan B, Latek R, Hossbach M, Tuschl T, Lewitter F (2004) siRNA selection server: an automated siRNA oligonucleotide prediction server. Nucleic Acids Res 32:W130–W134
Zhang X, Sato S, Ye X, Dorrance AE, Morris TJ, Clemente TE, Qu F (2011) Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene. Phytopathology 101:1264–1269
Zhang X, Yuan YR, Pei Y, Lin SS, Tuschl T, Patel DJ, Chua NH (2006) Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes Dev 20:3255–3268
Zrachya A, Kumar PP, Ramakrishnan U, Levy Y, Loyter A, Arazi T, Lapidot M, Gafni Y (2007) Production of siRNA targeted against TYLCV coat protein transcripts leads to silencing of its expression and resistance to the virus. Transgenic Res 16:385–398
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We are thankful to Jawaharlal Nehru University for providing DST-PURSE grant.
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Supplementary Fig. 1
A. Phylogenetic dendrogram of CMV2b sequences multiple aligned using CLC main workbench (GIF 82 kb)
Supplementary Fig. 2
A. Phylogenetic dendogram of ToLCV AV2 sequence multiple aligned using CLC main workbench (GIF 100 kb)
Supplementary Fig. 3
A. Phylogenetic dendrogram of ToLCV AC4 sequences multiple aligned using CLC main workbench (GIF 109 kb)
Supplementary Fig. 4
A. Phylogenetic dendrogram of ToLCV AC2 sequences multiple aligned using CLC main workbench (GIF 62 kb)
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Sharma, V.K., Kushwaha, N., Basu, S. et al. Identification of siRNA generating hot spots in multiple viral suppressors to generate broad-spectrum antiviral resistance in plants. Physiol Mol Biol Plants 21, 9–18 (2015). https://doi.org/10.1007/s12298-014-0264-0
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DOI: https://doi.org/10.1007/s12298-014-0264-0