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Evaluating the strength of RNA silencing suppressor proteins encoded by two geminiviruses using assay based on reversal of GFP silencing

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Abstract

Virus Induced Gene Silencing (VIGS) is conserved and dynamic gene regulation machinery, which plays an active role in shielding plants against viral infections. In counter-defense, viruses encode proteins that can suppress RNA silencing to ensure successful systemic invasion. The AC2 protein of Tomato Leaf Curl New Delhi Virus (ToLCV-AC2) and Mungbean Yellow Mosaic India Virus (MYMIV-AC2) are well-characterized suppressors of RNA silencing. In this study we have compared the structure and suppressor strength of ToLCV-AC2 and MYMIV-AC2, to gain insight on the conservation of the RNA silencing suppression activities. The relative suppressor strength was measured using an assay based on the reversal of GFP silencing and quantitated in terms of the amount of reversal of GFP expression and the sustenance of the reversal activity. The phenotypic observations in the reversal of silencing assay were further corroborated with molecular analysis. Our results indicate that ToLCV-AC2 exhibited weaker RNA silencing suppression than MYMIV-AC2. The suppression activity of ToLCV-AC2 is supplemented by related, though weak, activities encoded by ToLCV-AC4 and ToLCV-AV2 proteins. Collectively the three ToLCV proteins showed suppressor strength comparable to MYMIV-AC2. These results indicate that in all probability the ToLCV deploys more than one RNA silencing suppressor protein to synergistically counteract the host RNA silencing mechanism.

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References

  • Alison T, Lima M, Silva JCF, Silva FN, Castillo-Urquiza GP, Silva FF, Seah YM, Mizubuti ESG, Duffy S, Zerbini FM (2017) The diversification of begomovirus populations is predominantly driven by mutational dynamics. Virus Evol 3:vex005

    Google Scholar 

  • Anand A, Mukherjee SK, Sanan-Mishra N (2013) Tools for pathogenicity: virus encoded RNA silencing suppressors recent microbial pathogens and strategies for combating them: Science. Technol Educ Microbiol Book Series 1:751–763

    Google Scholar 

  • Bisaro DM (2006) Silencing suppression by geminivirus proteins. Virology 344:158–168

    CAS  PubMed  Google Scholar 

  • Brown JK, Zerbini FM, Navas-Castillo J, Moriones E, Ramos-Sobrinho R, Silva JCF, Fiallo-Olivé E, Briddon RW, Hernández-Zepeda C, Idris A, Malathi VG, Martin DP, Rivera-Bustamante R, Ueda S, Varsani A (2015) Revision of Begomovirus taxonomy based on pairwise sequence comparisons. Arch Virol 160:1593–1619

    CAS  PubMed  Google Scholar 

  • Buchmann RC, Asad S, Wolf JN, Mohannath G, Bisaro DM (2009) Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation. J Virol 83:5005–5013

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nuc Acids Res 31:3497–3500

    CAS  Google Scholar 

  • Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    CAS  PubMed  Google Scholar 

  • Chowda-Reddy R, Achenjang F, Felton C, Etarock MT, Anangfac M-T, Nugent P, Fondong VN (2008) Role of a geminivirus AV2 protein putative protein kinase C motif on subcellular localization and pathogenicity. Virus Res 135:115–124

    CAS  PubMed  Google Scholar 

  • Csorba T, Kontra L, Burgyán J (2015) Viral silencing suppressors: tools forged to fine-tune host-pathogen coexistence. Virology 479-480:85–103

    CAS  PubMed  Google Scholar 

  • Cui X, Zhou X (2004) AC2 and AC4 proteins of tomato yellow leaf curl China virus and tobacco curly shoot virus mediate suppression of RNA silencing. Chin Sci Bulletin 49:2607–2612

    CAS  Google Scholar 

  • Cui X, Li G, Wang D, Hu D, Zhou X (2005) A Begomovirus DNA beta-encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus. J Virol 79:10764–10775

    CAS  PubMed  PubMed Central  Google Scholar 

  • Czosnek H, Hariton-Shalev A, Sobol I, Gorovits R, Ghanim M (2017) The incredible journey of begomoviruses in their whitefly vector. Viruses. 9:273

    PubMed Central  Google Scholar 

  • Das SS, Sanan-Mishra N (2014) Comparative analysis of RNAi suppression activity of proteins from two disparate viruses. Am J Plant Sci 5:1789

    CAS  Google Scholar 

  • Dong X, van Wezel R, Stanley J, Hong Y (2003) Functional characterization of the nuclear localization signal for a suppressor of posttranscriptional gene silencing. J Virol 77:7026–7033

    CAS  PubMed  PubMed Central  Google Scholar 

  • Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Eramian D, Shen MY, Pieper U, Sali A (2006) Comparative protein structure modeling using Modeller. Curr Protoc Protein Sci 2:2.9

    Google Scholar 

  • Fauquet C, Bisaro DM, Briddon RW, Brown JK, Harrison BD, Rybicki EP, Stenger DC, Stanley J (2003) Virology division news: revision of taxonomic criteria for species demarcation in the family Geminiviridae, and an updated list of begomovirus species. Arch Virol 148:405–421

    CAS  PubMed  Google Scholar 

  • Fondong VN (2013) Geminivirus protein structure and function. Mol Plant Pathol 14:635–649

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gopal P, Pravin Kumar P, Sinilal B, Jose J, Kasin Yadunandam A, Usha R (2007) Differential roles of C4 and βC1 in mediating suppression of post-transcriptional gene silencing: evidence for transactivation by the C2 of Bhendi yellow vein mosaic virus, a monopartite begomovirus. Virus Res 123:9–18

    CAS  PubMed  Google Scholar 

  • Gouveia P, Nolasco G (2012) The p19. 7 RNA silencing suppressor from grapevine leafroll-associated virus 3 shows different levels of activity across phylogenetic groups. Virus Genes 45:333–339

    CAS  PubMed  Google Scholar 

  • Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D (2000) Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. Crit Rev Biochem Mol Biol 35:105–140

    CAS  PubMed  Google Scholar 

  • Islam W, Akutse KS, Qasim M, Khan KA, Ghramh HA, Idrees A, Latif S (2018) Bemisia tabaci-mediated facilitation in diversity of begomoviruses: evidence from recent molecular studies. Microb Pathog 123:162–168

    CAS  PubMed  Google Scholar 

  • Jeske H (2009) Geminiviruses. Curr Top Microbiol Immunol 331:185–226

    CAS  PubMed  Google Scholar 

  • Johansen LK, Carrington JC (2001) Silencing on the spot. Induction and suppression of RNA silencing in the agrobacterium-mediated transient expression system. Plant Physiol 126:930–938

    CAS  PubMed  PubMed Central  Google Scholar 

  • Karjee S, Islam MN, Mukherjee SK (2008) Screening and identification of virus-encoded RNA silencing suppressors. Methods Mol Biol 442:187–203

    CAS  PubMed  Google Scholar 

  • Kumar V, Mishra SK, Rehman J, Taneja J, Sundaresan G, Sanan-Mishra N, Mukherjee SK (2015) Mungbean yellow mosaic Indian virus encoded AC2 protein suppresses RNA silencing by inhibiting Arabidopsis RDR6 and AGO1 activities. Virology 486:158–172

    CAS  PubMed  Google Scholar 

  • Larsson P, Wallner B, Lindahl E, Elofsson A (2008) Using multiple templates to improve quality of homology models in automated homology modeling. Protein Sci 17:990–1002

    CAS  PubMed  PubMed Central  Google Scholar 

  • Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallography 26:283–291

    CAS  Google Scholar 

  • Lazarowitz SG, Shepherd R (1992) Geminiviruses: genome structure and gene function. Cri Rev Plant Sci 11:327–349

    CAS  Google Scholar 

  • Maniatis T (1989) In: Sambrook J, Fritsch EF, Maniatis T (eds) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Moissiard G, Voinnet O (2004) Viral suppression of RNA silencing in plants. Mol Plant Pathol 5:71–82

    CAS  PubMed  Google Scholar 

  • Moriones E, Praveen S, Chakraborty S (2017) Tomato leaf curl New Delhi virus: an emerging virus complex threatening vegetable and Fiber crops. Viruses 9:264

    PubMed Central  Google Scholar 

  • Omarov RT, Scholthof HB (2012) Biological chemistry of virus-encoded suppressors of RNA silencing: an overview. Methods Mol Biol 894:39–56

    CAS  PubMed  Google Scholar 

  • 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 224:390–404

    CAS  PubMed  Google Scholar 

  • Pandey P, Choudhury NR, Mukherjee SK (2009) A geminiviral amplicon (VA) derived from tomato leaf curl virus (ToLCV) can replicate in a wide variety of plant species and also acts as a VIGS vector. Virology J 6:1–13

    Google Scholar 

  • Preiss W, Jeske H (2003) Multitasking in replication is common among geminiviruses. J Virol 77:2972–2980

    CAS  PubMed  PubMed Central  Google Scholar 

  • Qu F, Morris TJ (2005) Suppressors of RNA silencing encoded by plant viruses and their role in viral infections. FEBS Letts 579:5958–5964

    CAS  Google Scholar 

  • Raja P, Wolf JN, Bisaro DM (2010) RNA silencing directed against geminiviruses: post-transcriptional and epigenetic components. Biochimica et Biophysica Acta (BBA) 1799:337–351

    CAS  Google Scholar 

  • Rigden J, Dry I, Krake L, Rezaian M (1996) Plant virus DNA replication processes in agrobacterium: insight into the origins of geminiviruses. Proc Natl Acad Sci U S A 93:10280–10284

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rodrigo G, Carrera J, Jaramillo A, Elena SF (2011) Optimal viral strategies for bypassing RNA silencing. J Royal Soc 8:257–268

    CAS  Google Scholar 

  • Rojas MR, Jiang H, Salati R, Xoconostle-Cázares B, Sudarshana M, Lucas WJ, Gilbertson RL (2001) Functional analysis of proteins involved in movement of the monopartite begomovirus, Tomato Yellow Leaf Curl Virus. Virology 291:110–125

    CAS  PubMed  Google Scholar 

  • Rojas MR, Hagen C, Lucas WJ, Gilbertson RL (2005) Exploiting chinks in the plant's armor: evolution and emergence of geminiviruses. Annu Rev Phytopathol 43:361–394

    CAS  PubMed  Google Scholar 

  • Ruiz MT, Voinnet O, Baulcombe DC (1998) Initiation and maintenance of virus-induced gene silencing. Plant Cell 10:937–946

    CAS  PubMed  PubMed Central  Google Scholar 

  • Saunders K, Lucy A, Stanley J (1991) DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication. Nuc Acids Res 19:2325–2330

    CAS  Google Scholar 

  • Selth LA, Randles JW, Rezaian MA (2004) Host responses to transient expression of individual genes encoded by tomato leaf curl virus. Mol Plant Microbe Interactions 17:27–33

    CAS  Google Scholar 

  • Sharma P, Ikegami M (2008) RNA-silencing suppressors of geminiviruses. J Gen Plant Pathol 74:189–202

    CAS  Google Scholar 

  • Singh DK, Karjee S, Malik PS, Islam N, Mukherjee SK (2007) DNA replication and pathogenecity of MYMIV. Communicating Current Research and Educational Topics and Trends in Applied Microbiology Formatex Research Center, Badajoz, pp 155–162

    Google Scholar 

  • Song L, Gao S, Jiang W, Chen S, Liu Y, Zhou L, Huang W (2011) Silencing suppressors: viral weapons for countering host cell defenses. Prot Cell 2:273–281

    CAS  Google Scholar 

  • Stenger DC, Revington GN, Stevenson MC, Bisaro DM (1991) Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA. Proc Natl Acad Sci U S A 88:8029–8033

    CAS  PubMed  PubMed Central  Google Scholar 

  • Thresh J, Otim-Nape G, Thankappan M, Muniyappa V (1998) The mosaic diseases of cassava in Africa and India caused by whitefly-borne geminiviruses. Rev Plant Pathol 77:935–945

    Google Scholar 

  • Trinks D, Rajeswaran R, Shivaprasad PV, Akbergenov R, Oakeley EJ, Veluthambi K, Hohn T, Pooggin MM (2005) Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes. J Virol 79:2517–2527

    CAS  PubMed  PubMed Central  Google Scholar 

  • Van WR, Dong X, Liu H, Tien P, Stanley J, Hong Y (2002) Mutation of three cysteine residues in tomato yellow leaf curl virus-China C2 protein causes dysfunction in pathogenesis and posttranscriptional gene-silencing suppression. Mol Plant-Microbe Interact 15:203–208

    PubMed  Google Scholar 

  • Vance V, Vaucheret H (2001) RNA silencing in plants--defense and counterdefense. Science 292:2277–2280

    CAS  PubMed  Google Scholar 

  • Vanitharani R, Chellappan P, Pita JS, Fauquet CM (2004) Differential roles of AC2 and AC4 of cassava geminiviruses in mediating synergism and suppression of posttranscriptional gene silencing. J Virol 78:9487–9498

    CAS  PubMed  PubMed Central  Google Scholar 

  • Varma A, Malathi V (2003) Emerging geminivirus problems: a serious threat to crop production. Annals Appl Biol 142:145–164

    CAS  Google Scholar 

  • Voinnet O, Pinto YM, Baulcombe DC (1999) Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96:14147–14152

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yadava P, Suyal G, Mukherjee SK (2010) Begomovirus DNA replication and pathogenicity. Curr Sci 98:360–368

    CAS  Google Scholar 

  • Zaidi SS, Martin DP, Amin I, Farooq M, Mansoor S (2016) Tomato leaf curl New Delhi virus: a widespread bipartite begomovirus in the territory of monopartite begomoviruses. Mol Plant Pathol 18:901–911

    PubMed  PubMed Central  Google Scholar 

  • Zhang Y, Skolnick J (2005) TM-align: a protein structure alignment algorithm based on the TM-score. Nuc Acid Res 33:2302–2309

    CAS  Google Scholar 

  • Zhang T, Xu X, Huang C, Qian Y, Li Z, Zhou X (2015) A novel DNA motif contributes to selective replication of a Geminivirus-associated Betasatellite by a helper virus-encoded replication-related protein. J Virol 90:2077–2089

    PubMed  Google Scholar 

  • Zrachya A, Glick E, Levy Y, Arazi T, Citovsky V, Gafni Y (2007) Suppressor of RNA silencing encoded by tomato yellow leaf curl virus-Israel. Virology 358:159–165

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank Abhishek Anand for help with correcting the manuscript. SSD acknowledges the fellowship received from CSIR, India. GS and AT acknowledge the fellowship support from Department of Biotechnology, Government of India. The research was supported by financial grants to NSM received from the Department of Biotechnology, Government of India.

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Authors and Affiliations

  1. Plant RNAi Biology Group, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India

    Geethaa Sundaresan, Sudhanshu Sekhar Das, Anita Tripathi, Sunil K. Mukherjee & Neeti Sanan-Mishra

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  1. Geethaa Sundaresan
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  2. Sudhanshu Sekhar Das
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  3. Anita Tripathi
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  4. Sunil K. Mukherjee
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  5. Neeti Sanan-Mishra
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Contributions

GS, SSD and AT performed the analysis. SKM and NSM were involved in various stages of the concept development. GS and NSM prepared the manuscript. All the authors read and approved the final manuscript.

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Correspondence to Neeti Sanan-Mishra.

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Key Message

ToLCV-AC2 exhibits weak RNA silencing suppression than MYMIV-AC2. The RSS activity of ToLCV-AC4 and ToLCV-AV2 supplement that of ToLCV-AC2. Collectively RSS strength of three ToLCV proteins is comparable to MYMIV-AC2.

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Sundaresan, G., Das, S.S., Tripathi, A. et al. Evaluating the strength of RNA silencing suppressor proteins encoded by two geminiviruses using assay based on reversal of GFP silencing. Australasian Plant Pathol. 49, 95–106 (2020). https://doi.org/10.1007/s13313-019-00678-4

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