pp 1–10 | Cite as

Screening of a multi-virus resistant RNAi construct in cowpea through transient vacuum infiltration method

  • K. Prasad BabuEmail author
  • Manamohan MaligeppagolEmail author
  • R. Asokan
  • M. Krishna Reddy
Original Article


Plant viruses are the most devastating pathogens causing substantial economic losses in many crops. Current viral disease management relies on prophylactics, roguing and insect vector control, since in most crops resistant gene pools for resistance breeding are unavailable. RNA interference, a sequence dependent gene silencing mechanism holds great potential in imparting virus resistance. In this study, the efficacy of a RNAi gene construct developed against four viruses commonly infesting tomato and chilli viz., capsicum chlorosis virus, groundnut bud necrosis virus, cucumber mosaic virus and chilli veinal mottle virus was evaluated. A 3546 bp dsRNA-forming construct comprising sense-intron-antisense fragments in binary vector pBI121 (hpRNAi-MVR) was mobilized into Agrobacterium tumefaciens. Cowpea (Vigna unguiculata) was used as an indicator plant for GBNV agroinfiltration to evaluate the efficacy of hpRNAi-MVR construct in conferring GBNV resistance. The type of agroinfiltration, bacterial concentration and incubation-temperatures were optimized. Vacuum infiltration of three pulses of 20–30 s at 66.66 kPa were effective than syringe infiltration. Of the five Agrobacterial concentrations, OD600 0.5 was more efficient. Incubation temperature of 31 ± 1 °C was favorable for development of disease symptoms than 20 ± 1 °C and 26 ± 1 °C. ELISA revealed a 35% decline in viral load in hpRNAi-MVR infiltrated plants compared to vector control plants. Quantitative real time PCR results have shown a viral gene silencing to the extent of 930–990 folds in hpRNAi-MVR infiltrated plants compared to vector control. This approach is simple, rapid and efficient to screen the efficacy of RNAi constructs developed for the RNAi mediated plant virus management.


Agroinfiltration Tospovirus Cowpea GBNV Bacterial concentrations 



We greatly thankful to ICAR-IIHR, for providing the facility to carry out research work and thankful to the Division of Plant Pathology, ICAR-IIHR for providing the space for virus maintenance/screening. This is the part of Ph.D. dissertation work of first author.

Supplementary material

13337_2018_509_MOESM1_ESM.docx (1.4 mb)
Supplementary material 1 (DOCX 1472 kb)


  1. 1.
    Anindya R, Joseph J, Gowri TS, Savithri HS. Complete genomic sequence of pepper vein banding virus (PVBC): a distinct member of the genus potyvirus. Arch Virol. 2004;149(3):625–32.CrossRefGoogle Scholar
  2. 2.
    Aragao FJL, Nogueira EOPL, Tinoco MLP, Faria JC. Molecular characterization of the first commercial transgenic common bean immune to the Bean golden mosaic virus. J Biotechnol. 2013;166:42–50.CrossRefGoogle Scholar
  3. 3.
    Berger PH, Pirone TP. The effect of helper-component on the uptake and localization of Potyviruses in Myzus persicae. Virology. 1986;153:256–61.CrossRefGoogle Scholar
  4. 4.
    Bhaskar PB, Venkateshwaran M, Wu L, Ané JM, Jiang J. Agrobacterium-mediated transient gene expression and silencing: a rapid tool for functional gene assay in potato. PLoS ONE. 2009;4(6):e5812.CrossRefGoogle Scholar
  5. 5.
    Bonfim K, Faria JC, Nogueira EOPL, Mendes ÉA, Aragao FJL. RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol Plant Microbe Interact. 2007;20:717–26.CrossRefGoogle Scholar
  6. 6.
    Bruinsona J. The quantitative analysis of chlorophyll a and b in plant extract. Photochem Phytobiol. 1963;2:241–9.CrossRefGoogle Scholar
  7. 7.
    Bucher E, Lohuis D, Pieter M, van Poppel JA, Geerts-Dimitriadou C. Multiple virus resistance at a high frequency using a single transgene construct. J Gen Virol. 2006;87:3697–701.CrossRefGoogle Scholar
  8. 8.
    Carrere I, Tepfer M, Jacquemond M. Recombinants of Cucumber mosaic virus determinants of host range and symptomatology. Arch Virol. 1999;144:365–79.CrossRefGoogle Scholar
  9. 9.
    Chatterjee A, Ghosh SK. Alterations in biochemical components in mesta plants infected with yellow vein mosaic disease. Braz J Plant Physiol. 2008;20(4):267–75.CrossRefGoogle Scholar
  10. 10.
    Chen Q, Lai H, Hurtado J, Stahnke J, Leuzinger K, Dent M. Agroinfiltration as an effective and scalable strategy of gene delivery for production of pharmaceutical proteins. Adv Tech Biol Med. 2013;1(1):103.CrossRefGoogle Scholar
  11. 11.
    Chen X, Equi R, Baxter H, Berk K, Han J, Agarwal S, Zale J. A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings. Biotechnol Biofuels. 2010;3:9.CrossRefGoogle Scholar
  12. 12.
    Clark MF, Adam AN. Characteristics of the microplate method of enzyme linked immuno sorbent assay for detection of plant viruses. J Gen Virol. 1977;34:475–83.CrossRefGoogle Scholar
  13. 13.
    Dantre RK, Keshwal RL, Khare MN. Biochemical changes induced by yellow mosaic virus in resistant and susceptible cultivars of soybean. Indian J Virol. 1996;12:47–9.Google Scholar
  14. 14.
    De Haan P, Kormelink R, Resende R, van Poelwijk F, Peters D, Goldbach R. Tomato spotted wilt L RNA codes a putative RNA polymerase. J Gen Virol. 1991;71:2207–16.CrossRefGoogle Scholar
  15. 15.
    De Haan P, Wagemakers L, Peters D, Goldbach R. The S RNA segment of tomato spotted wilt virus has an ambisense character. J Gen Virol. 1990;71:1001–7.CrossRefGoogle Scholar
  16. 16.
    Debat HJ, Grabiele M, Ducasse DA, Lambertini PL. Use of silencing reporter and agroinfiltration transient assays to evaluate the potential of hpRNA construct to induce multiple tospovirus resistance. Biol Plant. 2015;59(4):715–25.CrossRefGoogle Scholar
  17. 17.
    Duan CG, Wang CH, Guo HS. Application of RNA silencing to plant disease resistance. Silence. 2012;3(1):5.CrossRefGoogle Scholar
  18. 18.
    Dugdale B, Mortimer CL, Kato M, James T, Harding RM, Dale JL. Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants. Nat Protoc. 2014;9:1010–27.CrossRefGoogle Scholar
  19. 19.
    Emy S, Neena M, Shanna BN, Marilyn JR, Ralf GD. Host range, symptom expression and RNA 3 sequence analyses of six Australian strains of Cucumber mosaic virus. Australas Plant Pathol. 2004;33:505–12.CrossRefGoogle Scholar
  20. 20.
    Fazeeda NH, Adrian ML, Pathmanathan U. Optimization of an Agrobacterium-mediated transient assay for gene expression studies in Anthurium andraeanum. J Am Soc Hort Sci. 2012;137(4):263–72.Google Scholar
  21. 21.
    Ghanekar AM, Reddy DVR, Iizuka N, Amin PW, Gibbons RW. Bud necrosis of groundnut (Arachis hypogaea) in India caused by tomato spotted wilt virus. Ann Appl BioI. 1979;93:173–9.CrossRefGoogle Scholar
  22. 22.
    Gielen JJ, De HP, Kool AJ, Peters D, Van GMQ, Goldbach RW. Engineered resistance to tomato spotted wilt virus, a negative–strand RNA virus. Nat Biotechnol. 1991;9:1363–7.CrossRefGoogle Scholar
  23. 23.
    Hobbs HA, Reddy DVR, Rajeshwari R, Reddy AS. Use of direct antigen coating and protein A coating ELISA procedures for detection of three peanut viruses. Plant Dis. 1987;71:747–9.CrossRefGoogle Scholar
  24. 24.
    Jabeen A, Kiran TV, Subrahmanyam D, Lakshmi DL, Bhagyanarayana G. Variations in chlorophyll and carotenoid contents in tungro infected rice plants. J Res Dev. 2017;5:153.Google Scholar
  25. 25.
    Jain RK, Bag S, Umamaheswaran K, Mandal B. Natural infection by Tospovirus of cucurbitaceous and Fabaceous vegetable crops. Ind J Phytopathol. 2007;155:22–5.CrossRefGoogle Scholar
  26. 26.
    Jain RK, Pandey AN, Krishnareddy M, Mandal B. Immunodiagnosis of groundnut and watermelon bud necrosis viruses using polyclonal antiserum to recombinant nucleocapsid protein of groundnut bud necrosis virus. J Virol Met. 2005;130:162–4.CrossRefGoogle Scholar
  27. 27.
    Janssen BJ, Gardner RC. Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol. 1989;14:61–72.CrossRefGoogle Scholar
  28. 28.
    Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987;6:3901–7.CrossRefGoogle Scholar
  29. 29.
    Johansen LK, Carrington JC. Silencing on the spot induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol. 2001;126:930–8.CrossRefGoogle Scholar
  30. 30.
    Kalantidis K, Psaradakis S, Tabler M, Tsagris M. The occurrence of CMV-specific short Rnas in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus. Mol Plant Microbe Interact. 2002;15:826–33.CrossRefGoogle Scholar
  31. 31.
    Kapila J, DeRycke R, Van Montagu M, Angenon G. An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci. 1997;122:101–8.CrossRefGoogle Scholar
  32. 32.
    Kim HJ, Kim MJ, Pak JH, Im HH, Lee DH, Kim KH, Lee JH, Kim DH, Choi HK, Jung HW, Chung YS. RNAi-mediated Soybean mosaic virus (SMV) resistance of a Korean Soybean cultivar. Plant Biotechnol Rep. 2016;10:257–67.CrossRefGoogle Scholar
  33. 33.
    King JL, Finer JJ, McHale LK. Development and optimization of agroinfiltration for soybean. Plant Cell Rep. 2015;34:133–40.CrossRefGoogle Scholar
  34. 34.
    Kormelink R, DeHaan P, Meurs C, Peters D, Goldbach R. The nucleotide sequence of the M RNA segment of tomato spotted wilt virus, a bunyavirus with two ambisense RNA segments. J Gen Virol. 1992;73:2795–804.CrossRefGoogle Scholar
  35. 35.
    Kreuze JF, Klein IS, Lazaro MU, Chuquiyuri WJC, Morgan GL, Mejía PGC, Ghislain M, Valkonen JPT. RNA silencing-mediated resistance to a crinivirus (Closteroviridae) in cultivated sweet potato (Ipomoea batatas L.) and development of sweet potato virus disease following co-infection with a potyvirus. Mol Plant Pathol. 2008;9:589–98.CrossRefGoogle Scholar
  36. 36.
    Krishnareddy M, Usha Rani R, Anil Kumar KS, Madhavi RK, Pappu HR. Capsicum chlorosis virus (Genus Tospovirus) infecting chili pepper (Capsicum annuum) in India. Plant Dis. 2008;92:1469.CrossRefGoogle Scholar
  37. 37.
    Li J, Park E, Von AAG, Nebenfuhr A. The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Met. 2009;5:6.CrossRefGoogle Scholar
  38. 38.
    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR method. Methods. 2001;25(4):402–8.CrossRefGoogle Scholar
  39. 39.
    MacKenzie DJ, Ellis PJ. Resistance to tomato spotted wilt virus infection in transgenic tobacco expressing the viral nucleocapsid gene. Mol Plant Microbe Interact. 1992;5(1):34–40.CrossRefGoogle Scholar
  40. 40.
    Manamohan M, Sharath CG, Asokan R, Deepa H, Prakash MN, Krishna KNK. One-step DNA fragment assembly for expressing intron-containing hairpin RNA in plants for gene silencing. Anal Biochem. 2012;433:189–91.CrossRefGoogle Scholar
  41. 41.
    Manavella PA, Chan RL. Transient transformation of sunflower leaf discs via an Agrobacterium-mediated method: applications for gene expression and silencing studies. Nat Protoc. 2009;4:1699–707.CrossRefGoogle Scholar
  42. 42.
    Mandal B, Jain RK, Krishnareddy M, Krishna Kumar NK, Ravi KS, Pappu HR. Emerging problems of tospoviruses (Bunyaviridae) and their management in the Indian subcontinent. Plant Dis. 2012;96:468–79.CrossRefGoogle Scholar
  43. 43.
    Marilyn JR. Evolutionary history of Cucumber Mosaic Virus deduced by phylogenetic analyses. J Viro. 2002;76:3382–7.CrossRefGoogle Scholar
  44. 44.
    Moyer JW. Tospoviruses (Bunyaviridae). In: Webster R, Granoff A, editors. Encyclopedia of Virology. London: Academic Press Ltd.; 1999. p. 1803–7.CrossRefGoogle Scholar
  45. 45.
    Mubin M, Hussain M, Briddon RW, Mansoor S. Selection of target sequences as well as sequence identity determine the outcome of RNAi approach for resistance against cotton leaf curl geminivirus complex. Virol J. 2011;8:1–8.CrossRefGoogle Scholar
  46. 46.
    Ong CA, Varghese G, Poh TW. Aetiological investigation on a veinal mottle virus of chilli (Capsicum annuum L.) newly recorded from Peninsular Malaysia. MARDI Res Bull. 1979;7:78–88.Google Scholar
  47. 47.
    Ong CA, Varghese G, Poh TW. The effect of Chilli veinal mottle virus on yield of chilli (Capsicum annuum L.). MARDI Res Bull. 1980;8:74–9.Google Scholar
  48. 48.
    Palukaitis P, Avril J, Murphy A, Manjohn CP. Virulence and differential local and systemic spread of Cucumber mosaic virus in Tobacco are affected by the CMV 2b Protein. Am Phytopathol Soc. 1992;15(7):647–53.Google Scholar
  49. 49.
    Patil BL, Bagewadi B, Yadav JS, Fauquet CM. Mapping and identification of cassava mosaic geminivirus DNA-A and DNA-B genome sequences for efficient siRNA expression and RNAi based virus resistance by transient agro-infiltration studies. Virus Res. 2016;213:109–15.CrossRefGoogle Scholar
  50. 50.
    Patil BL, Fauquet CM. Light intensity and temperature affect systemic spread of silencing signal in transient agroinfiltration studies. Mol Plant Pathol. 2015;16(5):484–94.CrossRefGoogle Scholar
  51. 51.
    Peng JC, Chen TC, Raja JAJ, Yang CF, Chien WC, Lin CH. Broad-spectrum transgenic resistance against distinct tospovirus species at the genus level. PLoS ONE. 2014;9(5):e96073.CrossRefGoogle Scholar
  52. 52.
    Ramiah MP, Vidhyasekharan Kandaswamy TK. Changes in photosynthetic pigments of Bhindi infected by yellow vein mosaic disease. Madras Agric J. 1972;59:402–4.Google Scholar
  53. 53.
    Reddy DV, Buiel AA, Satyanarayana T, Dwivedi SL, Reddy AS, Ratna AS, Vijayalakshmi K, Ranga Rao GV, Naidu RA, Wightman JA. Peanut bud necrosis disease: an overview. In: Buiel AAM, Parlevliet JE, Lenne JM, editors. Recent studies on peanut bud necrosis disease. ICRISAT conference paper no. CP 994. ICRISAT Asia Centre, Hyderabad; 1995. p. 3–7.Google Scholar
  54. 54.
    Rupp JL. RNA interference mediated virus resistance in transgenic wheat (Doctoral dissertation, Kansas State University) 2015.Google Scholar
  55. 55.
    Santos-Rosa M, Poutaraud A, Merdinoglu D, Mestre P. Development of a transient expression system in grapevine via agroinfiltration. Plant Cell Rpt. 2008;27:1053–63.CrossRefGoogle Scholar
  56. 56.
    Schob H, Kunz C, Meins F Jr. Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing. Mol Gen Genet. 1997;256:581–5.CrossRefGoogle Scholar
  57. 57.
    Simmons CW, Vandergheynst JS, Upadhyaya SK. A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression. Biotechnol Bioeng. 2009;102:965–70.CrossRefGoogle Scholar
  58. 58.
    Simon-Mateo C, Garcia JA. Antiviral strategies in plants based on RNA silencing. Biochim Biophys Acta. 2011;1809:722–31.CrossRefGoogle Scholar
  59. 59.
    Singh A, Permar V, Basavaraj A, Bhoopal ST, Praveen S. Effect of temperature on symptoms expression and viral rna accumulation in groundnut bud necrosis virus infected vigna unguiculata. Iran J Biotechnol. 2018;16(3):227–34.CrossRefGoogle Scholar
  60. 60.
    Sparkes IA, Runions J, Kearns A, Hawes C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generations of stably transformed plants. Nat Protoc. 2006;1:2019–25.CrossRefGoogle Scholar
  61. 61.
    Tenllado F, Barajas D, Vargas M, Atencio FA, González-Jara P, Díaz-Ruíz JR. 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):149–58.CrossRefGoogle Scholar
  62. 62.
    Tenllado F, Diaz-Ruiz JR. Double-stranded RNA-mediated interference with plant virus infection. J Virol. 2001;75:12288–97.CrossRefGoogle Scholar
  63. 63.
    Tsuda K, Qi Y, Nyugen LV, Bethke G, Tsuda Y, Glazebrook J, Katagiri F. An efficient Agrobacterium-mediated transient transformation of Arabidopsis. Plant J. 2011;69:713–9.CrossRefGoogle Scholar
  64. 64.
    Vander HR, Laurent F, Roth R, De WPJ. Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/cf-9-induced and Avr4/Cf-4-induced necrosis. Mol Plant Microbe Interact. 2000;13:439–46.CrossRefGoogle Scholar
  65. 65.
    Vandergheynst JS, Guo HY, Simmons C. Response surface studies that elucidate the role of infiltration conditions on Agrobacterium tumefaciens-mediated transient transgene expression in harvested switchgrass (Panicum virgatum). Biomass Bioenergy. 2008;32:372–9.Google Scholar
  66. 66.
    Vargas M, Martínez-García B, Díaz-Ruíz JR, Tenllado F. Transient expression of homologous hairpin RNA interferes with PVY transmission by aphids. Virol J. 2008;5:42.CrossRefGoogle Scholar
  67. 67.
    Wang F, Li W, Zhu J, Fan F, Wang J, Zhong W. Hairpin RNA targeting multiple viral genes confers strong resistance to rice black-streaked dwarf virus. Int J Mol Sci. 2016;17(5):705.CrossRefGoogle Scholar
  68. 68.
    Waterhouse PM, Graham MW, Wang MB. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci. 1998;95:13959–64.CrossRefGoogle Scholar
  69. 69.
    Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 2001;27:581–90.CrossRefGoogle Scholar
  70. 70.
    Wroblewski T, Tomczak A, Michelmore R. Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol J. 2005;3:259–73.CrossRefGoogle Scholar
  71. 71.
    Yang Y, Li R, Qi M. In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J. 2000;22:543–51.CrossRefGoogle Scholar
  72. 72.
    Zhang X, Sato S, Ye X, Dorrance AE, Morris TJ, Clemente TE. Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene. Phytopathology. 2011;101:1264–9.CrossRefGoogle Scholar
  73. 73.
    Zhu CX, Song YZ, Yin GH, Wen FJ. Induction of RNA-mediated multiple virus resistance to Potato virus Y, Tobacco mosaic virus, and Cucumber mosaic virus. J Phytopathol. 2009;157:101–7.CrossRefGoogle Scholar

Copyright information

© Indian Virological Society 2019

Authors and Affiliations

  1. 1.Division of BiotechnologyICAR-Indian Institute of Horticultural ResearchBangaloreIndia
  2. 2.Division of Plant PathologyICAR-Indian Institute of Horticultural ResearchBangaloreIndia
  3. 3.Department of BiotechnologyCentre for Post-graduate StudiesBangaloreIndia

Personalised recommendations