Abstract
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.
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References
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.
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.
Berger PH, Pirone TP. The effect of helper-component on the uptake and localization of Potyviruses in Myzus persicae. Virology. 1986;153:256–61.
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.
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.
Bruinsona J. The quantitative analysis of chlorophyll a and b in plant extract. Photochem Phytobiol. 1963;2:241–9.
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.
Carrere I, Tepfer M, Jacquemond M. Recombinants of Cucumber mosaic virus determinants of host range and symptomatology. Arch Virol. 1999;144:365–79.
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.
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.
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.
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.
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.
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.
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.
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.
Duan CG, Wang CH, Guo HS. Application of RNA silencing to plant disease resistance. Silence. 2012;3(1):5.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Janssen BJ, Gardner RC. Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol. 1989;14:61–72.
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.
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.
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.
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.
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.
King JL, Finer JJ, McHale LK. Development and optimization of agroinfiltration for soybean. Plant Cell Rep. 2015;34:133–40.
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.
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.
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.
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.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR method. Methods. 2001;25(4):402–8.
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.
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.
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.
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.
Marilyn JR. Evolutionary history of Cucumber Mosaic Virus deduced by phylogenetic analyses. J Viro. 2002;76:3382–7.
Moyer JW. Tospoviruses (Bunyaviridae). In: Webster R, Granoff A, editors. Encyclopedia of Virology. London: Academic Press Ltd.; 1999. p. 1803–7.
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.
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.
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.
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.
Patil BL, Bagewadi B, Yadav JS, Fauquet CM. Map** 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.
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.
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.
Ramiah MP, Vidhyasekharan Kandaswamy TK. Changes in photosynthetic pigments of Bhindi infected by yellow vein mosaic disease. Madras Agric J. 1972;59:402–4.
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.
Rupp JL. RNA interference mediated virus resistance in transgenic wheat (Doctoral dissertation, Kansas State University) 2015.
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.
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.
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.
Simon-Mateo C, Garcia JA. Antiviral strategies in plants based on RNA silencing. Biochim Biophys Acta. 2011;1809:722–31.
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.
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.
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.
Tenllado F, Diaz-Ruiz JR. Double-stranded RNA-mediated interference with plant virus infection. J Virol. 2001;75:12288–97.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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.
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Prasad Babu, K., Maligeppagol, M., Asokan, R. et al. Screening of a multi-virus resistant RNAi construct in cowpea through transient vacuum infiltration method. VirusDis. 30, 269–278 (2019). https://doi.org/10.1007/s13337-018-00509-y
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DOI: https://doi.org/10.1007/s13337-018-00509-y