Abstract
Cucumber mosaic virus (CMV) infects a large number of plant species including Piper nigrum L. and related species. As natural resistance to CMV is absent in Piper spp., the study was undertaken to produce transgenic P. nigrum plants harboring the complete coat protein (CP) gene of CMV via Agrobacterium-mediated transformation and their evaluation for resistance against the virus. Among one hundred and nine hardened transformed plantlets, eight revealed the presence of the transgene in PCR. The production of transcript in these plants was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and buildup of CMV CP by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). Screening of all eight transgenic lines against CMV through cleft grafting revealed that all lines except one were symptomless or showed mild or moderate symptoms. The transgenic line with the highest resistance was vegetatively propagated and integration of transgene in these clones was validated by Southern hybridization. The presence of transcript in clones was affirmed by Northern blotting– and Western blotting–ratified translation of transgene. Furthermore, relative expression studies proved manifold expression of transgene compared to actin gene as analyzed by RT-qPCR. These studies validate the stable integration and expression of transgene which might be inhibiting the movement of virus to the scions in graft inoculated plants. This is the first report on CP-mediated resistance in P. nigrum and paves the way to the production of transgenic CMV–resistant P. nigrum using CP and other desirable genes, the only effective method to combat CMV attack in the crop.
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References
Acanda Y, Canton M, Zale J (2017) Kanamycin selection in temporary immersion bioreactors allows visual selection of transgenic citrus shoots. Plant Cell Tiss Org Cult 129:351–357
Abel PP, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy RN (1986) Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738–743
Apriasti R, Widyaningrum S, Hidayati WN, Sawitri WD, Darsono N, Hase T, Sugiharto B (2018) Full sequence of the coat protein gene is required for the induction of pathogen-derived resistance against sugarcane mosaic virus in transgenic sugarcane. Mol Biol Rep 45:2749–2758
Asurmendi S, Berg RH, Smith TJ, Bendahmane M, Beachy RN (2007) Aggregation of TMV CP plays a role in CP functions and in coat protein-mediated resistance. Virology 366:98–106
Beachy RN (1997) Mechanisms and applications of pathogen derived resistance in transgenic plants. Curr Opin Biotechnol 8:215–220
Bendahmane M, Chen I, Asurmendi S, Bazzini AA, Sczecsi JB, RN, (2007) Coat protein-mediated resistance to TMV infection of Nicotiana tabacum involves multiple modes of interference by coat protein. Virology 366:107–116
Bhat AI, Devasahayam S, Anandaraj M (2013) Viral disease of black pepper: current scenario. Focus on Pepper 4:33–48
Bhat AI, Hareesh PS, Madhubala R (2005) Sequencing of coat protein gene of an isolate of Cucumber mosaic virus infecting black pepper (Piper nigrum L.) in India. J Plant Biochem Biot 14:37–40
Cuozzo M, O’Connell KM, Kaniewski W, Fang R, ChuaTumer NNE (1988) Viral protection in transgenic tobacco plants expressing the Cucumber mosaic virus coat protein or its antisense RNA. Biotechnology 6:549–555
Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA cloning system for Gram–negative bacteria: construction of a gene bank of Rhizobium melliloti. Proc Natl Acad Sci USA 77:7347–7351
Domínguez A, Cervera M, Pérez RM, Romero J, Fagoaga C, Cubero J, López MM, Juárez JA, Navarro L, Peña L (2004) Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies. Mol Breed 14:171–183
Dubey VK, Chandrasekhar K, Srivastava A, Aminuddin SVP, Dhar K, Arora PK (2015) Expression of coat protein gene of Cucumber mosaic virus (CMV subgroup IA) Gladiolus isolate in Nicotiana tabacum. J Plant Interact 10:296–304
Gal On A, Wolf D, Wang Y, Faure J, Pilowsky M, Zelcer A (1998) Transgenic resistance to Cucumber mosaic virus in tomato; blocking of long distance movement of the virus in lines harboring a defective viral replicase gene. Phytopathology 88:1101–1107
Ghaderi I, Sohani MM, Mahmoudi A (2018) Efficient genetic transformation of sour orange Citrus aurantium L. using Agrobacterium tumefaciens containing the coat protein gene of Citrus tristeza virus. Plant Gene 14:7–11
Gielen J, Ultzen T, Bontems S, Loots W, Van Schepen A (1996) Coat protein mediated protection to Cucumber mosaic virus infections in cultivated tomato. Euphytica 88:139–149
Hareesh PS, Bhat AI (2008) Detection and partial nucleotide sequence analysis of Piper yellow mottle virus infecting black pepper in India. Indian J Virol 19:160–167
He F (2011) Laemmli–SDS–PAGE. Bio–protocol Bio 101:e80, https://doi.org/10.21769/BioProtoc80
Jacquemond M, Teycheney PY, Carrere I, Navas-Castillo J, Tepfer M (2001) Resistance phenotypes of transgenic tobacco plants expressing different Cucumber mosaic virus (CMV) coat protein genes. Mol Breed 8:85–94
Jiby MV, Bhat AI (2011) An efficient Agrobacterium-mediated transformation protocol for black pepper (Piper nigrum L.) using embryogenic mass as explants. J Crop Sci Biotechnol 14:247–254
Kester DE, Davies FT Jr, Geneva RL (2002) Hartmann and Kester’s plant propagation: principles and practices. 7thed 411–460
Kim E, Noh HM, Phat C, Lee GP, Kim JH, Park TS, Lee C (2016) Identification and safety assessment of cucumber mosaic virus coat protein in genetically modified pepper (Capsicum annuum). Korean J Hortic Sci Technol 34:924–939
Kunik T, Salomon R, Zamir D, Navot N, Zeidan M, Michelson I, Gafni Y, Czosnek H (1994) Transgenic tomato plants expressing the Tomato yellow leaf curl virus capsid protein are resistant to the virus. Nat Biotechnol 12:500–504
Li XG, Chen SB, Lu ZX, Chang TJ, Zeng QC, Zhu Z (2002) Impact of copy number on transgene expression in tobacco. Acta Botanica Sinica 44:120–123
Lardet L, Leclercq J, Bénistan E, Dessailly F, Oliver G, Martin F, Montoro P (2011) Variation in GUS activity in vegetatively propagated Hevea brasiliensis transgenic plants. Plant Cell Rep 30:1847–1856
Lindbo JA, Falk BW (2017) The impact of “coat protein-mediated virus resistance” in applied plant pathology and basic research. Phytopathology 107:624–634
Lindbo JA, Silva Rosales L, Proebsting WM, Dougherty WG (1993) Induction of a highly specific antiviral state in transgenic plants: Implications for regulation of gene expression and virus resistance. Plant Cell 5:1749–1759
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25:402–408
Marcel P, Margit L, Emanuela N, Jorg S, Michael W, Mark T (2008) Strategies for antiviral resistance in transgenic plants. Mol Plant Pathol 9:73–83
Nair RR, Gupta SD (2006) High frequency plant regeneration through cyclic secondary somatic embryogenesis in black pepper (Piper nigrum L.). Plant Cell Rep 24:699–707
Namba S, Ling K, Gonsalves C, Gonsalves D, Slightom JL (1991) Expression of the gene encoding the coat protein of Cucumber mosaic virus (CMV) strain WL appears to provide protection to tobacco plants against infection by several different CMV strains. Gene 107:181–188
Okuno T, Nakayama M, Yoshida S, Furusawa I, Koyima T (1993) Comparative susceptibility of transgenic tobacco plants and protoplasts expressing the coat protein of Cucumber mosaic virus to infection with virions and RNA. Phytopathol 83:542–547
Palukaitis P, García-Arenal F (2019) Cucumber mosaic virus. The American Phytopathological Society, St, Paul, MN, USA. https://doi.org/10.1094/9780890546109
Quemeda HD, Gonsalves D, Slightom JL (1991) Expression of coat protein gene from Cucumber mosaic virus strain C in tobacco: protection against infections by CMV strains transmitted mechanically or by aphids. Phytopathol 81:794–802
Pratap D, Kumar S, Raj SK, Sharma AK (2011) Agrobacterium mediated transformation of eggplant (Solanum melongena L.) using cotyledon explants and coat protein gene of Cucumber mosaic virus. Indian J Biotech 10:19–24
Ravindran PN (2000) Black pepper (Piper nigrum L.). Hardwood academic publishers, The Netherlands
Revathy KA, Bhat AI (2019) Designing of siRNAs for various target genes of Cucumber mosaic virus subgroup IB. Indian J Biotech 18:119–125
Revathy KA, Bhat AI (2017) Complete genome sequencing of Cucumber mosaic virus from black pepper revealed rare deletion in the methyltranferase domain of 1a gene. Virus Dis 28:309–314
Riaz T, Ashfaq M, Khan Z (2022) Plants expressing CP gene of chilli veinal mottle virus showed partial resistance against the virus. Braz J Biol 82:e243692
Sambrook J, Fritsch EF, Maniatis T (1989). Molecular cloning: a laboratory manual. Cold spring harbor laboratory press
Sanford JC, Johnston SA (1985) The concept of parasite derived resistance–deriving resistance genes from the parasites own genome. J Theor Biol 113:395–405
Sarma YR, Kiranmai G, Sreenivasulu P, Anandaraj M, Hema M, Venkataramana M, Murthy AK, Reddy OVR (2001) Partial characterization and identification of a virus associated with stunt disease of black pepper (Piper nigrum L.) in South India. Curr Sci 80:459–462
Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204
Siju S, Madhubala R, Bhat AI (2008) Sodium sulphite enhances RNA isolation and sensitivity of Cucumber mosaic virus by RT– PCR in black pepper. J Virol Methods 141:107–110
Singareddy V, Sheri VR, Muddanuru T, Tatineni R, Jain RK, Sankaraneni CR, Varaprasad S, Kodeboyina VS, Mulpuri S (2018) Genetic engineering of sunflower (Helianthus annuus L.) for resistance to necrosis disease through deployment of the TSV coat protein gene. Plant Cell Tiss Org Cult 135:263–277
Srivastava A, Raj SK (2008) Coat protein mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana. J Biosci 33:242–257
Su F, Hu P, Wang W, Lan Y, Du L, Zhou Y, Zhou T (2020) Rice stripe virus coat protein-mediated virus resistance is associated with RNA silencing in Arabidopsis. Front Microbiol 11:591619
Tricoli DM, Carney KJ, Russel PF, McMaster JR, Groff DW, Hadden KC, Himmel PT, Hubbard JP, Boeshore M, Quemeda HD (1995) Field evaluation of transgenic squash containing single or multiple virus coat protein gene constructs for resistance to Cucumber mosaic virus, Watermelon mosaic virus 2, and Zucchini yellow mosaic virus. Biotechnology 13:1458–1465
Umadevi P, Anandaraj M (2015) An efficient protein extraction method for proteomic analysis of black pepper (‘Piper nigrum’ L.) and generation of protein map using nano LC-LTQ Orbitrap mass spectrometry. Plant Omics 8:500
Acknowledgements
The authors are thankful to the Department of Biotechnology, Department of Science and Technology, Government of India (BT/PR14813/AGR/02/761/2010). We are also thankful to the Director and Head (Crop Protection), ICAR–Indian Institute of Spices Research, Kozhikode, Kerala, India, for the facilities.
Funding
This study received funding from the Department of Biotechnology, Government of India (BT/PR14813/AGR/02/761/2010), and the Department of Science and Technology, Government of India (SR/WOS-A/LS-1429/2015).
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RKA designed the resistance study, performed screening of the transgenic plants and evaluation of resistance, compiled the data, and wrote the original manuscript. JMV prepared the recombinant construct, and performed the transformation, regeneration, and hardening of plantlets. AIB conceived the project, designed the program, and analyzed the data. JMV and AIB provided critical feedback on previous versions of the manuscript. All the authors read and approved the final manuscript.
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Revathy, K.A., Jiby, M.V. & Bhat, A.I. Coat protein–mediated resistance to cucumber mosaic virus subgroup IB in black pepper (Piper nigrum L.). In Vitro Cell.Dev.Biol.-Plant 58, 351–360 (2022). https://doi.org/10.1007/s11627-022-10252-1
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DOI: https://doi.org/10.1007/s11627-022-10252-1