Symptom Severity of Nicotiana benthamiana Plants Inoculated with Agrobacterium Containing Infectious DNA-A Clones of Honeysuckle Yellow Vein Virus (HYVV)

  • Sung Oh Department of Biology & Medicinal Science, Pai Chai University
  • Chang Won Choi Department of Biology & Medicinal Science, Pai Chai University


To investigate the pathogenicity and virulence of the Honeysuckle yellow vein virus (HYVV) lacking betasatellites, PCR amplified unit-lengths of DNA-A genome of HYVV-[DJ] were cloned into binary vector pRI101-AN, and generated HYVV-[DJ]-1mer, -1.3mer and -2mer genomes. Each construct was transformed into Agrobacterium cells and agro-inoculated into young leaves of Nicotiana benthamiana. Except for the HYVV-[DJ]-1mer, HYVV-[DJ]-1.3mer and -2mer clones caused pronounced disease symptoms in N. benthamiana. HYVV-[DJ]-2mer agro-inoculated plants showed more severe plant stunting with downward leaf curling and crinkling than those of HYVV-[DJ]-1.3mer agro-inoculated plants. To discriminate the clone’s virulence quantitatively, SYBR Green-based real-time PCR was performed for the quantification of the target virulence gene DNA in agro-inoculated plants that were collected at weekly intervals for 4 weeks. Regression analysis was obtained from the standard curves by plotting Ct values over the logarithm of the amount of V1 protein gene DNA present in a dilution series of plasmid containing the full-length HYVV-[DJ] genome. Equation of the HYVV V1 DNA standard curve was used to quantify V1 gene DNA concentration in agro-inoculated plants with each clone. The accumulation of V1 gene DNA in HYVV-[DJ]-1.3mer agro-inoculated plants reached the peak level at 4 weeks post inoculation, while the accumulation of V1 gene DNA in HYVV-[DJ]-2mer agro-inoculated plants reached the peak level at 3 weeks post inoculation. The amount of V1 DNA in HYVV-[DJ]-1.3mer agro-inoculated plants was significantly more than that in HYVV-[DJ]-2mer agro-inoculated plants. Considering the results, there was a difference between the accumulation of virus DNA and the symptom severity of the analyzed plants agro-inoculated with each clone. It suggested that the infectious clones’ virulence is not necessarily correlated with the symptom severity.

Keywords: Honeysuckle yellow vein virus (HYVV), Symptom severity, Virulence of HYVV DNA-A clones, V1 protein gene, SYBR Green-based real-time PCR


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  1. H. Kim, S. Oh, T. -K. Oh, J. S. Park, S. C. Kim, S. H. Kim, Y. S. Kim, J. K. Hong, S. -Y. Sim, K. S. Park, H. G. Lee, K. J. Kim, C. W. Choi,Genetic diversity of tomato-infecting Tomato yellow leaf curl virus (TYLCV) isolates in Korea”, Virus Genes, vol. 42, pp. 117–127, 2011.

  2. Wang, J. Ji, T. -K. Oh, S. Oh, S. H. Kim, H. J. Lee, M. Y. Shim, C. W. Choi, S. H. Kim, I. -S. Kim, Y. S. Kim, “Occurrence of Honeysuckle yellow vein virus (HYVV) containing a monopartite DNA-A genome in Korea”, European Journal of Plant Pathology, vol. 129, pp. 361–370, 2011.

  3. Oh, S. Kim, N. Vinod, J. M. Koo, K. M. Jang, C. W. Choi, S. H. Kim, Y. S. Kim, “PCR-RFLP based typing for differentiation of Tomato yellow leaf curl virus (TYLCV) genotypes from infected host plants in Korea”, Virus Genes, vol. 47, pp. 579-583, 2013.

  4. A. Valverde, S. Sabanadzovic, J. Hammond, “Viruses that enhance the aesthetics of some ornamental plants: beauty or beast? ” , Plant Diseases, vol. 96, pp. 600-611, 2012.

  5. Lee, S. Kim, J. Jung, C. -K. Auh, E. Choi, M. Chang, S. Lee, “Agroinoculation of Nicotiana benthamiana with cloned honeysuckle yellow vein virus isolated from Lonicera japonica”, Archives of Virology, vol. 156, pp. 785–791, 2011.

  6. Ueda, M. Onuki, K. Hanada, Y. Takanami, “Unique grouping of the Far East Asian begomovirus complex based on sequence analysis of the DNA-A genome and associated DNAβ satellite molecules isolated from tomato, honeysuckle and Eupatorium plants in Japan”, Archives of Virology, vol. 153, pp. 417–426, 2008.

  7. Kumar, S. R. Palcheria, B. Mandak, S. Kadiri, “PCR based detection of betasatellite associated with the begomoviruses using improved universal primers”, Australasian Plant Pathology, vol. 47, pp.115–118, 2018.

  8. Swarnalatha, M. Krishna Reddy, “Duplex PCR for simultaneous detection of Begomovirus and phytoplasma from naturally infected tomato”, Pest Management in Horticultural Ecosystems, vol. 20, pp. 59–68, 2014.

  9. K. Brown, M. Z.Ur-Rehman, S. Avelar, N. Chingandu, U. Hameed, S. Haider, M. Ilyas, “Molecular diagnostic development for begomovirus-betasatellite complexes undergoing diversification: a case study”, Virus Research, vol. 241, pp.29–41, 2017.

  10. Noris, L. Miozzi, “Real-time PCR protocols for the quantification of the begomovirus Tomato yellow leaf curl Sardinia virus in tomato plants and in its insect vector”, Plant Virology Protocols, I. Uyeda, C. Masuta (Eds.), Methods in Molecular Biology, vol. 1236, pp. 61–72, 2015.

  11. C. Papayiannis, T. A. Iacovides, N. I. Katis, J. K, Brown, “Differentiation of Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus using real-time TaqMan® PCR”, Journal of Virological Methods, vol. 165, pp. 238–245, 2010.

  12. Becker, L. Rimbaud, F. Chiroleu, B. Reynaud, G. Thébaud, J. -M. Lett, “Rapid accumulation and low degradation: key parameters of Tomato yellow leaf curl virus persistence in its insect vector Bemisia tabaci”, Scientific Reports, vol. 5, pp. 17696, 2015.

  13. -L. Tsai, H. -T. Thomas Wang, H. -F. Grace Chang, C. -F., Tsai, C. -K., Lin, P. -H. Teng, C. Su, C. -C. Jeng, P. -Y. Lee, “Development of TaqMan probe-based insulated isothermal PCR (iiPCR) for sensitive and specific on-site pathogen detection”, PLos ONE, vol. 7, pp. e45278, 2012.

  14. Ferrer, S. Henriet, C. Chamontin, S. Lainé, M. Mougel, “From cells to virus particles: quantitative methods to monitor RNA packaging”, Viruses, vol. 8, pp. 239, 2016.

  15. Y. Wang, K. Yang, C. Bai, D. Yin, G. Li, K. Qi, G. Wang, Y. Li, “ Development of a SYBR Green I real-time PCR for the detection of the orf virus”, AMB Express, vol. 7, pp. 21, 2017.

  16. Tajadini, M. Panjehpour, S. H. Javanmard, “Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes”, Advanced Biomedical Research, vol. 3, pp. 85, 2014.

  17. W. Pfaffl, “Relative quantification: Real-time PCR”, T. Dorak (Ed.), International University Line, La Jolla, CA, USA, pp. 63–82, 2006.

  18. E. Vaghchhipawala, K. S.Mysore, “Agroinoculation: a simple procedure for systemic infection of plants with viruses”, Methods in Molecular Biology, vol. 451, pp. 555-562, 2008.

  19. Lapidot, G. Weil, L. Cohen, L. Segev, V. Gaba, “Biolistic inoculation of plants with Tomato yellow leaf curl virus DNA”, Journal of Virological Methods, vol. 144, pp. 143–148, 2007.

  20. M. Al Abdallat, H. S. Al Debei, H. Asmar, S. Misbeh, A. Quraan, A. Kvarnheden, “An efficient in vitro-inoculation method for Tomato yellow leaf curl virus”, Virology Journal, vol. 7, pp. 84, 2010.

  21. G. Poornima Priyadarshini, M. V. Ambika, R. Tippeswamy, H. S. Savithri, “Functional characterization of coat protein and V2 involved in cell to cell movement of Cotton leaf curl Kokhran virus-Dabawali”, PLos ONE, vol. 6, pp. e26929, 2011.

  22. Hallan, Y. Gafni, “Tomato yellow leaf curl virus (TYLCV) capsid protein (CP) subunit interactions: Implications for viral assembly”, Archives of Virology, vol. 146, pp. 1765-1773, 2001.

  23. Gorovitis, H. Czosnek, “The involvement of heatshock proteins in the establishment of Tomato yellow leaf curl virus infection”, Frontiers in Plant Science, vol. 8, pp. 355, 2017.

  24. N. Fondong, “Geminivirus protein structure and function”, Molecular Plant Pathology, vol. 14, pp. 635-649, 2013.

  25. -J. Shi, P. Palukaitis, R. H. Symons, “Differential virulence by strains of Cucumber mosaic virus is mediated by the 2b gene”, Molecular Plant-Microbe Interactions, vol. 15, pp. 947–955, 2002.

  26. Kong, J. Wu, L. Lu, Y. Xu, X. Zhou, “Interaction between Rice stripe virus disease-specific protein and host PsbP enhances virus symptoms”, Molecular Plant, vol. 7, pp. 691–708, 2014.

  27. Araya, E. Peňa, E. Salazar, L. Román, C. Medina, R. Mora, A. Aljaro, I. -M. Rosales, “Symptom severity and viral protein or RNA accumulation in lettuce affected by big-vein disease”, Chilean Journal of Agricultural Research, vol. 71, pp. 63–72, 2011.

  28. Lafforgue, J. Sardanyés, S. F. Elena, “Differences in accumulation and virulence determine the outcome of competition during Tobacco etch virus coinfection”, PLoS ONE, vol. 6, pp. e17917, 2011.

  29. Fraile, J. -M. Hily, I. Pagán, L. F. Pacios, F. García-Arenal, “Host resistance selects for traits unrelated to resistance-breaking that affect fitness in a plant virus”, Molecular Biology and Evolution, vol. 31, pp. 928–939, 2014.

  30. Froissart, J. Doumayrou, F. Vuillaume, S. Alizon, Y. Michalakis, “The virulence-transmission trade-off in vector-borne plant viruses: a review of (non-) existing studies”, Philosophical Transactions of the Royal Society B, vol. 365, pp. 1907–1918, 2010.

How to Cite
S. Oh and C. W. Choi, “Symptom Severity of Nicotiana benthamiana Plants Inoculated with Agrobacterium Containing Infectious DNA-A Clones of Honeysuckle Yellow Vein Virus (HYVV)”, Int. Ann. Sci., vol. 7, no. 1, pp. 12-20, Apr. 2019.
Research Article

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