Current Science

Open Access Open Access  Restricted Access Subscription Access

Development and Validation of Marker-Free Constructs Based on Nucleocapsid Protein Gene of Watermelon Bud Necrosis Orthotospovirus in Watermelon


Affiliations
1 Division of Crop Protection, ICAR-Indian Institute of Sugarcane Research, Biological Control Centre, Pravaranagar 413 712, India
2 Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
 

Marker-free (MF) constructs were developed using 825 bp and 426 bp N genes of watermelon bud necrosis orthotospovirus (WBNV). N genes were amplified and cloned in pDrive (825 bp) and/or pGEMT (426 bp) vectors and recombinants were confirmed. pCAMBIA 2300 was restricted with XhoI to release nptII gene and re-circularized; it is called MF-pCAMBIA 2300. GUS cassette with promoter and terminator from pBI 121 was cloned into the MCS region of MF-pCAMBIA 2300 with EcoRI and HindIII. Eluted PCR products of 825 and 426 bp were cloned in already developed MF-pCAMBIA 2300. These N gene constructs were then mobilized in Agrobacterium tumefaciens strain EHA 105. Agroinfiltration of A. tumefaciens harbouring MF-pCAMBIA 2300 N gene constructs was practised by syringe method in 20-day-old watermelon seedlings. PCR amplification showed 825 and 426 bp amplicons which confirmed their presence in agroinfiltrated seedlings. Further, three days of post agroinfiltration seedlings were challenge-inoculated with WBNV and kept under artificial light for 3–6 days. Agroinfiltrated and inoculated seedlings were tested by ELISA and resulted positive with OD ranging from 0.31 to 0.46 and 0.35 to 0.43 in full and partial N genes respectively. Six days post-inoculation, necrosis and complete drying of the leaves were observed. The systemic upper young leaves were subjected to DAC-ELISA and results revealed negative reaction with OD ranging from 0.11 to 0.19. The N gene constructs conferred resistance against WBNV in watermelon.

Keywords

Agroinfiltration, Bud Necrosis Disease, Orthotospovirus, Watermelon.
User
Notifications
Font Size

  • Jain, R. K., Bag, S., Umamaheswaran, K. and Mandal, B., Natural infection by tospovirus of cucurbitaceous and fabaceous vegetable crops. J. Phytopathol., 2007, 155, 22–25.

  • Pappu, H. R., Jones, R. A. C. and Jain, R. K., Global status of tospovirus epidemics in diverse cropping systems: Successes achieved and challenges ahead. Virus Res., 2009, 141, 219–236.

  • Krishnareddy, M. and Singh, S. J., Immunological and molecular based diagnosis of tospovirus infecting watermelon. In Golden Jubilee Symposium on Horticultural Research: Changing Scenario, Indian Institute of Horticultural Research Bangalore, India, 1993, pp. 247–248.

  • Singh, S. J. and Krishnareddy, M., Watermelon bud necrosis: a new tospovirus disease. Acta Hortic., 1996, 431, 68–77.

  • Jain, R. K., Pappu, H. R., Pappu, S. S., Krishanareddy, M. and Vani, A., Watermelon bud necrosis tospovirus is a distinct virus species belonging to serogroup IV. Arch. Virol., 1998, 143, 1637–1644.

  • Jain, R. K., Mandal, B., Pappu, H. R. and Holkar, S. K., ICTV taxonomic proposal 2014. 005aV. A. v2. Tospovirus_ sp. Create 1 new species in the genus Tospovirus, family Bunyaviridae. 2015; http://www.ictvonline.org/proposals-14/2014

  • Adams, M. J. et al., Changes to taxonomy and the international code of virus classification and nomenclature ratified by the International Committee on Taxonomy of Viruses. Arch. Virol., 2017, 162, 2505–2538.

  • Li, T. J. et al., Complete genome sequence of Watermelon bud necrosis virus. Arch. Virol., 2011, 156, 359–362.

  • Kunkalikar, S. R. et al., Importance and genetic diversity of vegetable-infecting tospoviruses in India. Phytopathology, 2011, 101, 367–376.

  • Kumar, R., Mandal, B., Geetanjali, A. S., Jain, R. K. and Jaiwal, P. K., Genome organisation and sequence comparison suggest intra-species incongruence in M RNA of Watermelon bud necrosis virus. Arch. Virol., 2010, 155, 1361–1365.

  • Gielen, J. J. L., de Haan, P., Kool, A. J., Peters, D., van Grinsven, M. Q. M and Goldb-ach, R. W., Engineered resistance to tomato spotted wilt virus, a negative-strand RNA virus. Biotechnology, 1991, 9, 1363–1367.

  • Prins, M., de Oliveira Resende, R., Anker, C., Van Schepen, A., Haan, P. D. and Goldbach, R., Engineered RNA-mediated resistance to Tomato spotted wilt virus is sequence specific. Mol. Plant Microbe Interact., 1996, 9, 416–418.

  • Stoeva, P. et al., Long-term resistance to Tomato spotted wilt virus in transgenic tobacco cultivars expressing the viral nucleoprotein gene: greenhouse and field tests. Mol. Breed., 1998, 4, 155–164.

  • Jan, F. J., Fagoaga, C., Pang, S. Z. and Gonsalves, D., A minimum length of N gene sequence in transgenic plants is required for RNA-mediated tospovirus resistance. J. Gen. Virol., 2000, 81, 235–242.

  • Raja, P. and Jain, R. K., Development of nucleocapsid gene mediated resistance in tomato against Groundnut bud necrosis virus. J. Plant Pathol., 2008, 90, 442.

  • David, G. R. and Shimat, V. J., Host plant resistance to Tomato spotted wilt virus (Bunyaviridae: Tospovirus) in Tomato. Hortic. Sci., 2011, 46, 1626–1633.

  • Mehdi, S. and Sudhakar, D. W., Tomato breeding for resistance to Tomato spotted wilt virus (TSWV): an overview of conventional and molecular approaches. Czech. J. Genet. Plant Breed., 2008, 44, 83–92.

  • Riley, D. G., Joseph, S. V., Srinivasan, R. and Diffie, S., Thrips vectors of tospoviruses. J. Integr. Pest Manage., 2011, 2, 1–10.

  • Sohrab, S. S., Bhattacharya, P. S., Rana, D., Kamal, M. A. and Pande, M. K., Development of interspecific Solanumlycopersicum and screening for tospovirus resistance. Saudi J. Biol. Sci., 2014, 22, 730–738.

  • Sundaraj, S., Srinivasan, R., Culbreath, A. K., Riley, D. G. and Pappu, H. R., Host plant resistance against Tomato spotted wilt virus in peanut (Arachishypogaea) and its impact on susceptibility to the virus, virus population genetics, and vector feeding behaviour and Survival. Phytopathology, 2014, 104, 202–210.

  • Pang, S. Z., Jan, F. J. and Gonsalves, D., Non-target DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants. Proc. Natl. Acad. Sci., 1997, 94, 8261–8266.

  • Duan, C. G., Wang, C. H. and Guo, H. S., Application of RNA silencing to plant disease resistance. Silence, 2012, 3, 5.

  • Catoni, M., Lucioli, A., Doblas‐Ibanez, P., Accotto, G. P. and Vaira, A. M., From immunity to susceptibility: virus resistance induced in tomato by a silenced transgene is lost as TGS overcomes PTGS. Plant J., 2013, 75, 941–953.

  • Peng, J. C., Chen, T. C., Raja, J. A. J., Yang, C. F. and Chien, W. C., Broad-spectrum transgenic resistance against distinct Tospovirus species at the genus level. PLOS ONE, 2014, 9, e96073.

  • Park, S. M. et al., Transgenic watermelon ischolar_mainstock resistant to CGMMV (Cucumber green mottle mosaic virus) infection. Plant Cell Rep., 2005, 24, 350–356.

  • Huang, Y. C., Chiang, C. H., Li, C. M. and Yu, T. A., Transgenic watermelon lines expressing the nucleocapsid gene of Watermelon silver mottle virus and the role of thiamine in reducing hyperhydricity in regenerated shoots. Plant Cell Tissue Organ. Cult., 2011, 106, 21–29.

  • Chettri, P. and Krishnaraj, P. U., Development of post-transcriptional gene silencing constructs for Groundnut Bud Necrosis Virus nucleocapsid protein gene. Res. J. Cell Mol. Biol., 2010, 4, 116–130.

  • Venkatesan, S., Raja, J. A. J., Maruthasalam, S., Kumar, K. K., Ramanathan, A., Sudhakar, D. and Balasubramanian, P., Transgenic resistance by N gene of a Peanut bud necrosis virus isolate of characteristic phylogeny. Virus Genes, 2009, 38, 445–454.

  • Yadav, R., Jain, R. K., Dobaria, J. R., Kumar, A., Thirumalaisamy, P. P. and Radhakrishnan, T., Transgenic resistance to Groundnut bud necrosis virus (GBNV) in peanut (ArachishypogaeaL.) plants expressing nucleocapsid protein gene of GBNV. In Proceedings of the 6th International Conference on Legume Genetics and Genomics, Hyderabad, India, 2012.

  • Kunkalikar, S., Marathe, R., Char, B. R., Zehr, U. and Anandlaxmi R., Transgenic approaches for virus resistance in plants. In Genetically Engineered Crops in Developing Countries (eds Reddy, D. V. R. et al.), Stadium Press, LLC, Houston, USA, 2014, pp. 271–307.

  • Hofgen, R. and Willmitzer, L., Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res., 1988, 16, 9877.

  • Wydro, M., Kozubek, E. and Lehmann, P., Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotiana benthamiana. Acta Biochim. Pol., 2006, 53, 289.

  • Bucher, E., Lohuis, D., Van-Poppel, P. M., Geerts-Dimitriadou, C., Goldbach, R. and Prins, M., Multiple virus resistance at a high frequency using a single transgene construct. J. Gen. Virol., 2006, 87, 3697–3701.

  • Yang, C. F., Chen, K. C., Cheng, Y. H., Raja, J. A., Huang, Y. L., Chien, W. C. and Yeh, S. D., Generation of marker-free transgenic plants concurrently resistant to a DNA geminivirus and a RNA tospovirus. Sci. Rep., 2014, 4, 5717.


Abstract Views: 331

PDF Views: 124




  • Development and Validation of Marker-Free Constructs Based on Nucleocapsid Protein Gene of Watermelon Bud Necrosis Orthotospovirus in Watermelon

Abstract Views: 331  |  PDF Views: 124

Authors

S. K. Holkar
Division of Crop Protection, ICAR-Indian Institute of Sugarcane Research, Biological Control Centre, Pravaranagar 413 712, India
Bikash Mandal
Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
R. K. Jain
Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India

Abstract


Marker-free (MF) constructs were developed using 825 bp and 426 bp N genes of watermelon bud necrosis orthotospovirus (WBNV). N genes were amplified and cloned in pDrive (825 bp) and/or pGEMT (426 bp) vectors and recombinants were confirmed. pCAMBIA 2300 was restricted with XhoI to release nptII gene and re-circularized; it is called MF-pCAMBIA 2300. GUS cassette with promoter and terminator from pBI 121 was cloned into the MCS region of MF-pCAMBIA 2300 with EcoRI and HindIII. Eluted PCR products of 825 and 426 bp were cloned in already developed MF-pCAMBIA 2300. These N gene constructs were then mobilized in Agrobacterium tumefaciens strain EHA 105. Agroinfiltration of A. tumefaciens harbouring MF-pCAMBIA 2300 N gene constructs was practised by syringe method in 20-day-old watermelon seedlings. PCR amplification showed 825 and 426 bp amplicons which confirmed their presence in agroinfiltrated seedlings. Further, three days of post agroinfiltration seedlings were challenge-inoculated with WBNV and kept under artificial light for 3–6 days. Agroinfiltrated and inoculated seedlings were tested by ELISA and resulted positive with OD ranging from 0.31 to 0.46 and 0.35 to 0.43 in full and partial N genes respectively. Six days post-inoculation, necrosis and complete drying of the leaves were observed. The systemic upper young leaves were subjected to DAC-ELISA and results revealed negative reaction with OD ranging from 0.11 to 0.19. The N gene constructs conferred resistance against WBNV in watermelon.

Keywords


Agroinfiltration, Bud Necrosis Disease, Orthotospovirus, Watermelon.

References





DOI: https://doi.org/10.18520/cs%2Fv114%2Fi08%2F1742-1747