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Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference

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Biologia Plantarum

Abstract

The coat protein (CP) gene of Tobacco streak virus (TSV) from sunflower (Helianthus annuus L.) was amplified, cloned and sequenced. A 421 bp fragment of the TSV coat protein gene was amplified and a gene construct encoding the hairpin RNA (hpRNA) of the TSV-CP sequence was made in the plasmid pHANNIBAL. The construct contains sense and antisense CP sequences flanking a 742 bp spacer sequence (Pdk intron) under the control of the constitutive CaMV35S promoter. A 3.6 kb Not I fragment containing the hpRNA cassette (TSV-CP) was isolated from pHANNIBAL and sub-cloned into the binary vector pART27. This chimeric gene construct was then mobilized into Agrobacterium tumefaciens strain LBA4404 via triparental mating using pRK2013 as a helper. Sunflower (cv. Co 4) and tobacco (cv. Petit Havana) plants were transformed with A. tumefaciens strain LBA4404 harbouring the hpRNA cassette and in vitro selection was performed with kanamycin. The integration of the transgene into the genome of the transgenic lines was confirmed by PCR analysis. Infectivity assays with TSV by mechanical sap inoculation demonstrated that both the sunflower and tobacco transgenic lines exhibited resistance to TSV infection and accumulated lower levels of TSV compared with non-transformed controls.

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Abbreviations

CP:

coat protein

PDR:

pathogen derived resistance

TSV:

Tobacco streak virus

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Authors and Affiliations

  1. Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore, 641 003, Tamil Nadu, India

    K. Pradeep, V. K. Satya, M. Selvapriya, A. Vijayasamundeeswari, D. Ladhalakshmi, V. Paranidharan, R. Rabindran, R. Samiyappan, P. Balasubramanian & R. Velazhahan

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  1. K. Pradeep
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  2. V. K. Satya
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  3. M. Selvapriya
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  5. D. Ladhalakshmi
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  9. P. Balasubramanian
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  10. R. Velazhahan
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Correspondence to R. Velazhahan.

Additional information

Acknowledgements: This research was supported by the Department of Biotechnology, Government of India. We thank the CSIRO Plant Industry, Australia for providing the vector pHANNIBAL. Our thanks are also due to Dr. S. Winter, DSMZ, Germany for providing us the TSV antibody.

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Pradeep, K., Satya, V.K., Selvapriya, M. et al. Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference. Biol Plant 56, 735–741 (2012). https://doi.org/10.1007/s10535-012-0111-5

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  • DOI: https://doi.org/10.1007/s10535-012-0111-5

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