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

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

Research output: Contribution to journalArticle

12 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)735-741
Number of pages7
JournalBiologia Plantarum
Volume56
Issue number4
DOIs
Publication statusPublished - Dec 2012

Fingerprint

Tobacco streak virus
RNA interference
Helianthus annuus
engineering
tobacco
coat proteins
RNA
Agrobacterium radiobacter
genes
amino acid sequences
genetically modified organisms
kanamycin
sap
transgenes
introns
plasmids
pathogenicity
promoter regions
genome

Keywords

  • Agrobacterium tumefaciens
  • Helianthus annuus
  • kanamycin
  • Nicotiana tabacum
  • PCR

ASJC Scopus subject areas

  • Plant Science
  • Horticulture

Cite this

Pradeep, K., Satya, V. K., Selvapriya, M., Vijayasamundeeswari, A., Ladhalakshmi, D., Paranidharan, V., ... Velazhahan, R. (2012). Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference. Biologia Plantarum, 56(4), 735-741. https://doi.org/10.1007/s10535-012-0111-5

Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference. / Pradeep, K.; Satya, V. K.; Selvapriya, M.; Vijayasamundeeswari, A.; Ladhalakshmi, D.; Paranidharan, V.; Rabindran, R.; Samiyappan, R.; Balasubramanian, P.; Velazhahan, R.

In: Biologia Plantarum, Vol. 56, No. 4, 12.2012, p. 735-741.

Research output: Contribution to journalArticle

Pradeep, K, Satya, VK, Selvapriya, M, Vijayasamundeeswari, A, Ladhalakshmi, D, Paranidharan, V, Rabindran, R, Samiyappan, R, Balasubramanian, P & Velazhahan, R 2012, 'Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference', Biologia Plantarum, vol. 56, no. 4, pp. 735-741. https://doi.org/10.1007/s10535-012-0111-5
Pradeep K, Satya VK, Selvapriya M, Vijayasamundeeswari A, Ladhalakshmi D, Paranidharan V et al. Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference. Biologia Plantarum. 2012 Dec;56(4):735-741. https://doi.org/10.1007/s10535-012-0111-5
Pradeep, K. ; Satya, V. K. ; Selvapriya, M. ; Vijayasamundeeswari, A. ; Ladhalakshmi, D. ; Paranidharan, V. ; Rabindran, R. ; Samiyappan, R. ; Balasubramanian, P. ; Velazhahan, R. / Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference. In: Biologia Plantarum. 2012 ; Vol. 56, No. 4. pp. 735-741.
@article{1f81a070e00d49edbbb58ac4ab9e131c,
title = "Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference",
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.",
keywords = "Agrobacterium tumefaciens, Helianthus annuus, kanamycin, Nicotiana tabacum, PCR",
author = "K. Pradeep and Satya, {V. K.} and M. Selvapriya and A. Vijayasamundeeswari and D. Ladhalakshmi and V. Paranidharan and R. Rabindran and R. Samiyappan and P. Balasubramanian and R. Velazhahan",
year = "2012",
month = "12",
doi = "10.1007/s10535-012-0111-5",
language = "English",
volume = "56",
pages = "735--741",
journal = "Biologia Plantarum",
issn = "0006-3134",
publisher = "Springer Netherlands",
number = "4",

}

TY - JOUR

T1 - Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference

AU - Pradeep, K.

AU - Satya, V. K.

AU - Selvapriya, M.

AU - Vijayasamundeeswari, A.

AU - Ladhalakshmi, D.

AU - Paranidharan, V.

AU - Rabindran, R.

AU - Samiyappan, R.

AU - Balasubramanian, P.

AU - Velazhahan, R.

PY - 2012/12

Y1 - 2012/12

N2 - 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.

AB - 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.

KW - Agrobacterium tumefaciens

KW - Helianthus annuus

KW - kanamycin

KW - Nicotiana tabacum

KW - PCR

UR - http://www.scopus.com/inward/record.url?scp=84862764446&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84862764446&partnerID=8YFLogxK

U2 - 10.1007/s10535-012-0111-5

DO - 10.1007/s10535-012-0111-5

M3 - Article

VL - 56

SP - 735

EP - 741

JO - Biologia Plantarum

JF - Biologia Plantarum

SN - 0006-3134

IS - 4

ER -