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Transgenic rice lines constitutively co-expressing tlp-D34 and chi11 display enhancement of sheath blight resistance

  • Original Papers
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Biologia Plantarum

Abstract

Transgenic rice (Oryza sativa L. subsp. indica cv. White Ponni) constitutively expressing the rice thaumatin-like protein gene (tlp-D34, PR-5) individually or in combination with the rice chitinase gene (chi11, PR-3) was generated using an Agrobacterium vir helper strain with multiple copies of pTiBo542 virB and virG. Transformation with the tlp-D34 gene alone and tlp-D34 + chi11 genes yielded five and seven single-copy transgenic lines, respectively. Southern blot analysis with two probes, one flanking the right T-DNA border and the second flanking the left T-DNA border, confirmed that all transgenic plants harboured single and complete T-DNA copies. Homozygous transgenic lines were first identified in the T1 generation by Southern blot analysis and were subsequently confirmed by segregation analysis of T2 plants. Accumulation of transcripts encoded by the transgenes was confirmed in T0 plants and homozygous T2 plants by Northern blot analysis. The homozygous T2 plants harbouring tlp-D34 + chi11 genes showed 2.8- to 4.2-fold higher chitinase activity. Western blot analysis revealed the accumulation of thaumatin-like protein and chitinase in the respective transgenic plants. Upon infection with Rhizoctonia solani, the disease index reduced from 100 % in control plants to 65 % in a T3 homozygous transgenic line T4 expressing the tlp-D34 gene alone. In a T2 homozygous transgenic line CT22 co-expressing tlp-D34 and chi11 genes, the disease index reduced to 39 %.

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Abbreviations

chi11:

rice chitinase gene

hph :

hygromycin phosphotransferase gene

Hygr :

hygromycin-resistant

Hygs :

hygromycin-sensitive

MS:

Murashige and Skoog

PR:

pathogenesis-related

PUbi1:

maize ubiquitin promoter intron

tlp-D34:

rice thaumatin like protein gene

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Authors and Affiliations

  1. Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, 625021, Tamil Nadu, India

    J. M. Shah, R. Singh & K. Veluthambi

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  1. J. M. Shah
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  2. R. Singh
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  3. K. Veluthambi
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Correspondence to K. Veluthambi.

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Acknowledgements: We gratefully acknowledge Dr. S. Muthukrishnan, Kansas State University, USA, for providing tlp-D34 and chi11 genes and for TLP and chitinase antibodies. We thank Dr. Toshihiko Komari, Japan Tobacco Inc, Japan, for providing pSB1 and Dr. M. Samiappan, Tamil Nadu Agricultural University, Coimbatore, India, for providing the Rhizoctonia solani strain RS7. N. Dhurigai is acknowledged for generating the tlp-D34-transgenic plant T7. This research was supported by funds from the Department of Biotechnology (DBT) and University Grants Commission (UGC), Government of India. JMS is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for Fellowship.

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Shah, J.M., Singh, R. & Veluthambi, K. Transgenic rice lines constitutively co-expressing tlp-D34 and chi11 display enhancement of sheath blight resistance. Biol Plant 57, 351–358 (2013). https://doi.org/10.1007/s10535-012-0291-z

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  • DOI: https://doi.org/10.1007/s10535-012-0291-z

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