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Virus-induced gene silencing in rice using a vector derived from a DNA virus

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Abstract

Virus-induced gene silencing (VIGS) is a method of rapid and transient gene silencing in plants using viral vectors. A VIGS vector for gene silencing in rice has been developed from Rice tungro bacilliform virus (RTBV), a rice-infecting virus containing DNA as the genetic material. A full-length RTBV DNA cloned as a partial dimer in a binary plasmid accumulated in rice plants when inoculated through Agrobacterium (agroinoculation) within 2 weeks and produced detectable levels of RTBV coat protein. Deletion of two of the four viral ORFs did not compromise the ability of the cloned RTBV DNA to accumulate in rice plants. To modify the cloned RTBV DNA as a VIGS vector (pRTBV-MVIGS), the tissue-specific RTBV promoter was replaced by the constitutively expressed maize ubiquitin promoter, sequences comprising the tRNA-binding site were incorporated to ensure reverse transcription-mediated replication, sequences to ensure optimal context for translation initiation of the viral genes were added and a multi-cloning site for the ease of cloning DNA fragments was included. The silencing ability of pRTBV-MVIGS was tested using the rice phytoene desaturase (pds) gene on rice. More than half of the agroinoculated rice plants showed white streaks in leaves within 21 days post-inoculation (dpi), which continued to appear in all emerging leaves till approximately 60–70 dpi. Compared to control samples, real-time PCR showed only 10–40% accumulation of pds transcripts in the leaves showing the streaks. This is the first report of the construction of a VIGS vector for rice which can be introduced by agroinoculation.

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Abbreviations

VIGS:

Virus-induced gene silencing

RTBV:

Rice tungro bacilliform virus

PDS:

Phytoene desaturase

RNAi:

RNA interference

GLH:

Green leafhopper

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Acknowledgments

This work was funded by Department of Biotechnology, Government of India, New Delhi, Grant no. BT/AB/03/FG-I/2003 to ID. AP wishes to thank University Grants Commission, New Delhi and SM, VV and SS wish to thank Council for Scientific and Industrial Research, New Delhi for Research Fellowships.

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Authors and Affiliations

  1. Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India

    Arunima Purkayastha, Saloni Mathur, Vidhu Verma, Shweta Sharma & Indranil Dasgupta

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  1. Arunima Purkayastha
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  2. Saloni Mathur
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  3. Vidhu Verma
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  4. Shweta Sharma
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  5. Indranil Dasgupta
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Corresponding author

Correspondence to Indranil Dasgupta.

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Suppl. Fig. S1

Dot blot of plants inoculated with pRTBV-Del-Inf, mock-inoculated controls and RTBV inoculated plants using GLH. Slots A1-D12, plants inoculated with pRTBV-Del-Inf, slots E1-E3 and E12, mock-inoculated plants, slot E4, plasmid pRTBV-Del-Inf DNA, slots E5 and E6, RTBV infected plants using GLH inoculation. No samples were loaded in slots E7-E11. Approximately 1 μg DNA was used in each case, except slot E4, which had 1 ng plasmid DNA and slots and E5-E6, which had 300 ng each. Probe used was a PCR amplified RTBV ORF III fragment. In samples A1-D12 and E5-E6, plants were at 15 dpi (PPT 252 kb)

Supplementary Table S1 (DOC 61 kb)

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Purkayastha, A., Mathur, S., Verma, V. et al. Virus-induced gene silencing in rice using a vector derived from a DNA virus. Planta 232, 1531–1540 (2010). https://doi.org/10.1007/s00425-010-1273-z

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