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Phenotyping of VIGS-mediated gene silencing in rice using a vector derived from a DNA virus

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Abstract

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Target genes in rice can be optimally silenced if inserted in antisense or hairpin orientation in the RTBV-derived VIGS vector and plants grown at 28 °C and 80% humidity after inoculation.

Abstract

Virus induced gene silencing (VIGS) is a method used to transiently silence genes in dicot as well as monocot plants. For the important monocot species rice, the Rice tungro bacilliform virus (RTBV)-derived VIGS system (RTBV-VIGS), which uses agroinoculation to initiate silencing, has not been standardized for optimal use. Here, using RTBV-VIGS, three sets of conditions were tested to achieve optimal silencing of the rice marker gene phytoene desaturase (pds). The effect of orientation of the insert in the RTBV-VIGS plasmid (sense, antisense and hairpin) on the silencing of the target gene was then evaluated using rice magnesium chelatase subunit H (chlH). Finally, the rice Xa21 gene, conferring resistance against bacterial leaf blight disease (BLB) was silenced using RTBV-VIGS system. In each case, real-time PCR-based assessment indicated approximately 40–80% fall in the accumulation levels of the transcripts of pds, chlH and Xa21. In the case of pds, the appearance of white streaks in the emerging leaves, and for chlH, chlorophyll levels and F v/F m ratio were assessed as phenotypes for silencing. For Xa21, the resistance levels to BLB were assessed by measuring the lesion length and the percent diseased areas of leaves, following challenge inoculation with Xanthomonas oryzae. In each case, the RTBV-MVIGS system gave rise to a discernible phenotype indicating the silencing of the respective target gene using condition III (temperature 28 °C, humidity 80% and 1 mM MES and 20 µM acetosyringone in secondary agrobacterium culture), which revealed the robustness of this gene silencing system for rice.

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Abbreviations

VIGS:

Virus induced gene silencing

pds:

Phytoene desaturase

Xoo :

Xanthomonas oryzae pv. oryzae

RTBV:

Rice tungro bacilliform virus

RNAi:

RNA interference

MLL:

Mean lesion length

%DLA:

Percentage diseased leaf area

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Acknowledgements

This work was funded by a grant of the Department of Biotechnology, Government of India. Grant No. BT/AB/FG-I(PH-II)/2009 to ID. Research fellowship from Indian Council of Medical Research, New Delhi to RK is gratefully acknowledged. ID also acknowledge the financial support from University of Delhi (R&D and DU-DST PURSE).

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

    Ravi Kant & Indranil Dasgupta

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  1. Ravi Kant
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  2. Indranil Dasgupta
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Correspondence to Indranil Dasgupta.

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Communicated by Renate Schmidt.

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Kant, R., Dasgupta, I. Phenotyping of VIGS-mediated gene silencing in rice using a vector derived from a DNA virus. Plant Cell Rep 36, 1159–1170 (2017). https://doi.org/10.1007/s00299-017-2156-6

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