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Induction of resistance to sugarcane mosaic virus by RNA interference targeting coat protein gene silencing in transgenic sugarcane

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Abstract

Sugarcane mosaic virus (SCMV) is a serious disease of monocotyledonous plants, including sugarcane, causing deterioration in both growth and productivity. RNA interference (RNAi) inhibits gene expression through RNA-mediated sequence-specific interactions and is considered an effective approach to control viral infection in plants. In this study, the SCMVCp gene encoding the coat protein (CP) was inserted into the pGreen-0179 plasmid in both sense and antisense orientations. Cauliflower mosaic virus (CaMV) and Zea mays ubiquitin (Ubi) promoters were selected to drive the transcription of the intron-hairpin constructs, called HpSCMVCp-CaMV and HpSCMVCp-Ubi, respectively. Transgenic sugarcane expressing these constructs was generated through Agrobacterium-mediated transformation. This transformation method produced a high percentage of transgenic plants for both HpSCMVCp-CaMV and HpSCMVCp-Ubi, as confirmed by PCR analysis. Southern blotting revealed a single stable insertion of the DNA target in the genome of transgenic sugarcane lines. After artificial virus infection, lines that developed mosaic symptoms were classified as susceptible, whereas those that remained green without symptoms were classified as resistant at 42 days post-inoculation. Immunoblotting revealed CP expression at 37 kDa in susceptible and non-transgenic sugarcane, but not in resistant lines. RT-PCR analysis confirmed viral Cp and Nib gene expression in susceptible lines and their absence in resistant lines. Interestingly, upon comparison of effectivity, CaMV and Ubi promoter-driven gene expression resulted in 57.69% and 82.35% resistant sugarcane lines, respectively. Thus, we concluded that RNAi is effective for inducing resistance against SCMV and that the Ubi promoter is an effective promoter for producing transgenic sugarcane.

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Data availability

All data generated or analysed during this study are included in this published article (and its supplementary files). Requests for material should be made to the corresponding authors.

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Acknowledgements

This research was supported by the Indonesian Ministry of Education and Culture through a research grant from the Islamic Development Bank (IDB) Project of The University of Jember and Master Thesis (Grant number 975/UN25.3.1/LT/2020).

Funding

This research was supported by the Indonesian Ministry of Education and Culture through a research grant from the Islamic Development Bank (IDB) Project of the University of Jember and Master Thesis (Grant Number 975/UN25.3.1/LT/2020).

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Authors and Affiliations

  1. Laboratory of Molecular Biology and Biotechnology, Center for Development of Advanced Science and Technology (CDAST), Jember University, Jln. Kalimantan 37, Jember, 68121, Indonesia

    Suvia Widyaningrum, Dwi Ratna Pujiasih, Wardatus Sholeha & Bambang Sugiharto

  2. Postgraduate Program in Biotechnology, Jember University, Jln. Kalimantan 37, Jember, 68121, Indonesia

    Suvia Widyaningrum & Bambang Sugiharto

  3. Department of Biology, Faculty of Mathematics and Natural Sciences, Jember University, Jln. Kalimantan 37, Jember, 68121, Indonesia

    Dwi Ratna Pujiasih, Wardatus Sholeha & Bambang Sugiharto

  4. Indonesian Institute of Sciences, Jl. Raya Jakarta-Bogor, Cibinong, Bogor, 16911, Indonesia

    Rikno Harmoko

Authors
  1. Suvia Widyaningrum
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  2. Dwi Ratna Pujiasih
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  3. Wardatus Sholeha
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  4. Rikno Harmoko
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  5. Bambang Sugiharto
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Contributions

DRP and WS performed sugarcane transformation, transformant screening, and acclimatization. RH generated RNAi constructs and collected data. SW performed artificial inoculation, observation of symptoms, molecular analysis, and data collection. SW, RH, and BS analysed the data and wrote the manuscript. BS supervised the experiments and acquired funding. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Bambang Sugiharto.

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Widyaningrum, S., Pujiasih, D.R., Sholeha, W. et al. Induction of resistance to sugarcane mosaic virus by RNA interference targeting coat protein gene silencing in transgenic sugarcane. Mol Biol Rep 48, 3047–3054 (2021). https://doi.org/10.1007/s11033-021-06325-w

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  • DOI: https://doi.org/10.1007/s11033-021-06325-w

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