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Virus-Induced Gene Silencing as a Tool to Study Regulation of Alkaloid Biosynthesis in Medicinal Plants

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Plant Secondary Metabolism Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2469))

Abstract

Advancements in genomics and transcriptomics have generated invaluable resources for the discovery of novel genes related to complex specialized metabolic pathways in plants. Virus-induced gene silencing (VIGS) has emerged as a powerful tool that is widely used for rapid functional characterization of genes in planta. VIGS has advantages over other reverse genetic approaches, such as RNAi-mediated suppression or T-DNA knockout, because it does not require the development of stable transgenic lines which is technically challenging and time consuming. Catharanthus roseus is an important medicinal plant that produces more than a hundred monoterpenoid indole alkaloids (MIAs), including the antineoplastic drugs vincristine and vinblastine. Biosynthesis of these alkaloids is strikingly complex, resulting in MIA accumulation in low quantities. Jasmonic acid (JA) is an elicitor of the MIA biosynthesis. Exogenous application of JA in C. roseus induces MIA pathway gene expression and increases MIA accumulation. The core JA signaling module comprises multiple components including the JA coreceptor Coronatine-Insensitive 1(COI1). COI1 plays a key role in JA-responsive gene expression in plants. Because generation of stable transgenic C. roseus plants is challenging, VIGS is being used for functional characterization of genes in the MIA pathway. Here we describe a detailed method for the VIGS-mediated suppression of C. roseus COI1(CrCOI1) expression to decipher the regulatory mechanism of JA-induced elicitation of MIA biosynthesis. When performing VIGS, gene silencing efficiency and the viral spread are monitored by the development of visible phenotype in the control plants. We use the C. roseus phytoene desaturase (CrPDS) and Protoporphyrin IX Mg-chelatase subunit H (CrChlH) as visual markers to access VIGS efficiency and viral spread. The protocol described here could be used for the functional characterization of genes involved in other metabolic pathways and in other medicinal plants.

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Authors and Affiliations

  1. Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY, USA

    Barunava Patra, Yongliang Liu, Sanjay Kumar Singh, Sitakanta Pattanaik & Ling Yuan

  2. South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China

    Yongliang Liu & Ling Yuan

  3. Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA

    Joshua J. Singleton & Ling Yuan

Authors
  1. Barunava Patra
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  2. Yongliang Liu
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  3. Joshua J. Singleton
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  4. Sanjay Kumar Singh
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  5. Sitakanta Pattanaik
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  6. Ling Yuan
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Corresponding authors

Correspondence to Barunava Patra or Ling Yuan .

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Editors and Affiliations

  1. Center for Biotechnology and Department of Botany, Plant Physiology Laboratory, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil

    Arthur Germano Fett-Neto

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Patra, B., Liu, Y., Singleton, J.J., Singh, S.K., Pattanaik, S., Yuan, L. (2022). Virus-Induced Gene Silencing as a Tool to Study Regulation of Alkaloid Biosynthesis in Medicinal Plants. In: Fett-Neto, A.G. (eds) Plant Secondary Metabolism Engineering. Methods in Molecular Biology, vol 2469. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2185-1_13

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  • DOI: https://doi.org/10.1007/978-1-0716-2185-1_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2184-4

  • Online ISBN: 978-1-0716-2185-1

  • eBook Packages: Springer Protocols

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