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

Barunava Patra, Yongliang Liu, Joshua J. Singleton, Sanjay Kumar Singh, Sitakanta Pattanaik, Ling Yuan

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

1 Scopus citations

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Pages155-164
Number of pages10
DOIs
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2469
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

Publisher Copyright:
© 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

  • Catharanthus roseus
  • Jasmonic acid
  • Medicinal plants
  • Monoterpenoid indole alkaloids
  • Phytoene desaturase (PDS)
  • Protoporphyrin IX magnesium-chelatase subunit H (ChlH)
  • Specialized metabolites
  • Virus-induced gene silencing (VIGS)

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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