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. 2022:2505:263-279.
doi: 10.1007/978-1-0716-2349-7_19.

A Rapid and Efficient Vacuum-Based Agroinfiltration Protocol for Transient Gene Overexpression in Leaves of Catharanthus roseus

Konstantinos Koudounas  1 , Ines Carqueijeiro  2 , Pamela Lemos Cruz  2 , Jennifer Perrin  2 , Arnaud Lanoue  2 , Audrey Oudin  2 , Sébastien Besseau  2 , Vincent Courdavault  3
Affiliations

A Rapid and Efficient Vacuum-Based Agroinfiltration Protocol for Transient Gene Overexpression in Leaves of Catharanthus roseus

Konstantinos Koudounas et al. Methods Mol Biol. 2022.

Abstract

Functional genomics analyses in planta can be hampered in non-model plants that are recalcitrant to the genetic transformation such as the medicinal plant Catharanthus roseus (Apocynaceae). No stable transformation and regeneration of plantlets have been achieved with a high efficiency in this plant to date. In addition, while virus-mediated transient gene silencing has been reported a decade ago in C. roseus, tools for transient overexpression remain scarce. Here, we describe an efficient and reliable methodology for transiently overexpressing any gene of interest in C. roseus leaves. This protocol combines a vacuum-based Agroinfiltration approach and the high translational efficiency of a deconstructed virus-based binary vector (pEAQ-HT). The described methodology is robust, easy to perform, and results in high amount of transient expression in C. roseus. This protocol is expected to serve as valuable tool to enhance the in planta characterization of gene functions or even transiently knock-in novel enzymatic activities.

Keywords: Agrobacterium tumefaciens; Agroinfiltration; Apocynaceae; Catharanthus roseus; Cowpea mosaic virus HT (CPMV-HT); Functional genomics; Madagascar periwinkle; Recombinant proteins; Transient expression; Vacuum infiltration.

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References

    1. O’Connor SE, Maresh JJ (2006) Chemistry and biology of monoterpene indole alkaloid biosynthesis. Nat Prod Rep 23(4):532–547. https://doi.org/10.1039/B512615K - DOI - PubMed
    1. Dugé de Bernonville T, Clastre M, Besseau S, Oudin A, Burlat V, Glévarec G, Lanoue A, Papon N, Giglioli-Guivarc’h N, St-Pierre B, Courdavault V (2015) Phytochemical genomics of the Madagascar periwinkle: unravelling the last twists of the alkaloid engine. Phytochemistry 113:9–23. https://doi.org/10.1016/j.phytochem.2014.07.023 - DOI - PubMed
    1. Liscombe DK, O’Connor SE (2011) A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus. Phytochemistry 72(16):1969–1977. https://doi.org/10.1016/j.phytochem.2011.07.001 - DOI - PubMed - PMC
    1. Carqueijeiro I, Masini E, Foureau E, Sepúlveda LJ, Marais E, Lanoue A, Besseau S, Papon N, Clastre M, Dugé de Bernonville T, Glévarec G, Atehortùa L, Oudin A, Courdavault V (2015) Virus-induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors. Plant Biol 17(6):1242–1246. https://doi.org/10.1111/plb.12380 - DOI - PubMed
    1. Caputi L, Franke J, Farrow SC, Chung K, Payne RME, Nguyen T-D, Dang T-TT, Soares Teto Carqueijeiro I, Koudounas K, Dugé de Bernonville T, Ameyaw B, Jones DM, Vieira IJC, Courdavault V, O’Connor SE (2018) Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle. Science 360(6394):1235–1239. https://doi.org/10.1126/science.aat4100 - DOI - PubMed

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