High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana

Søren Spanner Bach; Jean-Étienne André Bassard; Johan Andersen-Ranberg; Morten Emil Møldrup; Henrik Toft Simonsen; Björn Robert Hamberger

doi:10.1007/978-1-4939-0606-2_18

High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana

Søren Spanner Bach, Jean-Étienne André Bassard, Johan Andersen-Ranberg, Morten Emil Møldrup, Henrik Toft Simonsen, Björn Robert Hamberger

Section for Plant Biochemistry

26 Citations (Scopus)

Abstract

To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized to the native compartments, unlike microbial or prokaryotic expression systems. Two inherent drawbacks of plant-based expression systems, time-consuming generation of transgenic plant lines and challenging gene-stacking, can be circumvented by transient expression in Nicotiana benthamiana. In this chapter we describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps optimized for high throughput, this in planta expression platform is particularly suited for testing large panels of candidate genes in all possible permutations.

Original language	English
Title of host publication	Plant isoprenoids : methods and protocols
Editors	Manuel Rodríguez-Concepción
Number of pages	11
Volume	4
Publisher	Springer
Publication date	2014
Pages	245-255
Chapter	18
ISBN (Print)	978-1-4939-0605-5
ISBN (Electronic)	978-1-4939-0606-2
DOIs	https://doi.org/10.1007/978-1-4939-0606-2_18
Publication status	Published - 2014

Series	Methods in Molecular Biology
Volume	1153
ISSN	1064-3745

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10.1007/978-1-4939-0606-2_18

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Bach, S. S., Bassard, J-É. A., Andersen-Ranberg, J., Møldrup, M. E., Simonsen, H. T., & Hamberger, B. R. (2014). High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana. In M. Rodríguez-Concepción (Ed.), Plant isoprenoids: methods and protocols (Vol. 4, pp. 245-255). Springer. Methods in Molecular Biology Vol. 1153 https://doi.org/10.1007/978-1-4939-0606-2_18

High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana. / Bach, Søren Spanner; Bassard, Jean-Étienne André; Andersen-Ranberg, Johan et al.

Plant isoprenoids: methods and protocols. ed. / Manuel Rodríguez-Concepción. Vol. 4 Springer, 2014. p. 245-255 (Methods in Molecular Biology, Vol. 1153).

Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review

Bach, SS, Bassard, J-ÉA, Andersen-Ranberg, J, Møldrup, ME, Simonsen, HT & Hamberger, BR 2014, High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana. in M Rodríguez-Concepción (ed.), Plant isoprenoids: methods and protocols. vol. 4, Springer, Methods in Molecular Biology, vol. 1153, pp. 245-255. https://doi.org/10.1007/978-1-4939-0606-2_18

Bach SS, Bassard J-ÉA, Andersen-Ranberg J, Møldrup ME, Simonsen HT, Hamberger BR. High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana. In Rodríguez-Concepción M, editor, Plant isoprenoids: methods and protocols. Vol. 4. Springer. 2014. p. 245-255. (Methods in Molecular Biology, Vol. 1153). doi: 10.1007/978-1-4939-0606-2_18

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abstract = "To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized to the native compartments, unlike microbial or prokaryotic expression systems. Two inherent drawbacks of plant-based expression systems, time-consuming generation of transgenic plant lines and challenging gene-stacking, can be circumvented by transient expression in Nicotiana benthamiana. In this chapter we describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps optimized for high throughput, this in planta expression platform is particularly suited for testing large panels of candidate genes in all possible permutations.",

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AB - To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized to the native compartments, unlike microbial or prokaryotic expression systems. Two inherent drawbacks of plant-based expression systems, time-consuming generation of transgenic plant lines and challenging gene-stacking, can be circumvented by transient expression in Nicotiana benthamiana. In this chapter we describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps optimized for high throughput, this in planta expression platform is particularly suited for testing large panels of candidate genes in all possible permutations.

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High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana

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