TY - CHAP
T1 - High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana
AU - Bach, Søren Spanner
AU - Bassard, Jean-Étienne André
AU - Andersen-Ranberg, Johan
AU - Møldrup, Morten Emil
AU - Simonsen, Henrik Toft
AU - Hamberger, Björn Robert
PY - 2014
Y1 - 2014
N2 - 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.
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.
U2 - 10.1007/978-1-4939-0606-2_18
DO - 10.1007/978-1-4939-0606-2_18
M3 - Book chapter
C2 - 24777803
SN - 978-1-4939-0605-5
VL - 4
T3 - Methods in Molecular Biology
SP - 245
EP - 255
BT - Plant isoprenoids
A2 - Rodríguez-Concepción, Manuel
PB - Springer
ER -