Bach, Søren Spanner4; Bassard, Jean-Étienne André5; Andersen-Ranberg, Johan5; Møldrup, Morten Emil6; Simonsen, Henrik Toft4; Hamberger, Björn Robert4
1 Section for Plant Biochemistry, Department of Plant and Environmental Sciences, Faculty of Science, Københavns Universitet2 Plant Biochemistry, Department of Plant Biology, Faculty of Life Sciences, Københavns Universitet3 Molecular Plant Fysiology, Department of Plant Biology, Faculty of Life Sciences, Københavns Universitet4 Plant Biochemistry, Department of Plant Biology, Faculty of Life Sciences, Københavns Universitet5 Section for Plant Biochemistry, Department of Plant and Environmental Sciences, Faculty of Science, Københavns Universitet6 Molecular Plant Fysiology, Department of Plant Biology, Faculty of Life Sciences, Københavns Universitet
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.
Methods in Molecular Biology: Methods and Protocols, 2014, p. 245-255