Supporting Information: A Transient Expression Tool Box for Anthocyanin Biosynthesis in Nicotiana benthamiana

These tools include constructs for expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside and delphinidin 3-O-glucoside could be obtained in high amount in a few days. Additionally, co-infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins.

These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative, and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.