Transient expression of LaTPS7, LaTPS8, andLaCYP71D582 in N. benthamiana
Leaves of N. benthamiana collected from 4-week-old plants were infiltrated with Agrobacterium tumefaciens GV3101 harboring target genes. Meanwhile, GV3101 was mixed with TPS and CYP at respective ratios of 1:1 when OD600 of each strain corresponded to 1.0. Leaf tissues were collected after 5 days of infiltration, examined under UV light to ensure successful agroinfiltration and gene expression, and then ground in liquid nitrogen. Leaf powder was stored at -80°C, and volatiles were extracted by vortexing in 70% alcohol (1:4 m/v). Extracts were maintained at -20°C and oscillated before analysis via solid-phase microextraction (SPME, Agilent). The samples were heated in a water bath at 40°C for 30 min, and then a fibre coated with polydimethylsiloxane and divinylbenzene was used to collect volatiles under the same conditions for 30 min according to a previously published method (Ilc et al. 2017). Volatiles were then analysed using GC-MS.