Peaq ht dest1

The entire coding sequences (CDSs) of OeBAS, OeMAS, CYP716A48, and CYP716C67 were amplified from cDNA of olive leaves of cv Leccino, using iProof High‐Fidelity DNA Polymerase (Bio‐Rad) and gene‐specific primers (Table S2). CYP72A67 was amplified from Medicago truncatula Gaertn. root cDNA using Phusion polymerase (NEB, Ipswich, MA, USA; Table S2). The Centella asiatica L. CYP716C11 gene was synthesized with flanking Gateway® attB1 sites (IDT). OeBAS, OeMAS, and CYP716A48 amplification products were cloned into the Gateway vector pCR8‐GWTOPO‐T/A (Thermo Fisher). CYP716C67, CYP72A67, and CYP716C611 were cloned into pDONR207 using BP Clonase II Enzyme Mix (Thermo Fisher). All entry clones were sequenced to verify their integrity before LR reaction.
To generate constructs for yeast expression, OeBAS and OeMAS were subcloned into the vector pYES2‐DEST52, under the control of a galactose‐inducible promoter, using Gateway LR Clonase (Thermo Fisher), yielded the Saccharomyces cerevisiae expression constructs.
For the generation of constructs for Nicotiana benthamiana Domin. expression, OeBAS, OeMAS, CYP716A48, CYP72A67, and CYP716C611 were subcloned from the relevant entry vectors (pCR8 or pDONR 207) into pEAQ‐HT‐DEST1 (Sainsbury et al., 2009 (link)) vector using LR Clonase Mix (Thermo Fisher).

A synthetic gene containing the cDNA sequence of Pru p 3 fused to the CP gene of TuMV was ordered from GeneArt (ThermoFisher Scientific) and cloned in the pEAQ-HT-DEST1 expression vector (80 (link)). Both sequences (Pru p 3 and CP) were connected by one of two linkers (flexible or helicoidal) designed to provide physical separation between both parts of the fusion protein.
The pEAQ constructs were expressed in plants of Nicotiana benthamiana by agroinfiltration of the corresponding Agrobacterium tumefaciens (LB 4404 strain), for the production of the TuMV-derived VLPs (49 (link)).