Plasmid and vectors

To obtain the plasmid encoding NbAELP fused to the DYKDDDDKDYKDVDDYKDDDDK (3xFLAG) sequence (Ueda et al., 2011), a 3'-fragment of NbAELP containing the 3xFLAG encoding sequence was amplified using NbAELP(BglII+) and FL_SalI_r primers and subsequently digested with BglII and SalI. The 3xFLAG encoding sequence was generated after annealing the primers FL2_SalI_r and FL2_SalI_d, resulting in a fragment with overhangs corresponding to SalI and PstI “sticky” ends. These fragments were inserted into 35S-NbAELP via BglII/PstI sites to generate the 35S-NbAELP:3xFLAG plasmid. To obtain the proNbAELP (1000):GUS plasmid, NheI and SacI sites were introduced at the 5′- and the 3′-ends of proNbAELP, respectively, through PCR with primers “proNbAELP (NheI+)” and “proNbAELP (SacI−).” The proNbAELP (1,000) NheI/SacI fragment and the GUS-35S(term) fragment flanked with SacI/EcoRI sites was cloned into pBIN19, previously digested with XbaI/EcoRI, to generate the proNbAELP(1000):GUS construct. To obtain the proNbAELP(1500):GUS variant, the upstream region of proNbAELP was amplified using the primers “proNbAELP(SbfI+)” and “proNbAELP (HindIII−)” and subsequently inserted with the proNbAELP(1000):GUS fragment, flanked with HindIII/EcoRI sites, into pBIN19, previously digested with SbfI/EcoRI. To obtain proNbAELP(500):GUS, the 3′-proximal ~500 nt fragment of proNbAELP, digested with SalI/SacI, and the GUS-35S(term) fragment, flanked with SacI/EcoRI, were inserted into pBIN19 via SalI/EcoRI sites.

The proNtPME sequence was amplified using the primer pairs “PMEpr (HindIII+)” and “PMEpr (NcoI−)” and subsequently cloned into the pAL-TA vector (Evrogen, Russia). The obtained proNtPME fragment, with flanking 5′-HindIII and 3′-NcoI sites and the fragment containing GUS-35S(term), digested with NcoI/EcoRI, was inserted into pCampia1300 (CAMBIA, Australia) via HindIII/EcoRI sites, resulting in the proNtPME:GUS construct. For proNtPME:GFP construct, the second used fragment was GFP-35S(term) flanked with NcoI/EcoRI sites.

A full list of the oligonucleotides used for cloning is presented in Table S1.