Gateway Cloning for Molecular Studies

Unless stated otherwise, Phusion High-Fidelity DNA Polymerase (NEB, USA) was used to amplify all DNA sequences using the primer sets listed in S1 Table, and Gateway technology (Invitrogen, USA) was employed to generate plasmids. Coding sequences of HAC1p U, HAC1p S and IRE1p were amplified using yeast cDNA as described above. Coding sequences of bZIP60 U, bZIP60 S, bZIP60ΔN U, bZIP60ΔN S, bZIP60ΔC1, bZIP60ΔC2, IRE1A and IRE1B were amplified using Arabidopsis cDNA (cDNA from DTT-treated seedlings was used for amplification of bZIP60 S and bZIP60ΔN S, whereas cDNA from bzip60-2 seedlings for amplification of bZIP60ΔC2). Coding regions of P1, HC-Pro, P3, 6K1, CI, 6K2, NIaVPg, NIaPro, NIb and CP of TuMV were amplified from the TuMV infectious clone [40 (link)]. With the exception of pENTR^TM 1A Dual Selection vector (A10462, Invitrogen) used for IRE1B, all amplified coding sequences were recombined into pDONR221 via the BP reaction (Invitrogen, USA). The entry vector containing P3N-PIPO was described in our previous work [40 (link)]. To highlight the nucleus and to produce donor- and acceptor-only samples (used in FRET assays), constructs bearing 35S::NLS-CFP and 35S::NLS-YFP were created following the BP and LR reactions using the primers listed in S1 Table.