Bp reaction kit

The full-length coding sequence (CDS) of AtNRX1 was PCR-amplified from cDNAs using the primers AtNRX1-attB1_F and AtNRX1-attB2_R. The PCR product was extracted from the gel and used for secondary PCR amplification using the primers attB1_F and attB2_R. The DNA fragments amplified by the second PCR were cloned into Gateway entry vector pDONR221 using the BP reaction kit, according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA), to generate the pDONR-AtNRX1 construct. To generate Cys-to-Ser substitutions in AtNRX1M1, AtNRX1M2, and AtNRX1M3 proteins, the nucleotide sequence of AtNRX1 was mutated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) with the following primer pairs: AtNRX1-C55,58S_F/_R for AtNRX1M2 and AtNRX1-C375,378S_F/_R for AtNRX2. The AtNRX1 and AtNRX1M3 sequences were cloned to the pMDC43 vector [58 (link)] to generate the pMDC43-AtNRX1 and pMDC43-AtNRX1M3 constructs, respectively, which were then transformed into Agrobacterium tumefaciens strain GV3101 and further into Arabidopsis plants using standard protocols [59 (link)], thus generating transgenic AtNRX1^OE and AtNRX1M3^OE lines, respectively, using the atnrx1 mutant. Transgenic lines were selected on medium containing 30 mg/L hygromycin and confirmed by genomic DNA-based PCR and sqRT-PCR.

COP1 coding sequence minus the stop codon was PCR-amplified from Col-0 cDNA using COP1_attB1_F and COP1_attB2_R primers. The gel-eluted PCR product was used for a second round of PCR amplification with the attB1 and attB2 primers. The PCR product was cloned into pDONR221 using the BP reaction kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions, to generate the pDONR-COP1 construct. COP1^L105A, COP1^L170A, and COP1^MUT were generated by amplifying the pDONR-COP1 DNA (as a template) with specific primer sets (Table S1) using the QuickChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), as described previously [52 (link)]. Then, COP1 (WT), COP1^L105A, COP1^L170A, and COP1^MUT were cloned into the pMDC85 vector [53 (link)], obtained from ABRC (CD3-744), using an LR reaction kit (Invitrogen), according to the manufacturer’s instructions, to generate pMDC85-COP1, pMDC85-COP1^L105A, pMDC85-COP1^L170A, and pMDC85-COP1^MUT constructs, respectively. All of the above constructs were transformed into Agrobacterium tumefaciens strain GV3101, which was then used to transform Arabidopsis Col-0 plants using standard protocols [54 (link)]. Transgenic lines were selected on plates containing 30 μg/mL hygromycin (Duchefa) and 250 μg/mL cefotaxime (Duchefa), and were confirmed by immunoblot analysis.