Gene cloning
Orthologues of SlACRE75 and SlACRE180 were obtained from
CDS and protein sequences BLAST analysis against Arabidopsis genome
(TAIR10) for Arabidopsis sequences, or a reciprocal best BLAST hits
(RBH) (Ward and Moreno-Hagelsieb, 2014) test was performed (Sol Genomics
Network) for N. benthamiana , termed NbACRE75 andNbACRE180 , respectively. Best CDS and protein hits were
identified, being Niben101Scf03108g12002.1 and Niben101Scf12017g01005.1
for SlACRE75 and SlACRE180 respectively; termed NbACRE75 and NbACRE180
onwards. Flanking the (i) SlACRE75 , (ii) SlACRE180 , (iii)NbACRE180 and (iv) NbACRE75 coding sequences (CDS),
Gateway® cloning was used to design and produce overexpression
constructs for the gene candidates for a N-terminal GFP:ACRE fusion
protein per insert (Reece-Hoyes and Walhout, 2018). Briefly, pUC57
plasmids containing SlACRE75 , SlACRE180 , NbACRE75and NbACRE180 coding sequences were chemically synthesized by
GenScript. For SlACRE75 , SlACRE180, NbACRE75 andNbACRE180, cDNAs from pUC57 entry vector were transformed by
electroporation into Escherichia coli strain DH10B and
transferred by a recombinant LR reaction of Gateway cloning (Clonase II
enzyme mix Kit, Thermo Fisher) into pB7WGF2 (Karimi et al., 2002).