Plasmid constructions
The N-term sequence (first 240 bp) of the MtERF75 gene was
amplified by PCR reaction from M. truncatula cDNA template with
specific primers (MC80F and MC80R, Table S1) using
PhusionTM High Fidelity DNA-polymerase (New England
Biolabs Inc. USA). A second forward primer (MA80F) was also used to
introduce an Ala residue in place of the Cys residue in second position
(MA80 construction). Both sequences were cloned in Aat II andNco I restriction sites in 5’ and 3’ end respectively of pGEM®-T
(Promega, USA) vector by ligation. The firefly luciferase (luc) reporter
gene was obtained from gst1::luc pART7 vector (Grant et al. ,
2000) and introduced between the Nco I and Not I restriction
site of the previous constructs pGEMt-MA80 or pGEMt-MC80 by ligation.
The resulting pGE-MC/MA::LUC vectors have been digested withBam HI and Not I to transfer the constructions in a pENTR4
entry vector. Both constructs were then transferred in p2GW7 destination
vector with Gateway® LR Clonase™ II Enzyme Mix (Invitrogen, USA)
following the manufacturer’s recommendations.
For C-terminal GFP fusion, a PCR reaction was used to amplify the full
length ORF of ERF75 from cDNA of M. truncatula with
primers ERF75_MC_F and ERF75_GFP_R (lacking of stop codon). The PCR
product was cloned in pDONR207 vector by BP recombination. For
C-terminal GFP fusion, the resulting vector (pDO-ERF75Δstop) was
recombined into pK7FWG2 destination vector (carrying a GFP gene) by an
LR recombination reaction.
For the RNAi construct, a common region of 165 nt (Fig. S1), found in
the two target genes MtERF74 and MtERF75 was amplified
from the complete cDNA of M. truncatula by PCR using the primers
RNAi74/75F and RNAi74/75R. This sequence was first introduced into the
pJET1.2/blunt cloning vector (Thermo Fisher Scientific, USA), then in
the pENTR4 vector by ligation between the Xho I and Kpn I
sites, and finally in the pK7GWIW2D vector (Karimi et al. , 2002),
able to produce the hairpin structure for the gene silencing, by LR
recombination. As control for the RNAi experiment, a pk7WΔCnt (Marinoet al. , 2011) with a fragment of lacZ gene from E.
coli in pK7GWIW2D vector was used.
All the primers sequences and vectors are provided in Table S1 and Table
S2, respectively.