Constructing Binary Plasmids for Agroinfiltration

Two kinds of binary plasmids, pRI201-AN and pAT006, as well as their derivatives were used for agroinfiltration experiments (Supplementary Figure S1). pRI201-AN, which is commercially available from Takara Bio (Kusatsu, Japan), contains the CaMV 35S promoter and the heat shock protein (HSP) terminator derived from Arabidopsis thaliana. pAT006 has a pMDC background and contains the CaMV 35S promoter and terminator in the multiple cloning site (Tsuzuki et al. 2014 (link)). By cleaving pRI201-AN with two types of restriction endonucleases and self-ligating using DNA Ligation Kit Ver.2.1 (Takara Bio), four types of binary plasmids containing a shortened T-DNA region were prepared. The names of the prepared plasmids and the restriction enzymes used (in parentheses) are as follows: pRI-SA (SalI and ApaI), pRI-XA (XbaI and ApaI), pRI-AH (ApaI and HindIII), and pRI-EH (EcoRI and HindIII). Structures of these plasmids are shown in Supplementary Figure S1. The DNA fragments of two kinds of GFP genes, enhanced GFP (eGFP) and soluble modified GFP (smGFP), which are derived from the plasmids, pEGFP-1 (Takara Bio) and pBICGFP (Davis and Vierstra 1998 (link); Takeda et al. 2002 (link)), respectively, were cloned into pRI201-AN and pAT006 using the In-Fusion® HD Cloning kit or DNA Ligation Kit Ver.2.1 (Supplementary Figure S1).