Pentr vector

Human ERα, importin-α1, importin-α2, importin-α4, importin-β1 and TNPO2 were generated by polymerase chain reaction (PCR) amplification from total RNA from MCF-7 and HeLa cells using appropriate primers (Supplementary Table ), and subsequently subcloned into the pENTR vector (Thermo Fisher Scientific, Waltham, MA, USA). ERα mutants and importin-α ΔIBB were constructed by site-dir1ected mutagenesis and by inverse PCR mutagenesis with the KOD-Plus-Neo, Ligation high Ver.2, and T4 Polynucleotide Kinase enzymes (KOD-401, LGK-201, PNK-111, TOYOBO, Osaka, Japan) using primers (Supplementary Table 1). Then, using the Gateway system (Thermo Fisher Scientific), the genes were subcloned into p3 × FLAG-CMV DEST^{61 (link)}, pcDNA3.1/Zeo(+)-EGFP DEST^{62 (link)}, and pcDNA3.1/Zeo(+) DEST, pGEX-6P-2 DEST, which were generated by a method similar to^{62 (link),63 (link)} using pcDNA3.1/Zeo(+) (Thermo Fisher Scientific), pGEX-6P-2 (Cytiva, Piscataway, NJ, USA) and the Reading Frame Cassette of the Gateway Conversion System (Thermo Fisher Scientific). pCold II Ran Q69L was constructed by inserting the human Ran Q69L mutant into pCold II DNA (Takara Bio Inc., Shiga, Japan).

The lentiviral vector (Tet-CA-Src-GFP) for tetracycline-inducible expression of the GFP-tagged, constitutively active Src mutant, Src/Y527F, was obtained from Addgene (item no. 83469). The lentiviral vector for tetracycline-inducible expression of myristoylated constitutively active AKT2 mutant was constructed by first cloning the myristoylated HA-tagged AKT2 from pcDNA3 (Addgene, 9016) to pENTR vector (Thermo Fisher Scientific) and then to the destination vector pLIX403 (Addgene, 41395) by the Gateway cloning.

A lentiviral vector encoding Elk1 was generated by Gateway Technology (Thermo Fisher Scientific) as described previously (Suzuki et al., 2010). Briefly, Elk1 cDNA (a gift from H. Sugimoto, Dokkyo Medical University) was subcloned into the pENTR vector (Thermo Fisher Scientific) and subsequently transferred into the pCSII‐EF‐RfA lentiviral expression vector (a gift from H. Miyoshi, Keio University) by the LR recombination reaction (Thermo Fisher Scientific). GFP‐bearing CS‐CDF‐CG‐PRE plasmid was used as a control lentiviral vector. The 293FT cells were co‐transfected with the expression plasmids and packaging plasmids (pCMV‐VSV‐G‐RSV‐Rev and pCAG‐HIVgp) using Lipofectamine 2000 (11668019; Thermo Fisher Scientific). The viral supernatants were collected 48 h after transfection. For viral infection, 5.0 × 10⁴ TEC cells per well in 12‐well tissue culture plates were infected with lentiviral particles.

We used Golden Gate modular cloning to construct a vector to examine the expression pattern of PjGH9B3 during infection^{44 (link)}. The PjGH9B3 promoter region (1899 bp upstream of the ATG start codon) was cloned into the vector pAGM1311 as two fragments and then combined into the vector pICH41295 to generate the promoter module. This module was assembled into the vector pICH47751 containing 3xVenus-NLS and AtHSP18.2 terminator²² , then subsequently further assembled into pAGM1311 with pPjACT::3xmCherry-NLS²² . For RNAi experiments, we used the pHG8-YFP vector^{45 (link)}. Target sequences, from 1175 to 1474 in coding sequence, were PCR-amplified from P. japonicum genomic DNA and cloned into the pENTR vector (Thermo Fisher Scientific, Waltham, MA, USA), then transferred into the pHG8-YFP vector by the Gateway reaction using LR Clonase II Plus enzyme (Thermo Fisher Scientific). All primers used in this paper are listed in Supplementary Data 11.

The production of transgenic Arabidopsis lines (SvHKT2;1-1 and SvHKT2;2-15) expressing genes 35S-SvHKT2;1 and 35S-SvHKT2;2, respectively, was conducted according to the method described in our previous report [25 (link)]. In this study, cDNA for barley HvHKT2;1 (AEM55590.1) was amplified by PCR using primers HvHKT1F (5′-CACCATGGGTTGGGTGAAAAGATTTTACC-3′) and HvHKT2R (5′-GTATCATACTTTCCAGGATTTACC-3′), then cloned into a pENTR vector (Thermo Fisher Scientific, Tokyo, Japan) to produce the entry vector pENTR-HvHKT2;1. The entry vector was reacted with an LR enzyme (Thermo Fisher Scientific) with a destination vector pGH1 [25 (link)] to form pGH1-HvHKT2;1, in which the transgene was driven by a CaMV35S promoter. The transformation of Arabidopsis WT plants (ecotype Columbia) was performed by floral dipping [41 (link)] using Agrobacterium strain GV3101.