Pdest gbkt7

The yeast strains and media used for the yeast two-hybrid experiment were provided in the Matchmaker Golden Yeast Two-Hybrid System (Clontech). The open reading frames (ORF) of BlSPL genes, BlRGA (Genbank accession no. MF149049) and BlRGL (Genbank accession no. MF149050) were amplified and firstly cloned into pCR™8/GW/TOPO vector (Invitrogen). Then, these ORFs were cloned into pDEST-GBKT7 (GAL4 DNA binding domain, BD) or pDEST-GADT7 (GAL4 activation domain, AD; Rossignol et al., 2007 (link)) through LR recombination reaction (Invitrogen). The BD fused constructs and AD fused constructs were transformed into Y2HGold cells and Y187 cells by the lithium acetate-mediated method, respectively. The yeast two-hybrid assay was performed by yeast mating according to the user manual. The BD fused constructs and AD fused constructs were tested for their autoactivation and toxicity, and those constructs without autoactivation and toxicity in yeast cells were used for yeast two-hybrid assay (Supplementary Figure S1). To measure the transcription activation activity, β-Galactosidase activity was assayed using o-nitrophenyl β-D-galactopyranoside (ONPG) as described in Yeast Protocol Handbook (Clontech). The primers used for ORF amplification are listed in Supplementary Table S6.

The transactivation activity of CnTCP4 was tested with a yeast one hybrid assay [48 (link)]. The three different C’-deletion variants of the CnTCP4 coding region (Figure 3c) were amplified using the primer pair CnTCP4-pENTR1A-F plus one of CnTCP4-pENTR1A-R1/-R2/-R3 (sequences given in Table S1). The resulting fragments were inserted into pENTR™1A via the BamHI and NotI cloning sites. The resulting pENTR™1A-CnTCP4, pENTR™1A-CnTCP4-F1, pENTR™1A-CnTCP4-F2, and pENTR™1A-CnTCP4-F3 fusions were subsequently recombined with pDEST-GBKT7 via an LR reaction (Invitrogen) to form the constructs pDEST-GBKT7-CnTCP4, pDEST-GBKT7-CnTCP4-F1, pDEST-GBKT7-CnTCP4-F2, and pDEST-GBKT7-CnTCP4-F3. Each of these four constructs, plus pCL1 (positive control) and pDEST-GBKT7 (negative control), were introduced individually into Y2H Gold yeast cells (Clontech, Mountain View, CA, USA) following the manufacturer’s protocol. Selection for transformants (except for those carrying pCL1) was carried out by culturing the cells on SD/-Trp medium, while the pCL1 transformants were cultured on SD/-Leu medium. All of the transformant cell lines were finally plated on SD/-His/-Ade medium containing 20 mg/mL X-α-Gal, and incubated at 30 °C.