Pdest gadt7

For Y2H library screening, ASK13 was initially sub-cloned into the bait vector pGBKT7BD and transformed into the Y2H Gold strain (Clonetech) using an EZ-Yeast Transformation Kit (MP Biomedicals) according to the manufacturers’ instructions. The pGBKT7BD-ASK13 transformed cells were then mated with a normalized Arabidopsis Y2H cDNA library (purchased from Clonetech) and screening was carried out according to the manufacturer’s guidelines. Selection was performed on SD plates lacking histidine, tryptophan, leucine, and adenine but supplemented with X-α-Gal and aureobasidin A. For Y2H one-to-one interactions, the Gateway-compatible vectors pDEST-GBKT7 and pDEST-GADT7 (from TAIR) were used. Selected F-box, non-F-box, and several other ASK cDNAs were cloned into the pENTR/D-TOPO vector using Gateway cloning technology (Invitrogen) and eventually cloned in the pDEST-GADT7 Y2H vector. The resulting plasmids were co-transformed to the Y2H gold strain and then the transformed cells were selected on SD–Ade–His–Leu–Trp/X-α-Gal/aureobasidin agar media. pGADT7-T- and pGBKT7-Lam-transformed Y2H gold strain was used as a negative control and pGADT7-T- and pGBKT7-53-transformed Y2H gold strain was used as a positive control for interactions.

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.