Pet28b expression vector

The cDNA of the B domain of protein A was purchased from Fasmac Co., Ltd. (Kanagawa, Japan) and subcloned into a pET28b expression vector (Merck KGaA, Darmstadt, Germany). A recombinant B domain with an N-terminal hexahistidine tag was expressed in Escherichia coli strain BL21(DE3)-CodonPlus (Stratagene, San Diego, CA, USA). The hexahistidine-tagged B domain was then purified using a Chelating Sepharose Fast Flow column (GE Healthcare, Chicago, IL, USA). Subsequently, the hexahistidine tag was cleaved using thrombin, followed by gel filtration chromatography using a HiLoad 16/60 Superdex75 (GE Healthcare, Chicago, IL, USA) column with 50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl.

The groEL gene of B. glumae was amplified using the primers listed in Table S2. The amplified product was cloned into NdeI and HindIII restriction sites of a pET28b expression vector (Merck, Darmstadt, Germany), resulting in pET28b-groEL (see Table S1). His-GroEL was overexpressed in Escherichia coli BL21(DE3), which was induced by adding 0.5 mM isopropyl β-d-thiogalactopyranoside, followed by additional growth for 14 h at 20°C. His-GroEL was purified in a buffer A, which contained 50 mM Tris-Cl (pH 8.0), 100 mM NaCl, and 5% (vol/vol) glycerol, using an immobilized metal affinity column (GE Healthcare, Chicago, IL) equilibrated with buffer A and then eluted with buffer A containing 500 mM imidazole. Purified His-GroEL was dialyzed against buffer A overnight at 4°C.

The cDNA sequence corresponding to AKINβγ (At1g09020) was amplified with Pfu polymerase and cloned into the pGEM T-easy vector (Promega, WI USA). The plasmid was digested with NcoI and NotI enzymes and the released fragment was purified and cloned into a pET28b+ expression vector (Merck, Millipore USA). The cDNA sequence of AKINβ2 (At4g16360) was also amplified with Pfu polymerase and cloned directly into the pET101/D-TOPO vector (Invitrogen). The resulting expression vectors were transformed into the Escherichia coli BL21 (RIL) strain, and the recombinant proteins were induced and purified through a Ni-NTA column as indicated by the manufacturer (Qiagen, Mexico).