Amersham typhoon rgb biomolecular imager

avrXa27, tal6b, tal17, and tal6c were cloned into the pET-30a vector with a His tag to construct the plasmids pET30a-avrXa27, pET30a-tal6b/avrXa27A, pET30a-tal17/avrXa27B, and pET30a-tal6c/avrXa27C (Supplemental Table 1). After induction with isopropyl-β-D-thiogalactopyranoside (IPTG), the fusion proteins His-AvrXa27, His-Tal6b/AvrXa27A, His-AvrXa27B, and His-AvrXa27C were produced in E. coli BL21(DE3) cells containing pET30a-avrXa27, pET30a-tal6b/avrXa27A, pET30a-avrXa27B, and pET30a-avrXa27C, respectively. Ni-NTA HisBind Resin (Novagen, USA) was used to purify the proteins according to the manufacturer’s manual. Purified His-AvrXa27, His-Tal6b/AvrXa27A, His-AvrXa27B, and His-AvrXa27C were mixed with Cy5-labeled Xa27 or OsSWEET11a promoter fragments (probes synthesized by Shanghai DNA Bioscience) and loaded onto 4.5% nondenaturing polyacrylamide gels for electrophoretic separation. An Amersham Typhoon RGB Biomolecular Imager (Cytiva, Sweden) was used to scan the fluorescence patterns in the gels to detect the Cy5 fluorophore. Three independent experiments were performed with similar results.

Cy5-labeled promoter probes containing the UAS motif (TGTAAG-N6-CTTACA) in rsmY and rsmZ were synthesized commercially (Shanghai DNA Bioscience Co. Ltd). EMSA was performed using established protocols⁶⁸ . Briefly, the purified His-GacA was mixed with Cy5-labeled rsmY and rsmZ promoter fragments, respectively, then loaded on a 4.5% nondenaturing polyacrylamide gel for electrophoresis. The Cy5 fluorophore was detected using an Amersham Typhoon RGB Biomolecular Imager (Cytiva, Sweden). EMSA experiments were repeated three times independently.