Yeast two-hybrid assays were performed using the yeast strain Y2H Gold (Clontech), following the manufacturer’s instructions. For construction of the Gateway entry clones, PCR products were inserted into pDONR207 via BP reactions (Gateway BP clonase enzyme mix; Invitrogen), then cloned into the expression vectors (pGBKT7 or pGADT7) which contain the attR1-CmR-ccdB-attR2 fragment via LR reactions (Gateway LR clonase enzyme mix; Invitrogen) as previously described [46 (link)]. These vectors were transformed into Y2H Gold yeast (Clontech). The transformants were grown on SD/-Trp-Leu, SD/-Trp-Leu-His, and SD/-Trp-Leu-His-Ade dropout selective culture-media.
To perform firefly luciferase complementation imaging assays, the coding regions of the target genes were fused with either nLUC or cLUC and cloned into the pCAMBIA1300 vector as previously described [47 (link)]. These vectors were transformed into A. tumefaciens. The positive clones were injected into N. benthamiana as previously described [47 (link)], the bioluminescent signals were detected by NightSHADE LB985 system (Berthold).
To perform firefly luciferase complementation imaging assays, the coding regions of the target genes were fused with either nLUC or cLUC and cloned into the pCAMBIA1300 vector as previously described [47 (link)]. These vectors were transformed into A. tumefaciens. The positive clones were injected into N. benthamiana as previously described [47 (link)], the bioluminescent signals were detected by NightSHADE LB985 system (Berthold).
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