Figure 5.

CsBPC proteins bind to the CsABI3 promoter. (A) Yeast one-hybrid assay of interactions between CsBPCs and the CsABI3 promoter. The CsABI3 promoter was fused to the pLacZi plasmid that was then linearized and integrated into the genome of the YM4271 yeast strain. Four CsBPC genes were cloned into pGADT7 to obtain the CsBPC1/2/3/4-pGAD plasmids. The CsBPCs-pGAD plasmids were transformed into a YM4271 clone harboring the CsABI3 promoter. The transformants were grown on SD/-Ura-Leu dropout plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) for white/blue screening. Unmodified PGADT7 was used as a negative control. (B) A ChIP assay showed that the CsBPCs bound to the CsABI3 promoter in vivo. GFP enrichment is expressed relative to the chromatin input. One pool comprising three independent transgenic lines was analyzed for each construct. Genomic DNA was obtained via ChIP from cucumber transgenic lines over-expressing CsBPC-GFP fusion proteins (CsBPC1/3-GFP-cuOX) using an anti-GFP antibody and was then subjected to qPCR analysis. The fragment used for qPCR is indicated by a red bar in the schematic diagram of the CsABI3 promoter. A control experiment with the Tubulin gene was used to establish the ChIP specificity. The mean values ± SD are representative of three independent experiments. ^***p < 0.001 by Bonferroni post-hoc test.