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. 2016 Jan 8;12(1):e1005760. doi: 10.1371/journal.pgen.1005760

Fig 4. ERF1 directly binds to ASA1 promoter region in vitro and in vivo.

Fig 4

(a) EMSA assay for binding to GCC-box sequence in the promoter of ASA1 by ERF1 protein in vitro. Dig-labelled probes were incubated with ERF1-MBP protein. As indicated, unlabelled probes were used as competitors, unlabelled probes with mutated GCC-box sequence were used as non-competitors, and the ERF1-MBP protein bound probers were separated from free probes by an acrylamide gel. (b) Yeast-one-hybrid assay. pGADT7/ERF1 (AD-ERF1) and pHIS2/ASA1pro (BD-ASA1pro) constructs were co-transformed into yeast strain Y187. AD-empty and BD-empty, AD-empty and BD-ASA1pro, AD-ERF1 and BD-empty, AD-empty and BD-3*GCC-box were used as negative controls while AD-ERF1 and BD-3*GCC-box were used as a positive control. (c) Chromatin immunoprecipitation-PCR for ASA1 promoter. Roots of 5-day-old 35S:HA:ERF1 and Col-0 seedlings were used. Anti-HA antibodies were used for the enrichment of the DNA fragments containing GCC-box in the promoter of ASA1. The results were determined by real-time PCR. Tub8 was used as a negative control (NC). (d) Quantitative real-time PCR was performed using the same ChIP products and PCR primers flanking GCC-boxes in ASA1 promoter as in c. The region of ASA1 that do not contain GCC-box was used as negative control. Values are mean ± SD of three replicas (***P<0.001). Asterisks indicate Student’s t-test significant differences.