PvC3H69 was subcloned into the BD vector pGBKT7 to fuse PvC3H69 with the DNA-binding domain of GAL4. The pGBKT7-PvC3H69 and the control vector pGBKT7-GUS (UiDA gene) were then transformed into the yeast strain Y2HGold (Clonetech), separately. The pGBKT7-PvC3H72 was used as a positive control25 (link). The transformed positive clones grown well on SD/-Trp were then grown on plates containing SD/-Trp-Ade-His and SD/-Trp-Ade-His + 25 mM 3-AT for auto-transactivation assay.
For the transcriptional activity assay of PvC3H69 in plant cells, PvC3H69 was cloned into the 35S promoter-droven pZB370 vector to fuse with the yeast GAL4 DNA-binding domain (GAL4BD) as effector (pZB369-PvC3H69), while the vector without the target gene was used as the negative control. As a positive control, PvC3H72 has been reported to be a transcriptional activator25 (link). The internal control vector was pZB371-Luciferase under driven of 35S promoter as well. The reporter vector (pZB370-GUS) was constituted of four copies of GAL4 DNA-binding sites (GAL4(4x)-D1-3(4x)) to drive the GUS (UidA) reporter gene. Three plasmids (effector, reporter, and internal control) were co-transferred into Arabidopsis protoplasts at the ratio of 5:4:1. The transcriptional ability was assessed by the GUS/LUC ratio. Three biological replicates were included for each combination.
For the transcriptional activity assay of PvC3H69 in plant cells, PvC3H69 was cloned into the 35S promoter-droven pZB370 vector to fuse with the yeast GAL4 DNA-binding domain (GAL4BD) as effector (pZB369-PvC3H69), while the vector without the target gene was used as the negative control. As a positive control, PvC3H72 has been reported to be a transcriptional activator25 (link). The internal control vector was pZB371-Luciferase under driven of 35S promoter as well. The reporter vector (pZB370-GUS) was constituted of four copies of GAL4 DNA-binding sites (GAL4(4x)-D1-3(4x)) to drive the GUS (UidA) reporter gene. Three plasmids (effector, reporter, and internal control) were co-transferred into Arabidopsis protoplasts at the ratio of 5:4:1. The transcriptional ability was assessed by the GUS/LUC ratio. Three biological replicates were included for each combination.
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