ZmNF-YB16 interact with ZmNF-YC17 in vitro and in vivo. A, Transcriptional activation analysis of ZmNF-YB16. Numbers at the top indicate the position of AAs. The N-terminal 21–90 AA represents the histone-like domain. Different lengths of proteins were cloned and fused to the GAL4 DNA binding (BD) vector. Transformants were screened on SD/-Leu and SD/-His, and β-galactosidase (β-gal) activities were validated by X-Gal staining and quantified by the ONPG assay. B, Growth of yeast cells harboring ZmNF-YB16 and ZmNF-YC17 fused to the GAL4 BD domain (pGBKT7-BD) and GAL4 activity domain (pGADT7-AD). SD/-Leu/-Trp (DDO) is the nonselective medium, and SD/-Leu/-Trp/-Ade/-His (QDO) is the selective medium. C, In vitro pull-down assay of ZmNF-YC17-GST protein and ZmNF-YB16-His protein. The fractions pulled down were detected with an His antibody. D, Verification of the in vivo interaction between ZmNF-YB16 and ZmNF-YC17 by the BiFC system in N. benthamiana. Expression of ZmNF-YB16-cYFP and ZmNF-YC17-nYFP was observed in leaves. The YFP fluorescence signal was detected in the group co-transformed of ZmNF-YB16-cYFP and ZmNF-YC17-nYFP (lower row), but not in the control groups (Rows 1–3). bar = 20 µm. E, Co-IP assay to test the direct interaction between ZmNF-YB16 and ZmNF-YC17. IP: antibody, antibody used for immunoprecipitation, IB: antibody, antibody used for western blot.