Fig. 7: ELD1 interacts with phyB to regulate OsCCA1 AS.
a BiFC analysis revealed the interaction between ELD1 and phyB in N. benthamiana cells. The inset images display the magnified portions of YFP signal. U1-70K mCherry serving as nuclear speckles marker, Scale bars, 10 μm. b The in vivo Co-IP assay confirms the interaction between ELD1 and phyB in N. benthamiana. The immunoprecipitated samples were detected using the anti-GFP and anti-FLAG antibodies. The symbols “+” represent presence corresponding protein; symbols “−” represent empty construct. Three independent experiments were performed with similar results. c The AS pattern of A3SS1, IRS1, and IRS2 detected by sqRT-PCR after 2 h of red-light treatment in WT and phyBT822*. Continuous dark treatment serves as control. More than three independent experiments were performed with similar results. d–f The AS pattern of A3SS1 (d), IRS1 (e), and IRS2 (f) detected by RT-qPCR after 2 h of red-light treatment in WT and eld1 mutant. The indicated genotypes of 10-day-old seedlings were grown under CLD conditions, pretreated with darkness for 48 h, and then exposed to red light for 2 h. Continuous dark treatment serves as a control. Values are presented as means ± SD (n = 3 biological replicates). g RIP-qPCR assay shows the in vivo binding affinity of ELD1 to OsCCA1 mRNA. pUBI:ELD1-FLAG/eld1 transgenic seedlings grown for 10 days under CLD conditions were pretreated with darkness for 48 h, and then exposed to red light for 2 h. Continuous dark treatment serves as the control. Samples were immunoprecipitated with anti-FLAG Magnetic Beads. UBQ gene serves as an internal control. Values are presented as means ± SD (n = 3 biological replicates). For (d–g), two-sided Student’s t-test was used to calculate P values.
