Figure 4.

Nuclear FRH is required for Frq1 and Frq2 to function in nucleus. (A,B) Western blots for accumulation levelsof GFP and FRH::GFP in the total protein extracts of transgenic strain (TS3) and WT cultures at L:D 12:12 and of FRH::GFP in the total (T), cytoplasmic (C), and nuclear (N) protein extracts of 3-day-old TS3 cultures grown in SDBY at 25 °C and L:D 24:0 or 0:24. (C) Time-course western blots for accumulation levels of FRH::GFP in TS3 and of Frq1::GFP (F1) and Frq2::GFP (F2) in TS1::Δfrh and TS2::Δfrh strains. Nuclear and cytoplasmic protein extracts were isolated from the hyphal cultures exposed 0–24 h (3-h interval) to dark after 60-h light incubation (left panels) or to light after 60-h dark incubation (right panels) of a 10⁶ conidiamL⁻¹ suspension in SDBY at 25 °C. Each lane was loaded with 60 μg protein extract and probed by anti-GFP antibody. Nuclear standard: histone H3 probed by anti-H3 antibody. Cytoplasmic standard: β-tubulin (βT) probed by anti-β-tubulin antibody. Note that accumulation level of FRH::GFP in TS3 was steadily high in nucleus during the time-course exposure but undetectable in cytoplasm and that deletion of frh led to residual or undetectable levels of F1 and F2 in both nucleus and cytoplasm. (D) Conidial yields measured from the SDAY cultures of frh mutants and WT during a 10-day incubation at 25 °C in five L:D cycles. (E) Relative transcript levels of frq1, frq2, four photoreceptor genes and four developmental activator genes in the 3-day-old SDAY cultures of frh mutants versus WT grown at 25 °C under light and dark conditions. The dashed line denotes a significance for one-fold repression. Error bar: SD of the mean from three replicates (D) or cDNA samples (E) analyzed through qPCR with primers (Table S2).