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. 2020 Jul 13;184(1):487–505. doi: 10.1104/pp.20.00486

Figure 4.

Figure 4.

CRY1 and CRY2 physically interact with TOE1 and TOE2 in a BL-specific manner. A to D, Semi-in vivo pull-down assays showing BL-specific interactions of CRY1 with TOE1 (A) and TOE2 (B) and CRY2 with TOE1 (C) and TOE2 (D). His-TF-TOE1 served as bait in A and C, and GST-TOE2 served as bait in B and D. Myc-CRY1- and CRY2-containing protein extracts from 5-d-old etiolated Myc-CRY1-OX and Myc-CRY2-OX seedlings that were adapted to DK or exposed to BL (20 μmol m−2 s−1), RL (20 μmol m−2 s−1), or FRL (10 μmol m−2 s−1) for 1 h served as preys. The prey proteins pulled down by His-TF-TOE1 and GST-TOE2 were detected with anti-Myc antibody. The amount of His-TF-TOE1 and GST-TOE2 was shown in Coomassie Brilliant Blue staining gel. E to H, Co-IP assays showing BL-induced interaction of CRY1 with TOE1 (E) and TOE2 (F) and CRY2 with TOE1 (G) and TOE2 (H) in Arabidopsis. Five-day-old white-light-grown double transgenic seedlings coexpressing Myc-CRY1 and mTOE1-Flag (Myc-CRY1-OX/mTOE1-Flag) or Myc-CRY1 and mTOE2-Flag (Myc-CRY1-OX/mTOE2-Flag) or Myc-CRY2 and mTOE1-Flag (Myc-CRY2-OX/mTOE1-Flag) or Myc-CRY2 and mTOE2-Flag (Myc-CRY2-OX/mTOE2-Flag) were adapted in the dark for 3 d and then treated with 100 μm of MG132 for 4 h, and finally transferred back to DK or exposed to BL (30 μmol m−2 s−1) for 1 h. The IP (CRY1 and CRY2) and co-IP signals (TOE1 and TOE2) were detected by immunoblots probed with anti-Myc and -Flag antibodies. Transgenic Arabidopsis seedlings expressing the mutated TOE1 and TOE2 genes that comprise the mutated miR172 target sites but encode the same amino acid sequences as the wild-type TOE1 and TOE2 genes, which were fused to the DNA-fragment–encoding Flag tag under the control of the native promoters of TOE1 and TOE2 in wild-type background (mTOE1-Flag and mTOE2-Flag). We chose one line for each of the mTOE1-Flag and mTOE2-Flag transgenic Arabidopsis (mTOE1-Flag12 and mTOE2-Flag16) and genetically crossed with Myc-CRY1-OX or Myc-CRY2-OX plants to generate the double transgenic lines expressing both Myc-CRY1 or Myc-CRY2 and TOE1-Flag or TOE2-Flag (Myc-CRY1-OX/mTOE1-Flag, Myc-CRY2-OX/mTOE1-Flag, Myc-CRY1-OX/mTOE2-Flag, and Myc-CRY2-OX/mTOE2-Flag).