Pb42ad

To generate pLexA-phyB constructs for yeast two-hybrid assay, N-terminal (1-651 aa) and C-terminal (652-1172 aa) fragments of phyB CDS were amplified by Super-Fidelity DNA Polymerase (Vazyme) and inserted into the EcoR I/Xho I sites of pLexA vector (Clontech). For pB42AD-SMP2 plasmid, full-length SMP2 CDS was inserted into EcoR I/Xho I sites of pB42AD (Clontech).
For firefly luciferase complementation imaging (LCI) assays, the full-length phyB CDS were inserted into the Kpn I/Sal I sites of pCambia1300-nLUC, and SMP2 CDS were inserted into the BamH I/Sal I sites of pCambia1300-cLUC.
To generate overexpression of YFP-SMP2 construct, the Gateway cloning technology was used. Full-length SMP2 open reading frame was cloned into the pDONR-223 vector using Gateway BP Clonase Enzyme mix (Invitrogen), and introduced into the plant binary vector pEarley Gateway 104 under the control of the 35S promoter using Gateway LR Clonase Enzyme mix (Invitrogen).

FBL1 and Aux/IAA coding regions were cloned into the Y2H bait vector pGILDA and the prey vector pB42AD (Clontech), respectively, after amplifying using the primer pairs shown in Table S3. Bait and pray constructs were cotransformed into Saccharomyces cerevisiae strain EGY48[p8opLacZ] (Clontech), and transformants were selected on SD supplemented with –Ura/–His/–Trp dropout solution (BD Biosciences) and glucose medium. To test the interaction between FBL1 and Aux/IAA proteins, transformed yeast colonies were plated on SD‐galactose/raffinose‐inducing medium containing –Ura/–His/–Trp dropout supplement, 80 μg/mL X‐Gal and NAA in the different concentrations of 0 μm, 1 μm, 10 μm, 100 μm and incubated for 3‐4 days at 30 °C (Calderón Villalobos et al., 2012; Yu et al., 2015).

The Clontech™ one‐Hybrid System (Clontech, Dalian, China) was used in this study. The CDS of potential transactivators were fused with GAL4 AD domain in pB42AD (Clontech, Dalian, China), and the promoter region of Wx were cloned into pLacZ2u (Clontech, Dalian, China). Yeast strain EGY48 was transformed with indicated plasmids and grew on SD/‐Ura/‐Trp plates, and then strike on SD/‐Ura/‐Trp plates containing 2% glactose, 1% raffinose, 1 × BU salts and 80 mg/L X‐Gal (Clontech, Dalian, China). The interaction was confirmed by the visualization of blue colonies on the medium. NF‐YB1‐pB42AD and SUT4‐pLacZ2u were used as CK+ (Bai et al., 2015). The empty vectors pLacZ2u and pB42AD were used as negative control.

To analyze the specific binding of AaSPL2 to GTAC-box of the DBR2 promoters, the full length of AaSPL2 was fused into the EcoRI–XhoI sites of the activation domain of the vector pB42AD (Clontech). Equal volume of the GTAC-box forward primer (10 μM) and reverse primer (10 μM) (Table 1) were mixed and incubated at these conditions: 95°C for 1 min, 85°C for 1 min, 75°C for 1 min, 65°C for 1 min, 55°C for 1 min, 45°C for 1 min, 35°C for 1 min, 25°C for 1 min. The reaction primer products were inserted into P178 vector (gifted by Dr. Yang Hongquan of Shanghai Jiao Tong University, Shanghai, China) with a single restriction site XhoI to form the reporter vector. Vectors containing the transcription factor gene and 3 × GTAC-box were introduced into the yeast strain EGY48 by LiAc mediated transformation method (Gietz and Schiestl, 2007 (link)). Yeast cells were selected on the synthetic dropout (SD) base medium with deficient in Trp and Ura for 3 days at 30°C. Then yeast cells were resuspended with sterile water and dropped on an 5-bromo-4-chloro-3-indolyl-beta-Dgalactopyrano-side (X-gal) medium plate (Zhang et al., 2015 (link)). The sequences of the 3 × GTAC-box fragment are listed in Supplementary Table S1.

The coding sequences of ABI5 were inserted into pB42AD (Clontech), and the promoter of EM1 was inserted into pLacz (Clontech). The vectors were subsequently cotransformed into the yeast strain EGY48. Transformants were spotted on SD-Ura-Trp media. After 28 °C cultivation for 3 days, colonies were randomly selected and spotted on SD-Ura-Trp media supplemented with 2% (w/v) galactose, 1% (w/v) raffinose, 1 × salt buffer (7 g/L Na₂HPO₄·PH₂O, 3 g/L Na₂HPO₄, pH 7.0), and 80 mg/L 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside acid (Clontech). The plates were incubated for 1 day. The primers used in this assay are listed in Supplementary Data 2.

The reporter plasmids were constructed as described by Wang et al.75 (link). The PERE-like-element-containing fragment in TERF1 promoter was amplified with primer 5′-TCTTTAATTATAGATATTTTAAAC-3′ and 5′-CGTTAGTACTTATTTGAATGTATC-3′, and cloned as a trimer into the MCS upstream of HIS3 minimal promoter in pHISi-1 vector and that upstream of the lacZ minimal promoter in pLacZi vector (Clontech, USA). Then, these two plasmids were linearized and co-transformed into yeast stain YM4271 to obtain the reporter yeast. The cDNAs of LeEILs were cloned with following primers: 5′-TCATCCTGTGGAAGATGATGATGT-3′ and 5′-ACAACATGTCAACAGACTTCTGGC-3′ for LeEIL1, 5′-TGGCTGCCAAGATGATGATGTTTG-3′ and 5′-CTTGATGTTCATTTGAGTAATCGC-3′ LeEIL2, 5′-TGGTAAATGGGGATATTTGAAGAT-3′ and 5′-CAGTTTAATACTAGTACTAGTTCA-3′ for LeEIL3, and 5′-GAGTTTGTTCTTGTGAAGATGATG-3′ and 5′-ACGTTTCACCAATATCATGGCTAG-3′ for LeEIL4. Then, the coding sequences of LeEILs were fused into downstream of transcriptional activation domain of the yeast vector pB42AD (Clontech, USA), and introduced into the yeast reporter strain respectively. Transformants that could grow on the selective medium lacking histidine but containing 30 mM 3-AT were subjected to filter-lifted β-galactosidase activity according to the manufacturer’s protocol (Clontech, USA).