Plasmid Construction and Validation

All plasmids used in this study were generated by In-Fusion Cloning methods (https://www.takarabio.com/products/cloning/in-fusion-cloning). The sequences subcloned into plasmids were verified by Sanger sequencing. The mammalian cell expression vectors used in this study were made with pQCMV-Flag-GFP (Supplementary Fig. 4), pQCMV-GFP (Supplementary Fig. 4), and pCMV-Myc^{20 (link)}. pQCMV-Flag-GFP vector was modified from the commercial plasmid pEGFP-N1 (Clontech), which allows the inserted gene expressed under control of the CMV promoter and fused with dual in-frame tags of Flag and GFP. pQCMV-GFP vector was further modified from pQCMV-Flag-GFP by removing the coding sequence of Flag tag. pCMV-Myc vector was described previously^{20 (link)}. To generate plasmids expressing Flag-LRB1, Flag-LRB2, Flag-CRY2, and Flag-CRY2^D387A, the coding sequences (CDS) of all the above genes were amplified either from Arabidopsis cDNA or plasmids made before by PCR, the purified PCR products were then subcloned into SpeI/KpnI-digested pQCMV-Flag-GFP vector through in-fusion. Myc-LRB1, Myc-LRB2, Myc-CRY2, Myc-SPA1, and Myc-COP1 plasmids were prepared by cloning the CDS of the genes into the BamHI site of pCMV-Myc vector. For plasmids expressing HA-PPK1 and HA-PPK1^D267N, the coding sequences of PPK1 and PPK1^D267N were PCR-amplified from plasmids made before by using the primers, of which a 2x HA coding sequence was attached to the 5’ end of forward primer, and introduced into SpeI/KpnI-digested pQCMV-GFP vector by in-fusion. The gene-specific primers for constructs used for expressing recombinant proteins in HEK293T cells are listed in Supplementary Table 2.
The BiFC constructs were built with the vectors described previously^{47 (link)}. The coding sequences of LRB1 (AT2G46260), LRB2 (AT3G61600), CRY2 (AT1G04400), and CRY2^D387A were PCR-amplified and cloned into pDONR/Zeo entry vector through BP reaction (Gateway™ BP Clonase™ II Enzyme mix, Cat. # 11789020, Invitrogen). The entry constructs were then introduced into the destination vector pX-nYFP (N-terminus of YFP fused to C-terminus of genes) or pcCFP-X (C-terminus of CFP fused to N-terminus of genes) by LR reaction (Gateway™ LR Clonase™ II Enzyme mix, Cat. # 11791020, Invitrogen). The gene-specific primers of Gateway cloning for constructs used for BiFC assys are listed in Supplementary Table 2.
The split-LUC constructs were made with the vectors described previously^{48 (link)}. CRY2 were fused to the C-terminus of cLUC (the C-terminus of Luciferase). LRB1ΔBTB were generated by seamlessly connecting two fragments, 1-426 bp and 637-1683 bp of LRB1 coding sequence, with 15 bp overlaps, by infusion cloning method. LRB2ΔBTB were generated with two fragments, 1-432 bp and 751-1683 bp of LRB2 coding sequence through the same method as making LRB1ΔBTB. LRB1ΔBTB and LRB2ΔBTB were then fused to the N-terminus of nLUC (the N-terminus of Luciferase) by in-fusion. The primers for constructs used for Split-LUC assays are listed in Supplementary Table 2.
pFGFP (Supplementary Fig. 5) and pDT1H (Supplementary Fig. 5) binary vectors were used for creating overexpression transgenic lines. The pFGFP binary vector was modified from pCambia3301^{20 (link)}, which allows the interested genes to express under Actin2 promoter and fuse with 2x Flag and GFP tags. The pDT1H binary vector, possessing two independent expression cassettes that allows two genes to assemble into one vector, was modified from previously published vector pDT1^{49 (link)} by replacing the BAR gene with HPT gene to convert basta resistance to hygromycin resistance in plants. The CDS of LRB1, LRB2, CRY2, and CRY2^P532L were cloned into BamHI-digested pFGFP vector to generate FGFP-LRB1, FGFP-LRB2, FGFP-CRY2, FGFP-CRY2^P532L with 2x Flag and GFP tags fused to N-terminus of the genes. The native promoters of LRB1 and LRB2 were cloned into SacI/SpeI-digested FGFP-LRB1 and FGFP-LRB2 to generate ProLRB1::FGFP-LRB1 and ProLRB2::FGFP-LRB2, respectively. The CDS regions of LRB1, LRB2, and COP1 (AT2G32950) were cloned into the BamHI site of pDT1H vector to produce Myc-LRB1, Myc-LRB2, and Myc-COP1 with 4x Myc tags fused to N-terminus of the genes. The primers for constructs used for plant transformation are listed in Supplementary Table 2.