Pgbkt7

HEK293T cells or PK-15 cells (porcine kidney cells) were grown in Dulbecco's modified Eagle's medium (DMEM) (catalog no. C11995500BT; Gibco) containing 10% fetal bovine serum (FBS) (catalog no. 12007C; Sigma-Aldrich) and maintained at 37°C in 5% CO₂. The CSFV Shimen strain was propagated in PK-15 cells as described previously (13 (link)) and titrated using the Reed-Muench formula (29 ).
The bait construct pGBKT7-E2 (BD-E2) harboring the E2 gene without the transmembrane domain was generated from the CSFV Shimen strain by PCR and cloned into pGBKT7 (BD) or pGEX-6P-1. The E2 gene with the signal peptide sequence in the 5′ terminus and the Flag tag in the 3′ terminus was obtained by PCR and cloned into the pCAGGS vector (Addgene), giving rise to pCAGGS-E2-Flag. To construct the MEK2 expression vector, total cellular RNA was extracted from PK-15 cells using an RNeasy Plus minikit (catalog no. 74134; Qiagen). The gene encoding MEK2 (accession no. NM_001244550.1) was amplified by PCR and ligated into the pCMV-Myc vector (Clontech), creating pMyc-MEK2. The primers used in this study are shown in Table 1.

Total RNA was extracted from 14‐day‐old WT seedlings, and genomic DNA was removed after DNaseI treatment. cDNA was synthesized using the SMART cDNA Library Construction Kit (Clontech) and sent to Takara (Osaka, Japan) to construct the cDNA library. The yeast two‐hybrid assay was performed according to the manufacturer’s manual and the Matchmaker GAL4 Two‐Hybrid System 3 (Takara, Osaka, Japan). The CDS of ZmTE1 were cloned into the bait plasmid pGBKT7 (BD), and yeast‐two‐hybrid screening was conducted according to the manufacturer’s instructions. To confirm the identified protein–protein interactions, the CDS of candidate genes were cloned into the pGADT7 (AD). BD‐ZmTE1 and the AD‐candidate fusions (empty vector was used as negative control) were transferred together into yeast Y2HGold using the PEG/LiAc method. After culturing on synthetic medium plates (SD medium) lacking Trp and Leu (‐LW) for two days, the transformants were transferred onto SD‐Trp‐Leu‐His (‐LWH) and SD‐Trp‐Leu‐His‐Asp (‐LWAH) for an additional three or four days. Primers used in this assay are listed in Table S1.

A yeast two-hybrid (Y2H) assay was performed using the Matchmaker™ Gold Yeast Two-Hybrid System (Clontech, USA). The CDS of GmVQ58 was cloned into the vector pGBKT7 (BD) to produce the construct BD-GmVQ58. Additionally, the CDS of GmWRKY32 (Glyma.02g115200) was PCR-amplified from the leaf cDNA of the soybean cultivar Williams 82 at 1 d after CCW attack and was subcloned into the vector pGADT7 to produce the construct AD-GmWRKY32 (primers shown in Supplementary Table S1). The transformed yeast cells were first grown on plates containing double-dropout media (−Leu/−Trp) and then transferred to higher-stringency plates containing quadruple-dropout media (−Leu/−Trp/−His/−Ade). Combinations of pGBKT7-53 (BD-53) and pGADT7-T (AD-T) were used as positive controls, while the negative controls were pGBKT7-lam (BD-lam) and AD-T. Plates used for the identification of protein interactions were supplemented with X-α-Gal to visualize the interactions.

In Arabidopsis, previous works have shown that the C-terminal domain of AtCRY1 is an important region for the interaction between AtCRY1 and AtCOP1 through yeast two-hybrid (Y2H) assay (Wang et al., 2001 (link); Yang et al., 2001 (link)). Further, Holtkotte et al. (2017) (link) found that blue light enhanced the interaction intensity of AtCRY1 protein and AtCOP1 protein in yeast. To investigate the protein-protein interaction between FaCRY1 and FaCOP1, the Y2H assays were conducted as described by our previous study (Liu et al., 2022 (link)). The C terminal region (amino acids 479-673) of FaCRY1 was amplified and inserted into multiple cloning site (MCS) of bait vector pGBKT7 (BD). The full-length CDS of FaCOP1 was inserted into the MCS of prey vector pGADT7 (AD). The bait and prey constructs were co-transformed into yeast strain Y2HGold using the PEG/LiAc-based method. Following transformation, the yeast cells were spread onto synthetically defined medium (SD/-Trp-Leu) and incubated at 30 °C for 3 days. Then, 10 independent clones were selected and transferred to SD/-Trp-Leu-Ade-His medium supplemented with Aureobasidin A (100 ng/mL) and X-α-gal and cultured under blue light (50 μmol·m^-2·s^-1) for 4 days. All primers used for the vector construction are listed in Supplementary Table 1.

The full-length CDS of FaBBX22 was inserted to yeast expression vector pGBKT7 (BD). To verify the transcriptional activity of FaBBX22, the pGBKT7-FaBBX22 vector and negative control (pGBKT7-lam) were transformed to yeast strain Y2HGold, respectively. After 3 days, the growth of the yeast cells in the synthetically defined medium (SD/-Trp and SD/-Trp-Ade-His/X-α-gal) was observed.