Matchmaker gold yeast two hybrid system kit

The yeast two‐hybrid assays were performed using the Matchmaker^® Gold Yeast Two‐Hybrid System Kit (TaKaRa, Dalian, China) in accordance with the manufacturer's instructions with a minor modification. Briefly, 500 ng pGADT7 (AD) and 500 ng pGBKT7 (BD) vectors were simultaneously transformed into Y2HGold yeast cells using the polyethylene glycol/lithium acetate method. The transformants were selected on QDO/X plates (SD/‐Trp/‐Leu/‐His/‐Ade/X‐α‐gal). The pGADT7‐T and pGBKT7‐53 vectors provided with the kit were used as positive controls.

Y1H assays were performed using the Matchmaker Gold Yeast One-Hybrid System Kit (Takara) according to the manufacturer's protocol. Briefly, gene (PpMYB114, PpRAP2.4, and PpACS1) promoter fragments were ligated into the pAbAi vector, whereas the TF gene CDSs were cloned into the pGADT7 vector (gene-AD). The gene-AD vectors were then used to transform Y1HGold cells harboring the pAbAi-bait and then screened on SD/−Leu/AbA medium. Yeast two-hybrid assays were performed using the Matchmaker Gold Yeast Two-Hybrid System Kit (Takara) according to the manufacturer's protocol.

The yeast two-hybrid assays were performed, according to the manufacturer’s instructions, using the Matchmaker Gold Yeast Two-Hybrid System kit (Takara, Beijing, China). Briefly, FaBBX22 and its protein mutants were fused to the active domain of GAL4 (AD), and FaHY5 were fused to the DNA-binding domain of GAL4 (BD). The BD and AD plasmids were co-transformed into yeast strain Y2HGold, and, respectively, coated in synthetically defined medium (SD/-Trp-Leu and SD/-Trp-Leu-Ade-His/AbA/X-α-gal) for observation.
The point mutation constructs, FaBBX22-D20A, FaBBX22-D70A and FaBBX22-D79A, were generated with ClonExpress MultiS One Step Cloning Kit (Vazyme, Nanjing, China). The fragments of FaBBX22 containing D20A, D70A or D79A mutations were amplified using the primer pairs D20A-1/2, D70A-1/2 and D79A-1/2, respectively. After the PCR amplification, the products were ligated into pGADT7 vector, based on homologous recombination. The primer sequences used for the vector construction are listed in Table S1, Supplementary Materials.

The yeast two-hybrid assays were performed, according to the manufacturer’s instructions using the Matchmaker Gold Yeast Two-Hybrid System kit (Takara, Beijing, China). The coding sequence encoding the N-terminal of FaBBX24 (amino acids 1–99) and the C-terminal of FaBBX24 (amino acids 100–238) was cloned into pGBKT7 (Clontech) to generate the bait vector (BD-FaBBX24^N99, BD-FaBBX24^C139) containing the GAL4 DNA-BD sequence. The full-length coding sequence of FaMYB5 was individually cloned into pGADT7 vector (Clontech) to produce the prey vectors (AD-FaMYB5) containing the sequence encoding the GAL4 activation domain (AD). Yeast strain AH109 was co-transformed with the bait and prey vectors, and then protein interactions were evaluated on the basis of the ability of the cells to grow on synthetic defined (SD) medium lacking Leu, Trp, His, Ade and +X-α-gal after 4 days of growth at 28 °C.

The pGBKT7-NIa-pro construct containing the coding sequence of NIa-pro of PVY^NTN in-frame with the yeast GAL4 binding domain protein was used as a bait construct for the Y2H library screening. The mRNA was isolated from leaves of the N. benthamiana plants using Oligotex mRNA Mini Kit (Qiagen) and by following the manufacturer’s instructions. The N. benthamiana Y2H cDNA library was constructed using mRNA isolated from the leaves of the N. benthamiana plants and Matchmaker^® Gold Yeast Two-Hybrid System kit (Takara). Construction of N. benthamiana Y2H cDNA library and screening were performed as illustrated in the Matchmaker Gold Yeast Two-Hybrid System User Manual (Takara). The N. benthamiana Y2H library was screened using pGBKT7–NIa-pro as the bait in Saccharomyces cerevisiae strain Y2HGold. Yeast colonies containing potential interacting partners of NIa-pro were selected based on the ability of the co-transformed cells to grow on synthetic media containing aureobasidin and lacking Trp and Leu. Subsequently, those yeast cells were screened with high stringency and were selected based on their ability to grow on synthetic media lacking Trp, Leu, and His and containing 1mM 3-amino-1,2,4-triazole. The prey plasmids present in the yeast colonies that were able to grow on synthetic media lacking Trp, Leu, and His and containing 3-amino-1,2,4-triazole were rescued and sequenced.

Yeast two-hybrid assays were performed with the Matchmaker™ Gold Yeast Two-Hybrid System Kit (TaKaRa, Dalian, China). The full-length coding sequences encoding the prey and bait proteins were cloned into the pGADT7 (AD) and pGBKT7 (BD) vectors, respectively. First, the full-length PcMYB10-BD and PcMYC2-BD plasmids were inserted into Y2HGold cells with an empty AD vector, after which a lack of self-activation was confirmed for PcMYB10-BD and PcMYC2-BD. Y2HGold competent cells were co-transformed with the recombinant gene-AD and gene-BD plasmids and spread agar-solidified SD/−Leu/−Trp medium. To evaluate potential physical interactions, the co-transformed colonies were selected on SD medium lacking adenine, histidine, leucine, and tryptophan, supplemented with X-α-gal.