Matchmaker two hybrid system

For performing the yeast two-hybrid assay, the Matchmaker Two-Hybrid System (Clontech Laboratories, Inc., cat# 630489) was used. The yeast strain HA109 was used, and the construct was prepared as per methods described previously^{1 (link)} GID1 in pGBKT7 served as the bait, and SLR1 in pGADT7 was considered as the prey. Plate assays (-His) were performed according to the manufacturer’s instructions with modifications to the plates containing GA₁, GA₃, GA₄, and GA₇ concentrations ranging between 10⁻¹⁰ M and 10⁻⁵ M. Control plates contained no GA.

A Matchmaker two-hybrid system (catalog no. 630489; Clontech) was used to screen host proteins that interact with NS5A. Briefly, the bait construct pGBKT7-NS5A (BD-NS5A) was transformed into the yeast strain Y2HGold and hybridized with a cDNA library derived from porcine macrophages [39 (link)]. Transformants were selected for growth on synthetically defined (SD) medium lacking His, Leu, Trp and Ade (SD/-4) for 3 to 6 days at 30 °C. The clones were then transferred to SD/-4 medium containing 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-Gal) and aureobasidin A (Aba) (SD/-4/X-α-Gal/Aba). Blue colonies were selected and inoculated with 5 mL of SD/-4 medium for shaking culture at 30 °C for 1 to 3 days. Yeast plasmids were extracted using a yeast plasmid kit (catalog no. D3376; Omega, Guangzhou, China) according to the manufacturer’s instructions, and the target inserts were verified by sequencing using the Gal4 AD and 3ʹAD primers. To validate the interaction between NS5A and the cellular proteins, the bait and prey plasmids were cotransformed into the Y2HGold yeast strain using a yeast transformation system 2 (catalog no. 630439; Clontech). The interaction between murine p53 and SV40 large T-antigen was used as a positive control, and the human lamin C protein, which does not interact with the SV40 large T-antigen, was included as a negative control.

Saccharomyces cerevisiae strain AH109 and GAL4-based Matchmaker Two-Hybrid System (Clontech, Palo Alto, CA, USA) were used in a transactivation activity assay. The full-length ORF of TaMOC1 and N-terminal truncation versions were amplified, and inserted into pGBKT7 to produce in-frame fusions to the GAL4-binding domain. The TaMOC1 coding region was amplified using primers 5’-AGCTGAATTCATGATCGGCTCACTCCACTCTTC-3’ (EcoRI site underlined) and 5’-ACTCGGATCCCCTACTGCCACGCCGACAC-3’ (BamHI site underlined). Two pairs of primers, 5’-AATTGAATTCATGACGCGGGACCTCGTGCT-3’ (EcoRI site underlined) and 5’-ACTCGGATCCCCTACTGCCACGCCGACAC-3’ (BamHI site underlined), and 5’-AATTGAATTCATGCTGGCCGTGAACTGCGT-3’ (EcoRI site underlined) and 5’-ACTCGGATCCCCTACTGCCACGCCGACAC-3’ (BamHI site underlined), were used to amplify two N-terminal truncation versions, the former containing the whole GRAS domain, and the latter containing the PFYRE and SAW motifs of the GRAS domain in TaMOC1. The vectors were then transformed into yeast strain AH109 in liquid culture, which was diluted to an absorbance at 600 nm (A600) set at 1.0. Serial dilutions were inoculated onto tryptophan-, histidine- and adenine-negative synthetic dropout medium. The pGBKT7-vector was used as the negative control.

The cDNA sequences encoding the C-terminal of AhphyA (601–1125 aa), AhphyA-like (601–1125 aa) and AhphyB (626–1151 aa) were cloned into pGADT7 at NdeⅠ/SmaⅠ, NdeⅠ/SmaⅠ, BamHⅠ/XhoⅠ restriction sites, respectively. Full length of AhPIF3 with the removed N terminal 101–150 aa was cloned into pGBKT7 at EcoRⅠ/ BamHⅠ restriction sites. The primers used for these sequence amplifications were listed in S2 Table. The matchmaker two-hybrid system (Clontech, CA) was used for protein interaction analysis.

The yeast two-hybrid assay was performed using the Matchmaker Two-Hybrid System (Clontech¹). The full-length CDS and different truncations of OseIF3e, OsICKs, and other subunits of OseIF3, OseIF1, OseIF2, OseIF4, OseIF5, and OseIF6 were amplified by PCR using the primers listed in Supplementary Table S2. The fragments were cloned into the pGBKT7 or pGADT7 vector. Then co-transformed into yeast strain AH109 first selected on SD/-Leu/-Trp (DDO) plates at 30°C for 3 days, signal colony from yeast transformants including different pair of constructs were diluted in 0.9% NaCl, and a 1/10th dilution was spotted on SD/-Ade/-His/-Leu/-Trp (QDO) plates and incubate at 30°C for 3 days. Yeast cells co-transformed with pGBKT7-53 and pGADT7-T were used as the positive control, pGBKT7-Lam and pGADT7T were used as the negative control.