Split-ubiquitin membrane-based yeast two-hybrid system.

The MbY2H system was used to validate TSWV G_N-TIP interactions identified in the gel overlay assays. The MbY2H system enables the validation of interactions for soluble and integral membrane proteins. TSWV G_N coding sequence was cloned into the MbYTH vector pBT3-SUC, and the six TIP ORFs were cloned to vector pPR3N using the SfiI restriction site (Dualsystems Biotech, Schlieren, Switzerland). To identify the region of endoCP-G_N that binds to TSWV G_N using the MbY2H system, the amino acid sequence of endoCP-G_N (284 aa) was used to search against the NCBI nonredundant protein database using BLASTP. The conserved CHB4 domain was located at the C terminus of endoCP-G_N (amino acids 190 to 246). Therefore, the possible interacting domains, the nonconserved region of endoCP-G_N (amino acids 1 to 189) and the conserved CHB4 domain (amino acids 190 to 274), were individually cloned into pPR3N using the SfiI restriction site. Based on the BLASTP results, the homologous sequences from other insect species encompassed some additional amino acids in the N-terminal direction of the CHB4 domain; therefore, we made an alternative construct that included the conserved CBH4 domain starting from amino acid 177. Hence, the coding sequence of amino acids 1 to 176 and 177 to 284 of endoCP-G_N were also cloned to pPR3N using the SfiI restriction site. The primers used for cloning are available upon request.

The MbY2H assays were performed using the manufacturer’s instructions with recombinant plasmids that were confirmed by Sanger sequencing. Yeast (Saccharomyces cerevisiae strain NYM51) competent cells were freshly prepared, and the recombinant bait plasmid pBT3-SUC-G_N was transformed into yeast cells. Briefly, 1.5 μg of bait plasmid was added into 100 μl of yeast competent cells with 50 μg of denatured Yeastmaker carrier DNA (TaKaRa Bio USA, Mountain View, CA) and 500 μl polyethylene glycol-lithium acetate (PEG/LiAc). The mixture was incubated at 30°C for 30 min with mixing every 10 min. Twenty microliters of dimethyl sulfoxide (DMSO) was then added to each reaction mixture, and the cells were incubated at 42°C for 20 min with repeated mixing at 5-min intervals. After centrifugation at 14,000 rpm for 15 s, the supernatant was removed, and the pellet was resuspended in 1 ml of yeast-peptone-dextrose (YPD) medium. The resuspended cells were incubated at 30°C for 90 min with shaking at 200 rpm. Then, cells were centrifuged at 14,000 rpm for 15 s and resuspended in 500 μl of sterile 0.9% (wt/vol) NaCl, which was then spread and cultured on SD/–Trp dropout medium at 30°C until the colonies were visible. Several colonies from the same SD/–Trp plate were cultured for preparing yeast competent cells. Then, each individual recombinant plasmid, pPR3N-TIP or pPR3N-partial endoCP-G_N (1.5 μg/transformation reaction), was transformed into yeast competent cells expressing fused Nub-G_N. The transformants were cultured on both SD/–Leu/–Trp double-dropout (DDO) and SD/–Ade/–His/–Leu/–Trp quadruple-dropout (QDO) media. The positive controls included transformation of pOst1-NubI into the yeast strain NYM51 that already expressed fused Nub-G_N, as well as cotransformation of pTSU2-APP and pNubG-Fe65 into the yeast strain NYM51. The transformation of pPR3N (empty vector) into the yeast strain NYM51 that already expressed fused Nub-G_N was used as the negative control. Interactions between G_N-Cub and NubI and between G_N-Cub and NubG were used as positive and negative controls, respectively. All transformants were spread and cultured on both DDO and QDO media and cultured at 30°C in an incubator. The entire experiment was performed three times.

Expression of the reporter gene lacZ and the activity of expressed β-galactosidase in yeast cells derived from the MbY2H system was determined by a β-galactosidase assay kit, following the manufacturer’s protocol (Thermo Fisher Scientific).