Yeastmaker yeast transformation system

The SbMYB44 ORF was cloned in the EcoRI and XhoI sites of the yeast expression vector pYES2 (Invitrogen). The plasmids of pYES2-SbMYB44 and vector alone were transformed separately in yeast strain W303 using Yeastmaker yeast transformation system (Clontech). A stress tolerance assay of recombinant yeast cells was performed as described by Li et al. (2014) (link) with minor modifications. Yeast cells having pYES2-SbMYB44 and vector alone were grown in SD/-Ura broth for 24 h at 30 °C. After adjusting the OD₆₀₀ to 0.4, 500 µL of culture was added to 10 mL of induction medium (SD/-Ura broth supplemented with 2 % galactose) and grown for 36 h to promote the expression of SbMYB44 gene. The cultures were diluted to an OD₆₀₀ 0.6, and 500 µL of culture was inoculated in 10 mL SD/-Ura containing 2.5 M NaCl, 5.0 M NaCl, 15 % polyethylene glycol (PEG 6000) equivalent to −0.295 MPa of osmotic potential and 30 % PEG 6000 equivalent to −1.027 MPa of osmotic potential and incubated at 30 °C for 36 h. After stress treatment, the cultures were serially diluted (10⁰, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴) and 7 µL from each dilution were spotted on SD/-Ura medium and incubated at 30 °C for 3 days.

Human fetal hepatic MATCHMAKER cDNA library and YEASTMAKER Yeast Transformation system were purchased from CLONTECH Laboratories. Pierce GST Protein Interaction Pull-Down Kit and Pierce Human In Vitro protein expression kit for DNA templates were purchased from Thermo scientific. Mouse anti-MT-1X monoclonal antibody was purchased from Novus Biological, CO., Rabbit anti-FHL3 polyclonal antibody was purchased from ProteinTech Group, Inc.

The Arabidopsis Mate and Plate Library was screened using yeast
mating method according to the Matchmaker Gold Yeast Two-Hybrid System
manufacturer’s protocol (Clontech). Briefly, full-length NSswas amplified and inserted into the pGBKT7 vector by Gateway recombination, then
the constructs was transformed into yeast strain Y2HGold and testing for
autoactivation by using the Yeastmaker Yeast Transformation System (Clontech).
Then the Arabidopsis Mate and Plate Library and BD-NSs yeast
clones were mated in YPDA medium. After incubation, isolated destination clones
were selected from diploid-selection medium (SD/-Leu/-Trp). These primary
positive interactors were secondary screened on medium plates
(SD/-Leu/-Trp/-His) and third time screened on medium plates (SD/-Leu/-Trp/
-His/X-a-Gal). PCR and BLAST searches were used to obtain sequence information
on corresponding AD- and BD-clones per colony.
The interaction between TSWV NSs and AtMYCs were confirmed according to the
manufacturer’s protocol (Clontech). The pGBKT7-NSs and pGAD424-MYCs constructs
were co-transformed into yeast strain Y2HGold. Yeast cotransformed with the
indicated plasmids was spotted onto synthetic medium (SD-Leu-Trp-His) containing
10 mM 3-amino-1,2,4-triazole and 0.04 mg/mL X-α-gal. The empty vectors pGBKT7
(BD) and pGADT7 (AD) were used as negative controls [38 (link)].