Stress Tolerance Assay of SbMYB44 in Yeast

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.