Leu trp his ade

Yeast transformants were screened on SD media (-Leu/-Trp/-His/-Ade) according to the manufacturer’s instructions (Clontech, Palo Alto, CA, United States). To generate an fusion with the GAL4 activation domain, the McCOP1 coding region was cloned into pGBKT7 vector. McMYB10 coding sequence was cloned in pGADT7 to generate an fusion which contains the GAL4 DNA-binding domain (GBD). By lithium acetate method, two plasmids which constructed above were co-transformed into the yeast AH109 strain and cultured at 28°C. The resultant yeast transformants were screened on SD (-Leu/-Trp/-His/-Ade) medium according to the manufacturer’s instructions (Clontech, Mountain View, CA, United States) (Li et al., 2012 (link)). The positive transformants were transferred to medium (-Leu/-Trp/-His/-Ade) and containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside acid) at 28°C for 2 days, which selected by the medium lacking Trp and Leu (-Trp/-Leu) (Li et al., 2012 (link)).

Coding sequence of PI4Kγ5 was amplified (primers PI4Kγ5–11 and PI4Kγ5–12) and subcloned into pGBKT7 or pGADT7 vectors. The pGBKT7-ANAC078 construct was generated using primers ANAC078-9/ANAC078-10. For yeast two-hybrid screening, BD-PI4Kγ5 was used as bait to screen the candidate interacting proteins on SD (-Leu/-Trp/-His/-Ade, Clontech, USA) plates.
For auxotroph assays, candidate clones were streaked onto SD (-Leu/-Trp/-His) medium supplemented with X-α-Gal (80 mg/L) and grown at 30°C for 4 days.

The bacterial strains and yeast strains used in this study are listed in Table S1. Bacteria strains were grown in Luria-Bertani (LB) broth or Tryptic Soy Broth (TSB). Cultures were maintained in a 37 °C incubator with shaking at 250 rpm, or on LB agar plates in a standard stationary incubator. LB was purchased from Oxoid (Oxoid Ltd, England). TSB was purchased from Solarbio Company. Plasmids used in this study are listed in Table S2. For specific purposes, plasmids were transformed into P. aeruginosa and E. coli strains. The appropriate antibiotics were added into the medium to maintain the plasmid in P. aeruginosa or E. coli strains at the following concentrations: gentamicin, 50 μg/ml; tetracycline, 20 μg/ml; kanamycin, 50 μg/ml. Antibiotics were purchased from Sangon Biotech Co, Ltd. The yeasts were cultured with yeast extract-peptone-dextrose (YPD) medium or synthetic dropout (SD) medium in an incubator at 30 °C. YPD, SD, and Dropout (DO) supplements lacking Leu/Trp or Ade/His/Leu/Trp were purchased from Clontech (Clontech Laboratories, Inc). All plasmids used in this study were constructed by homologous recombination and confirmed by DNA sequencing. The plasmids were kept in the E. coli DH5α strains, and stored in a −80 °C refrigerator.