Auxin-inducible degradation

We modified pKT127 (56) plasmid by introducing AID-HPH (hygromycin B phosphotransferase, hygromycin resistance) fragment for endogenous tagging (42). AID-ymRFP pOQR4 plasmid was a gift from M. Lisby. pBabe Blast osTIR1-9Myc was a gift from A. Holland (Addgene plasmid #80073). We integrated OsTIR on pDK-HC plasmid. Pkc1 was endogenously tagged with AID or AID-ymRFP. Yeast cells were grown in SD media to mid-log phase, and Cu2⁺ was added to a final concentration of 10 μM to induce OsTIR expression. 1-Naphthaleneacetic acid (10 μM; Sigma) or vehicle was added for 3 hours to induce Pkc1 depletion. The cells were then shifted to media with or without glucose for 1 hour to induce SG formation.