4.6. Yeasts Two Hybrid (Y2H) Assay

The DNA fragments of CML12 and cytoplasmic domain of MLO4 (MLO4C) were amplified by PCR using the primer pairs NdeI-CML12/CML12-BamHI and NdeI-MLO4C/MLO4C-ECoRI listed in Table S3. The resulting fragments were inserted into the assay vector pairs pGADT7 (AD) and pGBKT7(BD) (Clontech, www.clontech.com/, accessed on 21 October 2020), respectively, as described previously [53]. The yeast strain AH109 was co-transformed with generated construct pairs. SD/-Leu-Trp plates were used to culture the transformed yeast cells for 3 days at 30 °C. Then, SD/-Trp-Leu-His-Ade plates were used to select the colonies with interacting protein pairs for 3 to 7 days.