Yeast Two-Hybrid Assay of TIR-TDL1A Interaction

For yeast two‐hybrid assays, the pGBKT7 vector expressing OsTMS15‐TIR, OsTMS15‐mTIR, L2‐TIR, L3‐TIR and L4‐TIR fused to the DNA‐binding domain (BD) and pGADT7 vectors expressing OsTDL1A^110–226 (truncated OsTDL1A) fused to the activating domain (AD) were cotransformed into the AH109 strain. Three independent clones were separately diluted into three concentrations. Dilutions were spread onto synthetic defined (SD) medium (‐Ade, ‐Leu, ‐His, ‐Trp) and spotted onto SD medium with X‐α‐Gal (Takara, Kyoto, Japan). For comparison of the growth rates of the yeast strains, five individual clones were selected from each SD⁻⁴ plate with TIR‐TDL1A or mTIR‐TDL1A. Each clone was cultured in SD⁻⁴ medium under different temperatures. Then, the OD₆₀₀ value of each clone was recorded every 2 h. Data were fitted using a DoseResp model of growth curves as previously described (Gao et al., 2019 (link)). The significance of the hill slope of the growth curve was analysed by t tests.