FIGURE 2.

RipI interacts with the bHLH93 transcription factor in Nicotiana benthamiana. (a) Yeast two‐hybrid assays showing the interaction between RipI and bHLH93. The transformants were prepared to a cell density of OD₆₀₀ = 1.0 and diluted in a 10‐fold series. For each concentration, 2 μl was spotted and incubated on synthetic defined SD −Ade −Leu −Trp −His plates supplemented with 20 μg/ml X‐α‐galactosidase (X‐α‐gal) for 4 days at 30 °C. The transformant containing BD‐RipI and empty vector pGADT7 served as a negative control. (b) RipI interacts with bHLH93 in vitro in glutathione‐S‐transferase (GST) pull‐down assays. Recombinant GST‐RipI and maltose binding protein (MBP)‐bHLH93 fusions were subjected to GST pull‐down analysis. GST tag and MBP‐bHLH93 fusions were used as the negative control. Gel stained with Coomassie brilliant blue (CBB) is shown. Interacting proteins were identified by immunoblotting using anti‐MBP antibodies. The experiment was repeated three times with similar results. (c) Subcellular localization of RipI and bHLH93 in Arabidopsis protoplasts. Arabidopsis protoplasts were transfected for coexpression of mCherry‐histone3.1 (serving as a nuclear localization marker) and either RipI or bHLH93 fused with yellow fluorescent protein (YFP). Images were recorded at 8 hr post‐transfection for visualization of YFP fluorescence at 488 nm and mCherry fluorescence at 580 nm. The colour of YFP fluorescence was set to green to facilitate detection of the fluorescence merged with mCherry fluorescence (green merged with red is shown as yellow, while yellow merged with red is shown as orange). Differential interference contrast (DIC) images were also photographed. Bars in all images represent 20 μm. (d) Immunoblot analysis of RipI expression in Arabidopsis protoplasts using green fluorescent protein (GFP) polyclonal antiserum (anti‐GFP). Total proteins were extracted from Arabidopsis protoplasts. Arabidopsis protoplasts transfected with empty vector expressing YFP were used as the control. The loading control was RuBisCO stained with Ponceau S. All experiments were replicated three times with similar results and representative results are shown