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. 2023 Sep 16;22(1):148–164. doi: 10.1111/pbi.14174

Figure 3.

Figure 3

Uv1809 physically interacts with OsSRT2. (a) Y2H analysis of the interaction between Uv1809 and OsSRT2. The interaction between BD‐53 and AD‐T was taken as the positive control, BD and AD‐OsSRT2 was taken as the negative control. SD‐3, SD‐Trp‐Leu‐His; SD‐4, SD‐Trp‐Leu‐His‐Ade; BD, pGBKT7; AD, pGADT7. (b) In vivo Co‐IP of Uv1809 interacts with OsSRT2. Co‐IP was performed on extracts of Nicotiana benthamiana leaves by co‐expression of OsSRT2‐Flag and Uv1809‐GFP. Uv1809‐GFP and OsSRT2‐Flag were detected by western blotting using anti‐GFP and anti‐Flag antibodies, respectively. (c) A GST pull‐down assay was used to detect the interaction between Uv1809∆SP‐His and OsSRT2‐GST. Uv1809 and OsSRT2 were fused to His and GST tags, respectively, and expressed in Escherichia coli. OsSRT2‐GST or GST‐bound resin was incubated with E. coli crude extracts containing Uv1809∆SP‐His and analysed by western blotting. Uv1809∆SP‐His and OsSRT2‐GST were detected using anti‐His and anti‐GST antibodies, respectively. (d) BiFC assays for the interaction between Uv1809 and OsSRT2. N. benthamiana leaves were infiltrated with a mixture of Agrobacterium tumefaciens strains co‐expressing the OsSRT2‐nYFP and Uv1809‐cYFP constructs. YFP signals were observed at 2 dpi. Infiltration with Agrobacterium co‐expressing the OsSRT2‐nYFP and cYFP, nYFP and Uv1809‐cYFP constructs were used as the negative control. No YFP signals was observed in these negative controls. Scale bar = 20 μm.