1. OsSHI1 Physically Interacts with IPA1.
OsSHI1 Physically Interacts with IPA1. (A) Schematic representation of the various truncated visions of OsSHI1 and IPA1 proteins. The conserved zinc finger, IGGH, and SBP domains are indicated. (B) Both the N- and C-terminal regions of OsSHI1 interact with the C terminus of IPA1 in yeast cells. Transformed cells were spotted on the control medium (DDO, SD/-Leu/-Trp) and selective medium (QDO, SD/-Leu/-Trp/-His/-Ade). The empty pGADT7 was used as the negative control. (C) In vitro pull-down assay confirms that OsSHI1-GST, but not GST itself, could precipitate IPA1 as detected by anti-MBP antibody. The symbols “–” and “+” indicate the absence and presence of the corresponding proteins. (D) BiFC assay verifies the interaction between OsSHI1 and IPA1 in the nuclei of epidermal cells of N. benthamiana. OsSHI1 and IPA1 were fused with the N- and C terminus of YFP, respectively. eYNE and eYCE were used as the negative controls. OsSPL16, a homologous protein of IPA1, was also used as a negative control to demonstrate the specific interaction between OsSHI1 and IPA1. DIC, differential interference contrast; Merged, merged images of YFP channel and DIC. Bar = 30 μm. (E) In vivo CoIP assay shows that OsSHI1 interacts with IPA1 in the axillary buds of wild-type seedlings. Total protein extracts were immunoprecipitated by the anti-IPA1–specific polyclonal antibodies and analyzed by immunoblot probed with the anti-IPA1 and anti-OsSHI1 polyclonal antibodies. Immunoglobulin G was used as the negative control.
Erchao Duan et al. Plant Cell 2019;31:
©2019 by American Society of Plant Biologists