Figure 1.
PP45 Interacts with DMI3 Both In Vitro and In Vivo.
(A) Y2H assay. AD vector and BD vector are Y2H vectors with no insert. SD-Trp-Leu-His-Ade/AbA/X-α-gal medium was used for testing the interaction. The combination of BD-P53 plus AD-SV40 was used as a positive control, and BD-Lam/AD-SV40 was used as a negative control.
(B) Pull-down assay. PP45-His and DMI3-GST proteins were expressed in Escherichia coli. For IB, PP45-His was detected with an anti-His antibody, and DMI3-GST was detected with an anti-GST antibody.
(C) BiFC analysis. Using the particle bombardment method, the constructs indicated were transiently expressed in onion epidermis cells. Coexpression of SYFPN-PP45 plus SYFPC or SYFPC-DMI3 plus SYFPN were used as negative controls. Scale bars, 90 μm.
(D) Co-IP test. After PP45-His and DMI3-Myc were cotransformed into rice protoplasts, total proteins of protoplasts were immunoprecipitated (IP) using an anti-Myc antibody and were detected with anti-His and anti-Myc antibodies. Protein input is shown by IB analysis of protein extracts before immunoprecipitation and antibodies against the respective tags. Molecular mass markers in kD are shown on the left. All the experiments were repeated at least three times with similar results.