Figure 8. XB15 Interacts with XA21K668 In Vitro and In Vivo.

(A) HIS-XB15ΔN was incubated with either glutathione alone (lane 1), glutathione-bound GST-XA21K668 (lane 2), or GST (lane3) in buffer containing 2 mM MgCl₂. After washing three times, proteins were eluted with 2× sample buffer, and analyzed by SDS-PAGE and immunoblotting with anti-GST or anti-HIS antibody. The experiment was repeated three times with similar results.

(B) Immunodetection of Myc-XA21 protein in transgenic plants carrying Myc-Xa21 under control of its native promoter before (Extract) and after immunoprecipitation (IP). Myc-XA21 and Myc-XA21^cp give bands at approximately 140 and 100 kDa, respectively, corresponding to the predicted mass of each protein [42]. A nonspecific band (n.s.) of 95 kDa was detected.

(C) Immunodetection of XB15 in Kitaake (Kit) and transgenic plants overexpressing NTAP-XB15 (17A and 19A). Anti-XB15 antibody detected at band of about 70 kDa of XB15 in Kit and of 95 kDa of in NTAP-XB15 in expressing plants (17A and 19A). Anti-actin antibody was used as a loading control.

(D) Co-immunoprecipitation of XB15 and XA21 after Xoo PR6 inoculation. XA21 was detected after immunoprecipitation with agarose conjugated anti-Myc antibodies. Total protein extracts from 15 g of leaf tissue of Kitaake (Kit), mock-treated Myc-XA21, and Xoo PR6-inoculated Myc-XA21 at the indicated time points were incubated with anti-Myc antibody to immunoprecipitate XA21 complex. The precipitates were used for protein gel blot analysis using anti-Myc antibody (top) or anti-XB15 antibody (bottom). Myc-XA21 and Myc-XA21^cp give bands at about 140 and 100 kDa, respectively, and XB15 is detected as a band of 70 kDa.