Figure 5. Transgenic Lines Overexpressing BiP3 Fail to Accumulate the XA21 and Its Cleavage Product.

(A) Lesion length measurements of F₂ population segregating for Myc-XA21 and overexpressed BiP3 (BiP3 ox). The F₂ segregants (Xa21/BiP3ox; +/−, +/+, −/+, −/−) were inoculated with Xoo strain PXO99Az and lesion lengths were measured 12 days post-inoculation. Nat Myc-XA21: Xa21 driven by the native promoter. Ubi Myc-XA21: Xa21 driven by the maize ubiquitin promoter. (B) RNA accumulation of the Myc-Xa21 and BiP3 transcripts in F₂ segregants 3A 1–5 and 3A 2–7 before and after Xoo strain PXO99Az inoculation. Lines are as described in (A). Total RNA was extracted and RT-PCR was performed using Myc-Xa21 and BiP3-specific primers. Control RT-PCR reactions were carried out with 18S rRNA-specific primers. (C) Protein accumulation of the Myc-XA21 and BiP3 in F₂ segregants 1–5 and 1–7 before and after Xoo strain PXO99Az inoculation. Equal amounts (100 µg) of total protein form Ubi Myc-XA21, Ubi Myc-XA21×BiP3ox3A 1-5, Nat Myc-XA21, and Nat Myc-XA21×BiP3ox3A 1–7 were extracted after Xoo strain PXO99Az inoculation, analyzed by SDS-PAGE, and immunoblotted with anti-Myc and anti-BiP antibodies. (D) Total protein was extracted from Kit, Nat Myc-XA21/BiP3 ox, and Nat Myc-XA21 plants after Xoo strain PXO99Az inoculation. Equal amounts (300 µg) of total protein were analyzed by SDS-PAGE and immunobloted with anti-Myc antibody. Equal total protein loading was confirmed with anti-actin antibody. A nonspecific band (n.s.) of 95 kD was detected. (E) After immunoprecipitation with anti-Myc antibody, western blot analysis was performed to visualize XA21 and its cleavage product. Immunoprecipitates from Nat Myc-XA21 were diluted ten-fold, resulting in similar amounts of XA21 in Nat Myc-XA21/BiP3 ox and in Nat Myc-XA21.