Figure 3.

The Dominant-Negative Effect of TGA2CT Is Dependent on Interaction with NPR1.

(A) Protein gel blot analysis of the TGA2CT protein in independent 35S::TGA2CT transformants 2 and 4 in the npr1-2 mutant background. Protein extracts were made from 4-week-old npr1-2 control and 35S::TGA2CT transgenic lines. The amount of His₆-tagged TGA2CT protein in each line was determined using an antibody against the His₆ tag.

(B) RNA gel blot analysis of PR-1 gene expression in wild-type (WT), npr1-2, and 35S::TGA2CT (npr1-2) transformants. Plants were grown for 2 weeks on MS medium with (+) or without (−) 20 μM INA. Total RNA (10 μg) was used for RNA gel blot analysis using probes specific for PR-1 and UBQ5.

(C) Copurification of TGA2CT and NPR1 in 35S::TGA2CT transgenic plants. Equal amounts of protein extract from the wild type and npr1-2 transformed with 35S::TGA2CT (lines 4 and 17, respectively) were loaded onto Ni-NTA columns. The input protein extracts (I) and the eluates (E) were run on a 6 to 16% gradient SDS-PAGE gel. The presence of TGA2CT and NPR1 proteins was detected by protein gel blot analysis using antibodies against the His₆ tag and NPR1, respectively.

(D) Effect of SA on TGA2CT-NPR1 copurification. Protein extracts were made from 35S::TGA2CT transformant line 17 in the wild-type background with (+) or without (−) a 24-h treatment of 1 mM SA. The amounts of the TGA2CT-NPR1 complex were measured as described in (C).