We have previously reported that OsBWMK1 over[removed](35S-OsBWMK1) in tobacco transgenic plants induced HR-like cell death, upregulated PR gene expression, and enhanced resistance to pathogens [15]. It has been known that plants showing HR-like lesions produce reactive oxygen species (ROS) and SA [1] and [20]. Therefore, we attempted to determine whether the HR-like lesion is associated with the production of hydrogen peroxide (H2O2), which is a ROS and SA in 35S-OsBWMK1 transgenic tobacco plants. As shown in Fig. 1A, total SA levels increased approximately 5.5- and 4.5-fold in the 35S-OsBWMK1 transgenic lines 4–11 and 6–2, respectively. H2O2 levels were higher in 35S-OsBWMK1 transgenic plants than in wild-type plants ( Fig. 1B). H2O2 production was approximately 5-fold higher in
Captisol of transgenic lines 4–11 and 6–2 than in those of wild-type plants. These results suggest that OsBWMK1-induced HR-like cell death can be mediated by the elevated production of H2O2 and SA. The promoters of various wound- and pathogen-responsive genes such as the PR genes contain cis-acting elements; an example is the W-box, to which WRKY proteins bind [1] and [21]. To understand the regulatory mechanism of PR gene
expression in 35S-OsBWMK1 transgenic plants, we tested the DNA-binding activity of OsWRKY33 to a W-box (TTGACCA) by an electrophoresis mobility shift assay (EMSA) using a 32P-labeled W-box motif. As shown in Fig. 1C, DNA-binding activity to a W-box was significantly higher in 35S-OsBWMK1 transgenic plants than in wild-type plants. This result suggests that the activation of MAP kinase can increase the DNA-binding activity of W-box binding factors.
