Characterization of wrky25 T-DNA insertion mutants and transgenic overexpressing plants. A. Diagram of WRKY25 gene and its T-DNA insertion mutants. B. RNA blot analysis of wrky25 mutant lines. Four hours after inoculation with PsmES4326 (OD600 = 0.0001), the inoculated leaves from two wild-type plants (Col-0) or four wrky25-1 and wrky25-2 mutant plants were harvested and total RNA was isolated. After separation and blotting to a nylon membrane, the blot was probed with a full-length WRKY25 cDNA fragment (upper panel) or a WRKY25 DNA fragment corresponding to the region downstream of the T-DNA insertion in the wrky25-2 mutant (lower panel). Ethidium bromide stained rRNA was used as a loading control. C. RNA blot analysis of WRKY25 expression in transgenic plants constitutively overexpressing WRKY25. RNA samples were prepared from leaves of two wild-type plants (Col-0) or four plants from each tansgenic 35S:WRKY25 line and probed with a WRKY25-specific DNA probe. Lines 12 and 18 expressed elevated levels of WRKY25 and contain a single T-DNA insertion based on the ratio of antibiotic-resistant progeny. F3 homozygous progeny plants of the two lines were used for further analyses.