FIGURE 1.

(A) AtWRKY50 activates the PR1 promoter. Arabidopsis protoplasts were co-transfected with a PR1::GUS construct together with empty pRT101 expression plasmid (minus sign) or with plasmids containing 35S::AtWRKY50 (50), 35S::AtWRKY51 (51) or 35S::AtWRKY59 (59). After incubation, GUS activity was measured spectrophotometrically. Expression levels (%) are given relative to the expression level without WRKY effector. (B) Effect of AtWRKY50, AtWRKY51, and AtWRKY59 on the expression of endogenous Arabidopsis genes. Expression of PR1, Act3 (actin) and Tub (tubulin) genes in Arabidopsis protoplasts was measured by qRT-PCR. Expression of each gene was measured in protoplasts transfected with the empty pRT101 vector (minus sign) or with the pRT101 vector containing the 35S::AtWRKY50 (50), 35S::AtWRKY51 (51), or 35S::AtWRKY59 (59) expression constructs. Bars represent the average level of mRNA accumulation observed in three experiments. mRNA levels in protoplasts transfected with the empty pRT101 vector were taken as 100%. Error bars represent the SEM. The experiment was repeated three times with similar results. Statistical differences among the samples is labeled with asterisk (p < 0.05). (C) Overexpression of AtWRKY50 enhances SA-induced expression of PR1 in plants. Three lines of transgenic seedlings overexpressing AtWRKY50 (W50#12, #13, #19) and a line expressing GUS were incubated for the indicated times (in hours) in liquid medium containing 0.3 mM SA, after which RNA was extracted and analyzed for accumulation of mRNA corresponding to genes AtWRKY50 and PR1. The blot hybridized with the AtWRKY50 probe was exposed for 1 and 4 h. A similar blot was hybridized with the PR1 probe. Arrows to the right of the blots indicate the expected positions of the respective mRNAs as deduced from the positions of the rRNAs. An Ethidium bromide stained gel showing total RNA is included as a loading control.