Identification of WRKY22 direct targets under submergence in Arabidopsis with ChIP. Arabidopsis thaliana

To identify direct targets of WRKY22, we created a transgenic Arabidopsis line that expresses a c-myc epitope-tagged WRKY22 and used ChIP followed by microarray hybridization (ChIP-chip) to screen for candidates and validate the in vivo protein-DNA interactions with ChIP followed by quantitative PCR (ChIP-Q-PCR). The WRKY22 and c-myc epitope tag fusion construct was generated and transformed into wrky22-ko2 plants. The resulting transgenic lines should have better ChIP efficiency than the wild-type background, due to the reduced competition for WRKY22 binding sites from endogenous WRKY22. ChIP-enriched DNA fragments were identified using criteria of a window of +300 to －1200 of a gene for a promoter, a width of 4 probes or more, and a false discovery rate (FDR) 2 or < 0.5-fold induction in any time point under submergence treatments in expression array data. Overall design: Comparison of c-myc tagged WRKY22 transgenic plants vs wild-type (Columbia) plants. Both materials were submergence treated for 3 hours.