DAP followed by qPCR analysis

DAP followed by qPCR was performed following a protocol modified from Bartlett et al., 2017.

For each condition, genomic DNA was extracted from leaves of three independent 5-week-old plants using the CTAB (hexadecyl trimethyl-ammonium bromide) protocol (Doyle and Doyle, 1990). Then, 5 μg of genomic DNA were sonicated to obtain DNA fragments of around 200–300 bp long. Sonicated DNA was then precipitated and resuspended in EB buffer (10 mM Tris-HCl, pH = 8.5).

For each TF or control expressing GST alone, 200 ml cultures of E. coli Rosetta strains, carrying pGEX-TW-WRKY or empty vector for the GST alone, at a OD_600nm = 0.3–0.6 were induced with 1 mM IPTG during 1 hr at 37°C. Expression of the WRKYs was then confirmed on a Coomassie gel.

Bacterial pellets were lysed and sonicated for 10 cycles of 10 s. The supernatants containing expressed proteins were incubated with washed MagneGST beads (Promega) (25 µl per TF and per DNA sample tested) during 1 hr at room temperature with gentle rotation. After five steps of washing with 1× PBS + NP40 (0.005%) and three steps with 1× PBS, beads bound with GST-WRKYs were mixed with 200 ng of sonicated DNAs and incubated 1 hr at room temperature with gentle rotation. Samples were then washed five times with 1× PBS + NP40 (0.005%) and two times with 1× PBS. Beads were then resuspended in 25 μl EB (10 mM Tris-HCl, pH = 8.5) and the samples heated during 10 min at 98°C. Supernatant was kept for further qPCR analysis.

The equivalent of 1 μl of immunoprecipitated DNA was then amplified by real time PCR reactions using Takyon SYBR Green Supermix (Eurogentec) and gene specific primers, and normalized to the input DNA.