AtNIGT1/HRS1 TARGET approach

This work studies the impact of AtNIGT1/HRS1-GR entrance in the nucleus upon DEX treatment in protoplasts. AtNIGT1/HRS1 TARGET. The whole procedure has been performed as previously described in Bargmann et al Mol Plant 2013. In brief, the protoplasts were transfected with the plasmid pBeaconRFP_GR-HRS1 that trigger the expression of HRS1 protein fused with the glucocorticoid receptor under control of CaMV35S promoter. Protoplasts were treated with 35µM cycloheximide (CHX) to inhibit translation and to select only direct target genes and 10µM dexamethasone (DEX) to induce HRS1-GR entry in the nucleus. Nitrate is maintained during the whole TARGET procedure. The Red Fluorescent Protein was used as marker selection for fluorescent-activated cell sorting (FACS) of successfully transformed protoplasts. RNA were extracted and amplified in order to be tested with ATH1 Affymetrix™ chips.