INDETERMINATE-DOMAIN 4 (IDD4) coordinates immune responses with plant-growth in Arabidopsis thaliana
Fig 6
IDD4 associates to the promoter region of SAGT1 and phospho-modified IDD4 versions show a distinct DNA binding ability.
(A) Genome Browser snapshots of IDD4 (blue) and single GFP (green) ChIP-SEQ peak on the genomic regions of chromosome 2 [chr2:18,151,900–18,152,300]. Schematic diagram of the SAGT1 promoter and gene model, upper panel shows the position of the DNA region (P1, P2, G1) used in the ChIP assay and the putative core binding sequences consisting of two ID1 motifs (red letters) used for EMSA (shown in Fig. 6G) are indicated. (B-C) ChIP-qPCR by using three biological replicates of pUBI10::IDD4:GFP (B) and pIDD4::IDD4:YFP (C) expressing plants. Binding of IDD4 to genomic regions close to SAGT1 was tested with three primer pairs (P1, P2, G1) for each locus. Y-axis shows either the fold enrichment in the pUBI10::IDD4:GFP lines normalized to GFP immunoprecipitation, driven by the pUBI10 promoter (B) or in (C) the fold enrichment in the pIDD4::IDD4:YFP lines normalized to YFP immunoprecipitation, driven by the pIDD4 promoter. (D-E) Evaluation of the SAGT1 expression in idd4 (D) and IDD4ox lines (E) before and 4 hrs after flg22 treatment compared to WT. The expression of SAGT1 was reduced under both conditions in the idd4 mutant (D), while it increased in IDD4ox lines (E) after flg22-application. (F) Assessment of the DNA binding activity of IDD4 phospho-modified versions under mock and flg22-treated conditions. Recruitment of IDD4-AA and IDD4-DD to SAGT1 promoter as determined by ChIP-qPCR. The results are presented as INPUT/IP ratios obtained by signals from ChIP with RFP antibody. Fourteen-day-old seedlings from WT, IDD4-AA:RFP and IDD4-DD:RFP transgenic plants were used for chromatin isolation. ChIP- and input-DNA samples were quantified by qPCR using primer pair P1; results shown represent the average of three biological replicates. The protein amount of the different IDD4 variants in transgenic plants is shown by immunoblot assays in S3D Fig. (G) Electrophoretic mobility shift assay (EMSA) using truncated IDD4-AA, IDD4-DD and IDD4. Competition experiments were performed using increased amounts (0.5μM, 100x excess) of the indicated unlabeled competitor (spe, specific; mut, mutated). As probe, we used the 35bp sequence inside SAGT1 promoter that contains two ID1 motifs, depicted in Fig 6A (H) ChIP-based binding study of IDD4 before and after flg22-treatment, represented here by the average of 3 biological replicates. The binding of IDD4 to the SAGT1 promoter region was increased after 1h of flg22 treatment when compared to untreated samples. IDD4 binding was assessed by using ChIP-qPCR primer P1 (399a/as). (B-F, H) Error bars show ± SEM, statistical significance was analyzed by Student’s test. Asterisks indicate significant differences **p≤ 0.05, **p≤ 0.01, ***p≤ 0.001, letters above bars represent significant groups p≤0.05.