Fig 1.
Expression analysis of IDD4 and functional characterization of idd4 mutants.
(A-C) pIDD4::GUS reporter lines under the control of the 2.5kb upstream region of the translational start sequence of IDD4 showing GUS staining in the cotyledons, hypocotyl, root tip (A), as well as in rosette leaves (B) and flower organs (C) (see also S1 Fig) Scale bar = 2.5 mm. (D) Genomic structure of IDD4 locus showing the position of T-DNA insertion. The idd4 mutant allele resulting from the T-DNA insertion in the 1st exon was confirmed to be homozygous (see also S2A Fig). (E) RT-PCR based IDD4 transcript evaluation in idd4 compared to wild-type (WT) by using oligo-nucleotides 352s/as, loading control cACTIN P107/108 (S7 Table). (F) Phenotype of 18 day-old WT compared to idd4 mutant plants and gain-of-function mutant IDD4ox1 (pUBI10::GFP:IDD4), cultivated on Murashige and Skoog basal medium under long-day conditions. Scale bar = 10 mm. (G-H) Fresh weight of shoot and root of 18 day-old WT plants compared to idd4 and IDD4ox lines. Boxes represent the 25th and 75th percentiles and the inner rectangle highlights the median, whiskers show the SEM, letters above boxes represent significance groups as determined by multiple comparison Student’s test p≤0.01. Plants of three biological replicates (n = 30) were analysed (see also S2B–S2E Fig). (I-J) WT plants, idd4 mutant, IDD4ox line and idd4 complementation line (pIDD4::IDD4:YFP) were treated by PstDC3000 (I) and PstDC3000 hrcC- (J). Boxes represent the 25th and 75th percentiles and the inner rectangle highlights the median, whiskers show the SEM, and outliers are depicted by dots (Min/Max range), letters above boxes represent significance groups as determined by multiple comparison Student’s test p≤0.05. Plants of three biological replicates (n = 30), were spray-inoculated with a bacterial suspension of OD600 0.2, the density of colony-forming units (cfu) was analyzed 2 and 72 hours post inoculation (hpi).
Fig 2.
Transcriptome composition of idd4, IDD4ox and idd4 flagellin22-treated lines associated with the characterization of pathogenicity-associated traits.
(A) Transcriptome comparison of WT and idd4 mutant with and without flg22 treatment. The original FPKM values were adjusted by normalized genes/rows and subsequently processed by hierarchical clustering by means of average linkage method using MeV4.0. Blue and yellow color indicates relatively low and high expression levels, respectively. For complete gene list see S1 Table. (B-E) Transcriptome composition and gene ontology annotations of up-regulated genes (p<0.05) in idd4 mock (B-C) and idd4 flagellin22 treated samples (D-E), GO terms were determined by using the AgriGo database (TAIR9, genomic locus). For complete gene list see S2 Table. (F) Evaluation of H2O2 levels by 3,3'- diaminobenzidine staining (DAB) in untreated idd4 mutants compared to WT. Scale bar = 2.75 mm. Boxes represent the 25th and 75th percentiles and the inner rectangle highlights the median, whiskers show the SEM, and outliers are depicted by dots (Min/Max range). Statistical significance was analyzed by Student’s test, asterisks indicate significant difference, ***p<0.001. (G) flg22-induced ROS burst assay of idd4, IDD4ox and WT plants, 5 week old plants were treated with 1 μM flg22 treatment for 45 min, values indicate mean ± SE, n = 36 (3 biological replicates). (H-I) Transcriptome composition and gene ontology annotations of down-regulated genes (p<0.05) in IDD4 gain-of-function mutant (pUBI10::GFP:IDD4). GO terms were determined by using the agriGo database (see also S2 Table).
Fig 3.
Genome-wide identification of IDD4 chromatin-bound regions and functional categorization of primary target genes.
(A) The number of significant peaks per biological replicate with an FDR <0.005 and the number of gene annotations is depicted for the independent biological samples. Qualitative evaluation of the single biological replicates was performed by co-occurance matrix generation shown in S3C Fig. (B) IDD4 target sequences share common DNA-binding motifs. The most frequent site consists of the core motif AGACAA (ID1 motif) and can be found in 31.73% of IDD4 targets (p = 1e-305). (C) Mean profile of IDD4 ChIP-SEQ read density of three independent biological samples within the ±2 kb region with respect to a gene model from transcriptional start sequence (TSS) to transcriptional end sequence (TES). Predominant binding of IDD4 in the region 500bp upstream of the TSS could be observed. (D) Gene ontology annotations of significant ChIP-SEQ targets (S3 Table) (E, H-J) Protein interaction networks derived from the IDD4 ChIP-SEQ targets. All significant IDD4 ChIP-SEQ targets were pooled and used to generate a network using STRING (version 10.0). Minimum required interaction score defined as high confidence 0.700, Meaning of network edges “evidence”. (F-G) Binding profiles of IDD4 to the AP2C1 (F) and ERF104 (G) loci. ChIP-SEQ profiles and ChIP-qPCR evaluations are depicted for each locus. The TAIR annotations of the genomic loci are shown at the bottom of each panel. The genomic locus indicated above the scale represents forward (+) orientation, while the one below represents reverse orientation. In each case, the enrichment was found to be in the upstream region of the respective genomic locus. (K) Binding profiles of IDD4 to the REV, SAUR15, PHYB and PIF6 loci. The TAIR annotation of the genomic loci is shown at the bottom of each panel. The genomic locus indicated above the scale represents forward (+) orientation, while the one below represents reverse orientation. In every case, the enrichment was found to be in the upstream region of the respective genomic locus.
Fig 4.
Hierarchical clustering of IDD4 target gene collection, differentially regulated in idd4 mutant and IDD4ox lines.
(A) Cluster I shows GO annotations and selected IDD4 target genes being predominately up-regulated in idd4 mutant and/or down-regulated in IDD4ox lines. (B) Cluster II displays aligned IDD4 target genes that are prevalently down-regulated in idd4 mutant and/or up-regulated in IDD4ox line; furthermore, GO annotations and selected genes are presented. (A-B) Log2 (FC) (p<0.05, idd4/WT, IDD4ox/WT) of individual genes was used for clustering by using the average linkage method under Pearson Correlation (MeV4.0) (for complete gene list see S4 Table).
Fig 5.
Interaction analysis and kinase assay of IDD4 with MPK6.
(A) In-vitro pull-down assays of MBP-tagged IDD4 showed prevalent interaction with MPK6 and minor association to MPK3 and MPK4. The negative control GST and MBP did not show interaction with MPK3,-4,-6. (B) Nuclear interaction study by Bimolecular Fluorescence Complementation of IDD4 with MPK6 in Nicotiana benthamiana epidermal cells. SCL3 served as positive control, UBIQUITIN10 and empty vector (EV) acted as negative control. Scale bar = 50 μm. (C-D) in-vitro kinase assays were performed by using recombinant IDD4 and constitutively active MPK6 and analyzed by LC/MS-MS. Depicted are the spectra of the obtained phosphorylated IDD4 peptides containing SERINE-73 (Ser-73) and THREONINE-130 (Thr-130). (E) Domain map of IDD4. IDD4 contains a nuclear-localisation signal (NLS) at the very N-terminus and a highly conserved ID domain that comprises 4 zinc finger (ZF). MPK6-targeted phospho-peptides reside in front of ZF1 (S73) and inside of ZF2 (T130).
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.
Fig 7.
Phospho-modified IDD4 versions exert a distinct transcriptional activity, SA accumulation and susceptibility against PstDC3000 hrcC- infection.
(A) Phospho-modified IDD4 lines show a distinct SAGT1 expression 4 hrs after flg22 treatment. (B) Pst DC3000 hrcC- infection levels in two independent IDD4-AA and IDD4-DD lines. Plants of three biological replicates (n = 30) were spray-inoculated with a bacterial suspension at OD600 0.2. Density of colony forming units (cfu) was analyzed 2 and 72 hours post inoculation (hpi). (C-D) GO term analysis of down-regulated (C) and up-regulated genes (D) in the IDD4-DD line (p≤0.05). (E-F) GO term analysis of down-regulated (E) and up-regulated genes (F) in the IDD4-AA line (p≤0.05). GO analysis was performed by using AgriGo (TAIR10). (G) Transcriptome compositions of IDD4-AA and IDD4-DD lines with respect to significant differentially regulated genes (p<0.05). Three main clusters generated illustrate a distinct and partly opposite expression of differentially expressed genes in the particular genotypes. (H-I) Quantitative analysis of free SA levels in WT, IDD4-AA and IDD4-DD lines (H) and as well as in WT and idd4 mutant plants (I) analysed by LC-MS/MS, before and 24hrs after Pst DC3000 hrcC- infection. Plants of three biological replicates were spray-inoculated with a bacterial suspension at OD600 0.2. Boxes represent the 25th and 75th percentiles and the inner rectangle highlight the median, whiskers show the SEM, and outliers are depicted by dots (Min/Max range). (A, B, H, I) Error bars show ± SEM, statistical significance was analyzed by Student’s test, letters above bars represent significance groups, p≤0.05.