Expression analysis of IDD4

So far, it was reported that IDD4 is expressed in the root ground tissue of the basal meristem [20]. To investigate the contribution of IDD4 in defense response, its expression was analysed by generating stably transformed Arabidopsis lines expressing GUS or NLS:3xGFP under a 2.5 kb IDD4 promoter sequence. Intense staining of the GUS reporter was observed in cotelydons, root tips and in all stages of rosette leaf development (Fig 1A and 1B). In leaves, we detected GUS/GFP signals in the trichomes, stomata, epidermal cells (S1A–S1D Fig) and mesophyll cells (S1E Fig). Moderate GUS staining was observed in sepals and petals (Fig 1C) as well as in ovules embedded in carpels of Arabidopsis flowers (S1F Fig). Various public microarray datasets (Genevestigator) (S1G Fig) showing endogenous IDD4 transcript abundance corresponds to our histological results and reveal the expression of IDD4 in a wide range of tissues throughout the life cycle in Arabidopsis.

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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 1^st 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).

https://doi.org/10.1371/journal.ppat.1007499.g001

Enhanced plant-growth and pathogen-resistance of idd4 mutants

The functional contribution of IDD4 in response to bacterial pathogen attack was investigated by challenging idd4 mutant, a complementation line (idd4/pIDD4::IDD4:YFP) and an overexpressor line IDD4ox1 (pUBI10::GFP:IDD4) with the virulent hemi-biotrophic plant pathogen Pst DC3000. We used an IDD4 insertion line (Salk_148352) containing the T-DNA insertion in the first exon that could be confirmed by sequencing as a true knockout line (Fig 1D and 1E and S2A Fig). The idd4 complementation line expresses IDD4:YFP driven by the 2.5 kb upstream sequence of the IDD4 ORF. Interestingly, by determining the fresh weight of 18 day-old plants, we consistently found an increase of approximately 20% biomass in the aerial part of the idd4 mutant. By contrast, the shoot fresh weight of the four analyzed IDD4ox lines showed a reduction of about 11.5% in IDD4ox1 (Fig 1F and 1G) up to approximately 65% in IDD4ox4 when compared to WT (S2B–S2D Fig). Interestingly, the growth reduction seems to be in accordance with the ectopic expression of IDD4 in IDD4ox1-4 lines (S2B Fig). The differences in shoot growth also corresponded to altered root formation in the idd4 and IDD4ox lines. The root biomass in idd4 is increased by about 36% whereas that of the IDD4ox lines show a reduction of approximately 14% (Fig 1F and 1H) in IDD4ox1 and 75% in IDD4ox4 (S2C and S2D Fig). The enhanced growth of idd4 could be reverted to WT by the expression of the complementation construct (S2E Fig). The restored phenotype in idd4/pIDD4::IDD4:YFP shows that the improved growth can be traced back to the mutation in IDD4. The differences in shoot and root growth in idd4 and IDD4ox lines suggest a function of IDD4 as a regulator of growth-associated processes.

Two hours after spray infection by PstDC3000, the infection levels in the different transgenic lines corresponded to those in WT plants indicating that stomatal immunity was not affected (Fig 1I). However, 72 hours after spay-infection, the proliferation levels of Pst DC3000 in the idd4 mutant were significantly reduced when compared to WT. Furthermore, the bacterial titer in the idd4 complementation line was indistinguishable from WT, suggesting that the reduced susceptibility in idd4 mutants is due to the lack of IDD4 protein function. By contrast, the IDD4ox line exhibited increased susceptibility to Pst DC3000 (Fig 1I). Therefore, we reasoned that IDD4 acts as a negative regulator of basal resistance to hemi-biotrophic pathogen infection. To evaluate the PTI response, the idd4 mutant was challenged by Pst DC3000 hrcC-. The Pst DC3000 hrcC- strain is compromised in virulence due to its inability to inject any of its type III-secretion system-dependent effectors, one function of which is to suppress plant immunity. In this way, infection with Pst DC3000 hrcC- principally induces only PTI-mediated defense responses. In comparison to WT plants and idd4 complementation lines, the proliferation levels of the bacteria 72 hrs after spray-infection were reduced in idd4 mutants and elevated in IDD4ox (Fig 1J). The higher resistance of the idd4 mutant indicates an enhanced PTI response thereby suggesting that IDD4 also functions to regulate PTI-mediated defense responses.

IDD4 regulates genes that coordinate specific processes in plant growth and immunity

In order to analyse the transcriptome composition of idd4 and IDD4ox lines, we performed RNA-Hiseq analysis on 3 biological replicates of 14 day-old idd4, IDD4ox and WT seedlings without and after flg22 application (1μM flg22, 1hr). A close to linear correlation coefficient of WT and idd4 (0.85), WT and idd4 (flg22) (0.87), as well as WT and IDD4ox (0.98) was obtained when considering the expression profiles for all transcripts. The strict correlation suggests that IDD4 does not affect general gene expression, but rather influences subsets of genes in particular biological processes. Hierarchical clustering of significant genes (p<0.05) in idd4 before and after flg22 treatment, by using normalized FPKM values, revealed distinct differences in gene expression patterns suggesting altered gene induction in idd4 after flg22 perception (Fig 2A, S1 Table). At a stringency of p<0.05 in untreated idd4 mutants, 2244 differentially expressed genes (DEGs) (S2 Table) could be identified that show a log₂-fold change from 0.27 (1.21 FC) to 4.05 (16.57 FC) of positively regulated genes and from -4.95 (30.88 FC) to -0.27 (1.21 FC) of negatively regulated genes. Among these 2244 genes, 621 genes are up- and 1623 genes are down-regulated. To categorize DEGs in functional modules, gene ontology (GO) terms were determined by using the AgriGO platform [30] (TAIR9) (Fig 2B, S2 Table). The up-regulated genes can be grouped in very different GO terms describing gene functions in different hormonal pathways and a multitude of cellular biological processes. GO terms are highlighted for defense response, response to salicylic acid stimulus, oxidative stress and response to other organism thereby indicating a function of IDD4 in the repression of defense-related genes and factors contributing to growth and development. Intriguingly, transcript levels of CALMODULIN-BINDING PROTEIN 60g (CBP60g) [31] that regulates expression of the rate-limiting enzyme ICS1 in SA biosynthesis [32] and the SA marker gene PATHOGENICITY-RELATED FACTORS PR2 were expressed at significantly higher levels in untreated idd4 plants compared to WT (Fig 2C and S2F Fig). Similarly, enhanced expression was found in idd4 mutant plants for the pattern-triggered immunity-responsive marker gene FLG22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1) [33], and the early-defense marker transcription factor WRKY22 (Fig 2C and S2F Fig).

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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).

https://doi.org/10.1371/journal.ppat.1007499.g002

Interestingly, the enhanced growth phenotype of the idd4 mutant corresponds with GO terms describing gene functions for response to auxin stimulus, glucosinolate metabolic process and anatomical structure development. The enrichment of auxin response genes, including AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE (ARGOS) and several members of the SMALL AUXIN UPREGULATED RNA (SAUR)-like auxin-responsive protein family (Fig 2C, S2 Table), promoting plant growth and architecture [34], in idd4 mutant plants might partly explain its elevated biomass. In addition, the idd4 transcriptome was enriched in genes of the glucosinolate metabolism that participates both in defense and growth [9, 35]. After flg22 treatment, 2048 DEG (p<0.05) were obtained in idd4 mutant, with 1063 genes showing enhanced while 985 genes reduced transcript abundance (S2 Table). In accordance with the untreated idd4 transcriptome, the GO analysis of up-regulated genes after flg22 application emphasized GO terms for hypersensitive response, oxidative stress and defense response (Fig 2D). In particular, the expression of the defense markers WRKY33, WRKY49 and FRK1 is elevated as well as components of the kinase signaling cascades represented by MKK6 and MPK11 (Fig 2E). Additionally, macromolecular biosynthesis process and protein metabolic process are among the most significant GO terms (Fig 2D, S2 Table).

By contrast to the idd4 mutant, the overexpression of IDD4 (IDD4ox) reduces defense-related gene expression depicted by the GO analysis of significantly down-regulated genes (p<0.05) (S2 Table). These DEGs are functionally grouped in GO terms for respiratory burst during defense response, salicylic acid stimulus and innate immune response (Fig 2H). Prominent representatives are WRKY38 [36], PR5 as well as ERF4 and ERF5 [37] (Fig 2I). The deregulation of genes and functional groups dedicated to immunity in the idd4 mutant and IDD4ox lines confirms the role of IDD4 as a regulator of defense-related gene expression.

In accordance, the GO analysis before and after flg22 treatment show DEGs involved in the response to reactive oxygen species (ROS) which belong to the first line of defense upon pathogen invasion [38]. Intriguingly, genes coding for the H₂O₂-scavenging enzymes CATALASE 1 (CAT1) and CAT2, which catalyze the reduction of photo-respiratory generated hydrogen peroxide and protect cells from its toxicity [39], are down-regulated in idd4 (S2 Table). Interestingly, SA-mediated suppression of CAT2 results in increased H₂O₂ levels and resistance against biotrophic pathogens [40]. Furthermore, PEROXIDASE CA (PRXCA) [41], ASCORBATE PEROXIDASE 1 (APX1) [42] and L-ASPARTATE OXIDASE (AO) [43] are up-regulated after flg22-treatment in idd4 and have been reported to promote H₂O₂ metabolism and turn-over. By comparing the levels of the ROS H₂O₂ by 3,3'-diaminobenzidine (DAB) staining in untreated idd4 with WT plants, we observed that the H₂O₂ levels in idd4 were strongly elevated already before infection (Fig 2F). In the mock-treated control without applied DAB, the staining value is about 180 of an arbitrary unit for WT and idd4 and can be considered as default level. After DAB staining, WT-plants showed a staining value of 165 and idd4 of about 156. This staining difference indicates a higher H₂O₂ accumulation in the idd4 mutant. Additionally, in a comparative flg22-triggered reactive oxygen species burst assay, we detected strikingly enhanced ROS levels in idd4 mutants and reduced ROS efflux in IDD4ox lines when compared to WT (Fig 2G). Notably, maximum ROS levels were observed in WT at 17 minutes whereas the idd4 mutants achieved maximal ROS production already at 7 minutes after flg22 perception. These results show that the GO term annotations response to oxidative stress in idd4 mutant and respiratory burst during defense response in IDD4ox plants correspond with their biochemical traits. In summary, the constitutive activation of ROS and defense-related traits and the enrichment of factors that promote structural development indicate a role of IDD4 in growth and immunity-related processes.

Primary target genes of IDD4 contribute to growth and defense-associated processes

In order to identify the genomic regions targeted by IDD4 in vivo, Chromatin-Immunoprecipitation (ChIP) was performed followed by deep sequencing on the Illumina High-Sequencing platform (SEQ). In order to evaluate the reproducibility of the obtained genome-wide IDD4 binding profiles, ChIP-SEQ was performed on 3 independent lines carrying the IDD4:GFP construct under the control of the UBI10 (At4g05320) promoter. The retrieved binding patterns were normalized by comparison with data obtained from pUBI10::GFP plants. GFP antibodies were used to immunoprecipitate protein-DNA complexes that were verified by Western-Blot analysis before sequencing (S3A Fig). Sequence coverage at each position on the genome was plotted to identify peaks in the Arabidopsis genome. A significant number of peaks per biological replicate with an FDR <0.005 could be annotated (Fig 3A) in the 500 bp region upstream of the translational start sequence in the genic region. A highly consistent read pattern in all three IDD4:GFP lines showed predominate IDD4 binding in the vicinity upstream of the transcriptional start site (Fig 3C), which includes the 5’-UTRs and promoter regions. Sequence analysis of the 500 bp regions upstream of the TSS of IDD4 targets revealed the in vivo enrichment of the ID1 motif on a genome-wide scale in about 31% of target genes (Fig 3B) and was ranked as the primary binding site. Previously, the core sequence of the ID1 motif (AGACAA) was initially suggested as binding site of the maize ID1 protein [44], and further in vitro characterized for its association with IDD3 and IDD8 in Arabidopsis [16, 45]. To compare the independent transgenic lines, the binding of IDD4:GFP to the same target sequences was monitored by co-occurrence matrix analysis (S3C Fig). The comparison illustrates that replicates 1 and 3 provide the most reliable overlapping binding sites. After removing genes that showed similar sequence peaks in the negative control, 837 genes could be defined as significant binding targets of IDD4 (S3 Table).

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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.

https://doi.org/10.1371/journal.ppat.1007499.g003

A GO term analysis of the 837 major IDD4 target genes highlighted gene functions in innate immune response, response to salicylic acid stimulus, hypersensitive response and respiratory burst during defense response (Fig 3D). These GO terms demonstrate that a significant part of the direct IDD4 targets are assigned to functions in defense response and pathogenicity (S3 Table). On the other hand, IDD4 exhibits binding preferences on genes that mediate cellular processes governing growth and development, including auxin and glucoside metabolic process as well as response to gibberellin and light stimulus. To gain a more precise view on global IDD4 function, target genes were grouped into protein interaction networks by performing gene cluster analyses using STRING [46]. Versatile functional networks could be generated with IDD4 in various distinct biological processes (Fig 3E and 3H–3J). Several genes are integrated into a ramified functional network that contributes to defense-associated processes (Fig 3E), including WRKY33, whose phosphorylation by MPK6 promotes the transcriptional induction of camalexin biosynthesis genes [47]; ERF104, a key regulator of basal immunity whose stability and interaction with MPK6 is compromised in response to flg22 [48]; AP2C1, which interacts and dephosphorylates MPK6 and consequently suppresses immunity [49]; CAF1a, a mRNA deadenylase targeting transcripts for post-transcriptional modification with temporal specificity during plant defense response [50], and BIR1, encoding a BAK1-interacting receptor-like kinase and activates plant defense responses [51]. IDD4 binding profiles of AP2C1 and ERF104 generated by ChIP-SEQ and ChiP-qPCR confirmed the association of IDD4 to the 5’upstream region of the respective gene loci (Fig 3F and 3G). The association of IDD4 to these target genes indicates its direct influence on factors shaping immunity response (Fig 3E).

Furthermore, a prominent hub describes IDD4 function in the ad/abaxial pattern specification and leaf blade formation by regulating REVOLUTA (REV), KANADI (KAN), PHABULOSA and ABNORMAL FLORAL ORGANS (Fig 3H and 3K) [18]. A further functional node combining factors involved in auxin-signaling was formed and comprises the AUXIN RESPONSE FACTOR ARF9 and ARF10, and the INDOLE-3-ACETIC ACID INDUCIBLE IAA18 and IAA26, as well as one member of the SAUR family, suggesting a regulatory role of IDD4 in auxin-mediated growth processes (Fig 3I and 3K). Moreover, recently published data indicate IDD4 as a transcriptional activator of nuclear-encoded photosynthetic gene expression and photomorphogenesis [52]. In this context, chloroplast maturation and import machinery, as well as chlorophyll biogenesis, seems to be targeted by IDD4 (Fig 3J), as shown by the binding to the promoter regions of PHYTOCHROME B, PHYTOCHROME INTERACTION FACTOR (PIF) 3, PIL1, PIL2/PIF6, PIL5/PIF1 [53] (Fig 3K) and Chloroplast heat shock protein 70–1 (cpHsc70-1) [54] together with DE-ETIOLATED 1 (DET1) [55]. Moreover, RNA POLYMERASE SIGMA SUBUNIT 2 (SIG2) [56] encodes a subunit of chloroplast RNA polymerase, and REDUCED CHLOROPLAST COVERAGE 2 (REC2) [57] which contributes to the size establishment of the chloroplast compartment are two of several further IDD4 target genes that are involved in the chloroplast biogenesis and function (S3 Table).

To pinpoint whether IDD4 acts as a positive or negative regulator of major ChIP-SEQ target-genes, we compared DEGs identified in the transcriptome analysis of the idd4 mutant and IDD4 overexpression lines. The comparison of CHIP-SEQ targets with DEGs in idd4 and IDD4ox revealed an overlap of 11.7% and 6.7%, respectively. Altogether, the search for direct targets yielded 135 genes (S4 Table) that can be grouped by GO terms for glucoside metabolic process (p = 1.00e^-08), defense (p = 1.10e^-06) and immune response (p = 8.10e^-05). The predominantly opposite regulation of these target genes in the two genetic backgrounds suggests that IDD4 functions as a direct regulator of the respective genes. In this regard, hierarchical clustering of differentially regulated major targets yielded 49 genes matching the criteria of being predominantly up-regulated in idd4 and down-regulated in IDD4ox which indicates IDD4 as transcriptional repressor of this cluster (Fig 4A, S4 Table). The corresponding GO terms emphasise hormone-mediated signaling, response to other organism, defense response and developmental process. In addition, transcriptionally repressed targets of IDD4 are involved in various defense processes including AP2C1, CPK28, CAF1a and SERK1, thereby indicating IDD4 as a direct regulator of genes in immunity. STRING-based protein interaction network of these 49 genes created clusters combining target genes involved in immunity like AP2C1, CAF1a, CPK28 and ERF2 with those involved in vesicle-transport, like EXO70H7 [58] (S5A Fig).

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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).

https://doi.org/10.1371/journal.ppat.1007499.g004

On the other hand, 86 genes identified in the DEGs and CHIP-SEQ overlap predominantly correspond to down-regulated genes in idd4 but enhanced expressed in the IDD4ox lines (Fig 4B, S4 Table). The selected genes can be grouped in GO terms for response to blue light, response to salt and carbohydrate biosynthesis process. Furthermore, the supported GO terms indoleacetic acid biosynthesis, and glucoside metabolic process refer to IDD4 function as a regulator of genes that participate in growth and development. Gene cluster analyses using STRING created several functional modules (S5B Fig) for light response/ photorespiration/ photo-morphogenesis and chloroplast import machinery emphasizing again the contribution of IDD4 in processes mediating growth and development. Noteworthy, the expression of IDD4 is compromised by defects in the chloroplast import machinery and the retrograde transport, and it is postulated to act as a transcriptional activator of nuclear-encoded photosynthetic gene expression [52]. In this context, the expression of TRANSLOCON AT THE OUTER MEMBRANE OF CHLOROPLASTS 64-III (TOC64-III) [59], Ankyrin repeat-containing protein 2 (AKR2) [60], EARLY LIGHT-INDUCABLE PROTEIN (ELIP1) [61], Tonoplast dicarboxylate transporter (TDT) [62] and VARIEGATED 1 (VAR1) [63] which collectively contribute to chloroplast maturation is subject to IDD4 function (S5B Fig). In addition, our data indicate that IDD4 exerts transcriptional regulation of the blue light photoreceptor PHOTOTROPIN2 (PHOT2) [64] and the J-DOMAIN PROTEIN (JAC1) [65, 66] with assigned functions in the adaptation to light stress (blue light) and photo-morphogenesis in a signal-transduction pathway for photo-chloroplast movement and accumulation (S5B Fig, S4 Table). Altogether, our data indicate that IDD4 is embedded in widely-ramified regulatory pathways thereby exerting transcriptional control on key factors that shape and balance growth and defense.

IDD4 is a substrate of MPK6

To test the interaction between the immune MAPKs MPK3, 4 and 6 with IDD4, we searched for MAPK docking sites in IDD4 that are essential for the binding of substrates to MAPKs. Analysis of the IDD4 amino acid sequence revealed a highly conserved MAPK docking motif that lies between ZF1 and ZF2 (S4A Fig) [67]. The interaction of IDD4 with the immune MAPKs MPK3, MPK4 and MPK6 was tested by in vitro pull-down assays using MBP-His-tagged IDD4 and GST-tagged MAPKs (Fig 5A). IDD4 predominately interacted with MPK6. The interaction with MPK3 and MPK4 was at background intensity and the negative controls, single GST and MBP, did not associate with the respective proteins. The association of IDD4 with MPK6 was then evaluated by bifluorescence complementation experiments (BiFC) in Nicotiana benthaniama. A strong fluorescence signal was detected in the nucleus of Nicotiana epidermis cells that were co-transfected with YFP^N-IDD4 and YFP^C-linked MPK6 (Fig 5B). As a positive control, the interaction of SCL3 with IDD4 could be confirmed [16], whereas no association could be detected for UBI10 and as well the empty vector control. The data suggest that among the three immune MAP kinases MPK6 is the principal interaction partner of IDD4.

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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).

https://doi.org/10.1371/journal.ppat.1007499.g005

MPK6-mediated phosphorylation of IDD4

To evaluate the capability of MPK6 to phosphorylate IDD4, in vitro kinase assays followed by LC-MS/MS analysis were performed with a constitutively active version of MPK6 and recombinant IDD4 protein as a substrate. The MS/MS spectra for IDD4 (Fig 5C and 5D) show that MPK6 targets the same amino acid residue (Ser-73) that was identified in several phosphoproteomic studies [43–48]. Moreover, using an inducible MAPKK-activation system, Hoehenwarter et al. (2013) [29] identified the equivalent phosphosite as a putative target of the MAPKs MPK3 and MPK6. Intriguingly, an additional phosphosite in IDD4 (Thr-130) is phosphorylated by MPK6 in the highly conserved N-terminal ID domain (Fig 5E, S4A Fig). Recently, it was shown that MPK6 targets besides the common SP and TP sites PT sites after flg22 treatment [68], suggesting a biological function of Thr-130 after bacterial perception. Notably, the first phosphorylation site resides 11 amino acids upstream of ZF1 whereas the second phosphorylation site is located inside ZF2 (Fig 5E). The post-translational modifications in this region of the ID domain suggest an inherent phosphorylation-dependent regulation mechanism for DNA-binding of IDD4 [17].

IDD4 binds to the ID1 motif of the SAGT1 promoter

The elevated resistance of the idd4 mutant accompanied by the elevated expression of genes involved in salicylic acid (SA) metabolism and signaling suggest a function of IDD4 in these processes. The 500 bp upstream region of the translational start sequence (TSS) of the SALICYLIC ACID GLUCOSYLTRANSFERASE 1 (SAGT1) gene has two ID1 motifs in the promoter region between positions -259 and -224 upstream of the TSS (Fig 6A) and becomes bound by IDD4 with a log₂(FC) of 2.20. This finding was confirmed in vivo by ChIP_qPCR using transgenic lines expressing IDD4-GFP under the UBI10 promoter (Fig 6B) and its native promoter (Fig 6C) showing consistently that IDD4 binds to the SAGT1 promoter region P1 and P2 (-300 to 0) close to the TSS. SAGT1 converts SA to the biologically inactive storage forms SA-2-l-β-D-glucoside (SAG) and SA-glucose-ester (SGE) [69]. In agreement with this function, transgenic SAGT1ox plants exhibited increased susceptibility to Pst DC3000 and showed reduced levels of free SA [70, 71].

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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.

https://doi.org/10.1371/journal.ppat.1007499.g006

IDD4 regulates expression of SAGT1

The expression of SAGT1 is induced by Pst DC3000 infection and Methyl-SA [69, 71]. Genome-wide DNA binding and ChIP-qPCR analysis showed that IDD4 binds to the SAGT1 promoter and consequently identifies SAGT1 as a direct target of IDD4. Indeed, SAGT1 transcript levels are reduced in the idd4 mutant (log₂(FC) -0.39, p<0.01) in untreated conditions and after flg22 treatment (log₂(FC) -0.4, p<0.05) (S2 Table) when compared to WT plants. These results could be confirmed by qPCR (Fig 6D). On the other hand, SAGT1 transcript levels are enhanced in IDD4ox (Fig 6E) after flg22-treatment, indicating that IDD4 acts as a positive regulator of SAGT1 expression.

IDD4 phosphorylation determines ID1-motif DNA-binding

To study the biological function of IDD4 phosphorylation in planta, we replaced Ser-73 and Thr-130 by aspartic acid (D) to generate a phospho-mimicking IDD4 version S-73-D, T-130-D (DD) or by alanine (A) to produce a phospho-dead IDD4 S-73-A, T-130-A (AA) version. As shown in S4E and S4F Fig, both phospho-modified IDD4 versions still interact with the M5GAI (RGA/DELLA) protein in Y2H and SCL3 in BiFC analysis. These results suggest that phosphorylation of IDD4 in the first 2 ZFs does not hamper the interaction with these partners. To test the influence of phosphorylation on the DNA binding capability of IDD4, we produced stable transgenic plants expressing the phospho-modified IDD4:RFP versions controlled by the UBIQUITIN10 promoter. For further studies, transgenic lines were selected that exhibit comparable transcript levels of IDD4-modified-versions IDD4-AA and IDD4-DD thereby accumulating equal amounts of the encoded protein (S3D Fig). The phenotype of the IDD4 phospho-modified plants is indistinguishable from WT and the shoot fresh weight of 18 day-old IDD4-AA lines is slightly reduced compared to IDD4-DD lines but insignificant to WT. The root fresh weight of IDD4-AA and IDD4-DD lines is comparable to WT (S2G–S2I Fig). These lines were used for ChIP-PCR to determine binding preferences of phosphorylated IDD4. Quantitative PCR of DNA that was immunoprecipitated by RFP antibody confirmed that the phospho-mimicking version IDD4-DD shows higher binding affinity to the SAGT1 promoter compared to the IDD4-AA line or WT control under both flg22 and mock-treated conditions (Fig 6F). The inability of IDD4-AA to increase DNA binding to the SAGT1 promoter after flg22-treatment demonstrates the importance of the flg22-mediated post-translational modification of IDD4 at the phosphorylation sites. In order to analyze whether the binding ability enhancement of the unmodified IDD4 to the ID1 element also occurs in vivo in the context of PAMP signaling, ChIP of IDD4:GFP lines was performed 1 hour after flg22 treatment (Fig 6H). Previously, we tested whether flg22 perception influences IDD4 protein stability by analyzing IDD4ox lines for IDD4:GFP degradation at 30 and 60 min after flg22 treatment in 10 day-old seedlings. No change of stability was seen under these conditions by Western blotting or fluorescence microscopy in roots (S3E and S3F Fig) demonstrating that flg22-perception does not compromise IDD4 protein stability. However, the association of IDD4:GFP to the SAGT1 promoter in the ChIP approach was significantly elevated after flg22 application unlike the IDD4:GFP mock-treated control (Fig 6H). This finding suggests that PAMP-treatment determines the binding ability of IDD4 to the ID1 motifs of the SAGT1 promoter.

These studies were extended by DNA-shift experiments using the recombinant N-terminal domain of IDD4 (IDD4-WT, IDD4-AA and IDD4-DD), containing the 2 phosphorylation sites and the 4 zinc-finger-containing ID domain previously shown to be necessary for DNA binding and interaction with other partners, respectively. As a probe, the sequence of the SAGT1 promoter region -259 to -224 (Fig 6A) was used that harbours 2 ID1 motifs and was shown to be targeted by IDD4:GFP in the ChIP-SEQ and ChIP-qPCR approaches (Fig 6A–6C). Predominant binding of IDD4-DD to the biotinylated ID1 motifs could be confirmed; whereas IDD4-AA and unmodified IDD4 exhibited much weaker binding preferences (Fig 6G). By adding an unlabeled specific competitor to the reaction, the interaction between IDD4-DD and the ID1 probe could be significantly diminished. By contrast, the addition of an unlabeled probe containing mutated ID1 motifs only resulted in a minor competition on IDD4-DD binding. Taking altogether, these results demonstrate the enhanced DNA-binding ability of the phospho-mimiking IDD4 version.

Phospho-modified IDD4 versions show opposite transcriptional activity in vivo

After obtaining indications that the DNA-binding property of IDD4 might depend on the phosphorylation status in vivo and in vitro, we analyzed whether the phosphorylation status of IDD4 determines the transcriptional activity as well. To prove the biological relevance of the transcriptional activity of IDD4, the expression of SAGT1 was studied in the phospho-modified versions expressing lines under untreated conditions and after flg22 application (after 4 hrs). The relative gene induction level of SAGT1 after flg22 treatment was significantly induced in the IDD4-DD line, reminiscent to the IDD4ox line (Fig 6E), however, unlike to the IDD4-AA line that does not show an increased expression level when compared to WT (Fig 7A). These findings suggest that IDD4-DD acts as an activator of SAGT1 gene expression. By contrast, the expression of SAGT1 in the phospho-modified lines under untreated conditions was indistinguishable from WT, accordingly to the WT-like expression of SAGT1 in the IDD4ox line (Fig 6E). Altogether, these results imply the importance of the IDD4 phosphorylation status to initiate target gene expression in a flg22-dependent manner.

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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.

https://doi.org/10.1371/journal.ppat.1007499.g007

Phospho-modified IDD4 versions show different PTI responses and transcriptional composition

After showing that phosphorylation of IDD4 coordinates gene expression, the biological relevance of this mechanism was assessed after hemi-biotrophic pathogen infection. Therefore, we measured Pst DC3000 hrcC- infection levels in two independent transgenic lines harbouring IDD4-AA and IDD4-DD. At 2 hrs after spray-infection of the different phospho-modified line, similar bacterial cfu were obtained, indicating that the accessibility of the bacteria to the leaf apoplast tissue was not affected. However, 72 hrs after plant infection, the proliferation levels of the bacteria were significantly elevated in both IDD4-DD lines compared to IDD4-AA lines and WT. Furthermore, the bacterial titers in the IDD4-AA lines were reduced even below WT levels indicating that the posttranslational-modification of IDD4 impacts bacterial growth and defense response (Fig 7B). In order to analyse the susceptibility of the modified IDD4 expressing lines at the molecular level, the transcriptomes of 14 day-old seedling were studied by RNA-Hiseq of 3 biological replicates. The transcriptome composition of IDD4-DD lines revealed 429 genes as being down-regulated and 354 up-regulated (p<0.05) when compared to WT or IDD4-AA lines. GO terms of down-regulated genes could be determined for defense response, respiratory burst and innate immunity (Fig 7C, S6 Table). GO terms of the up-regulated genes in IDD4-DD highlighted gene functions in energy metabolism, RNA biosynthesis process and photosynthesis (Fig 7D). On the other hand, GO terms of down-regulated genes in IDD4-AA (p≤0.05, 211 genes) referred to response to starvation, root system development and metal ion transport (Fig 7E). Furthermore, GO terms for up-regulated genes in IDD4-AA (p≤0.05, 169 genes) emphasize gene functions for sugar and carbohydrate response (Fig 7F, S6 Table). In summary, the GO term analysis indicates a distinct molecular setting between IDD4-AA and IDD4-DD lines, e.g. favouring growth and energy metabolism at the expense of defense response in the case of the phospho-mimicking (DD) line. The transcriptome compositions of IDD4-AA and IDD4-DD lines with respect to significant differentially regulated genes (p<0.05) resulted in 3 main clusters illustrating a distinct and partly opposite expression of DEGs in the particular genotypes (Fig 7G). Cluster I and II show a profound differential regulation of downstream targets in the IDD4-DD lines whereas their deregulation in IDD4-AA lines is rather moderate and oppositely to the IDD4-DD line, such as in cluster I. To assess the biological functions of DEGs in the respective clusters, we carried out a GO term analysis. Cluster I highlights among others GO terms for response to water deprivation (5.4e^-13) and abiotic stress stimulus (2.1e^-08), whereas genes in cluster II were dedicated to the GO terms plant-type cell wall organisation (6.6e^-10) and defense response (1.7^e-07). By contrast, cluster III emphasises genes that are down-regulated in the IDD4-AA lines and moderately up-regulated in the IDD4-DD lines supporting additionally the notion of a distinct regulation of subsets of target genes indicating a distinct transcriptome composition (Fig 7G). In this context, GO terms for cluster III could be assigned to gene function in response to starvation (4.6e^-08) and inorganic cation transport (3.1e^-07). The molecular characterisation by qPCR of the phospho-modified lines 4 hours after flg22 treatment showed differential regulation of the SA response marker PR1 being higher expressed in IDD4-AA lines than in IDD4-DD and WT (S4B Fig). By contrast, the expression of the SA-signaling inhibitors WRKY38 [72] and NIMIN1 [73] are elevated in the IDD4-DD lines and the expression of NIMIN1 is reduced in IDD4-AA compared to WT and IDD4-DD line (S4C and S4D Fig). These expression patterns of SA-signaling markers indicate a reduced SA response in the IDD4-DD lines. Therefore, to determine the concentration of free SA in the IDD4-phospho-modified lines after Pst DC3000 hrcC- infection, hormonal measurements were conducted. Quantitative analysis of free SA in 3 biological replicates of WT, IDD4-AA and IDD4-DD transgenic lines before pathogen infection (Fig 7H) revealed comparable amounts of about 0.42 ng/mg dry weight. However, 24 hrs after being challenged by Pst DC3000 hrcC-, WT and IDD4-AA lines showed strongly increased comparable levels of free SA, whereas the IDD4-DD lines exhibited significantly lower amounts. These findings are compatible with the notion that the higher susceptibility of the IDD4-DD lines to Pst DC3000 hrcC- is to some extent caused by its diminished SA levels as reflected by the elevated expression of the negative SA regulators NIMIN1, WRKY38 and SAGT1. If IDD4 and in particular its phosphorylation status contributes to SA homeostasis, then the SA accumulation after pathogen treatment should as well as be compromised in the idd4 mutant. Therefore, eventually, we determined the free SA levels in the idd4 mutant 24 hrs after Pst hrcC- infection. Interestingly, we found already a moderately elevated SA level in the idd4 mutant (Fig 7I). Furthermore, the pathogen-induced SA rise was also higher than in infected WT plants. Taking into account the higher resistance of idd4 against hemi-biotrophic pathogens (Fig 1I and 1J) and the differential regulation of components contributing to SA metabolism/ catabolism such as SAGT1 (Fig 6D and S2F Fig), then these results corresponds with a function of IDD4 in the modulation of SA homeostasis. All in all, these findings suggest a profound biological significance of IDD4 phosphorylation to coordinating plant defense with growth and developmental processes.

IDD4 contributes to distinct functional networks that coordinate growth and defense

Plant response to pathogen attack often requires “trade-off” processes in which resources initially dedicated to growth and pattern formation will be redistributed to pay the high metabolic costs of defence. It follows that plants have to precisely regulate resources to be allocated to fight against a pathogen [74]. In this regard, it was shown that the members of the PHYTOCHROME-INTERACTING FACTORS (PIFs) play a pivotal role in the regulation of growth defense trade-offs to adapt to changing environmental conditions [75]. PIFs redundantly inhibit skotomorphogenesis and individually regulate other light-mediated processes such as shade avoidance responses, chloroplast differentiation and seed germination but their own transcriptional regulation is poorly understood [76]. In addition to the light responses, some PIF members including PIF4 are involved in the hormonal responses (gibberellic acid (GA), brassinosteroid (BR), and auxin) [53]. In this regard, PIF4-mediated thermosensory growth and architecture adaptations are directly linked to suppression of immunity at elevated temperature. Accordingly, the pif4-101 and pifQ (pif1, pif3, pif4, pif5) quadruple mutant exhibited increased resistance to PstDC3000, demonstrating PIF4 as a positive regulator of growth and development and negative regulator of immunity [77]. Interestingly, we found that IDD4 binds to the promoter region of several PIF members (PIL1, PIL2/PIF6, PIL5/PIF1, PIF3) and photoreceptor PHYTOCHROME B (PHYB) that regulates PIF protein levels through promoting light-dependent protein degradation [75] thereby supporting resistance against PstDC3000 [77]. The association of IDD4 to these promoter regions shows an important function in the transcriptional regulation of these system integrators in plant development. In addition, our ChIP-SEQ analysis revealed the association of IDD4 to the G-Box, also called PIF4-box (Fig 3B), which is highly enriched in PIF4 target promoters previously shown by PIF4 ChIP-SEQ analysis [78]. The PIF4 DNA binding ability depends on its capacity to form hetero- and homodimers. The shared usage of the G-Box cis-regulatory elements by PIF4 and IDD4 might be mediated by heterodimer formation of these factors to shape the DNA-binding ability and expression of particular gene subsets. We have shown that IDD4, in accordance with PIF4, acts as repressor of immunity against hemi-biotropic pathogens. However, the enhanced growth of the idd4 mutant demonstrates that IDD4 acts as a suppressor of plant growth and development, unlike PIF4 that positively regulates these processes. Consequently, we suspect that IDD4 and PIF4 cooperatively mediate the expression of defense-related genes that might antagonistically contribute to processes promoting growth and development. Moreover, IDD4 was previously reported being transcriptionally repressed by KAN and REV, and IDD4ox lines are compromised in leaf blade formation and leaf size [18]. The overexpression of IDD4 causes downward leaf curling, resembling reduced HD-ZIPIII gene function which indicates a negative feedback regulatory network of the three factors. The discovered binding of IDD4 to promoter regions of KAN and REV suggest an up to now uncharacterized feedback regulation. In addition, pathogen attack has been shown to suppress components of photosynthesis at the level of gene expression and protein abundance; and defense exerts a negative impact on photosynthesis which results in a reduction of components essential for light harvesting and carbon fixation [74]. We show that IDD4 affects the expression of genes involved in chloroplast maturation, localization in response to changing light conditions and photo-morphogenesis. Furthermore, the auto-immune phenotype of idd4, reflected by the internal increase of H₂O₂, indicates a role of IDD4 in the photo-respiratory H₂O₂ accumulation.

Indications for flg22-mediated phosphorylation of IDD4

We showed that IDD4 is phosphorylated by the immune MAPK MPK6 whose activation is triggered in planta upon PAMP-perception to modulate immune reaction [33]. We found that the IDD4 phospho-modified versions behave differently regarding the DNA-binding ability, target-gene activation and response to pathogen-attack. In this context, the IDD4-phospho-mimiking version (IDD4-DD) shows stronger recruitment to the DNA and acts as transcriptional activator of SAGT1 expression. By contrast, the phospho-dead version (IDD4-AA) displays weak DNA-binding and low transcriptional activation after flg22-treatment. By exploiting the native IDD4 version, we found that IDD4-binding and transcriptional activity are increased upon PAMP-perception. After flg22-application, the recruitment of IDD4 to the DNA and the transcriptional activity are elevated in accordance with the results of the phospho-mimicking IDD4-DD version. Moreover, the opposite behavior of IDD4-AA and its unresponsiveness to flg22-treatment support the notion of a post-translational modification-based mechanism to regulate IDD4 DNA-binding properties. Phosphosite-dependent transcriptional deactivation of IDD8 mediated by AKIN 10 in the process of carbon metabolism was recently reported [79]. The phosphorylation of IDD8 at Ser-178 and Ser-182, which reside both in the fourth ZF domain did not affect the subcellular localization and DNA-binding property of IDD8 but diminished the transcriptional activation activity. Interestingly, the function of a putative transactivation domain might be compromised by the phosphorylation of the closely adjacent ZF4 [45]. Recently, the importance of ZF4 for protein-protein interaction of IDD10 and IDD3 with the SCR-SHR complex was reported [17]. Furthermore, the transcriptional activity of IDD10 is modulated by reciprocal interactions with IDD3, SCR and SHR [80]. Therefore, it is conceivable that the phosphorylation of ZF4 in IDD8 interferes with the association of coactivators. In summary, the post-translational modification of particular ZFs in IDD4 and IDD8, as the discussed phosphorylation events, change their characteristic traits and can be considered as a general IDD/BIRD-regulation mechanism to modulate DNA-binding ability, protein-protein interaction and transcriptional activity.

IDD4 and DELLA/RGA might synergistically modulate immunity

Recently, the gibberellin (GA)-inhibitory DELLAs were introduced to control plant immune responses by modulating the balance of JA and SA [81]. Notably, DELLAS cannot directly bind to the DNA because of the lack of a DNA interaction—domain. However, they act as co-activators or repressors, respectively, by binding to transcription factors in a stress-dependent manner in order to coordinate target gene expression. We showed the interaction of IDD4 with the DELLA/GAI. The IDD family members 2, 3, 4, 5, 9 and 10 serve as transcriptional scaffolds to enable transactivation activity of the DELLA/RGA of the GRAS-family [16] [22]. In vitro studies have confirmed the transcriptional activation of DNA-bound IDDs upon the association of RGA and the subsequent expression of the SCL3 locus [16]. Interestingly, genome-wide DNA-binding studies on DELLA/RGA revealed the enrichment of the ID1 cis-motif in about 28% of target sequences underpinned by a total p value of 2.3e^-10. The occurrence of the ID1 motif indicates a common set of RGA and IDD4 controlled genes [82]. In this context, we identified a substantial overlap of 20.4% of RGA and IDD4 target genes and found associated GO terms, among others, for response to salicylic acid stimulus, light stimulus and regulation of defense response (S3 Table). Interestingly, the introduced quadruple-DELLA mutant is more resistant to Pst DC3000 compared to WT and accumulates higher levels of free SA after pathogen attack [81], demonstrating that DELLAs promote disease susceptibility to hemi-biotrophic pathogens and repress the SA-defense pathway. Therefore, the reduced susceptibility to bacterial infection in the association with the elevated SA levels of the idd4 mutant, and the reduced SA accumulation in the IDD4-DD lines after Pst HrcC- infection suggest a synergistic interaction of IDD4 and DELLA/RGA proteins in the regulation of selected defense processes. Moreover, GAI-ASSOCIATED FACTOR1/IDD2 is involved in the regulation of GA homeostasis and signaling in Arabidopsis for binding to genes which are part of GA feedback regulation. GA converts the IDD2 complex consisting of DELLA, IDD2 and TOPLESS RELATED from a transcriptional activator to a repressor upon the degradation of DELLA [22]. Similarly, the co-repressor SCL3 interacts competitively with DELLAs for the binding to IDD proteins to antagonistically regulate downstream gene expression to control GA signaling pathways [21].

IDD4 specificity to ID1 motif binding

To assess the distribution and abundance of the core ID1 motif (AGACAA), we performed a genome-wide in silico search of the 500 bp upstream regions in the promoter regions of IDD4-bound genes for the occurrence of at least two ID1-core motifs. Previous publications suggested the ID1-cis motif as the main binding sequence of different IDD family members, including IDD4 [22] [44] and it was shown that IDD4 binds to the ID1-core sequence inside the SCL3 promoter in vitro [16]. Noteworthy, our in vivo ChIP-SEQ data confirm the binding of IDD4 to the upstream regulatory sequence of SCL3 (S3B Fig, S3 Table). Our analysis revealed 2840 genes harbouring this element up to 8 times whereas 82.9% of the genes contained two ID1-core motifs (S5 Table). This high number of genes with two ID1-core motifs in their promoter regions is surprising and raises the question of the specificity of IDD4 DNA-binding and how target gene activation can be achieved. We provide evidence that phosphorylation of IDD4 is a post-translational regulatory mechanism to trigger IDD4 DNA-binding. In order to coordinate the binding of IDD4 to the ID1 motif, binding partners might be involved in a developmental- and stress-dependent manner modulating the accessibility of the binding sites to IDD4. For example, IDD4 activity could be orchestrated by the formation of IDD4 homo- and heterodimers with other transcriptional regulators in a developmental or stress-dependent manner. Further mechanisms might include different splice variants as shown for IDD14, which are generated by cold stress to form a competitive inhibitor regulating starch metabolism. IDD14β lacks one functional DNA binding domain but is still able to create heterodimers with the functional IDD14 form (IDD14α). However, IDD14α–IDD14β heterodimers have diminished DNA-binding activity to their target promoter [83]. Slight modifications in the ratio of functional transcription factors and alternatively spliced variants could sustainably affect the expression of target genes. In this context, it is worth mentioning that IDD4 also forms a second splice variant (IDD4.2) (TAIR-database), which contains the 4 zinc finger-containing ID domain, but excludes the phosphosite Ser-73. This means that IDD4 can presumably form heterodimers consisting of IDD4.1-IDD4.2 as well as homodimers with each of the two splice variants. It is well conceivable that the IDD4.2 homodimer without the phosphorylation site could act as a competitive inhibitor of the phosphorylated IDD4.1 protein and further research is necessary to pursue these possibilities.

Plant material and growth conditions

Experiments were performed by using Arabidopsis thaliana of the Columbia accession grown on soil in plant growth chambers (Percival Scientific) under short-day conditions (8h light/ 16 h dark) at 22°C. Nicotiana benthamiana were grown under long day conditions (16 h light + 8 h darkness) at 28 °C. idd4 (Salk_148352C) seeds were obtained from NASC.

RNA Extraction and real-time quantitative PCR analysis

Total RNAs were extracted from 14 day-old seedlings, grown on sugar-free Murashige and Skoog (MS) plates under long-day conditions. We used the NucleoSpin RNA Plant (MACHEREY-NAGEL) kit, according to the manufacturer’s instructions. First strand cDNA was synthesised from 5μg of total RNAs using SuperScript First-Strand Synthesis System for RT-PCR according to the manufacturer’s instructions. The cDNA stock was diluted to a final concentration of 25ng/ul. Subsequently, 500nM of each primer was applied and mixed with LightCycler 480 Sybr Green I Master mix for quantitative PCR analysis, according to the manufacturer’s instructions. Products were amplified and fluorescent signals acquired with a LightCycler 480 detection system. The specificity of amplification products was determined by melting curves. GADPH was used as internal control for signals normalisation. Exor4 relative quantification software automatically calculates relative expression level of the selected genes with algorithms based on ΔΔCt method. Data were from duplicates of at least three biological replicates. All the sequences of primers used are given in S7 Table.

Immunoblot and ChIP Analyses

Nuclear proteins were extracted from 14 day-old on half MS-grown seedlings. After quantification with the Bradford method, equal amounts of proteins were resolved by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane using a Mini-Protean 3 Cell. Immunoblot analysis was performed using 1 μg/mL primary polyclonal antibodies raised against GFP and then with secondary antibodies conjugated to alkaline phosphatase. Antibody complexes were detected by chemiluminescence using the Immun-Start AP Substrate kit. ChIP assays were performed on 14 day-old in half MS-grown seedlings using Anti-GFP antibody—ChIP Grade and RNA polymerase II antibodies. Briefly, after plant material fixation in 1% (v/v) formaldehyde, the tissues were homogenised, and the nuclei were isolated and lysed. Cross-linked chromatin was sonicated using a water bath Bioruptor (Diagenode; 15-s on/15-s off pulses, 15 times). The complexes were immunoprecipitated with antibodies overnight at 4°C with gentle shaking and incubated for 1 h at 4°C with 50 μL of Dynabeads Protein A. Immunoprecipitated DNA was then recovered and analysed by qRT-PCR. An aliquot of untreated sonicated chromatin was processed in parallel and used as the total input DNA control.

Bioinformatics analysis of ChIP-SEQ data

Sequencing of Col-0, IDD4:GFP sample material was performed in Illumina Hi-Seq2000 platform. About 47 million and 53 million paired-end reads (insert size of 300 bp) with 125 bp read length were obtained for Col-0 and IDD4 respectively. Quality statistics of reads were analyzed using FASTQC [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/]. Trimming and filtering of reads were performed using trimmomatic [84]. Parameters for read quality filtering were set as follows: Minimum length of 36 bp; Mean Phred quality score greater than 30; Leading and trailing bases removal with base quality below 3; Sliding window of 4:15. Filtered reads were aligned to TAIR10 using Bowtie (Unique mapping of reads was adopted) and enriched regions were identified using MACS2 [85]. Parameters for peaks detection were set as follows: Number of duplicate reads at a location: 1; Bandwidth: 300; mfold of 5:30; q-value cutoff: 0.05. Peaks were identified against input DNA sequence. Results from all three peak callers (MACS2, SISSR, HOMER) have shown very high correlation. Bedtools was used for manipulation of these genomic peak intervals [86]. Identification of putative IDD4 binding motifs (p-value < 0.05) at called peak positions was done using HOMER [87]. Coverage and enrichment of functional elements from TSS to TES and their flanking region were visualized using NGSplot [88]. The complete ChIP-SEQ data sets are available at the GEO repository (GEO accession GSE120068).

Bioinformatics analysis of RNAseq data

Sequencing was performed on each library to generate 101-bp paired-end reads on Illumina HiSeq2500 Genome Analyzer platform. Read quality was checked using FastQC and low quality reads were trimmed using the Trimmomatic version 0.32 (http://www.usadellab.org/cms/?page=trimmomatic) with the following parameters: Minimum length of 36 bp; Mean Phred quality score higher than 30; Leading and trailing bases removal with base quality below 3; Sliding window of 4:15. After pre-processing the Illumina reads, the transcript structures were reconstructed using a series of programs, namely, TopHat (ver. 2.1.1; http://tophat.cbcb.umd.edu/) for aligning with the genome, and Cufflinks (ver. 2.2.1; http://cufflinks.cbcb.umd.edu/) for gene structure predictions. For TopHat, the Reference-Arabidopsis thaliana (TAIR10) genome (https://www.arabidopsis.org) was used as the reference sequences with a maximum number of mismatches as 2. To identify the differentially expressed genes, the following parameters were used: p-value of 0.05 with a statistical correction using Benjamini Hochberg FDR of 0.05 in cuffdiff. A cut-off of 2 fold up- or down-regulation has been chosen to define the differential expression. After processing the data, visualisation of differential expression was done using cummeRbund v2.14.0 (http://bioconductor.org/packages/release/bioc/html/cummeRbund.html). Differentially regulated genes that are common among the samples were identified using Venny. The complete RNAseq data is available at the GEO repository (GEO accession GSE120068).

Bioinformatic analyses/ GO term analysis

For the GO term analysis, AGRIGO analysis tool was used (http://bioinfo.cau.edu.cn/agriGO/, [30] by using significantly differentially expressed genes between the tested conditions. Protein-network analysis was performed by using STRING [46]. Putative MAPK docking site of IDD4 was searched using the ELM program (http://elm.eu.org/).

Plasmid construction

Whole seedling Arabidopsis cDNA library was used to amplify the coding sequence (cds) of the IDD4 and SCL3. Subsequently, the entry clone was generated by introducing the cds either in the pENTR- or pCR8/GW/TOPO-vector. Subsequently, entry clones were used to generate protein expression constructs (pDEST-MBP [89]) and protein localisation vectors, fused to GFP/RFP driven by the Ubiquitin promoter [90]. Site-direct mutagenesis of IDD4 was performed in a 2 step approach. First oligo-nucleotide based introducing of the base pair exchange results in two truncated cDNA fragments with an overlapping region. Secondly, both fragments were fused and amplified by using oligos that bind in the end and beginning of the total cDNA. GAI (amino acids 148 to 533), RGA, IDD4, IDD4-AA, IDD4-DD cDNA inserted into pDONR207 by Gateway cloning were recombined with pGADT7 (AD) or pGBKT7 (BD) to generate BD-GAI, AD-IDD4, AD-IDD4-AA and AD-IDD4-DD. All the sequences of primers used are given in S7 Table.

Yeast two-hybrid assays

Direct interaction assays in yeast were carried out following the small-scale LiAc yeast transformation procedure. The N-terminal part of the DELLA proteins is subject to autoactivation in yeast two-hybrid assays; therefore, only the C-terminal domain of GAI (amino acids 148 to 533) was recombined by Gateway with pGBKT7 to generate BD-GAI. Yeast strain AH109 was co-transformed with BD-GAI and AD-IDD4/IDD4-AA/IDD4-DD or empty vector (pGADT7), and interaction tests were surveyed on selective media lacking Leu, Trp, Ade and His.

Quantification of SA

Plant materials were lyophilised and ground in a bead beater. Aliquots (about 5 mg dry weight) of powdered tissues were extracted with 400 μL of 10% methanol containing 1% acetic acid and internal standards (11.1 ng of 2H₄-SA). The samples were extracted in the bead beater for 1 min, placed in ice for 30 min, and then centrifuged at 13,000 g for 10 min at 4°C. The supernatant was carefully removed and the pellet re-extracted with 400 μL of 10% methanol containing 1% acetic acid. Following further 30 min incubation in ice, the extracts were centrifuged and the supernatants combined. The samples were filtered through 0.22 μm PTFE filters before LC-MS/MS analysis. Analysis SA was performed by comparing retention times and mass transitions with the standards using an Agilent 1200 HPLC coupled to an Q-TRAP 5500 MS with an electrospray source. Chromatographic separation was carried out at 35°C on a Phenomenex Gemini C18 (150×2.0 mm, 5 μm) column with the solvent system formic acid/acetonitrile/water (0.1/94.9/5, v/v/v; mobile phase A) and formic acid/ acetonitrile/water (0.1/5/94.9, v/v/v; mobile phase B). The gradient used was 0–20 min, 0%-100% A; 20–25 min, 100% A; 25–26 min, 100%-0% A; 26–36 min, 0% A. To reduce contamination of the MS, the first 5 min of the run was directed to waste using the inbuilt Valco valve. Analysis of SA was based on appropriate Multiple Reaction Monitoring (MRM) of ion pairs for labelled and endogenous SA using the following mass transitions: 2H₄SA 141>97, SA 137>93. The MS was operated in negative ionization mode. The conditions were as follows: Temperature 500 °C, Ion source gas 1 50 psi, Ion source gas 2 60 psi, Ion Spray Voltage -4500 V, curtain gas 40 psi, Collision Gas Medium; DP (-25 V), EP (-9) and CXP (-2) were the same for all compounds. CE (-38), and DT (50) were used for 2H₄SA and SA. Data were acquired and analysed using Analyst 1.4 software.

Pseudomonas syringae infections

Plants were spray-inoculated with Pseudomonas syringae DC3000 and Pseudomonas syringae DC3000 hrcC- at OD₆₀₀ = 0.2 and sampled 2 h and 72 h after inoculation to determine the level of colonisation (colony-forming units (cfu)) as described previously [91]. In three biological replicates, a total of 30 plants were sampled for each plant genotype by each taking 3 leaf discs per plant.

Quantitation of immunoblot membranes

Bradford assays were used to quantify protein levels in extracts and ensure equal loading of total proteins for gels used for immunoblot analysis and EMSA.

Electro-mobility shift assay

Recombinant truncated protein of IDD4, IDD4-AA and IDD4-DD (each from amino acid 20–219) fused to MBP-tag was affinity-purified from E.coli Rosetta cells and enriched by Ion Exchange Purification.

3´ End-Biotinylated oligos were ordered. Biotinylated DNA (20fmol) was mixed with 1 μg of the indicated proteins after the instructions of the Lightshift Chemiluminescent DNA EMSA. These experiments were repeated at least three times with similar results.

Kinase assays and phospho-site identification

Purified recombinant proteins and constitutively active MAPKs were mixed together in kinase reaction buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 5 mM EGTA, 1 mM DTT and 50 μM ATP) and incubated at ambient temperature for 30 min. SDS-sample buffer was added to stop the reaction followed by boiling at 95°C for 10 min. Protein samples were resolved by SDS-PAGE. The gel was stained with SimplyBlue SafeStain and the band corresponding to the protein of interest was excised out, cut into small pieces of 0.5 mm3 and destained with four successive washes of 15 min each with ACN and 100 mM NH₄HCO₃. Proteins were reduced with 10 mM Tris(2-carboxyethyl)phosphine (TCEP) in 100 mM NH₄HCO₃ at 37°C for 1 h followed by alkylation with 20 mM S-Methyl methanethiosulfonate (MMTS) at ambient temperature for 30 min. Proteins were then digested with trypsin (Porcine trypsin) at 37°C overnight. The digestion was stopped by the addition of 1% formic acid, and the peptides were recovered by incubating the gel pieces in ACN. The recovered peptide solution was desalted using C18 ZipTip and analysed by LC-MS/MS. Briefly, peptide samples were separated on a C18 connected to an LTQ-Orbitrap Velos or a Q-Exactive HF instrument. The LC gradient ramped from 5% solvent B (water/ACN/formic acid, 20/80/0.1, v/v/v) to 45% solvent B over 45 min, then to 90% solvent B for 10 min. The MS instrument acquired fragmentation spectra on the top 10 peptides using CID fragmentation in the LTQ-Orbitrap or HCD in the Q-Exactive instrument. RAW data files obtained were converted to MGF files using Proteome Discoverer interface (version 1.4). Database searches were performed with the Mascot server v2.4 specifying the following parameters: database TAIR10 (release 2010/12/14, 35386 sequences); enzymatic specificity: trypsin permitting two allowed missed cleavages; fixed modification of cysteine residues (Methylthio(C)); possible variable modifications of phosphorylation on S, T and Y residues; 5 ppm tolerance on precursor masses and 0.5 Da tolerance on fragment ions. The results were filtered based on Mascot scores and MD-scores.

Bimolecular fluorescence complementation (BiFC)

To obtain the expression vectors, coding sequences of candidate genes and MAPKs (kindly provided by J. Colcombet) were cloned in fusion with the N- and C-terminal parts of YFP, either as N- or C-terminal fusions, under the control of the cauliflower mosaic virus 35S (CaMV-35S) promoter in the pBIFC1,2,3 and 4 vectors [92]. Appropriate positive and negative controls were carried out for all combinations. Recombined vectors were individually transformed in Agrobacterium tumefaciens C58C1 strain by electroporation. Agrobacterium cultures from glycerol stocks were inoculated in 10 ml of LB medium with appropriate antibiotics and incubated for 24 h at 28°C with agitation. Each culture was pelleted and resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES pH 5.7, 150 μM acetosyringone) to an OD₆₀₀ of 1.5 and kept in the dark for 3 h. The P19 viral suppressor of gene silencing was co-expressed with each combination to prevent silencing of transiently expressed proteins [93]. 500 μl of each bacterial culture was mixed before infiltration. For fluorescence complementation, all eight possible combinations between a candidate gene and a MAPK were agro-infiltrated into 3-week-old Nicotiana benthamiana leaves. After 3 days, an upright confocal microscope with a 20X objective (Plan-Apochromat, NA 1.0) was used to visualise fluorescence. All images were acquired using Argon laser with 514-nm excitation.

GUS staining

3 weeks old plants were incubated in GUS-Staining solution (NaPO₄ (pH 7.2, 50mM), EDTA (pH8.0 10mM), TritonX100 (0.1%), Ferrocyanid (2mM), Ferricynaid (2mM), X-Gluc (1mg/ml)) for 12hr at 37°C. Subsequently, chlorophyll was bleached by applying 70% ETOH.

DAB staining

Stainings were conducted by using 14-day old seedlings grown under sterile conditions on half MS medium, in accordance to [94].

ROS burst assay

ROS burst assay was performed as described by [95].

Accession numbers

IDD4 (AT2G02080), SAGT1 (AT2G43820), MPK6 (AT2G43790).