Plant materials and growth conditions
A.thaliana Columbia accession (Col-0) was used as wild-type plant in this study. The DEK2 T-DNA insertion mutants dek2-1 (SALK_1375152C) was provided by Claudia Jonak and dek2-2 (SALK_033428) was obtained from the Nottingham Arabidopsis Seed Centre (NASC) and genotyped by PCR for homozygosity.
A. thaliana seeds were surface sterilized in a solution of bleach: ethanol: H2O (V:V:V::1:4:3) for 10 min and washed four times with sterile water. The seeds were then stratified by placing them at 4°C for 48 h. Seedlings were grown on 1/2 MS plates in a plant growth chamber for 14 days at 21°C with 75% relative humidity under 16 h of daylight. A. thaliana plants were grown on Jiffy-7 pots to prevent contaminations from garden soil for four weeks at 23°C, 60% relative humidity with an 8 h light/16 h dark short-day photoperiod and 16 h light/8 h dark long day photoperiod in environmentally controlled growth cabinet (Percival). 2g/L of fertilizer were added on the second and fourth weeks. N.benthamiana plants were grown in the green house under these conditions: humidity 70%, temperature 28°C, long day.
Pathogen strains and growth conditions
Escherichia coli (E. coli) bacteria was grown on LB broth or agar medium for cloning and protein expression purposes. The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and hrcC− mutant (defective in type III secretion system) strains were grown on NYGA agar medium supplemented with 50 mg/ml rifampicin at 28°C for 48h for infection assay hormone quantification and RNA-seq experiment. Agrobacterium tumefaciens (C58C1) was grown in 10 ml LB medium with appropriate antibiotics overnight at 28°C for generating transgenic lines and transient expression studies. The necrotrophic fungus Botrytis cinerea strain BS05.10 was grown on potato dextrose plates at 22°C for two weeks in dark.
Plant transformation of A. thaliana by floral dip method
About five-week-old A. thaliana plants were grown under long days in pots. The transformation was done with Agrobacterium tumefaciens (C58C1) by using the floral dip method80. Transgenic plants were selected on MS agar with appropriate selection agent (BASTA 100 µg/mL, Hygromycin 10 µg/mL). Genotypes of survival transgenic plants were validated by PCR in the T1 generation, and confirmed in the following 2–3 generations. For all the transformation, four transgenic lines were obtained and were used for phenotypic analysis.
Infection of A. thaliana with Pseudomonas syringae pv. tomato DC3000
Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) bacteria were resuspended in 10 mM of MgCl2 in the presence of 0.04% Silwet L-77 and adjusted to OD600 = 0.2. Four-week-old A. thaliana plants were spray-inoculated with Pst DC3000 bacteria and the bacterial growth was quantified as described earlier38. The infection assays were repeated three times with reproducible results.
Infection of A. thaliana with Botrytis cinerea
The necrotrophic fungus B.cinerea was cultivated for 14 days and spores were collected in Vogel buffer (for 1 L: 15 g of Sucrose, 3 g of Na-citrate, 5 g of K2HPO4, 0.2 g of MgSO4·7H2O, 0.1 g of CaCl2·2H2O, and 2 g of NH4NO3) as previously described81. Spore number was determined using a hemocytometer and adjusted to a final conc of 5 ×105 spores/ml. Five leaves of four-week-old plants were drop inoculated with 5 µL of the spore suspension and the trays were cover to maintain humidity. 6–8 plants were used per genotype. Photographs were taken two days after inoculation, and the lesion diameter/area was determined using ImageJ software.
Oxidative burst Measurement
Luminol-HRP-based luminescence method was carried out to quantify ROS in treated leaves. Briefly, 4-mm leaf discs from four-week-old A. thaliana were floated in 150µL dH2O overnight in a 96-well plate in continuous light. Water was then replaced by 100 µl of elicitation reaction mixture containing 1 µM flg22 (QRLSTGSRINSAKDDAAGLQIA) synthesized peptide by GenScript or 100 µM chitin from shrimp shells (Sigma, C9752), 170 µg/ml luminol (Sigma, A4685) and 100 µg/ml horseradish peroxidase (Sigma, P6782). Addition of the same solutions without flg22 or chitin served as controls. Plates were placed immediately into Promega GloMax navigator plate reader and luminescence was recorded at one min intervals over 40 min. Every time point is the mean value of 8 seedlings.
β -Glucuronidase (GUS) staining and subcellular localization
To study the in planta expression of AtDEK2, 1 kb of the promoter region of DEK2 was PCR amplified from RIKEN TAC clone (pdg02820) and cloned into pENTR-D/Topo (Invitrogen) and consequently recombined with the pGWB433 vector by LR reaction to generate ProAtDEK2::Gus construct. The construct was introduced into A. thaliana (Col-O) by Agrobacterium tumefaciens-mediated transformation C58C1. Progeny of these transgenic plants were selected on MS agar plates supplemented with 50 µM kanamycin. Histochemical detection of GUS activity was performed using 5–10 day-old seedlings incubated in the GUS staining solution as described in82 at 37°C for 1–3 hours followed by destaining in Visikol (Phytosys LLC).
Subcellular localization assay was performed on 6-day-old AtDEK2 transgenic A. thaliana complemented stable lines under native promoter fused to YFP. For Subcellular localization in N. benthamiana, the gene of interest was cloned in fusion with GFP at their N- or C-terminal part under the control of the CaMV-35S promoter (in the pGWB5 vector), or under the control of ubiquitin promoter in the pUBIN-GFP, pUBIC-GFP respectively. Plasmids were transformed into A. tumefacies C58C1 and CFP–serrate as a marker for nucleus were infiltrated into leaves of four-week-old N. benthamiana plants using needle less syringes. After three days, fluorescence signals were excited at 488 nm and detected using an upright Zeiss LSM880 laser scanning confocal microscope with a 20X/40X objective (Plan-Apochromat, NA 1.0). All images were acquired using Argon laser.
Bimolecular fluorescence complementation (BiFC)
BiFC was used to visualize the protein-protein interaction and determine the subcellular localization of the interacting proteins. The three MAPKs and the proteins of interest were fused to the N- or C- terminal fragment of YFP that produces a fluorescent readout upon reconstruction of YFP. In the pBIFC1, 2, 3 and 4 vectors used, the expression is under the control of the cauliflower mosaic virus 35S (CaMV-35S) promoter. To test interaction between two proteins by BiFC, eight different combinations of N- or C-terminally tagged YFP fragments were tested. Appropriate positive and negative controls were carried out for all combinations. The constructs were transiently expressed in N.benthamiana leaves by co-infiltration with Agrobacterium. To visualize the fluorescence of the reconstituted YFP expression in leaves, an upright Zeiss LSM880 laser scanning confocal microscope with a 20X/40X objective (Plan-Apochromat, NA 1.0) was used and all images were acquired using Argon laser with 514-nm excitation.
Quantification of Phytohormones
Plant hormones (ABA, JA and SA) were extracted and quantified as previously reported83. Four-week-old A. thaliana plant were harvested 24h after spray inoculation either with 10mM of MgCl2 containing 0.04% of Silwet L-77 as control or infected with Pst DC3000 hrcC− pathogen as described above. Subsequently, plant materials were lyophilized and ground in a Gino grinder (for 2 cycles of 45 sec each, at 1150rpm). 10 mg of plant materials were weighed and extracted with 1 mL of extraction solution containing 70% methanol and the respective phytohormone internal standards (d6-ABA, d6-JA, and d4-SA). Five replicates were prepared for each condition. Extraction procedure and hormone quantification were performed as described84. Samples were analyzed using an Agilent 1100 HPLC system (Agilent Technologies, Böblingen, Germany) connected to a LTQ Ion trap mass spectrometer (Thermo Scientific, Bremen, Germany), and the quantification of phytohormones was based on a calibration curve using original SA, JA and ABA standards.
flg22-induced callose deposition
Fourteen day old A. thaliana seedlings were grown on 1⁄2 MS agar plates and then transferred to 1⁄2 MS liquid medium in 12 wells plate before treating with water as control or 1 µM flg22 as a PAMP for 24 h. Then seedlings were fixed in acetic acid: ethanol (1:3 v/v) over night, rehydrated with ethanol (50% v/v) for 1 h, ethanol (30% v/v) for 1 h and then twice with sterilized H2O. Cleared seedlings were stained with 0.01% aniline blue dissolved in 150 mM K2HPO4. Stained leaves were mounted using 50% glycerol and imaged under a UV microscope (Nikon). Callose deposits were estimated after processing the images by Photoshop and ImageJ software (http://rsb.info.nih.gov/ij/). Six seedlings were analyzed for each treatment.
Isolation of RNA from Plant Tissue
50–100 mg of A. thaliana plant leaves or 14 days old seedling grown on ½ MS plates were taken in 2 ml tubes containing 2 steel beads, frozen in liquid Nitrogen and homogenized 2X using a Tissue Lyser (Eppendorf) for 1 min at 20 Hz to obtain a fine powder. NucleoSpin® Plant RNA kit (Macherey Nagel, 7740949) was used to extract the total RNA from the plant. The procedure was performed according to the manufacturer instructions. The final RNA was eluted in 60 µl of RNAse free water. Finally, the RNA was quantified using Nanodrop and the to assess the purity, the ratio of A260/A280 > 2.0 was considered. The RNA was flash frozen in liquid Nitrogen and stored at -80 oC to prevent degradation.
cDNA synthesis and qRT-PCR analysis
Reverse transcription was performed using SuperScript™ III First-Strand Synthesis SuperMix (Invitrogen 18080400). The amount of starting material can vary from 0.1pg to 5µg of total RNA and the procedure was performed according to the manufacturer’s protocols. The normalized complementary DNA (cDNA) was used for qRT-PCR reactions. qRT-PCR was carried out on the CFX96/CFX384 real-time PCR machine (Bio-Rad) using SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad,172–5270) with the following parameters: 50oC for 2 min, 95oC for 10 min, 39 cycles at (95oC for 10 sec, 60oC for 40 sec) and 65°C for 30 sec to obtain the melting curve. The qRT-PCR was performed using gene-specific primers listed in the Supplementary Table S3, with A. thaliana Actin (AT3g18780) and ubiquitin (At4g05320) as internal references for normalization. The resulting data was then analyzed using CFX software and values were normalized to WT. Relative expression levels of genes of interest were calculated using the 2 − ΔΔCT method. qRT-PCR experiments were repeated in three independent biological replicates, each with three technical replicates.
RNA-seq
mRNA libraries were prepared using the illumina Truseq Stranded mRNA Sample Preparation LS (low sample) kit following the manufacturer’s protocol. Briefly, the mRNA was purified from 1ug of total RNA using poly-T oligo-attached magnetic beads then purified, fragmented and primed. Subsequently, both first and second strands of cDNA were synthesized using SuperScript II reverse transcriptase, followed by adenylation on the 3’ ends and ligation of adapters. The DNA fragments with adapters were amplified by 15 PCR cycles. Finally, the libraries were validated using the 1000 DNA kit on 2100 Bioanalyzer (Agilent Technologies), quantified using qubit (Life Technologies) then barcoded libraries were normalized and pooled in equal volumes. Stranded mRNA libraries were sequenced on an Illumina Hiseq 4000 system using paired-end method. The length of the read was around 150 bp. The RNA-seq was done on three independent biological replicates.
Approximately 40 million reads were obtained for each sample. Reads were quality checked using FASTQC v0.11.585. Adapters and reads with low sequencing quality were filtered using Trimmomatic 0.3686, retaining first 100 bps and by using other default settings for paired-end sequences. The trimmed reads were then aligned to the A. thaliana reference genome (TAIR10) using Tophat v2.1.187–89 with –N 2 –g 1. The annotation file was provided as reference for reads alignment. MultibamSummary from deepTools2 package90 was used on the bam files derived from the previous step, to check for the correlation between the replicates. Summary of read counts at gene level was calculated using feature Counts v1.5.191. Cufflinks v.2.2.189 was used to calculate the FPKM values for individual replicates and CuffDiff v2.2.1 with quartile normalization to find the significant differential gene expression89. Genes with 2-fold change and P value < = 0.05 were considered as significantly different between samples with and without treatment. Hierarchical clustering of these genes was performed using Mev v4.8.192. GO term enrichment in each gene list was carried out using AgriGO93 with a cutoff for significant enrichment is P value < 0.01 and calculation false discovery rate < 0.5.
Dual-luciferase transient expression analysis in tobacco leaves
The transcriptional repression activity of DEK2 was assayed using a dual-luciferase transient expression system in tobacco (Nicotiana benthamiana) leaves as described94. Briefly, pMYC2 was fused to a Firefly Luciferase reporter gene, the regulator/effector (DEK2 and TCP20 as positive control) were fused to RFP and expressed under a CaMV 35S promoter. The expression of RFP was used to confirm the expression of the regulator/effector. Renilla-Luciferase expressed under the CaMV 35S promoter was used to normalize the transformation efficiency. The reporter and effector constructs were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. The transformed Agrobacterium cells were injected into the abaxial side of 4-week-old tobacco leaves using a 1 ml syringe. After 48 hours, D-luciferin substrate was sprayed on the leaves and the reporter gene activity was measured using a CCD Luminescence camera and images acquired.
Recombinant protein expression in E. coli and purification
10ml of an overnight culture of E. coli BL21-AI or Rosetta harboring His6-tagged, His6-MBP-tagged, GST-tagged proteins or constitutively active MAPKs were diluted in 500 mL LB medium. The culture was grown at 37°C until the OD600 reached 0.8, and then induced with L-Arabinose (Sigma, A3256) for BL21-AI cells or 0.5 mM IPTG (Invitrogen 15529-019) for Rosetta cells at 20°C and incubated overnight. Cells were harvested by centrifugation at 4000×g at 4°C for 15 min. The bacterial pellet was resuspended in 40 ml of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10mM NaCl, pH 8) containing a protease inhibitor cocktail (Complete mini EDTA-free, Roche, 4115449). The cells were lysed by treatment with 2 mg/ml of lysozyme (Thermo scientific, PI89833) for 1 hour on ice, followed by sonication (Branson Digital Sonifier 250–450, runtime 3 min, amplitude 20%, pulse on 2 sec, pulse off 1 sec). The cell lysate was then centrifuged at 20,000xg at 4°C and the supernatant was purified using glutathione sepharose 4B beads (GE Healthcare,17-0756-01) for purification of GST-tagged proteins or Ni2+-NTA beads (Invitrogen, R901-15) for the purification of His-tagged proteins. The proteins were purified according to the manufacturer’s protocol. The eluted protein was desalted on PD-10 Desalting columns (GE Healthcare, 17-0851-01). Laemmli 2X protein loading dye (0.5M Tris-HCl pH 6.8, 20% Glycerol, 4% SDS, 2% β-Mercaptoethanol and 0.01% bromophenol blue in distilled water) was added to 20ul of proteins and heated for 10 min at 95°C before analysis on SDS PAGE.
SDS PAGE and Western blotting
The denatured proteins were resolved on a 10% SDS–polyacrylamide gel for 1h at 100V. PageRuler™ Prestained protein marker (Thermo Scientific, 26616) was used as a size standard in SDS-PAGE and western blotting. The gel was stained with Coomassie SimplyBlue Safe Stain (Life Technologies, LC6065).
For Western blot analysis, proteins were transferred from a gel to an ethanol-activated PVDF membranes (GE Healthcare) in a transfer apparatus for 1 h at 100 V. Subsequently, blots were blocked in 5% skimmed milk in Tris buffered saline with tween 20 (1X TBST) (20mM Tris HCL pH7.5, 150mM NaCl, 0.1% tween 20) for 1 hour followed by incubation with the appropriate primary antibody in 2% skimmed milk on a shaker at 4°C overnight. Blots were then washed three times in 1X TBST and appropriate HRP-conjugated secondary anti-rabbit or anti-mouse antibodies were added. Blots were washed again three times in TBST. The blot was visualized using chemiluminescence (ECl prime detection reagent (GE Healthcare, RPN2232) on an imaging system (ChemiDoc MP Bio-Rad). Equal loading was verified by staining the blot with Ponceau S solution (Sigma; P7170).
Chromatin immuno-precipitation (ChIP)
ChIP seq was performed as described in 47. Briefly, ChIP was performed on 14-day-old seedlings (grown in short day condition, in ½ MS agar plates) using anti-GFP (Santa Cruz). ChIP was performed as previously described95. Briefly, plant materials were cross-linked with 1%(v/v) formaldehyde under vacuum. Chromatin was isolated and fragmented by sonication (30 sec on/off pulses, at high intensity for 60 min) using a water bath Bioruptor UCD-200 (Diagenode, Liège, Belgium) Later, the sonicated protein/DNA was incubated with antibodies (overnight at 4°C with gentle shaking) and then incubated with 50 µL of Dynabeads Protein A (Invitrogen, Ref. 100-02D) for 1h at 4°C. Immunoprecipitated DNA was then recovered by dissociating the complexes using the IPure kit (Diagenode, Liège, Belgium) and analyzed by RTqPCR. An aliquot of untreated sonicated chromatin was kept as a total input DNA control.
For ChIP-seq, libraries were prepared and sequenced as described in 96. ChIP-Seq reads were aligned to A.thaliana genome TAIR10 by Bowtie97 v0.12.7 on A. thaliana genome TAIR10. MACS (Model-based Analysis of ChIP-Seq) was used to identify peaks using a q-value cutoff of 0.05 (http://liulab.dfci.harvard.edu/MACS/)79 while GPAT was used for gene annotation and peak distribution relative to annotated A. thaliana transcription start site (http://bips.u-strasbg.fr/GPAT/Gpat_home.html).
Microscale thermophoresis (MST)
Fluorophore-labeled protein was used for the quantitative binding assay using microscale thermophoresis. His6-MBP-DEK2 and His6-MBP (used as control) protein were labeled using Monolith His-tag labeling kit RED-tris-NTA (nanotemper; MO-L008) following the manufacturer's protocol. Briefly, 400 nM of the protein of interest was incubated with 100 nM of the His-tag labeling dye in MST buffer (20 mM HEPES; pH 7.9, 150 mM NaCl, 0.05% Tween-20) for 30 min at room temperature.
The fluorophore-labeled protein was titrated with differentially modified histone H3 peptides (H3K4me1/3, H3K9me1/3, H3K27me1/3). The data points obtained from three different measurements were fitted using the following equation derived from law of mass action to get the dissociation constant:
[BL]/[B0] = [([L0]+[B0]+Kd)−√{(([L0]+[B0]+Kd)2−4⋅[L0]⋅[B0])]/ 2[B0]}
K d is the dissociation constant, [B0] is the total concentration of the binding sites. [L0] stands for the amount of added ligand at each data point and [BL] is the concentration of formed complexes between the binding sites, [B], and the ligand, [L].
Chromatin states analysis DEK2
The chromatin state analysis was based on the topology established by Sequeira-Mendes et al.37. The genomic coordinates of the chromatin states were overlapped with the DEK2 peaks identified as previously described. A DEK2 binding site was considered to be in a certain chromatin state if it overlapped with a domain of that state for at least 150bp. This was tested using the Genomic Ranges package (version 1.44.0)98. The control sets were generated by shifting the coordinates of the DEK2 binding site up- or downstream by the indicated number of base pairs. The significance was tested by a permutation test using the peakPermTest() function from the ChIPpeakAnno package (version 3.26.0)99 with 1000 permutations.
For the DEK2 target genes analysis, the DEK2 targets identified as described previously were divided into 3 sets depending on whether they were up- or downregulated or did not present a change in expression in the dek2 mutant. A gene was considered as being in a certain state if at least 150 bp of its gene body overlapped with said state. For each chromatin state, the lists of DEK2 targets were overlapped with the list of genes containing a domain of at least 150bp of said state within its gene body, using the genomic coordinates provided by Sequeira-Mendes et al.37. The proportion of genes of interest in that chromatin state was compared to the proportion of all nuclear genes in said state and the significance of the difference betwee the two proportions was examined using the Marascuilo procedure with a confidence level of 0.95. This procedure was performed using R code adapted from the NIST/SEMATECH tutorial (NIST/SEMATECH e-Handbook of Statistical Methods, Section 7.4.7.4, http://www.itl.nist.gov/div898/handbook/).