The Arabidopsis active demethylase ROS1 cis-regulates defence genes by erasing DNA methylation at promoter-regulatory regions
- Institut de Biologie de l’Ecole Normale Supérieure (IBENS), Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Université de recherche Paris, Sciences & Lettres (PSL), France
Figures
Figure 1
The enhanced Pto DC3000 disease susceptibility observed in ros1-infected mutants is mainly dependent on DCL2 and/or DCL3 functions.
(A) Increased Pto DC3000 vascular propagation in the two independent ros1 mutant alleles, ros1-3 and ros1-4. Secondary veins of 5-week-old Col-0, ros1-3 and ros1-4 mutants were inoculated with a …
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Figure 1—source data 1
Original data of bacterial propagation assays for Figure 1A,C,D, and F.
- https://cdn.elifesciences.org/articles/62994/elife-62994-fig1-data1-v2.xlsx
Figure 2 with 3 supplements
The TNL gene RMG1 contributes to apoplastic and vascular resistance against Pto DC3000 and its flg22-triggered induction is negatively regulated by RNA-directed DNA methylation (RdDM) in the absence of ROS1.
(A) RMG1 mRNA levels in Col-0 and the two rmg1 mutant alleles, namely rmg1-1 and rmg1-2, were monitored by RT-qPCR at 6 hr after syringe-infiltration of mock (water) or 1 μM of flg22 peptide. (B) RMG…
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Figure 2—source data 1
Original qRT-PCR data for Figure 2A and F, and bacterial propagation data for Figure 2B and C.
- https://cdn.elifesciences.org/articles/62994/elife-62994-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Scheme representing T-DNA insertion sites at the RMG1 locus.
The T-DNA insertions SALK_007034 (rmg1-1 allele) and SALK_023944 (rmg1-2 allele) are located in the second exon and in the first intron of RMG1 (AT4G11170), respectively.
Figure 2—figure supplement 2
FRK1 and bZIP60 promoters are not hypermethylated in ros1-3 mutants.
IGV snapshots showing mRNA levels (mRNA-seq) after syringe-infiltration of mock (water) or 1 μM of flg22 peptide in 5-week-old rosette leaves of Col-0 and ros1-3, and cytosine methylation levels …
Figure 2—figure supplement 3
The flg22-triggered induction of RMG1 and RBA1 is restored, while RLP43 remains in a repressed state, in the ros1dcl23-elicited mutant.
Second biological replicate of RT-qPCR analyses depicting RMG1, FRK1, RLP43, and RBA1 mRNA levels in 5-week-old rosette leaves of Col-0, ros1-3, dcl23, and ros1dcl23 treated with either mock (water) …
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Figure 2—figure supplement 3—source data 1
Original qRT-PCR data for Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/62994/elife-62994-fig2-figsupp3-data1-v2.xlsx
Figure 3 with 3 supplements
Genome-wide identification of flg22-responsive ROS1 targets and characterisation of the role of RNA-directed DNA methylation (RdDM) in the methylation status of these genes in the absence of ROS1.
(A) Proportion of flg22-responsive genes that are regulated by ROS1. One hundred and two flg22-responsive genes that are ‘less-induced’ and 115 flg22-responsive genes that are ‘less-repressed’ in ros…
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Figure 3—source data 1
Original qRT-PCR data for Figure 3G.
- https://cdn.elifesciences.org/articles/62994/elife-62994-fig3-data1-v2.xlsx
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Figure 3—source data 2
Original transcriptomic data used for the heatmap presented in Figure 3B.
- https://cdn.elifesciences.org/articles/62994/elife-62994-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
RNA sequencing experiment in 5-week-old rosette leaves of Col-0 and ros1-3 syringe-infiltrated with either mock or flg22 for 6 hr.
(A) Heatmap depicting the relative expression of the 102 less-induced genes in Col-0 and ros1-3 mutants upon 6 hr of mock or flg22 treatments. AGI numbers and gene names are shown for all individual …
Figure 3—figure supplement 2
Analysis of 23–24 nt siRNA levels for the groups of 102 less-induced genes and 2943 genes carrying hyperDMRs in the ros1-3 mutant background.
(A) Heatmaps depicting 23nt- and 24nt-long siRNA levels at the 102 less-induced genes (left panel) and at the 2943 genes presenting hyperDMR in ros1-3 (right panel) on replicate 1. Global siRNA …
Figure 3—figure supplement 3
Gain of 21-nt siRNAs and P4RNAs at the RLP43 promoter in the ros1dcl23 background might contribute to the maintenance of DNA methylation levels in this triple mutant.
(A) Snapshot representing levels of DNA methylation and siRNA species classified per size at the RLP43 promoter, in 5-week-old rosette leaves of untreated Col-0, ros1-3, and ros1dcl23. (B) Snapshot …
Figure 4 with 5 supplements
Several WRKY transcription factors bind to the demethylated region of the RLP43 promoter, which contains a functional and flg22-responsive W-box cis-element.
(A) A subgroup of the ROS1 targets exhibits an over-representation of WRKY DNA binding at the promoter regions corresponding to the hyperDMRs that were retrieved in ros1-3. GAT analysis performed on …
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Figure 4—source data 1
Original qRT-PCR data of GUS transcript quantification for Figure 4E.
- https://cdn.elifesciences.org/articles/62994/elife-62994-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
GAT analysis of all the transcription factor classes binding to the 26 stringent less-induced ROS1 target promoters.
(A) GAT analysis performed on publicly available DNA affinity purification sequencing (DAP-seq) data (O'Malley et al., 2016) for WRKY TFs at the 26 stringent ROS1 primary targets. This analysis was …
Figure 4—figure supplement 2
GAT analysis of all the transcription factor classes binding to the 28 stringent less-repressed ROS1 target promoters.
(A) GAT analysis performed on publicly available DNA affinity purification sequencing (DAP-seq) data (O'Malley et al., 2016) for WRKY TFs at the 28 stringent less-repressed ROS1 targets, from which …
Figure 4—figure supplement 3
WRKY DNA binding peaks are present at the RMG1 promoter region that is subjected to ROS1-directed demethylation.
Snapshots representing, from top to bottom, RNA-seq, BS-seq, and DNA affinity purification sequencing (DAP-seq) data sets at the RMG1 locus (left panel) with an enlargement surrounding the hyperDMR …
Figure 4—figure supplement 4
Position of WRKY DNA binding peaks at the 11 flg22-induced ROS1 targets.
Snapshots depicting RNA-seq (rep1), BS-seq (rep1 and rep2), and the position of WRKY-binding sites observed in the DNA affinity purification sequencing (DAP-seq) data (O'Malley et al., 2016) in …
Figure 4—figure supplement 5
Flg22-triggered chromatin association of WRKY18-HA and WRKY40-HA at the promoters of five less-induced ROS1 targets and at the promoter of the positive control bZIP60.
Snapshots representing mRNA-seq and BS-seq of Col-0 and ros1 untreated rosette leaves and ChIP-seq data for wrky18 WRKY18-HA and wrky40 WRKY40-HA seedlings subjected to either mock (medium without …
Figure 5 with 1 supplement
ROS1-directed demethylation is crucial for the DNA binding of WRKY18 and WRKY40 at the RLP43 promoter.
(A) DAP-seq data from O'Malley et al., 2016 showing that WRKY family members are generally sensitive to DNA methylation. Graph representing ratio of binding capacity in DAP versus ampDAP data at …
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Figure 5—source data 1
Original DAP-qPCR data for Figure 5B.
- https://cdn.elifesciences.org/articles/62994/elife-62994-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
ROS1-directed demethylation is crucial for the binding of WRKY18 and WRKY40 at the RLP43 promoter.
The ability of WRKY18 and WRKY40 to bind DNA corresponding to the demethylated region of the RLP43 promoter is abolished in the ros1-3 mutant background. Second biological replicate of the DAP-qPCR …
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Figure 5—figure supplement 1—source data 1
Original DAP-qPCR data for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/62994/elife-62994-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 1 supplement
The artificial siRNA-directed targeting of remethylation at the RMG1 and RLP43 promoters impairs the flg22-triggered inducibility of these genes and enhances susceptibility towards Pto DC3000.
(A) Scheme depicting the chimeric inverted repeat (IR) construct designed to simultaneously direct DNA remethylation at the RMG1 and RLP43 promoter regions. The IR-RMG1p/RLP43p contains the …
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Figure 6—source data 1
Original McrBC-qPCR, qRT-PCR, and bacterial propagation data for Figure 6B–E.
- https://cdn.elifesciences.org/articles/62994/elife-62994-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Artificial siRNA-directed remethylation of RMG1 and RLP43 promoters in the Col-0 background limits flg22-triggered induction of these genes.
(A) Accumulation of siRNAs at RLP43 and RMG1 promoters in IR-RMG1p/RLP43p lines was detected by low molecular weight northern blot analysis. U6 was used as a loading control. (B) Result from another …
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Figure 6—figure supplement 1—source data 1
Original qRT-PCR data for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/62994/elife-62994-fig6-figsupp1-data1-v2.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | ROS1 | Arabidopsis.org | AT2G36490 | |
| Gene (Arabidopsis thaliana) | DCL2 | Arabidopsis.org | AT3G03300 | |
| Gene (Arabidopsis thaliana) | DCL3 | Arabidopsis.org | AT3G43920 | |
| Gene (Arabidopsis thaliana) | RMG1 | Arabidopsis.org | AT4G11170 | |
| Gene (Arabidopsis thaliana) | RLP43 | Arabidopsis.org | AT3G28890 | |
| Strain, strain background (Pseudomonas syringae pv. tomato) | Pto DC3000-GFP | Gift from Pr. Sheng Yang He (Duke University, US). | ||
| Genetic reagent (Arabidopsis thaliana) | ros1-3 | Penterman et al., 2007 | ||
| Genetic reagent (Arabidopsis thaliana) | ros1-4 | Nottingham Arabidopsis stock center (NASC) | N682295 | |
| Genetic reagent (Arabidopsis thaliana) | dcl2-1 dcl3-1 | Xie et al., 2004 | ||
| Genetic reagent (Arabidopsis thaliana) | ros1-3 dcl2-1 dcl3-1 | This study | ||
| Genetic reagent (Arabidopsis thaliana) | rmg1-1 | Nottingham Arabidopsis stock center (NASC) | N678063 | |
| Genetic reagent (Arabidopsis thaliana) | rmg1-2 | Nottingham Arabidopsis stock center (NASC) | N674117 | |
| Commercial assay or kit | MagneGST Pull-Down System | Promega | V8870 | |
| Commercial assay or kit | McrBC | New England Biolab (NEB) | M0272 |
Additional files
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Supplementary file 1
DMRs position of 102 less-induced and 115 less-repressed ROS1 targets.
- https://cdn.elifesciences.org/articles/62994/elife-62994-supp1-v2.xlsx
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Supplementary file 2
Primers used in this study.
- https://cdn.elifesciences.org/articles/62994/elife-62994-supp2-v2.jpg
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62994/elife-62994-transrepform-v2.docx
