Direct ETTIN-auxin interaction controls chromatin states in gynoecium development
- Department of Crop Genetics, John Innes Centre, Norwich Research Park, United Kingdom
- Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, United Kingdom
- Institute of Science and Technology, Austria
Figures
Figure 1 with 2 supplements
ETT directly binds auxin (IAA).
(a) DARTS assay suggests that ETT binds IAA. Seedlings from an inducible two-component ETT-FLAG line were used for the protein isolation. Samples were treated with DMSO (mock), IAA and BA and …
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Figure 1—source data 1
Parameters for HSQC NMR.
- https://cdn.elifesciences.org/articles/51787/elife-51787-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Original western blot images for DARTS assays.
The red boxes indicate the regions used in Figure 1a.
Figure 1—figure supplement 2
Parameters for HSQC NMR experiment.
Figure 2 with 1 supplement
ETT regulates target gene expression independently of TIR1/AFB auxin receptors.
Expression of the canonical auxin responsive IAA19 gene (a) and the ETT-target genes HEC1 (b) and PID (c) in control-treated (dark grey) or 100 µM IAA-treated (light grey) gynoecia assayed using …
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Figure 2—source data 1
Oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/51787/elife-51787-fig2-data1-v2.xlsx
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Figure 2—source data 2
Output of statistical tests.
- https://cdn.elifesciences.org/articles/51787/elife-51787-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Expression of HEC1 and PID in Col-0 and ett-3.
In wild-type gynoecia HEC1 and PID are up-regulated upon auxin treatment while both genes are constitutively up-regulated in ett-3. Treatment with 100 µM IAA does not affect HEC1 and PID expression …
Figure 3 with 3 supplements
ETT interacts with members of the TPL/TPR co-repressor family in an auxin-sensitive manner.
(a) Schematic representation of full-length ETT protein highlighting an EAR-like motif and tryptophan in position 505 (W) in the C-terminal ETT-specific domain. DBD, DNA-binding domain. (b) Y2H …
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Figure 3—source data 1
Output of statistical tests.
- https://cdn.elifesciences.org/articles/51787/elife-51787-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
ETT can interact with several members of the TPL/TPR co-repressor family through a conserved EAR-like motif.
(a) Alignment of ETT protein sequences of 22 species identified a conserved repressive motif (RLFGF) at its C-terminal domain. A logo of the sequence surrounding the conserved W505 is also shown. (b)…
Figure 3—figure supplement 2
Interaction between ETT and TPL, TPR2 and TPR4 is auxin-sensitive and specific to IAA.
(a) In Y2H increasing concentrations of IAA lead to reduction of yeast growth abolishing the interaction between ETT and its partners. The interactions are, therefore, auxin-sensitive. (b) Y2H to …
Figure 3—figure supplement 3
Original western blot images for Co-immunoprecipitation assays.
The red boxes indicate the regions used in Figure 3c,d and Figure 3—figure supplement 2d.
Figure 4 with 1 supplement
ETT, TPL/TPR2 and HDA19 co-operatively regulate gene expression to facilitate gynoecium development.
(a–d) Promoter GUS expression analysis of pETT:GUS (a), pTPL:GUS (b), pTPR2:GUS (c) and pTPR4:GUS (d) revealed that ETT, TPL and TPR2 but not TPR4 are co-expressed in the Arabidopsis style. Scale …
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Figure 4—source data 1
Output of statistical tests.
- https://cdn.elifesciences.org/articles/51787/elife-51787-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Expression of TPL, TPRs and HDAs genes in the gynoecium.
Expression analysis using qRT-PCR in wild-type gynoecia showed that TPL and TPR2 are more strongly expressed than TPR1,3 and 4. Likewise, HDA19 exhibits higher expression compared to HDA6. …
Figure 5
ETT, TPL and HDA19 co-operatively regulate HEC1 and PID by modulating chromatin acetylation.
(a–c) Chromatin immunoprecipitation (ChIP) shows ETT (a), TPL (b) and HDA19 (c) binding to conserved regions of HEC1 and PID loci in the absence of IAA (dark grey columns). In the presence of IAA …
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Figure 5—source data 1
Output of statistical tests.
- https://cdn.elifesciences.org/articles/51787/elife-51787-fig5-data1-v2.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information | ||||
|---|---|---|---|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | ETTIN (ETT) | The Arabidopsis Information Resource | AT2G33860 | |||||
| Gene (Arabidopsis thaliana) | TOPLESS (TPL) | The Arabidopsis Information Resource | AT1G15750 | |||||
| Gene (Arabidopsis thaliana) | TOPLESS- RELATED 1 (TPR1) | The Arabidopsis Information Resource | AT1G80490 | |||||
| Gene (Arabidopsis thaliana) | TOPLESS-RELATED 2 (TPR2) | The Arabidopsis Information Resource | AT3G16830 | |||||
| Gene (Arabidopsis thaliana) | TOPLESS-RELATED 3 (TPR3) | The Arabidopsis Information Resource | AT5G27030 | |||||
| Gene (Arabidopsis thaliana) | TOPLESS-RELATED 4 (TPR4) | The Arabidopsis Information Resource | AT3G15880 | |||||
| Gene (Arabidopsis thaliana) | HISTONE DEACETYLASE 6 (HDA6) | The Arabidopsis Information Resource | AT5G63110 | |||||
| Gene (Arabidopsis thaliana) | HISTONE DEACETYLASE 19 (HDA19) | The Arabidopsis Information Resource | AT4G38130 | |||||
| Gene (Arabidopsis thaliana) | HECATE 1 (HEC1) | The Arabidopsis Information Resource | AT5G67060 | |||||
| Gene (Arabidopsis thaliana) | PINOID (PID) | The Arabidopsis Information Resource | AT2G34650 | |||||
| Genetic reagent Arabidopsis thaliana | Col-0 | widely distributed | ||||||
| Genetic reagent Arabidopsis thaliana | hda19-4 | Kim et al., 2008 | SALK_13944 | |||||
| Genetic reagent Arabidopsis thaliana | tir1-1 afb2-3 afb3-4 | Parry et al., 2009 | ||||||
| Genetic reagent Arabidopsis thaliana | tpl | European Arabidopsis Stock Centre | SALK_034518C | |||||
| Genetic reagent Arabidopsis thaliana | ett-3 | Simonini et al., 2017; Sessions et al., 1997 | AT2G33860 | |||||
| Genetic reagent Arabidopsis thaliana | pETT:GUS | Ng et al., 2009 | AT2G33860 | |||||
| Genetic reagent Arabidopsis thaliana | pETT:ETT-GFP | Simonini et al., 2016 | AT2G33860 | |||||
| Genetic reagent Arabidopsis thaliana | pTPL:TPL-GFP | Pi et al., 2015 | AT1G15750 | |||||
| Genetic reagent Arabidopsis thaliana | p35S:HDA19-GFP | Pi et al., 2015 | AT4G38130 | |||||
| Genetic reagent Arabidopsis thaliana | pTIR1:ccvTIR1 | Szemenyei et al., 2008 | ||||||
| Genetic reagent Arabidopsis thaliana | tpl tpr2ge | this paper | AT1G15750, AT3G16830 | Further details in the Materials and methods section | ||||
| Genetic reagent Arabidopsis thaliana | pTPL:GUS | this paper | AT1G15750 | Further details in the Materials and methods section | ||||
| Genetic reagent Arabidopsis thaliana | pTPR2:GUS | this paper | AT3G16830 | Further details in the Materials and methods section | ||||
| Genetic reagent Arabidopsis thaliana | pTPR4:GUS | this paper | AT3G15880 | Further details in the Materials and methods section | ||||
| Genetic reagent Arabidopsis thaliana | p35S:CDGVG p6xGAL4UAS:ETT-FLAG | this paper | Further details in the Materials and methods section | |||||
| Antibody | Mouse anti-FLAG | Abcam | ab49763 | monoclonal, conjugated with HRP | ||||
| Antibody | Mouse anti-HA | Abcam | ab173826 | monoclonal, conjugated with HRP | ||||
| Antibody | Mouse anti-GFP | Roche | 11814460001 | monoclonal | ||||
| Antibody | Rabbit anti-H3K27ac | Abcam | ab4729 | polyclonal | ||||
| Antibody | Rabbit anti-H3 | Abcam | ab1791 | polyclonal | ||||
| Recombinant DNA reagent | pGWB14; p35s:HA-TPL | Espinosa-Ruiz et al., 2017 | ||||||
| Recombinant DNA reagent | pICH47732; p35s:ETT-3xFLAG | this paper | Backbone Addgene #48000 | |||||
| Recombinant DNA reagent | pICH47732; p35s: ETTW505A-3xFLAG | this paper | Backbone Addgene #48000 | |||||
| Recombinant DNA reagent | pGADT7; ETT | this paper | Further details in the Materials and methods section | |||||
| Recombinant DNA reagent | pGADT7; ETTW505A | this paper | Further details in the Materials and methods section | |||||
| Recombinant DNA reagent | pGADT7; ETTL552S; F553S | this paper | Further details in the Materials and methods section | |||||
| Recombinant DNA reagent | pGBKT7; TPL | Causier et al., 2012 | ||||||
| Recombinant DNA reagent | pGBKT7; TPR1 | Causier et al., 2012 | ||||||
| Recombinant DNA reagent | pGBKT7; TPR2 | Causier et al., 2012 | ||||||
| Recombinant DNA reagent | pGBKT7; TPR3 | Causier et al., 2012 | ||||||
| Recombinant DNA reagent | pGBKT7; TPR4 | Causier et al., 2012 | ||||||
| Recombinant DNA reagent | pESPRIT; ES388-594 | Simonini et al., 2018 | ||||||
