Direct ETTIN-auxin interaction controls chromatin states in gynoecium development

  1. André Kuhn
  2. Sigurd Ramans Harborough
  3. Heather M McLaughlin
  4. Bhavani Natarajan
  5. Inge Verstraeten
  6. Jiří Friml
  7. Stefan Kepinski
  8. Lars Østergaard  Is a corresponding author
  1. Department of Crop Genetics, John Innes Centre, Norwich Research Park, United Kingdom
  2. Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, United Kingdom
  3. Institute of Science and Technology, Austria
5 figures, 1 table and 1 additional file

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 …

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 …

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 …

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 …

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. …

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 …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene (Arabidopsis thaliana)ETTIN (ETT)The Arabidopsis Information ResourceAT2G33860
Gene (Arabidopsis thaliana)TOPLESS
(TPL)
The Arabidopsis Information ResourceAT1G15750
Gene (Arabidopsis thaliana)TOPLESS- RELATED 1 (TPR1)The Arabidopsis Information ResourceAT1G80490
Gene
(Arabidopsis thaliana)
TOPLESS-RELATED 2 (TPR2)The Arabidopsis Information ResourceAT3G16830
Gene (Arabidopsis thaliana)TOPLESS-RELATED 3 (TPR3)The Arabidopsis Information ResourceAT5G27030
Gene (Arabidopsis thaliana)TOPLESS-RELATED 4 (TPR4)The Arabidopsis Information ResourceAT3G15880
Gene (Arabidopsis thaliana)HISTONE DEACETYLASE 6
(HDA6)
The Arabidopsis Information ResourceAT5G63110
Gene (Arabidopsis thaliana)HISTONE DEACETYLASE 19
(HDA19)
The Arabidopsis Information ResourceAT4G38130
Gene (Arabidopsis thaliana)HECATE 1
(HEC1)
The Arabidopsis Information ResourceAT5G67060
Gene (Arabidopsis thaliana)PINOID (PID)The Arabidopsis Information ResourceAT2G34650
Genetic reagent Arabidopsis thalianaCol-0widely distributed
Genetic reagent Arabidopsis thalianahda19-4Kim et al., 2008SALK_13944
Genetic reagent Arabidopsis thalianatir1-1 afb2-3 afb3-4Parry et al., 2009
Genetic reagent Arabidopsis thalianatplEuropean Arabidopsis Stock CentreSALK_034518C
Genetic reagent Arabidopsis thalianaett-3Simonini et al., 2017Sessions et al., 1997AT2G33860
Genetic reagent Arabidopsis thalianapETT:GUSNg et al., 2009AT2G33860
Genetic reagent Arabidopsis thalianapETT:ETT-GFPSimonini et al., 2016AT2G33860
Genetic reagent Arabidopsis thaliana pTPL:TPL-GFPPi et al., 2015AT1G15750
Genetic reagent Arabidopsis thalianap35S:HDA19-GFPPi et al., 2015AT4G38130
Genetic reagent Arabidopsis thalianapTIR1:ccvTIR1Szemenyei et al., 2008
Genetic reagent Arabidopsis thaliana tpl tpr2gethis paperAT1G15750, AT3G16830Further details in the Materials and methods section
Genetic reagent Arabidopsis thalianapTPL:GUSthis paperAT1G15750Further details in the Materials and methods section
Genetic reagent Arabidopsis thalianapTPR2:GUSthis paperAT3G16830Further details in the Materials and methods section
Genetic reagent Arabidopsis thalianapTPR4:GUSthis paperAT3G15880Further details in the Materials and methods section
Genetic reagent Arabidopsis thalianap35S:CDGVG p6xGAL4UAS:ETT-FLAGthis paperFurther details in the Materials and methods section
AntibodyMouse anti-FLAGAbcamab49763monoclonal, conjugated with HRP
AntibodyMouse anti-HAAbcamab173826monoclonal, conjugated with HRP
AntibodyMouse anti-GFPRoche11814460001monoclonal
AntibodyRabbit anti-H3K27acAbcamab4729polyclonal
AntibodyRabbit anti-H3Abcamab1791polyclonal
Recombinant DNA reagentpGWB14; p35s:HA-TPLEspinosa-Ruiz et al., 2017
Recombinant DNA reagentpICH47732; p35s:ETT-3xFLAGthis paperBackbone Addgene
#48000
Recombinant DNA reagentpICH47732; p35s:
ETTW505A-3xFLAG
this paperBackbone Addgene
#48000
Recombinant DNA reagentpGADT7; ETTthis paperFurther details in the Materials and methods section
Recombinant DNA reagentpGADT7; ETTW505Athis paperFurther details in the Materials and methods section
Recombinant DNA reagentpGADT7; ETTL552S; F553Sthis paperFurther details in the Materials and methods section
Recombinant DNA reagentpGBKT7; TPLCausier et al., 2012
Recombinant DNA reagentpGBKT7; TPR1Causier et al., 2012
Recombinant DNA reagentpGBKT7; TPR2Causier et al., 2012
Recombinant DNA reagentpGBKT7; TPR3Causier et al., 2012
Recombinant DNA reagentpGBKT7; TPR4Causier et al., 2012
Recombinant DNA reagentpESPRIT; ES388-594Simonini et al., 2018

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