In this work, we address the hierarchical function of the ECF σ factor paralogs EcfG1 and EcfG2 in triggering the GSR in Sphingopyxis granuli TFA and describe the role of EcfG2 as global switch of this response. In addition, we define a GSR regulon for TFA and use in vitro transcription analysis to study the relative contribution of each EcfG paralog to the expression of selected genes. We show that the features of each promoter ultimately dictate this contribution, though EcfG2 always produced more transcripts than EcfG1 regardless of the promoter. These first steps in the characterisation of the GSR in TFA suggest a tight regulation to orchestrate an adequate protective response in order to survive in conditions otherwise lethal.Under ever-changing environmental conditions, the General Stress Response (GSR) represents a lifesaver for bacteria in order to withstand hostile situations. In  One direct way to alter the transcription is the swapping of σ factors, small switchable subunits of the RNA polymerase (RNAP) whose main functions are the promoter recognition and the double strand unwinding to form the transcription bubble2. The general classification of σ factors comprises two main groups: the σ70 and the σ54 families. Among all of them, the vegetative σ70 factor, responsible for transcribing the bulk of bacterial genes, is the only essential representative3. The σ70 family can be subdivided into four groups based on their domain architecture. This include the σ70 housekeeping Group I, and the non-essential structurally-related alternative σ factor Groups II–IV, which despite their large variation in size, all keep the σ2 and σ4 domains, responsible for binding the -10 and -35 boxes, respectively, in a promoter region3. Extracytoplasmic function σ factors (ECFs), belonging to Group IV, are the minimal expression of this family, since they are only formed by the σ2 and σ4 domains, but are also the most abundant and diverse in the bacterial world1. From a physiological point of view, they participate in regulating the transcription in response to a wide range of environmental signals4.In an environment where conditions are constantly changing, bacteria need to adapt their physiology in order to increase their chances of survival. These adaptations are not only mediated by particular responses to certain stimuli, but also by global changes in the transcriptional profile. These changes allow bacteria to thrive in conditions that otherwise would be considered hostile habitats for them. Global transcriptional adaptations can be exerted by a number of mechanisms, all of them ending up in a regulator that allows or prevents the expression of certain genesα-proteobacteria, ECFs play a central role in the regulation of the so-called General Stress Response (GSR). This protective response is activated by a number of non-specific stresses, such as desiccation, osmotic shock, oxidative stress or carbon starvation among others5. Three main regulatory elements are required to control the GSR in α-proteobacteria: an EcfG-type ECF σ factor (G standing for GSR), its cognate anti-σ factor NepR and the response regulator PhyR4. In favourable growth conditions, EcfG is sequestered by NepR, thus preventing the expression of the GSR regulon. When some kind of stress appears in the environment, one or more GSR-specific histidine kinases belonging to the HWE or HisKA2 family start the signal transduction by autophosphorylation in a His residue and transfer the phosphoryl group to an Asp residue in PhyR6. This phosphorelay causes a conformational change in PhyR, so that a σ-like domain is exposed. Upon this, by a partner-switching mechanism, NepR is sequestered by the σ-like domain presented by PhyR, thus releasing EcfG to activate the expression of the GSR target genes6. This model may display a diverse variety of organizations, with different levels of complexity according to the number of PhyR, NepR and EcfG paralogs encoded in the genome10 and the presence of accessory regulators with different levels of essentiality for the activation of the GSR13.In 15, although more baroque models, such as the one for Methylobacterium extorquens, have been described10. With respect to the function and the distribution of the target genes among the different EcfG copies, there are diverse examples described, such us in Rhizobium etli, where the two EcfG proteins show some sort of functional specialisation between them by controlling different sets of genes14, or Caulobacter crescentus, in which the additional EcfG copy σU controls to some extent the expression of a small subset of genes belonging to the regulon of the main regulator σT, putatively to amplify its response16. In other models, the number of EcfG elements can be even higher, such as the case of M. extorquens, where up to six of these regulators were annotated. Nevertheless, a main regulator could be identified as EcfG110.Regarding EcfG, most of the described models only have one or two paralogsSphingopyxis granuli TFA is one of the few strains able to use the organic solvent tetralin as a carbon and energy source. Tetralin catabolism has been intensively characterised in this bacterium at biochemical, genetic and regulatory level17. Moreover, TFA is the only strain within its genus that has been reported to anaerobically grow while respiring nitrate19. Recently, its genome has been totally assembled and annotated, showing some characteristics of oligotrophic bacteria18.The α-proteobacterium In this work, we have identified in the TFA genome two paralogous genes encoding EcfG σ factors and defined their hierarchical role as regulators of the GSR. We have also identified the members of the GSR regulon in TFA using transcriptomic analyses.2 and σ4, connected by a short unstructured linker (<50 amino acids). TFA bears a total of 25 genes encoding putative ECF σ factors that might play a regulatory role in its physiology. Using the classification tool developed by Staron et al.1, which allows the analysis of the ECF by sequence similarity for further assignment to distinct functional categories, two of them were classified in the ECF15 category. This unique group is formed exclusively by EcfG-type σ factors whose main function is the activation of the GSR in α-proteobacteria, given that this taxonomic group lacks rpoS homologs20. Therefore, the corresponding genes SGRAN_1161 and SGRAN_1163 were subsequently designated as ecfG1 and ecfG2, respectively. One of the main features of the EcfG group is a conserved genomic context consisting in the EcfG-like σ factor preceded by an anti-σ factor (NepR-like proteins) and with a PhyR-like response regulator in the vicinity, which functions as the anti-anti-σ factor5. Although nepR genes are not usually automatically annotated due to their small size, the TFA genome revealed that it contains two short orfs that could actually code for NepR-like proteins and were therefore designated as nepR1 (SGRAN_0992) and nepR2 (SGRAN_1162). The one designated nepR2 is located upstream ecfG1, shares the same transcription start site (TSS)21 and actually overlaps with ecfG1 by 4 bp  was found upstream nepR2, suggesting that these genes form an operon that could be auto-regulated, an organization previously reported for other GSR regulatory σ factors in α-proteobacteria6. Divergently from this transcriptional unit, the ecfG2 gene is located, which does not bear the proposed consensus for EcfG-type promoters and does not present a cognate anti-σ factors upstream  elements present in TFA is found, completing the typical synteny conserved in α-proteobacteria, in which at least one representative of each GSR regulatory element is encoded in the same locus6. On the other hand, the second putative nepR paralog, nepR1, is located elsewhere in the genome divergently from a second phyR element .We have analysed the recently annotated TFA genome searching for candidates with a common signature of ECF σ factors: the characteristic architecture with only domains σ5. However, the relationships among the different regulators when multiple copies of them are present seem to be species-specific. To start the characterisation of the GSR cascade in TFA, we first focused on the ECF σ factors EcfG1 and EcfG2.The presence of paralogous regulatory elements in the α-proteobacterial GSR cascades has emerged as an usual feature based on the analysis of multiple of α-proteobacterial genomesα-proteobacteria is mediated by EcfG homologs, which belong to the ECF15 class. In order to evaluate whether EcfG1 and EcfG2 play a protective role in response to environmental stresses in TFA and whether they are redundant in function, ΔecfG1, ΔecfG2 and double ΔecfG1ΔecfG2 deletion mutants were constructed and their resistance to different stresses, such us, heavy metals, osmotic stress, desiccation and exposure to hydrogen peroxide, was tested. No growth differences were observed between the wild type and the ΔecfG1 mutant on agar plates in the presence of copper 3.5 mM, NaCl 600 mM  or ecfG2 upstream the lacZ gene in an integrative vector. After the introduction of the fusions in TFA, as shown in Fig. ecfG1 was observed during the exponential phase of growth, which was induced over 6-fold when stationary phase was reached. However, the expression of ecfG2 was constitutive regardless of the growth stage  in the ΔecfG1ΔecfG2 mutant compared to the wild type  distributed in 79 putative operons was defined for S. granuli TFA . Among the genes directly regulated by the GSR, some obvious targets could be found, such as most of the regulatory elements of the response , as described in the literature4. Noticeable examples of genes directly regulated by the GSR among the genes with annotated function are the stress inducible csbD (SGRAN_3063), two katA catalases (SGRAN_1770 and SGRAN_2521)4, the ecnAB toxin-antitoxin system29 (SGRAN_1922), an mscS mechanosensitive ion channel30 (SGRAN_0895), an osmC peroxiredoxin31 (SGRAN_3924), an RND efflux system component15 (SGRAN_4134) and the ku-ligD non-homologous DNA repair system (SGRAN_4136 and SGRAN_4135)32, some of them recently found GSR-dependent in the sphingomonad S. melonis13. In addition, a large representation of envelope related elements also appear in this regulon 4. However, some other elements, such as the sphingosine kinase coding gene (SGRAN_0919)33 or the putative egtB and egtD genes (SGRAN_4050 and SGRAN_4049), which may be involved in ergothioneine biosynthesis34 are reported as part of a GSR regulon for the first time.Several GSR regulons have been defined in diverse α-proteobacterial species, including fleQ (SGRAN_4107) and ctrA (SGRAN_3324), and some elements involved in chemotaxis, such as cheA (SGRAN_2956), cheB (SGRAN_2953) or cheR (SGRAN_2952)35, together with a putative bdlA biofilm dispersal regulator36. This phenomenon may imply a mechanism to escape from unfavourable situations, such as carbon starvation, because of the absence of a functional stress response.Together with the genes directly regulated by the GSR, a number of genes altered their expression independently of this response, maybe as an indirect regulation mediated by the GSR or perhaps as an adaptation to a hostile situation that could not be properly overcome because of the double deletion. As shown in Supp. Fig. a priori presented as direct GSR targets, revealed the same promoter consensus sequence, this may suggest a model in which both ECFs regulate the same genes but with one of them having a secondary role. To address this, we next focused on the capability of EcfG1 and EcfG2 to recognise selected promoters and their differences in the regulation.Altogether, our transcriptional analysis shows that many cellular processes may be affected to cope with stress, having the GSR a key role in the protection. Also, given that all the genes manually inspected, ecfG1 expression is dependent on EcfG2, it may be considered that the absence of stress protection in an ecfG2 mutant  Ptrc promoter, was constructed and introduced into the ΔecfG1ΔecfG2 double mutant containing the nepR2::lacZ translational fusion inserted in the chromosome. As control, a wild type strain and the ΔecfG1ΔecfG2 mutant carrying the parental plasmid were also included in the analysis. Activity from ecfG1 promoter in the complemented strain was determined in cultures grown to stationary phase in the presence of IPTG. Figure ecfG1 own promoter was induced to similar levels to the wild type strain. These results confirm that EcfG1 is able to substitute EcfG2 in activating its own expression if ectopically produced in sufficient amounts in the cell.As ned Fig. . To do tecfG1 resulted in an increased resistance to heavy metal and salt stresses when compared to the ΔecfG1ΔecfG2 double mutant. However, the complemented strain did not reach the levels of tolerance observed in the wild type strain, indicating only a partial stress resistance. These results indicate that EcfG1 has the capability to partially activate the GSR in TFA on its own when produced in sufficient amounts, thus suggesting that it may play a secondary role in this stress resistance pathway.Coherently with the results mentioned above, Fig. ecfG mutant backgrounds, and also in the ΔecfG1ΔecfG2 mutant with ecfG1 ectopically expressed, and compared to the respective wild type strain in stationary phase. For that, ten genes, identified as members of the GSR regulon, were selected, including nepR2, whose expression pattern was already described by lacZ translational fusions. As control, we tested the expression of ecfG2, which had already been demonstrated to be constitutive and GSR-independent  assays in order to distinguish between EcfG1 and EcfG2 at the molecular level.As mentioned above, many species encode multiple EcfG paralogs and much effort has been made trying to establish possible regulatory differences among them, either by transcriptomic studies or at the level of gene expression phenotype. Since these approaches did not always result in obvious differences, we have introduced in vitro transcription assays were performed with either EcfG1 or EcfG2 forms of the RNAP using five putative promoter regions, representatives of the most frequent -10 boxes, as templates. In order to restrict the regulatory inputs to the effect of the σ factor alone, the DNA regions used as templates would only extend from the putative TSS21 to the -35 sequence. Among the chosen templates, nepR2 and gsp  were selected for having a consensus CGTT -10 box, but also for differing in the -35 element , the putative transglycosylase mltA2 (SGRAN_1260) and SGRAN_2273, which putatively contains an EF-hand Ca-binding domain, harbouring the -10 boxes TGTT, CATT and GGTT, respectively. For doing that, an IVT system was first set up by purifying both σ factors and the TFA core RNAP to reconstitute each RNAP holoenzyme. To know their functionality as σ factors, a titration of both EcfG1 and EcfG2 was assayed in IVT reactions using as template the promoter of mltA2. As shown in Supp. Fig. PmltA2in vitro. However, transcription driven by EcfG2 was much higher than the one driven by EcfG1, which pointed out that EcfG2 has a greater potential to direct the transcription, at least with mltA2 promoter.All the results shown above indicate that EcfG2 has a major role in the GSR in TFA both at the level of gene regulation and in resistance to different stresses, thus relegating EcfG1 to a secondary role as an accessory activator. In order to know the specific contribution of EcfG1 and EcfG2 to the transcription of different GSR promoters, in vitro when equal amounts of either EcfG1 or EcfG2 (2.22 µM) were added to IVT reactions using the selected GSR promoter sequences as templates. As a first observation, the natural variability in the -35 box does not seem to affect the transcription in the tested promoters when comparing nepR2 and gsp. The levels of RNA driven by EcfG1 were modest in absolute terms, but different transcript levels could be observed when comparing among them, reaching up to 5.11-fold compared to the reference (transcription obtained for PnepR2 using EcfG1). Transcription driven by EcfG2 was always higher than that mediated by EcfG1 regardless of the promoter used, again confirming its role as the master regulator of the GSR. However, a broader range of transcription levels was observed for EcfG2, reaching up to 13.59-fold compared to the reference (transcription obtained for PnepR2 using EcfG2). Thus, the EcfG2/EcfG1 transcription ratio ranged from 38.53-fold for PmltA2 to 2.77-fold for Pgsp. Considering this, transcription driven by EcfG1 at some promoters may represent a considerable fraction of the transcriptional output when the transcription exerted by EcfG2 is only moderately higher than that obtained with EcfG1 .After this, in order to discard σ factor concentration effects, saturating concentrations of each EcfG protein were used in the subsequent IVT assays. Figure in vivo when EcfG1 is ectopically produced, whereas others need the presence of EcfG2 to be transcribed at a fully functional level. The cause of this, taking into account the function of a σ factor, might be either the capability of each protein to bind the promoter region or their efficiency to unwind the double strand to start the transcription2. Also, this phenomenon endorses that the relative contribution of each EcfG paralog depends on the characteristics of each promoter.This may explain why some promoters completely recover their transcription levels Pgsp promoter to CATT or TGTT, and (ii) mutating the CATT and TGTT -10 boxes in the PmltA2 and PyiaD promoters, respectively, to the consensus CGTT box. As shown in Fig. Pgsp promoter. Coherently with this result, a change toward the consensus -10 box in the PmltA2 and PyiaD promoters clearly increased the transcription levels  mutating the CGTT consensus sequence in the els Fig. .Figure 7These results indicate that for each individual promoter, having a consensus -10 box would produce a maximum amount of transcripts. Considering the variability observed in that sequence among the GSR regulon, two interpretations may be drawn: firstly, having a non-consensus -10 box might be an intrinsic regulatory mechanism. Secondly, other features in the promoter region may contribute to the final output of the transcription, since promoters diverging from the consensus -10 box show higher levels of transcription than others bearing it. This strategy may allow each gene to be expressed at the level required for its physiological activity. In agreement with this observation, equal -35 and -10 surrounded by different spacer sequences between them and between the -10 box and the TSS produced diverse levels of transcription with both σ factors, but more pronouncedly with EcfG2.E. coli σ70 factor, the spatial conformation of the spacer region, even when altered in one nucleotide, may affect the transcription37. Another example of this can be found in Bacillus subtilis, where transcription led by ECFs σM, σV, σW and σX is modulated by a T-stretch that affects the spatial conformation of the spacer region38. In a scenario in which these four ECFs regulate overlapping sets of genes, the properties conferred by the T-stretch aid in the promoter selectivity, although it is not the only feature that allows the promoter specificity in that case40. Considering the variability in these spacer sequences among all the promoters within the GSR regulon described in TFA, the composition and the curvature in this region may explain the differences in the contribution of each EcfG protein to the transcription as a feature of each particular promoter. Even though we could not find a correlation among the genes that are better activated by EcfG1 and the ones that are more restricted to EcfG2 nor a functional specialisation between the two regulators, another possibility would be that the differentiation came from upstream in the signalling. Since there are two putative GSR regulatory elements (nepR and phyR) annotated in the TFA genome, the organisation of the pathway may define the EcfG protein that is predominantly active under certain conditions and thus, the subset of genes that is more highly expressed. Altogether, our results suggest that each gene in the GSR regulon presents a promoter sequence adapted to produce the appropriate level of transcription to exert its protective role in response to stress. The possible modulation exerted by the GSR regulatory elements upstream in the signalling cascade will be further investigated in future work.Other studies have demonstrated that the properties of the spacer region between the -35 and -10 boxes affect the transcriptional output. For instance, for S. granuli TFA responds to general stress by activating the transcription of 104 genes that are organised in 79 transcriptional units. We have identified the two σ factors governing this response, with EcfG2 being the master regulator, and EcfG1 acting as an accessory regulator. Even though EcfG1 may be dispensable to protect against stress, it is able to partially protect from adverse conditions when produced in sufficient amounts. The IVT assays of selected GSR-regulated genes have demonstrated that the intrinsic characteristics of each promoter determine the relative contribution of each EcfG protein to the transcriptional output, thus defining the differences between EcfG1 and EcfG2. These features lie mainly on the -10 box and the properties of the spacer region between the -35 and the -10 of each GSR-dependent promoter, suggesting that each gene has evolved to be expressed at the optimal level to exert its function in the protection against stress.In this work, we show that Escherichia coli and Sphingopyxis granuli strains were routinely grown in LB rich medium at 37 °C or MML mineral medium at 30 °C, respectively. When indicated, S. granuli strains were grown in minimal medium supplemented with β-hydroxybutyrate, (BHB), as a carbon source in concentrations 8, 20 or 40 mM, depending on the experimental conditions.ecfG1 and ecfG2 scarless deletions, the protocol described in41 was followed with some modifications. ecfG1 upstream and downstream regions were amplified by PCR using oligonucleotide pairs del1161-1/del1161-2 and del1161-3/del1161-4, respectively. The resulting fragments were digested with BamHI and XhoI and ligated into pEMG cut with the same enzymes, resulting in plasmid pMPO1407. In the case of ecfG2, upstream and downstream regions were amplified using oligonucleotide pairs del1163-11/del1163-2 and del1163-3/del1163-4, respectively. The fragments were assembled together by overlapping PCR, digested with SacI and XbaI and cloned into pEMG cut the same to obtain pMPO1409. The same procedure was followed for the construction of pMPO1410, but using oligonucleotide del1163-12, with MPO855 chromosomic DNA as template instead of wild type DNA. As a first step for the deletion of ecfG1 and ecfG2, pMPO1407 and pMPO1409, respectively, were transformed into TFA, and the single recombination event was selected selecting for kanamycin resistant colonies. Positive candidates were picked and transformed with pSW-I, and then selected for its resistance to ampicillin The second recombination event, which generates the in-frame deletion, was selected checking for both ampicillin-resistant and kanamycin-sensitive candidates, eventually resulting in the deletion mutants MPO855 and MPO859. The same protocol was used for MPO860 (ΔecfG1ΔecfG2 double mutant) construction, using MPO855 as initial strain and pMPO1410 as vector.Bacterial strains plasmids used in this work are described in Supp. Table nepR2, gsp and ecfG2 translational fusions to lacZ, 1 kbp fragments upstream the ORF of each gene, including the promoter region and the first 5–8 codons, were amplified with oligo nucleotide pairs nepR2-lacZ fw/nepR2-lacZ rv, gsp-lacZ fw/gsp-lacZrv and del1163-11/del1163-2 . These amplifications were clone in pJES379 digested with SmaI, resulting in vectors pMPO1408, pMPO1417 and pMPO1426, respectively. In order to monitor in vivo the expression of nepR2 and gsp in the wild type and the mutant backgrounds, plasmids pMPO1408 and pMPO1417 were transformed in the wild type TFA, MPO855, MPO859 and MPO860, resulting in strains MPO858, MPO857, MPO863 and MPO864, respectively, for the nepR2::lacZ fusion, and MPO875, MPO876, MPO877 and MPO878, respectively, for the gsp::lacZ fusion. The same procedure was followed to introduce the ecfG2::lacZ fusion in the wild type TFA and MPO860, using pMPO1426, resulting in strains MPO891 and MPO892 respectively.For the generation of the ecfG2 mutation, a PCR fragment amplified with oligonucleotides ecfG2-1 EcoRI/ecfG2-2 BamHI and cut with EcoRI and BamHI was ligated into pLAFR3 digested with the same enzymes, generating pMPO1411. For the complementation of the ecfG2 mutation by heterologous expression of ecfG1, plasmid pMPO1433, which carries ecfG1 downstream the Ptrc promoter and its own Shine-Dalgarno sequence , was constructed. To do this, PCR fragments were amplified with primers nepR2 del1/nepR2 del2 and nepR2 del3/nepR2 del4, ensemble together by overlapping PCR, digested with XbaI and cloned into pMPO1412 cut with SmaI and XbaI, resulting in pMPO1413. Using this plasmid as template, a fragment was amplified using primers del1163-12/ecfG1-XbaI 5′, digested with SacI, blunted with T4 DNA polymerase (New England Biolabs) and digested with XbaI was ligated into pSEVA224 cut with HindIII, blunted with Klenow and digested cut with XbaI, obtaining pMPO1433, which carries ecfG1 downstream its own Shine-Dalgarno sequence and Ptrc promoter.To construct a vector to complement the To insert a 6xHis tag at the C-terminus end of the β′ subunit of the RNA polymerase for the purification of the whole core RNA polymerase, plasmid pMPO998 was constructed by cloning into vector pBluescript II KS a PCR fragment amplified with primers BetaB SacI/BetaB XbaI (the latter containing the 6xHis coding sequence) using TFA chromosomic DNA as template. Both fragment and vector were digested with SacI and XbaI and ligated. The resulting plasmid was introduced into the wild type TFA and inserted into the chromosome by recombination, obtaining strain MPO700.For overexpression prior to purification of EcfG1 and EcfG2, pMPO1431 and pMPO1432, respectively, were constructed following the IMPACT kit’s manual (New England Biolabs). PCR fragments were amplified using ORF-ecfG1 fw/ORF-ecfG1 rv BamHI and ORF-ecfG2 fw/ORF-ecfG2 rv BamHI respectively, digested with BamHI and cloned into the expression vector pTYB21 digested with SapI, blunted with Klenow and digested again with BamHI.in vitro transcription template vectors , primers containing both strands  and they left to air-dry in a laminar flow cabin for 5 h (5 min in the control assay). Then, filters were placed on MML rich medium plates supplemented with bromophenol blue 0.002% and incubated for 5 days at 30 °C. In the case of resistance to oxidative shock, late-exponential phase cultures were diluted to an OD600 of 0.1. When an OD600 of 0.5 was reached, H2O2 was added to the medium up to a concentration of 10 mM. Recovery from the shock is represented by a percentage of the OD600 reached by treated cultures after 5 h of growth compared to non-treated cultures. At least three independent replicates of each experiment were performed, and most representative examples are shown.To test the resistance to osmotic stress and copper, 10 μl spots of serial dilutions of late-exponential phase cultures were placed on solid MML rich medium plates supplemented with NaCl 0.6 M or CuSO600 of 0.05 in minimal medium supplemented with β-hydroxybutyrate 40 mM and incubated at 30 °C in an orbital shaker for 16 h. Then, 20 ml of minimal medium with β-hydroxybutyrate 8 mM were inoculated at OD600 0.1 of the strain to be assayed. In the case of strains ectopically expressing ecfG1, IPTG 1 mM was added from the beginning of the growth. 1 ml aliquots were withdrawn 72 h after starting the culture to measure growth and GSR activation by β-galactosidase activity from nepR2. Averages of three independent replicates are represented. To measure the expression from the ecfG2 promoter, the procedure mentioned above was followed, but samples were taken at different time points since the expression did not change over time.Saturated preinocula were diluted to an OD600 of 0.1 in minimal medium supplemented with β-hydroxybutyrate 20 mM and incubated at 30 °C in an orbital shaker for 30 h. 10 ml of each culture were centrifuged and the pellets were snap-frozen in liquid nitrogen. Total RNA purification was carried out according to the protocol previously described42 from the different strains at the beginning of the stationary phase. DNA was removed by DNase I treatment . Once the DNA removal was confirmed by PCR amplification, RNA samples were purified using the RNeasy purification kit . RNA integrity was checked by agarose gel electrophoresis.Late-exponential phase cultures grown in minimal medium with β-hydroxybutyrate 40 mM were diluted to an ODhttp://www.ascidea.com/lifesciences-services.html) for cDNA library preparation and high-throughput sequencing using an Illumina HiSeq2000 machine.Equimolecular amounts of three independent RNA extraction replicates were mixed for the wild type TFA strain and strain MPO860. Samples were sent to the company ASCIDEA (http://hannonlab.cshl.edu/fastx_toolkit/index.html) and ASCIDEA specific perl scripts were run as low quality region filters. Adaptors and low quality bases at the ends of sequences and reads with undetermined bases or with 80% of their bases with less than 20% quality score were trimmed. Reads that passed these filters were mapped using43 to generate read alignments using the Sphingopyxis granuli strain TFA genome (RefSeq: NZ_CP012199.1) as ref. 18. Differential transcript expression was then computed using DESeq2. Genes downregulated more than 3-fold comparing strain MPO860 to TFA were selected for further analysis.The normalization and the analyses of the dRNA-seq results were performed by ASCIDEA. In summary, quality of the reads obtained by HiSeq2000 sequencing was checked with FastQC software FASTX-Toolkit .450 bp sequences upstream the start codon of genes more than 3-fold downregulated in MPO860 compared to the wild type TFA were subjected to motif search using the online tool MEMETo validate the data obtained from the dRNA-seq, total RNA was extracted from three independent replicates of strains TFA, MPO855, MPO859, and MPO860 in the same conditions performed for the dRNA-seq experiment (see above). cDNA samples were generated using the High-Capacity cDNA Archive Kit (Applied Biosystems) and purified with the QIAquick PCR purification kit (Ambion). RT-qPCR experiments were performed using the universal kit FastGene ICGreen  and the oligonucleotide pairs listed in Supp. Table S. granuli core RNA polymerase purification was based on Burgess protocol45 with modifications from Hager46 and Patek47. Saturated cultures of S. granuli MPO700 grown in MML were diluted to OD600 0.1 in 1 L of MML. The culture was incubated at 30 °C and 180 rpm up to OD600 0.6–0.9. Then, the grown cells were collected by centrifugation 8.000 g 20 min at 4 °C, and the pellet was kept at −80 °C. A total of 22.5 OD600 unit were used for the purification. The biomass was resuspended in 200 mL of grinding buffer , NaCl 0.2 M, DTT 0.1 mM, β-mercaptoethanol 1 mM, PMSF 0.1 mM) and broken by sonification . The cell extract was centrifuged during an hour at 13.000 g 4 °C. The soluble fraction was fractioned with 0.35% Polimin P pH 7.9 (Sigma). The mixture was incubated 15 min with shaking at 4 °C and then pelleted during 20 min at 7630 g 4 °C. The supernatant was discarded and the pellet was resuspended in 150 mL of cold TGED , DTT 0.1 mM) with NaCl 0.5 M and centrifuged 20 min at 7630 g 4 °C. This step was repeated twice. The pellet was resuspended in 100 mL of cold TGED NaCl 1 M and two tablets of proteases inhibitor . The mixtures were stirred during 30 min at 4 °C and centrifuged 30 min at 4290 g 4 °C. The supernatant was precipitated overnight at 4 °C using 35.5 g of ammonium sulphate. After that, the sample was centrifuged 30 min at 7630 g 4 °C and the supernatant was discarded. The sediment was resuspended in 50 mL of cold TGED and two tablets of proteases inhibitor. The mixture was centrifuged 20 min at 7630 g 4 °C and the supernatant was dialysed at 4 °C to reduce salinity using cellulose membranes (33 mm of pore diameter). The dialyses was performed in 4 steps using 2 L of buffer P , 3 mM β-mercaptoethanol, PMSF 0,1 mM pH 8) with decreasing amount of salt, from NaCl 0.5 M to NaCl 0.3 M with imidazol 10 mM. The last step of dialysis was performed overnight. The desalted sample was incubated overnight at 4 °C with 5 mL of nickel agarose beads  previously equilibrated in buffer P NaCl 0.3 M imidazol 10 mM. The mixture was decanted by gravity using a Glass Econo-Column (BioRad) and the resin was washed with 50 mL of buffer P with NaCl 300 mM imidazole 10 mM preincubated 20 min. This step was performed twice. The next wash was done with 25 mL of the same buffer but with 20 mM of imidazole. The washing was eluted by gravity. Finally, the resin was incubated 30 min with 5 mL of buffer P NaCl 0.3 M imidazol 0.5 M and was eluted by gravity. A second elution was performed with 3 mL of the same buffer. The eluted fractions were analysed by SDS-PAGE and concentrated using 10 KDa centricons . Simultaneously, the buffer was change to TGED NaCl 100 mM and finally conserved in 50% of glycerol at −80 °C.ecfG1 and ecfG2 respectively) were transformed into E. coli strain ER2566. Saturated preinocula of each strain were diluted to an OD600 of 0.1 in 400 ml of LB medium and incubated at 37 °C in an orbital shaker until OD600 0.5 was reached. Then, cultures were ice-cooled for 30 min. After that, cultures were induced with IPTG 0.5 mM and incubated at 14 °C in a shaker for 16 h. Cultures were centrifuged and the induction was confirmed by SDS-PAGE. Cells were resuspended in binding buffer  and mechanically lysed by sonication. Lysates were clarified by centrifugation. Once the chitin resin was packed in a purification column and washed with binding buffer, the respective clarified lysates were loaded on the column and left to flow through the resin by gravity. The column was flushed with 100 ml of binding buffer prior to the induction of the on-column protein cleavage. To release the target protein, the resin was incubated with binding buffer supplemented with DTT 50 mM at 18 °C for 40 h approximately. The proteins were eluted and the enrichment of the target protein was confirmed by SDS-PAGE. The eluate was dialysed against modified TEDG buffer 2X at 4 °C overnight. Protein solutions were concentrated to a volume of approximately 300 μl using Amicon Ultra centrifugal filters (Millipore) of different pore size. Then samples were diluted with 1 vol of glycerol 99% to a final glycerol concentration of 50%. After protein quantification , protein aliquots were conserved at −80 °C.EcfG1 and EcfG2 proteins were purified using the IMPACT kit (New England Biolabs) following the manufacturer’s instructions. Briefly, pMPO1431 and pMPO1432  were run in a final volume of 22.5 μl in vitro transcription buffer  at 30 °C. RNA core polymerase was preincubated at 30 °C for 5 min, then the appropriate amount of EcfG σ factor diluted in TEDG buffer without glycerol was added to the reaction mixtures and incubated for other 5 min. Then 0.5 μg of the appropriate plasmid were added as circular template. 5 min later, a mix of ATP, GTP, CTP , UTP (0.07 mM) and [α−32P]-UTP  was added start the reaction. After 10 min, reaction reinitiation was prevented by adding heparin to a final concentration of 0.1 mg/ml, and 10 min later reactions were stopped by adding 5 μl of stop/loading buffer . Samples were boiled for 3 min and run on 4% polyacrylamide-urea denaturing gels in Tris-borate-EDTA buffer at room temperature. Gels were dried and exposed in a phosphoscreen. Results were visualised in an Amersham Typhoon scanner and analysed using the ImageQuant software .Multi-round Supplementary information.Supplementary information2.Supplementary information3.


Hormonal signalling in animals often involves direct transcription factor-hormone interactions that modulate gene expression. In contrast, plant hormone signalling is most commonly based on de-repression via the degradation of transcriptional repressors. Recently, we uncovered a non-canonical signalling mechanism for the plant hormone auxin whereby auxin directly affects the activity of the atypical auxin response factor (ARF), ETTIN towards target genes without the requirement for protein degradation. Here we show that ETTIN directly binds auxin, leading to dissociation from co-repressor proteins of the TOPLESS/TOPLESS-RELATED family followed by histone acetylation and induction of gene expression. This mechanism is reminiscent of animal hormone signalling as it affects the activity towards regulation of target genes and provides the first example of a DNA-bound hormone receptor in plants. Whilst auxin affects canonical ARFs indirectly by facilitating degradation of Aux/IAA repressors, direct ETTIN-auxin interactions allow switching between repressive and de-repressive chromatin states in an instantly-reversible manner. Tight temporal and spatial regulation of the genes involved in these processes is essential for proper development of the organism. Changes in gene expression are often controlled by mobile signals that translate positional information into cell type-specific transcriptional outputs  complex towards its downstream targets . This mea as IAA. Treatments with IAA but not BA protected ETT-FLAG from pronase-induced degradation consistent with direct, specific interaction between IAA and ETT in planta.ETT can interact with a diverse set of TFs and these interactions are sensitive to the naturally occurring auxin, indole 3-acetic acid (IAA) . To test388-594, sufficient for mediating IAA-sensitivity in ETT-protein interactions, was produced recombinantly and shown to be intrinsically disordered  domain. A protein fragment containing 207 amino acids of the ES domain, ESsordered . The senharacter . In addiharacter . We founharacter . These sharacter . Since td value for the interaction, which may be due to the interaction being weak as well as potential protein aggregation. This could reflect that the ETT-auxin interaction is stabilised by interacting protein partners in planta that are not present in this in vitro experiment.We also used the recombinant ETT fragment in an Isothermal Titration Calorimetry (ITC) assay, which characterises binding of ligands to proteins by determining thermodynamic parameters of the interaction as heat exchange. This experiment revealed interaction between ETT and IAA, while control experiments titrating IAA into buffer without protein and titrating buffer without IAA into the ETT fragment showed no heat exchange . We wereTogether, these three independent biochemical methods demonstrate that ETT binds IAA directly thus revealing a key molecular aspect of the non-canonical auxin-signalling pathway.PINOID (PID) (HECATE1 (HEC1)   and HECA1 (HEC1)  were ide1 (HEC1)  and bothild type . We alsockground . This ET mutants .tir1 afb2 mutant background, the canonical pathway will only respond to the addition of cvxIAA and not IAA  thereby repressing STM expression  affects the direct interaction between the ETT protein and auxin. To test the role of W505 in the auxin-sensitive interaction with TPL/TPRs, we constructed a mutated version of ETT (W505A) and assessed the effect in protein interaction assays. Both Y2H and Co-IP experiments revealed that W505 is required for the ETT-TPL/TPR interaction to be sensitive to auxin . The keyett loss-of-function mutants are observed during gynoecium development  using reporter lines expressing GFP fusion protein. Although only ETT is expected to bind DNA, ChIP followed by qPCR revealed that all three proteins associate with DNA elements in the same regions of the promoters of and HEC1 . Moreoveand HEC1 . This sussociate . In agreth auxin .PID expression at the gynoecium apex where PID is repressed at early stages of development to allow symmetry transition, but subsequently de-repressed as auxin levels rise to facilitate polar auxin transport  disassociate from ETT, promoting H3K27 acetylation . This moransport . Whilst STM expression  (pETT:GUS (pETT:ETT-GFP in ett-3 (pTPL:TPL:GFP (p35S:HDA19:GFP (pTIR1:ccvTIR1 in tir1-1 afb2-3 (tir1-1 afb2-3 afb3-4(tpl mutant (SALK_034518C) was obtained from the European Arabidopsis Stock Centre.Plants were grown in soil at 22°C in long day conditions (16 hrs day/8 hr dark). All mutations were in the de ett-3 , hda19-4_139443) , pETT:GUpETT:GUS , pETT:ETin ett-3 , pTPL:TP:TPL:GFP , p35S:HDDA19:GFP , pTIR1:c1 afb2-3  and tir1-3 afb3-4. The tplCol-0 and ett-3 inflorescences with a solution containing 100 µM IAA (Sigma) or cvxIAA and 0.015% Silwet L-77 (De Sangosse Ltd.). Treated samples were returned to the growth room and incubated for two hours.For both expression and ChIP analysis, auxin treatments were applied by spraying bolting -ΔΔCt method (POLYUBIQUITIN 10 (UBQ10/AT4G05320) expression.Quantitative Real time PCR (qRT-PCR) was used for expression analysis. RNA was extracted from floral buds using the RNeasy mini kit (Qiagen). Using the SuperScript IV First-Strand Synthesis kit (ThermoFisher), cDNA was synthesised from 1 µg of total RNA. Subsequently, qRT-PCR was carried out using SYBR Green JumpStart Taq ReadyMix (Sigma) using the appropriate primers . Relativt method . Data weETT sequences of 22 Angiosperm species were retrieved from Phytozome version 12 (Published rsion 12 . Nucleotrsion 12 . The EARrsion 12 .getpl tpr2 mutant was generated using CRISPR/Cas9 technology by a method previously described (TPR2 (AT3G16830). DNA-oligonucleotides  and dotted on YSD medium lacking Tryptophan (W), Leucin (L), adenine (A) and Histidine (H) to test for interaction. To examine interaction strength 3-amino-1,2,4-triazole (3-AT) was supplemented to the YSD (-W-L-A-H) medium with different concentrations . To determine the effect of auxinic compounds on the protein-protein interactions benzoic acid (BA), IAA, NAA and 2,4D  were dissolved in ethanol and added directly to the medium at the desired concentrations. Pictures were taken after 3 days of growth at 28°C.For Yeast-two-Hybrid (Y2H) assays coding sequences were cloned into pDONR207 and recombined into the pGDAT7 and pGBKT7 (Clontech). Using the co-transformation techniques  these co600 of 0.5. The cells were then harvested and lysed using 150 µL Buffer Z with β-mercaptoethanol , 50 µL chloroform and 20 µL of 0.1% SDS. After lysis, the sample was incubated with 700 µL pre-warmed ONPG solution  prepared in Buffer Z without β-mercaptoethanol at 37°C until a yellow colour developed in the samples without auxin treatment. After stopping all reactions (using 500 µL Na2CO3) the supernatant was collected and OD405 determined. The β-Galactosidase activity was calculated as follows: (A405*1000)/(A600*min*mL).For the β-Galactosidase assay, transgenic yeast was grown in liquid YSD (-W-L) medium supplemented with /-out 100 µM IAA or NAA, to an ODETT  and dehydrated through an ethanol series (70% to 100%) as described previously . The constructs were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation, followed by plant transformation by floral dip into Col-0 (For the construction of the promoter:GUS reporter plasmids of to Col-0 .3Fe(CN)6 and 1 mM K4Fe(CN)6 (both Sigma) at room temperature. Subsequently, samples were vacuum infiltrated for 5 min with X-Gluc solution 6, 3 mM K4Fe(CN)6, 0.1% Triton X100) containing 1 mg/ml of ß-glucoronidase substrate X-gluc  and incubated at 37°C. pTPL:GUS samples were incubated for 20 min and pTPR2:GUS lines for 45 min to prevent overstaining. pTPR4:GUS lines were incubated for 16 hr. After staining, the samples were washed in 70% ethanol until chlorophyll was completely removed. Gynoecia were dissected and mounted in chloral hydrate (Sigma). Samples were analysed using a Leica DM6000 light microscope.The GUS histochemical assay was performed in at least three individual lines per construct. Inflorescences of each GUS line were pre-treated with ice cold acetone for 1 hr at −20°C and washed two times for 5 min with 100 mM sodium phosphate buffer followed by one wash with sodium phosphate buffer containing 1 mM KpETT:ETT:GFP, pTPL:TPL:GFP and p35S:HDA19-GFP lines and data analysed as described previously (WUS gene fragment (WUSp4 amplicon) was used as a negative control for ETT binding  and anti-H3 . All antibodies were validated by the manufacturers.Histone acetylation ChIP was carried out and data were analysed as described previously . The expHEC1 or PID divided by H3 at HEC1 or PID).In all ChIP experiments, DNA enrichment was quantified using quantitative PCR (qPCR) with the appropriate primers . In caseqPCR data were analysed using two-way ANOVA with post hoc Bonferroni multiple comparison test. All output of statistical tests can be found in the source data files. All statistical tests were carried out using GraphPad Prism Version 5.04 .In all graphs error bars represent the standard deviation of the mean for all numerical values. QRT-PCR and ChIP experiments have been carried out at least in triplicate. The data presented here show an average of three replicates. For qRT-PCR data were analysed using one-way ANOVA with post-hoc Tukey multiple comparison test. ChIPETT  in a 1:1 (w/v) ratio, followed by a centrifuging step to discard the plant debris. Protein concentration was determined with Quick Start Bradford 1x reagent (Bio-Rad) and adjusted to 5 mg/mL by adding extraction buffer. The lysate was then split into different reaction tubes and incubated with the respective chemical  at the indicated concentrations for 30 min at room temperature with slow mixing. The treated aliquots were further aliquoted and mixed with different dilutions of Pronase (Roche), prepared in Pronase buffer  to achieve the aimed for ratio of total enzyme to total protein. After incubation for 30 min at room temperature, the proteolytic digestion was terminated by adding protease inhibitor cocktail  and the samples were kept on ice for 10 min. The protein samples were then mixed with 4x NuPAGE LDS sample buffer (Invitrogen) and heated at 80°C for 10 min. The protein samples were analysed by Western blot , visualized using an anti-FLAG-HRP antibody (1:1000 – Sigma A8592). HRP activity was detected using the Supersignal Western Detection Reagents (Thermo Scientific) and imaged with a GE Healthcare Amersham 600 RGB system.The DARTS assay to evaluate the stability of ETTIN-FLAG in the presence of IAA was performed as described previously  with plaETT  with a 6388-594, protein was isotopically labelled in preparation for NMR analysis. The ES domain was expressed for as a fusion protein with a 6x Histidine tag in minimal media with 15N ammonium chloride. The 15N isotope labelling of the expressed protein involved a 125-fold dilution of cell culture in enriched growth media into minimal media with 15N ammonium chloride and grown for 16 hr (37 °C / 200 rpm); followed by a further 40-fold dilution into minimal media for the final period of cell growth and protein expression (induced with L-arabinose 0.2 % w/v / 18 °C / 200 rpm and grown for a further 12 hr). The fusion protein was isolated from soluble cell lysate by Co-NTA affinity chromatography with two His-Trap 1 mL TALON Crude columns . Chromatography buffers contained sodium phosphate 20 mM pH 8.0, NaCl 500 mM and either no-imidazole or 500 mM imidazole for wash and elution buffers respectively. The majority of the non-specifically bound protein was removed by passing 20 mL of the wash buffer through the columns. The protein eluted on a gradient of increasing imidazole concentration of up to 30% elution buffer over 20 mL.The ES domain, ES388-594 protein was grown in LB medium, cells harvested and lysed as described above. After centrifugation of the sonicated cell suspension, the pellet was resuspended and washed in Wash Buffer  Glycerol; 0.1 mM DTT; 50 mM NaCl; 2% (w/v) NaDOC) for 1 hour at 4°C followed by centrifugation . The resulting pellet was again washed in Wash Buffer and centrifuged as described previously. The pellet was resuspended in 20mL of Solubilisation Buffer  Glycerol; 0.1 mM DTT; 50 mM NaCl; 0.25% Sarkosyl (N-Lauroyl sarcosine) and stirred at 4°C for 1h. Subsequently the resuspension was dialysed overnight in 2L Dialysis Buffer  Glycerol; 0.1 mM DTT; 50 mM NaCl). The Dialysis Buffer was exchanged for fresh Dialysis Buffer after at least 2 hours of dialysis. After dialysis the lysate was centrifuged . The resulting supernatant contained the required protein which was concentrated to 2 mL using Amicon Ultra-15 centrifugal filters with a 10kDa cut-off (Millipore (UK) Ltd.) and further purified using size exclusion chromatography. The protein was eluted in Chromatography Buffer and concentrated to 2 mL as previously described. Protein concentration was determined with Quick Start Bradford 1x reagent (Bio-Rad) and adjusted using Chromatography Buffer.For ITC, the ES388-594, protein was analysed by NMR at 5°C under reducing conditions (DTT 10 mM), buffered at pH 8.0 (Tris 20 mM). 1H-15N HSQC was performed at 950 MHz, TCI probe, Bruker following the parameters described in 388-594 and 500 µM IAA ligand (i.e. 1:10).The ES domain, ES388-594 protein. Titration of Buffer A into 50 µM ES388-594 protein and IAA (2 mM) into Buffer A served as negative controls. Measurements of the binding affinity of all the titration data were analysed using the MicroCal Software .ITC was carried out on a MicroCal PEAQ-ITC  at 25°C in a Buffer A . Ligand (2 mM IAA) was injected (19 × 4.0 μl) at 150 s intervals into the stirred (500 rpm) calorimeter cell (volume 270 μl) containing 50 µM ESETT, AT2G33860; TPL, AT1G15750; TPR1, AT1G80490, TPR2, AT3G16830; TPR3, AT5G27030; TPR4, AT3G15880; HDA6, AT5G63110; HDA19, AT4G38130; HEC1, AT5G67060; PID, AT2G34650; WUS, AT2G17950. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:eLife can provide a forum for this exciting set of data.This is an outstanding study that deepens our mechanistic understanding of a non-canonical auxin perception mechanism and we are very pleased that Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Christian Hardtke as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Direct ETTIN-auxin interaction controls chromatin state in gynoecium development" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife in principle, they also see certain shortcomings, which prevents publication in its present form.While both reviewers highlighted the suitability of your work for Essential revisions include:HDA19 to ETT and ETT interacts with other transcription factors that in turn can also recruit co-repressors. It is currently unclear whether auxin-mediated disruption of the ETT/TPL interaction directly induces changes in HDA19 interaction or chromatin recruitment. This should be tested directly in the presence of auxin in transient interaction assays and by ETT/TPL/HDA19 ChIP +/- IAA at target loci (see further comments to this aspect by reviewer #1).1) The reviewers see a big gap between the in vitro ETT/TPL interaction and the H3K27ac changes in vivo that needs to be bridged. TPL is not the only co-repressor complex that may recruit 2) The reviewers also request additional experiments, consolidating the auxin binding to ETT. They encourage the authors to carry out additional MST analyses with ideally using full length ETTIN vs ettin mutant protein (W505). Please quantify binding ETT binding to auxin (see further comments to this aspect by reviewer #2).Please, see also additional comments by the reviewers below, which may guide you to further strengthen your manuscript.Reviewer #1:PID expression based on in situ hybridization in mutant gynoecia. Mutations in IND that disrupted interaction with ETT led to reduced accumulation of PID, which was interpreted as a role in transcriptional activation of PID by IND and ETT in the absence of auxin.This study picks up where Simonini et al., 2016 had left off, which had showed that multiple TFs including IND interact with ETT in an auxin -dependent manner. ETT-IND were found to repress HEC1 and PID are induction, suggesting that Aux/IAA degradation is not required for this processes. Critiques that should be addressed are in the numbered/lettered paragraphs below.As I am not an expert on HSQC-NMR or ITC, I will let other reviewers comment on these data. The authors show that nicely TIR1 is neither necessary nor sufficient for HDA19 interaction with ETT in N. benthamiana. Using ETT and HDA19 co-transfection into plant cells, ETT was recently shown to interact with HDA19 via endogenous co-repressors (such as TPL or SEU) by co-IP and by BiFC . In addition, the ETT interaction partner FIL also interacts with HDA19 (likely via different co-repressors). So it is critical to assess whether auxin disrupts ETT complex formation with HDA19 to show that the auxin disrupted ETT/TPL interaction is important for HDA19 association with ETT. As an aside, it should be mentioned in the text that ETT/FIL were shown to recruit HDA19 to STM in the above-mentioned study .1) They next test physical interaction between ETT and TPL/TPRs in Y2H and show it is abolished by addition of IAA. They also test interaction by co-IP in 35S:OE lines after transient transfection into N. benthamiana. In plants, the IAA treatment seems to lead to a both strong reduction in TPL accumulation as well as interaction. This needs to be addressed. It is moreover critical to assess the HEC1 and PID, this was true to a lesser degree for HEC1 in the hda19 mutant. In addition, HEC1 and PID expression is elevated in ett, tpl and hda19 mutants relative to wt. Although not based on inducible mutants, these data suggest that in the wild type ETT and TPL/R repress HEC1 and IND.In ett or tpl /tpr mutant gynoecioa, auxin was not able to induce HEC1 and HDA19. There are several issues with the data provided.2) Figure 5 attempts to show a possible mechanisms for the observed effects by using chromatin immunoprecipitation of ETT, a) First, from a technical perspective, the authors should show the factor binding as% input (to give the reader an idea of the occupancy compared to other TFs or chromatin regulators). More importantly, H3K27ac should be presented as fraction of H3. The latter is critical as a change in histone modification is only informative if we know that the nucleosome occupancy has not changed.HDA19 occupancy upon auxin treatment at PID or HEC1, although this is a key tenet of their model. ETT occupancy at PID and HEC1 should also be assayed plus minus auxin.b) Second, there was no test for a possible change in TFL or HDA19 occupancy at the loci being tested. This is important, because changes in gene expression lead to changes in histone modifications and vice -versa.Third, the authors reported lower H3K27ac in the non auxin-treated wild type. This fits with the reduced gene expression, yet cause and consequence are not clear – especially in the absence of test of disruption of ETT- HDA19 interaction by auxin or 3) Finally, the title and main conclusion are misleading and oversell the findings.HDA19 in the absence of IAA. In the presence of IAA this complex may fall apart. This does not show that ETT toggles between activation and repression. It would show that ETT 's ability to recruit co-repressors is abrogated by auxin.a) According to the author's data and with some additional support as specified above, the manuscript could show that ETT serves to recruit TPL and PID and HEC1. This suggest ETT acts as a repressor. Likewise, for genes bound by MP where loss of MP function results in loss-of-expression, MP is an activator, even though MP does associate with different types of chromatin regulators in the absence or presence of auxin at these loci.b) I know the authors suggested this activator/repressor model in the Simonini paper based on dominant mutant proteins, which are tricky to interpret. Besides there being no evidence for such a dual role of ETT in the current paper, I think it is important to remember that the ett loss of function mutant has elevated Reviewer #2:The authors deal with the idea that ETT is a particular ARF protein, that engages directly not only other transcriptional regulators, but the family of TPL co-repressors, and more importantly binds directly auxin. Two previous manuscripts from the same research group  had brought up the very intriguing hypothesis that ETT-binds auxin by a non-canonical mechanism, independently of TIR1/AFB-AUX/IAA auxin perception. The hypothesis is fascinating, and the authors previously used genetics, plant physiology and developmental genetics to validate their claim.HEC1 and PID expression. Kuhn et al. work is good and straightforward, in the sense that their hypotheses were addressed by few, but robust experiments. The manuscript would tremendously profit from additional biochemical or biophysical experiments though, using preferably the full length of ETT, to evidence auxin binding. Is that feasible?In Kuhn et al., the authors seek to: show biochemically direct auxin binding by ETT; connect the ETT "auxin binding role" to specific non-canonical gene expression; bring evidence of ETT directly binding TPL and TPRs for auxin-triggered responses in the gynoecium; show that ETT interactions with TPL and histone deacetylases are sensitive to auxin levels, regulating thereby Since the authors identified W505 as important putative auxin binding site, why was not an ETT W505 mutant version, and other mutations, tested in iTC or MST, for instance? This reviewer is concerned that since the interaction between ETT and auxin is weak, one could almost account for random peptide interaction, via charges or the similar, for the poor ITC data. Can the authors please quantify binding?– Along the same lines and about Figure 1 and interactions experiments: Why did the authors not estimate the binding affinity or Kd of ETT-IAA? could the weak (not saturable) ETT-IAA signal be the result of transient peptide interactions?– When the authors implemented the synthetic auxin-TIR1 tool developed in Keiko Torii's lab, they showed that ETT interaction is highly specific for IAA, but not cvxIAA even though the interaction occurs in an IDR. Can the authors please explain the findings?– Have the authors evaluated whether there is any sequence conservation for IAA binding around ETT W505?– Can the authors make the binding comparison of ETT-IAA vs. ETT-BA, or vs. ETT-other auxins, or ETT-tryptophan via iTC in order to make a more robust claim about auxin binding?– Regarding Model Figure 5D. Do the authors have any hypothesis about what happens with ETT upon auxin binding?Specific comments:– The Abstract includes references, is that not a bit unusual?– Abstract fifth sentence: Sentence too long. Split and/or rephrase– Abstract final sentence: Giving the outcome of the paper, I recommend to state "ETT might be able to switch chromatin locally…”– Introduction first paragraph: The word "ubiquitinylation" is incorrect, as it does not refer to ubiquitin conjugation of proteins. Please use "ubiquitination" or "ubiquitylation" instead– Please rephrase the statement about ES domain being disordered, as it is disconnected, and the reason for bringing it up on that section of the Introduction is not clear.– The authors used the word "sensitivity", which is a broad claim, given they used a single IAA concentration of 100 µM in their yeast-two hybrid assay.– HSQC is not the typical experimental approach to proof an X ligand binds to a putative receptor. If this approach is implemented because of the solely disordered nature of ETT, please say it so or rephrase.– "Helical character" indicated by what? It would be nice to have a visualization help for non-NMR experts.– “…found that a number of residues shifted…". Please elaborate.– Why did the authors use benzoic acid (BA) as a control ligand in their experiments? Why not to use tryptophan as the obvious non-active auxin precursor?– "The HSQC experiment demonstrates that ETT binds IAA directly". I recommend the authors to be cautious here with this statement. While high-field NMR is a powerful tool for investigating transiently forming protein-ligand complexes and for the identification of protein binding partners, there are a number of strategies that the authors must implement first that will specifically enable the physicochemical characterization of ETT-IAA interactions.– iTC experiments are unfortunately weak e.g. no saturation reached, extremely low signal to noise ratio. Therefore, the authors again should be careful about their statements on direct biochemically relevant ETT-IAA binding.– Please specify if the ccvTIR1 can accommodate the native IAA.– Please use here "…independently of the canonical TIR1/AFB1-5 signalling pathway."– Why IAA = auxin? This is unnecessary and not accurate, stick to "IAA" please.– Could the authors elaborate on a hypothesis for the role of TPR4? Could its action and interaction with ETT or an ETT-like protein in a different tissue be also auxin sensitive?– "low levels of auxin" do not maintain per se ETT-TPL/TPR2 associations. Please rephrase.– The authors have referred twice about the "instantly reversible" event. What do they mean by this? There is no data on timing! "Instantly" as when a [IAA] threshold has been reached?– The inclusion of an auxin insensitive ett mutant might have shed light on the true nature of the ETT-IAA responsive genes.Comments on Figures:– Figure 1. Which IAA concentration was used for these experiments? Please include relevant info in the Figure legend.– Figure 1. The color of NMR data is hard to distinguish, particularly the blues and the blacks.Figure 1D-F. Are the control panels E and F necessary as extra figure? Cannot they be included in the D?– Figure 2. Colors in panel d are difficult to be distinguished. Please recolor.– Figure 3A. Please improve, resize and label residues. Mark and depict W505.– Figure 3B. What is WTL? Give info in Legend, please.eLife. Your article has been reviewed by one peer reviewers, and the evaluation has been overseen by Reviewing Editor Jürgen Kleine-Vehn and Christian Hardtke as the Senior Editor The reviewers have opted to remain anonymous.Thank you for submitting your article "Direct ETTIN-auxin interaction controls chromatin states in gynoecium development" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our policy on revisions we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Both reviewers agreed that you convincingly revised your manuscript and that your manuscript is in principle ready for publication. Reviewer #1 asks you to modify some of your statements and your overall model depiction (see point 2 below).Reviewer #1:The authors have addressed the majority of my concerns. I do not recommend additional experiments, but provide a clarification based on the author response. In addition, I ask for changes in the text and model to address my comments in point 2 below.Previous reviewHDA19 interaction with ETT in N. benthamiana. Using ETT and HDA19 co-transfection into plant cells, ETT was recently shown to interact with HDA19 via endogenous co-repressors (such as TPL or SEU) by co-IP and by BiFC . In addition, the ETT interaction partner FIL also interacts with HDA19 (likely via different co-repressors). So it is critical to assess whether auxin disrupts ETT complex formation with HDA19 to show that the auxin disrupted ETT/TPL interaction is important for HDA19 association with ETT.1) It is moreover critical to assess the HDA19 interaction with ETT in N. benthamiana must be assessed. As mentioned, this experiment was recently published in Chung et al., 2019, and showed that HDA19 and ETT do not interact directly, but via other proteins. Unless we have misunderstood the reviewer's comment, we do not see the need to carry this out again.Authors: We are confused about the first sentence of this section that N. benthamiana assays also probe indirect interactions between proteins via host proteins. Perusal of the pulldowns in Chung et al. does indeed reveal a weak HDA19 pulldown by ETT (and a very strong ARF4 pulldown by HDA19). BiFC using Arabidopsis protoplasts showed strong (indirect) interactions between ETT and HDA19. The current study suggests that these indirect interactions should be impaired in the presence of high levels of auxin.Reviewer: Current reviewHDA19 containing co-repressor complexes to repress KNOX genes in regions of local auxin maxima at the inflorescence shoot apex. In looking at the literature and the current manuscript, a difference in effective auxin levels became apparent. The authors used a rather high auxin concentration to disrupt the ETT – HDA19 co-repressor complex in planta: 100 µM IAA for Figure 4 and Figure 5. By contrast 10 µM IAA is commonly used in inflorescences. Gynoecia may indeed have higher auxin levels.2) The authors should reconcile their data with the previously published data that implicates ETT/ARF4/FIL in recruitment of HDA19-containing co-repressors, while at higher auxin levels, those present in the gynoecium, this interaction is blocked. The authors should revise the Discussion and their model accordingly.This suggests a different model and a different conclusion from that currently proposed by the authors are needed. At intermediate auxin levels, for example those of the inflorescence shoot apex, ETT (together with other TFs) is able to recruit  Essential revisions include:1) The reviewers see a big gap between the in vitro ETT/TPL interaction and the H3K27ac changes in vivo that needs to be bridged. TPL is not the only co-repressor complex that may recruit HDA19 to ETT and ETT interacts with other transcription factors that in turn can also recruit co-repressors. It is currently unclear whether auxin-mediated disruption of the ETT/TPL interaction directly induces changes in HDA19 interaction or chromatin recruitment. This should be tested directly in the presence of auxin in transient interaction assays and by ETT/TPL/HDA19 ChIP +/- IAA at target loci (see further comments to this aspect by reviewer #1).HDA19/H3K27ac was not established in the original submission. We performed the ChIP experiments suggested showing that the interaction of both HDA19 and TPL with the promoters of HEC1 and PID is disrupted in the presence of IAA, while ETT binding remains . These data support that HDA19 is recruited by ETT via TPL and agree with the disruption of protein interactions between ETT and TPL by IAA. Whilst HDA19 does not directly interact with ETT , the data support that HDA19 forms part of the repressive ETT/TPL complex that is disrupted by IAA. The data are presented in the revised manuscript.We agree that this direct connection between ETT/TPL and HDA19 interaction assay in N. benthamiana but refer to Chung et al., 2019, where our experiments on this are published. Since no interaction was observed, we suggest it may not be that informative to carry this out +/- IAA.We did not carry out the ETT-2) The reviewers also request additional experiments, consolidating the auxin binding to ETT. They encourage the authors to carry out additional MST analyses with ideally using full length ETTIN vs ettin mutant protein (W505). Please quantify binding ETT binding to auxin (see further comments to this aspect by reviewer #2).Over several years, we tried a range of expression systems and hosts to produce full-length ETT for biochemical studies with no success. Additional attempts could take a long time with no guarantee for success. However, we have previously shown that the ES domain is sufficient for the IAA-sensitivity  in agreement with numerous other studies reporting a biophysical interaction using an individual protein domain in isolation  Science 352, 986-90).d value. This is now discussed in the revised manuscript.We have not been able to improve on the ITC data presented in the first version. This is due to different factors. Firstly, even with a further optimisation of ES-domain production, we are unable to obtain large enough amounts of fully soluble and stable protein. Secondly, the protein we use seems to aggregate during the ITC measurements making it difficult to obtain a KIn the revised manuscript, we instead include an additional independent experiment using the DARTS assays providing additional evidence for the direct interaction between ETT and IAA .Finally, we also show that the tryptophan in position 505 (W505) is required for the IAAsensitivity of ETT in both Y2H and transient assays in N. benthamiana.Reviewer #1:1) They next test physical interaction between ETT and TPL/TPRs in Y2H and show it is abolished by addition of IAA. They also test interaction by co-IP in 35S:OE lines after transient transfection into N. benthamiana. In plants, the IAA treatment seems to lead to a both strong reduction in TPL accumulation as well as interaction. This needs to be addressed.We agree with the reviewer that the previous figure had lower levels of TPL at higher concentrations of IAA. We have carried this out several times without an effect on TPL levels, but showing the reduction in ETT-TPL interaction. We have now included the results from a more representative experiments .It is moreover critical to assess the HDA19 interaction with ETT in N. benthamiana. Using ETT and HDA19 co-transfection into plant cells, ETT was recently shown to interact with HDA19 via endogenous co-repressors (such as TPL or SEU) by co-IP and by BiFC . In addition, the ETT interaction partner FIL also interacts with HDA19 (likely via different co-repressors). So it is critical to assess whether auxin disrupts ETT complex formation with HDA19 to show that the auxin disrupted ETT/TPL interaction is important for HDA19 association with ETT. As an aside, it should be mentioned in the text that ETT/FIL were shown to recruit HDA19 to STM in the above-mentioned study .HDA19, we have performed ChIP with HDA19/TPL/ETT +/-IAA and found that the interaction of HDA19 and TPL with the HEC1 and PID promoters is sensitive to IAA, while ETT remains bound independently of IAA levels. This supports the model that ETT recruits HDA19 via interactions with TPL and that the ETT/HDA19/TPL complex is disrupted by IAA. These data are included in the manuscript .As mentioned above, to assess whether auxin disrupts ETT complex formation with HDA19 interaction with ETT in N. benthamiana must be assessed. As mentioned, this experiment was recently published in Chung et al., 2019, and showed that HDA19 and ETT do not interact directly, but via other proteins. Unless we have misunderstood the reviewer’s comment, we do not see the need to carry this out again.We are confused about the first sentence of this section that HDA19 to the STM promoter as reported in Chung et al., 2019. This is now included in the revised version of the manuscript.We agree with the reviewer about specifically mentioning the previous finding that ETT is required to recruit In ett or tpl /tpr mutant gynoecioa, auxin was not able to induce HEC1 and PID, this was true to a lesser degree for HEC1 in the hda19 mutant. In addition, HEC1 and PID expression is elevated in ett, tpl and hda19 mutants relative to wt. Although not based on inducible mutants, these data suggest that in the wild type ETT and TPL/R repress HEC1 and IND.2) Figure 5 attempts to show a possible mechanisms for the observed effects by using chromatin immunoprecipitation of ETT, HEC1 and HDA19. There are several issues with the data provided.a) First, from a technical perspective, the authors should show the factor binding as% input (to give the reader an idea of the occupancy compared to other TFs or chromatin regulators). More importantly, H3K27ac should be presented as fraction of H3. The latter is critical as a change in histone modification is only informative if we know that the nucleosome occupancy has not changed.We agree with the technical concerns raised by the reviewer and have revised the figure as suggested .b) Second, there was no test for a possible change in TFL or HDA19 occupancy upon auxin treatment at PID or HEC1, although this is a key tenet of their model. ETT occupancy at PID and HEC1 should also be assayed plus minus auxin.HDA19 occupancy at PID and HEC1. The new data have been included in the revised figure .We agree with this point and have performed additional ChIP experiments plus/minus auxin to test ETT, TPL and Third, the authors reported lower H3K27ac in the non auxin-treated wild type. This fits with the reduced gene expression, yet cause and consequence are not clear – especially in the absence of test of disruption of ETT- HDA19 interaction by auxin or HDA19 occupancy at the loci being tested. This is important, because changes in gene expression lead to changes in histone modifications and vice -versa.HDA19 occupancy at PID and HEC1 by ChIP as requested by the reviewer above. Moreover, we have inserted a sentence to highlight that no direct interaction between ETT and HDA19 was detected in both Y2H and Co-IP ; however, an indirect interaction via a co-repressor was suggested.We agree that this is an important point and have addressed experimentally the 3) Finally, the title and main conclusion are misleading and oversell the findings.a) According to the author's data and with some additional support as specified above, the manuscript could show that ETT serves to recruit TPL and HDA19 in the absence of IAA. In the presence of IAA this complex may fall apart. This does not show that ETT toggles between activation and repression. It would show that ETT 's ability to recruit co-repressors is abrogated by auxin.We do not agree that the title is misleading and would prefer to keep as is. However, we have modified the concluding remarks to take the reviewer’s comments on repressive/de-repressive ability of ETT on board. This has led to a section in which we suggest the possibility that auxin either renders ETT a passive DNA-binding protein or has a role in recruiting activating components.b) I know the authors suggested this activator/repressor model in the Simonini paper based on dominant mutant proteins, which are tricky to interpret. Besides there being no evidence for such a dual role of ETT in the current paper, I think it is important to remember that the ett loss of function mutant has elevated PID and HEC1. This suggest ETT acts as a repressor. Likewise, for genes bound by MP where loss of MP function results in loss-of-expression, MP is an activator, even though MP does associate with different types of chromatin regulators in the absence or presence of auxin at these loci.As above to 3a.Reviewer #2:[…] In Kuhn et al., the authors seek to: show biochemically direct auxin binding by ETT; connect the ETT "auxin binding role" to specific non-canonical gene expression; bring evidence of ETT directly binding TPL and TPRs for auxin-triggered responses in the gynoecium; show that ETT interactions with TPL and histone deacetylases are sensitive to auxin levels, regulating thereby HEC1 and PID expression. Kuhn et al. work is good and straightforward, in the sense that their hypotheses were addressed by few, but robust experiments. The manuscript would tremendously profit from additional biochemical or biophysical experiments though, using preferably the full length of ETT, to evidence auxin binding. Is that feasible?Despite using a large range of different expression systems over several years, we are unable to produce full length ETT in any recombinant system. As described in Simonini et al., 2018, we can make an almost full version of the Cterminal ES domain (only lacking 14 amino acids at the terminus), which is sufficient for the ETT-sensitivity.Since the authors identified W505 as important putative auxin binding site, why was not an ETT W505 mutant version, and other mutations, tested in iTC or MST, for instance? This reviewer is concerned that since the interaction between ETT and auxin is weak, one could almost account for random peptide interaction, via charges or the similar, for the poor ITC data. Can the authors please quantify binding?As explained above, we have not been able to improve on our ITC data despite several attempts . However, we agree with the reviewer on exploiting the potential importance of the tryptophan in position 505 (W505) in the ETT-auxin interaction. Therefore, we developed mutant versions substituting tryptophan for alanine (W505A) and tested them in interaction assays ± IAA (Y2H and Co-IP with TPL). In agreement with a central role of W505, the ETT(W505A)-TPL interaction was not disrupted by IAA .– Along the same lines and about Figure 1 and interactions experiments: Why did the authors not estimate the binding affinity or Kd of ETT-IAA? could the weak (not saturable) ETT-IAA signal be the result of transient peptide interactions?Possibly due to insufficiently high protein concentration and/or aggregation, we were unable to obtain ITC data allowing quantification of the binding affinity.– When the authors implemented the synthetic auxin-TIR1 tool developed in Keiko Torii's lab, they showed that ETT interaction is highly specific for IAA, but not cvxIAA even though the interaction occurs in an IDR. Can the authors please explain the findings?Without a protein structure it is hard to explain the specific requirement for IAA over other auxinic compounds in this pathway. However, this is not just IAA vs cvxIAA. We have previously reported that neither NAA nor 2,4-D are able to mediate the effect on ETTprotein interactions .– Have the authors evaluated whether there is any sequence conservation for IAA binding around ETT W505?This is an excellent point. We have found that the tryptophan in position 505 is highly conserved among the 25 ETT proteins that we have identified. In contrast, very little sequence conservation is observed among other residues in that region. We have included a sequence logo of the W505 region in the alignment in Figure 3—figure supplement 1A to point this out.– Can the authors make the binding comparison of ETT-IAA vs. ETT-BA, or vs. ETT-other auxins, or ETT-tryptophan via iTC in order to make a more robust claim about auxin binding?In the revised manuscript, we use IAA vs BA in the DARTS and NMR experiments and IAA vs. NAA in the Y2H and Co-IP in N. benthamiana. We did not test all variations in ITC due to difficulty in obtaining sufficient protein as described above.– Regarding Model Figure 5D. Do the authors have any hypothesis about what happens with ETT upon auxin binding?Structurally, we do not yet have any idea what changes are occurring in the ETT protein upon IAA binding, although, our recent data included in this revised manuscript suggest that the tryptophan in position 505 may play an important role. Moreover, in response to the comments from reviewer 1, we have modified the speculations to include the possibility that ETT functions primarily as a repressor and that IAA binding induces a conformational change in ETT to make it unable to bind the co-repressive complex. An alternative hypothesis is that IAA-bound ETT is required for de-repression by recruiting activators of gene expression. Both possibilities are presented in the revised version.Specific comments:– The Abstract includes references, is that not a bit unusual?eLife papers have references in the Abstract whereas others do not.We are happy to exclude if the journal requests this. However, it seems some – Abstract fifth sentence: Sentence too long. Split and/or rephraseWe agree and have modified the sentence.– Abstract final sentence: Giving the outcome of the paper, I recommend to state "ETT might be able to switch chromatin locally…”We have modified this sentence.– Introduction first paragraph: The word "ubiquitinylation" is incorrect, as it does not refer to ubiquitin conjugation of proteins. Please use "ubiquitination" or "ubiquitylation" insteadThis has been corrected.– Please rephrase the statement about ES domain being disordered, as it is disconnected, and the reason for bringing it up on that section of the Introduction is not clear.We agree and have modified the sentence.– The authors used the word "sensitivity", which is a broad claim, given they used a single IAA concentration of 100 µM in their yeast-two hybrid assay.We disagree and prefer to use the term ‘sensitivity’. We have previously shown that the interactions of ETT with the IND TF occurs with several IAA concentrations .– HSQC is not the typical experimental approach to proof an X ligand binds to a putative receptor. If this approach is implemented because of the solely disordered nature of ETT, please say it so or rephrase.NMR was used to investigate the binding of auxin to ETT principally because it is a powerful technique for analysing interactions that are relatively low affinity, as was suspected in this case. Such HSQC experiments examine the interaction from the perspective of the receptor rather than the ligand (as is the case in WaterLOGSY NMR experiments) and provide information on the number of residues of the receptor that are affected by the binding of the ligand, indicated by changes in the position of HN cross peaks within the spectra.– "Helical character" indicated by what? It would be nice to have a visualization help for non-NMR experts.The helical character is typically indicated by the dispersed HN cross peaks between 7.8 to 7.0 ppm on the 1H chemical shift axis of the HSQC spectrum. This has now been indicated in Figure 1B, C.– “…found that a number of residues shifted…". Please elaborate.Perturbations in the position of the HN cross peaks in the HSQC is due to changes in the local chemical environment of the amide bonds within the protein. This is caused either by being in close proximity to bound ligand and/or due to conformational change to the protein induced by the binding event. These IAA specific induced changes are shown in Figure 1D.– Why did the authors use benzoic acid (BA) as a control ligand in their experiments? Why not to use tryptophan as the obvious non-active auxin precursor?Benzoic acid was selected as the control because it has no auxinic activity but like IAA, is a weak acid.– "The HSQC experiment demonstrates that ETT binds IAA directly". I recommend the authors to be cautious here with this statement. While high-field NMR is a powerful tool for investigating transiently forming protein-ligand complexes and for the identification of protein binding partners, there are a number of strategies that the authors must implement first that will specifically enable the physicochemical characterization of ETT-IAA interactions.The IAA specific induced changes in the HSQC spectrum is the effect of IAA binding directly to ETT, either by direct interaction with the binding interface or due to more distal conformational changes as a result of binding. This protein-ligand complex is further supported by ITC and DARTS assays. These results do not rule out possibility that the bioactive complex involves further binding partners.– iTC experiments are unfortunately weak e.g. no saturation reached, extremely low signal to noise ratio. Therefore, the authors again should be careful about their statements on direct biochemically relevant ETT-IAA binding.We agree with the reviewer and have included text to explain the problems we have experienced with the ITC. In addition to the HSQC-NMR data we now also show data from a DARTS experiment that provide additional evidence of direct interaction .– Please specify if the ccvTIR1 can accommodate the native IAA.This is described in Uchida et al., 2018 and their data show that ccvTIR1 does not respond to IAA. This is now specifically mentioned in the manuscript.– Please use here "…independently of the canonical TIR1/AFB1-5 signalling pathway."This has been modified.– Why IAA = auxin? This is unnecessary and not accurate, stick to "IAA" please.We have changed to (primarily) use IAA and have specified this earlier in manuscript.– Could the authors elaborate on a hypothesis for the role of TPR4? Could its action and interaction with ETT or an ETT-like protein in a different tissue be also auxin sensitive?This is an entirely possible scenario and we have included a sentence in the revised manuscript to that extent.– "low levels of auxin" do not maintain per se ETT-TPL/TPR2 associations. Please rephrase.We agree and have modified this sentence.– The authors have referred twice about the "instantly reversible" event. What do they mean by this? There is no data on timing! "Instantly" as when a [IAA] threshold has been reached?We have modified the text at both places in response to the reviewer’s comment. In fact, we like the ‘threshold’ formulation so much that we have included this in the revised manuscript.– The inclusion of an auxin insensitive ett mutant might have shed light on the true nature of the ETT-IAA responsive genes.We are not entirely sure what the reviewer means by “the true nature of theD30G) that makes the ETT-IND interaction insensitive leads to constitutive repression of PID (shown by in situ hybridisation in the gynoecium).ETT-IAA responsive genes”; however, we refer to the paper by Simonini et al., 2016, where expressing a version of the TF IND , Leucin (L), Adenine (A) and Histidine (H) to assess interaction. This is now specified in the legend.Reviewer #1:The authors have addressed the majority of my concerns. I do not recommend additional experiments, but provide a clarification based on the author response. […]Reviewer: N. benthamiana assays also probe indirect interactions between proteins via host proteins. Perusal of the pulldowns in Chung et al. does indeed reveal a weak HDA19 pulldown by ETT (and a very strong ARF4 pulldown by HDA19). BiFC using Arabidopsis protoplasts showed strong (indirect) interactions between ETT and HDA19. The current study suggests that these indirect interactions should be impaired in the presence of high levels of auxin.p35S:HDA19:GFP line in presence of auxin also addresses this point showing that auxin leads to dissociation of HDA19 from ETT-target loci, while ETT remains bound.We thank the reviewer for clarifying this comment. We believe the new ChIP data provided using a Current review2) The authors should reconcile their data with the previously published data that implicates ETT/ARF4/FIL in recruitment of HDA19 containing co-repressor complexes to repress KNOX genes in regions of local auxin maxima at the inflorescence shoot apex. In looking at the literature and the current manuscript, a difference in effective auxin levels became apparent. The authors used a rather high auxin concentration to disrupt the ETT – HDA19 co-repressor complex in planta: 100 µM IAA for Figure 4 and Figure 5. By contrast 10 µM IAA is commonly used in inflorescences. Gynoecia may indeed have higher auxin levels.This suggests a different model and a different conclusion from that currently proposed by the authors are needed. At intermediate auxin levels, for example those of the inflorescence shoot apex, ETT (together with other TFs) is able to recruit HDA19-containing co-repressors, while at higher auxin levels, those present in the gynoecium, this interaction is blocked. The authors should revise the Discussion and their model accordingly.This is an excellent point and we agree with the reviewer that different auxin concentrations may have different effects in diverse developmental contexts. It is, moreover, possible that different ETT-containing complexes require different auxin concentration thresholds to generate auxin responses. We have modified the Discussion accordingly (first paragraph); however, we think that Figure 5F depicts our model appropriately.