No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.
Cloning of in-vitro auxin sensors
The TrpR sensors Trp-CTY and Trp-CTYT were gifts from W. Frommer (Addgene plasmids 13533 and 13534). The fluorophores tested were also donated: Aquamarine by F. Merola (Addgene plasmid 42888), Clover and mRuby2 by K. Beam (Addgene plasmid 49089), mKO1 by K. Thorn (Addgene plasmid 44642) and mCherry by M. Bayer29. eGFP was amplified from pGIIK NLS:3xEGFP30. mNeonGreen, mWasabi and mTFP1 were purchased from Allele Biotechnology and Pharmaceuticals, mKate2 and TagRFP were from Evrogen.
For the initial screening, we first used the Trp-CTY sensor and mutated the residues T44 and S88 individually to all possible amino acids. To this end, we generated primers with 15–16 bp overlaps around the exchanged amino acid codons. We first mutated the amino acids sequence randomly with a degenerate primer and screened 96 clones. Variants not found were then generated by targeted mutagenesis. For example, to generate all T44 variants we first used the primers CCTGATGCTGnnnCCAGATGAGCGCG and CGCGCTCATCTGGnnnCAGCATCAGG, to generate the missing T44C variant we then used the primers CCTGATGCTGtgtCCAGATGAGCGCG and CGCGCTCATCTGGacaCAGCATCAGG. The most promising candidates were introduced by targeted mutagenesis into Trp-CTYT and TrpR without fluorescent proteins, which were analysed by isothermal titration calorimetry (ITC) and structural studies. Trp-CTYT with mutations T44L and S88Y was generated in two steps: we first used the primers CCTGATGCTGctaCCAGATGAGCGCG and CGCGCTCATCTGGtagCAGCATCAGG to generate Trp-CTYT with T44L and then GATTACGCGTGGATCTAACtac-CTGAAAGCCGCGCCC and GGGCGCGGCTTTCAGgtaGTTAGATCCACGCGTAATC to generate Trp-CTYL with the mutations T44L and S88Y. To produce the recombinant proteins, we cloned the TrpR domain into the pET21 expression vector and generated the variants by targeted mutagenesis. This procedure was repeated with the most promising variants after each round. The primer sequences are available upon request.
The final variant was then codon-optimized for Arabidopsis and synthesized by Thermo Fisher Scientific GENEART. To allow an easy exchange of the fluorophores we added restriction enzyme sites at the ends: BamH1 and XhoI around the first fluorophore and ApaI and HindIII around the second. All fluorescent proteins were tested in the fluorophore I–TrpR–fluorophore II–TrpR configuration. Having identified Aquamarine and mNeonGreen as the optimal pair, we introduced all final binding-pocket variants into the backbone by site-directed mutagenesis.
Ligands used for screening and testing
IAA, TRP, IAN, indole-3-carboxaldehyde, indole, indole-3-acetyl alanine, indole-3-acetyl aspartic acid, indole-3-acetamide, indole-3-ethanol, l-kynurenine, 2-oxindole-3-acetic acid, phenylalanine, picloram, tryptamine, (NH4)2SO4, CaCl2, DTT, H2O2, NH4NO3, KNO3 and yucasin were purchased from Sigma-Aldrich; 4-hydroxyindole-3-carbaldehyde and 5-hydroxyindole-3-carboxylic acid from Santa Cruz Biotechnology; 1-naphthaleneacetic acid, KCl and DMSO from Carl-Roth; 2,4-dichlorophenoxyacetic acid (2,4-D) from Alfa Aesar; indole-3-acetyl glucose from TRC; indole-3-butyric acid from Serva; NaCl from Merck; NPA from Supelco; and BFA from Thermo Fisher Scientific.
Trp-CTY variants were generated by site-directed mutagenesis with degenerate or specific oligonucleotides purchased from Sigma-Aldrich. Amplification was carried out using Pfu polymerase (Thermo Fisher Scientific).
More than one thousand oligonucleotides were used; the sequences and resulting vector maps are available upon request. Each variant was sequenced and screened in crude extract of sonicated bacteria for IAA binding; promising candidates were confirmed as purified proteins. The linkers were generated by site-directed mutagenesis, including linkers with 15–16 bp overlap and 3–9 degenerated nucleotides in the middle. To generate linkers shorter than the original ones, parts were deleted, whereas for longer linkers a fixed sequence was inserted into the middle to reduce the risk of generating stop codons by having too many degenerate nucleotides in the sequence.
Protein expression and purification for screening
For protein expression, bacteria were grown in the dark on plates with LB-agar supplemented with ampicillin for 3 days at room temperature. To measure crude extracts, we resuspended the bacteria in 20 mM MOPS pH 7.2, sonicated the suspension with an MS 73 probe (Bandelin) and centrifuged the sample with a tabletop centrifuge (Eppendorf). Protein extraction was performed with His Spin trap columns according to the manufacturer’s instructions (GE Healthcare). We resuspended the bacteria of one Petri dish in 2 ml binding buffer and sonicated with a MS 73 probe. Buffer exchange was performed with illustra NAP-25 columns (GE Healthcare). All measurements were performed in 20 mM MOPS (Sigma-Aldrich) with an Infinite F200 plate reader (Tecan).
Ligands used for crystallization and ITC
IAA, IAN and TRP used for crystallization and ITC were dissolved in 50 mM Tris/300 mM NaCl pH 8 buffer containing 1% DMSO if necessary.
Protein purification for crystallography and ITC
After subcloning to pET21b(+), wild-type TrpR and all variants were expressed in E. coli BL21(DE3) and purified with a NiIMAC column and a subsequent Superdex-S75 gel-filtration column. All purification steps and measurements were based on the above 50 mM Tris/300 mM NaCl pH 8 buffer.
Crystallization, data collection and processing
Crystals of wild-type TrpR and variants with different ligands were obtained by standard vapour diffusion in sitting drop plates. The crystals were cryoprotected if needed and flash-cooled in liquid nitrogen. Data for single crystals were collected at the synchrotron beamline PXII (Swiss Light Source) at 100 K and 0.5 degree images were recorded on a Pilatus 6 M detector. Only variant TrpR(M42F/T44L/T81M/N87G/S88Y)–IAA was recorded at MX Beamlines BL14.1 at BESSY II (Helmholtz-Zentrum Berlin für Materialien und Energie). Data were indexed, integrated and scaled with the program XDS and converted with XDSCONV31. Molecular replacement was performed with Phenix using the coordinates of wild-type TrpR (PDB ID: 1WRP32 or 1TRO33) as search model. Model building was performed using the program Coot34, and refinement was performed using Phenix35. Details on crystallization conditions, data and refinement statistics for all structures are summarized in Extended Data Table 2b and Supplementary Table 1.
ITC was performed using a VP-ITC (MicroCal). The protein concentration was adjusted to 74 μM and 730 μM ligand solutions were prepared using the above buffer containing 1% DMSO. Measurements were performed at 20 °C with a stirring speed of 300 rpm, reference power 15 μcal s−1 and spacing of 300 s between injections. The data were analysed using the MicroCal program. Binding data were derived from sigmoidal fits based on a one-site binding model from two measurements for each variant. Heat-of-dilution baselines for the ligands alone were subtracted from the experimental data as previously described36. The pH dependence of IAA binding to the variant TrpR(M42F/T44L/T81M/N87G/S88Y) (AuxSen) was measured on a NanoITC LV device with a stir rate of 300 rpm, 15 injections with 2 μl and 300 s spacing between injections. The 170 μl cell was overfilled with 400 μl protein to ensure air-free filling. The protein was added at a concentration of 100 μM and the ligand IAA at 1 mM at 20 °C. For each pH the data were recorded in the respective buffer: (i) 50 mM Tris pH 8.5, 300 mM NaCl; (ii) 50 mM Tris pH 8.0, 300 mM NaCl; (iii) 50 mM Tris pH 7.5, 300 mM NaCl; (iv) 50 mM Tris pH 7.0, 300 mM NaCl; (v) 50 mM MES pH 6.5, 300 mM NaCl; (vi) 50 mM MES pH 6.0, 300 mM NaCl; (vii) 50 mM MES pH 5.5, 300 mM NaCl; (viii) 50 mM citrate buffer pH 5.0, 300 mM NaCl; and (ix) 50 mM citrate buffer pH 4.5, 300 mM NaCl.
Test of different FRET pairs
We tested pairs of Aquamarine, mCerulean3, mTFP1 and mTurquoise2 with Clover, Ypet and mNeonGreen; Aquamarine was additionally tested with eGFP and mWasabi, and mTFP1 with TagRFP. Furthermore, we tested mNeonGreen, Clover and Ypet with TagRFP and mRuby2. mKO1 was tested with mCherry, mKate2, mNeonGreen and mWasabi. TagRFP was also tested with mTFP1, mWasabi, mKate2 and mCherry.
Constructs for in-vivo AuxSen experiments
For protoplast expression, we cloned the final version of the sensor into pJIT6022 (pJIT60-2xp35S:NLS:AuxSen).
For expression in transgenic plants, we cloned the in-vitro optimized AuxSen in constructs for conditional two-component expression, using the pBay-bar vector (a gift from M. Bayer37). pRPS5a-mGAL4-VP16-GR-UAS_NLS was amplified from a pGII plasmid and inserted into pBay-bar digested with KpnI and BamHI with Gibson Assembly (In-Fusion Cloning, Takara Bio Europe SAS) according to the manufacturer´s instructions. In a second step, AuxSen and ocs terminator were inserted into pBay-bar pRPS5a-mGAL4-VP16-GR_UAS_NLS digested with BamHI. To obtain the individual spectra, we replaced AuxSen by mNeonGreen or Aquamarine. These constructs were used for transforming plants and as a template for the unmix matrix in Fiji. To generate the ER-localized auxin sensor SP:AuxSen:HDEL, we removed the NLS from the nuclear AuxSen construct and inserted the signal peptide of an Arabidopsis vacuolar basic chitinase and the HDEL ER retention sequence38 in frame with the coding sequence N-terminally and C-terminally, respectively.
Flow cytometry of protoplasts transiently expressing AuxSen
Protoplasts were prepared from suspension cell cultures and transfected as previously described39, using 12-ml PP tubes and 12 μg of construct pJIT60-2xp35S:NLS:AuxSen per 120 μl of protoplasts (3.5 × 106 per ml) per transfection. On the next day, transfected protoplasts were pooled, filtered through 100-μm nylon mesh and split into 200-μl aliquots. Each IAA stock (in DMSO) was added 1:100 with a timing offset to account for the 5-min measurement cycle, ensuring a 1-h treatment for each sample, performed in triplicate. Cytometric analysis was set up on a Beckman Coulter MoFlo XDP (100 μm CytoNozzle, 30.5/30.0 psi, PBS sheath) to excite mNeonGreen at 488 nm (70 mW, elliptical focus) and capture peak FL1 (534/30) and shoulder FL2 (585/29) emission; Aquamarine at 405 nm excitation (100 mW, spherical focus) and capture peak FL9 (465/30) and shoulder FL10 (529/28) emission. Data were collected and processed using Summit 5.5 (Beckman Coulter). The main light-scattering gate was determined by identifying the population expressing the greatest amount of reporter. The FRET response was the ratio mean of FL10/FL9, with the auxin response moving towards FL10, directly calculated in Summit 5.5. Representative plots were drawn with FCS Express v.6.06.0033 (deNovo).
Plant material and growth conditions
Wild-type Arabidopsis thaliana (accession Col-0) plants were used for transformation. Plants were grown on soil at 24 °C, 65% relative humidity under long-day conditions (16-h illumination and 8-h dark period). Seeds were surface-sterilized, stratified for 2 days at 4 °C and grown on half-strength Murashige and Skoog agar plates containing 1% sucrose (0.5MS + S) (Serva). After 1 week plants were transferred to soil.
For imaging, 4-day-old seedlings were transferred to 0.5MS+S 25 μM DEX agar plates, and 16 h later to microscope slides on which they were incubated in 0.5MS+S + IAA or DMSO (control) at the specified concentrations and for the indicated periods of time. To preserve field-of-view and optimal buffer exchange we fixed the cover slip and root with double-sided adhesive tape (Tesa, type 05338, Beiersdorf). To exchange the buffer, we completely emptied the slide on a paper tissue and refilled from the side with the pipette. For BFA treatment, seedlings were transferred to 0.5MS+S 25 μM DEX plates containing either 10 μM BFA or 0.1% (v/v) DMSO (control), and mounted on microscope slides 10 h later.
The imaging of seedling roots was performed with an LSM780 confocal laser scanning microscope, running ZEN 2.3 black SP1 as acquisition software (Zeiss) and using a Zeiss LD C-Apochromat 40×/1,1 W Korr for all experiments except the gravitropism experiments, which were recorded with a Zeiss Plan-Apochromat 20×/0.8. Spectral imaging was performed using the QUASAR detection unit on the same system: Aquamarine was excited for FRET ratio measurement at 405 nm using 5 fluorescent channels (419–455 nm, 454–491 nm, 490–526 nm, 525–562 nm and 561–598 nm); subsequently, mNeonGreen was imaged for segmentation of the regions of interest with excitation at 488 nm and detection of 3 fluorescent channels (490–526 nm, 525–562 nm and 561–598 nm). Gravitropism imaging requiring control over the direction of gravity was performed with a custom-made horizontal imaging kit that can be equipped on most inverted microscope stands for wide-field and confocal imaging. The kit consists of two pieces: a holder for the objective with a mirror for changing the direction of the optical axis of the system from a vertical direction to a horizontal direction (components from Thorlabs). The second piece is a rotatable, vertical sample holder that can be mounted into a standard multiwell plate holder (components from Fischertechnik).
We used spectral FRET40 to be able to control for influences from sources of autofluorescence, which can be abundant in plant tissues41. Spectral FRET therefore also has the advantage that the method can be adapted by adjusting the number of acquisition channels if other sources of autofluorescence are present in different plant tissues.
All analyses were performed using a current version of Fiji42. First, signals generated were linearly unmixed43 using J. Walter’s spectral unmixing plugin (https://imagej.nih.gov/ij/plugins/spectral-unmixing.html). The unmixing matrix was generated with mNeonGreen, and Aquamarine as fluorophore controls and a wild-type Col-0 control for background autofluorescence. After this, the images were manually thresholded on all channels to remove unspecific signals and saturated areas, regions of interest (ROIs) for the cell nuclei were automatically generated based on the 488 nm/490–526 nm-channel data using an adaptive threshold plugin (by Q. Tseng, https://sites.google.com/site/qingzongtseng/adaptivethreshold) and the ‘Watershed’ and ‘Analyze Particles’ functions of ImageJ. We analysed all pixels of the image only for the ER. The FRET ratio was calculated by spectral unmixing of the channels using ‘Spectral Unmix’ version 1.3 (by J. Walter, https://imagej.nih.gov/ij/plugins/spectral-unmixing.html) and a precomputed unmixing matrix (see above) yielding the Aquamarine and mNeonGreen emission for division. Unmixed ROIs were colour-coded using the ‘ROI Color Coder’ plugin (BAR library, by T. Ferreira, http://imagejdocu.tudor.lu/doku.php?id=macro:roi_color_coder). In general, the colour scales were adapted for each experiment best reflecting the differences. Nuclei consisting of areas that were too small or those that had unrealistic high FRET ratios (owing to insufficient Aquamarine signal) were omitted (reflected as black in the colour coding).
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.