Mechanistic insights into the regulation of plant phosphate homeostasis by the rice SPX2 – PHR2 complex

doi:10.21203/rs.3.rs-653544/v1

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Mechanistic insights into the regulation of plant phosphate homeostasis by the rice SPX2 – PHR2 complex

https://doi.org/10.21203/rs.3.rs-653544/v1

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published 24 Mar, 2022

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Phosphate (Pi) is a key macronutrient limiting plant growth and crop productivity. In response to the nutrient deficiency, Pi starvation response (PHR) transcription factors activate Pi starvation induced (PSI) genes. PHR transcription factors are negatively regulated by stand-alone SPX proteins, cellular receptors for inositol pyrophosphate (PP-InsP) nutrient messengers. How PP-InsP-bound SPX domains interact with PHR transcription factors is poorly understood. Here, we report crystal structures of the rice SPX2/InsP₆/PHR2 complex and of the PHR2 DNA binding (MYB) domain in complex with its target DNA at resolutions of 3.1 Å and 2.7 Å, respectively. Inositol polyphosphate binding causes SPX2 to assemble into a domain-swapped dimer. The signalling-active SPX2 dimer binds two copies of PHR2, targeting both its coiled-coil (CC) oligomerisation domain and its MYB domain. Structural comparisons, biochemical analyses and genetic characterizations reveal that the SPX2 senses InsP₆ / PP-InsPs to inactivate PHR2 by establishing severe steric clashes with the PHR2 MYB domain, preventing DNA binding, and by disrupting oligomerisation of the PHR2 CC domain, attenuating promoter binding. The complex structure rationalizes how PP-InsPs activate SPX receptor proteins to target PHR family transcription factors and provides a mechanistic framework to engineer crops with improved phosphate use efficiency.

Plant Molecular Biology and Genetics

Plant Physiology and Morphology

plant phosphate homeostatis

PHR

PP-InsPs

Phosphorus is an essential nutrient limiting plant growth, development, and crop productivity¹^,². Plants have developed sophisticated signal systems to perceive, uptake, transport, and store phosphate (Pi) for the maintenance of Pi homeostasis^3-10. During Pi limitation, a broad range of Pi starvation induced (PSI) genes are expressed in response to the nutrient deficiency^11-14. Central regulators responsible for the transcriptional activation of PSI genes are the highly conserved Pi starvation response (PHR) transcription factors^14-18. Under Pi starvation, rice (Oryza sativa) PHR2 (OsPHR2) binds to a cis-element (P1BS) in the promoter of various PSI genes and up-regulates their transcription, thus optimizing rice Pi acquisition and utilisation^19-21. In contrast, under Pi sufficient conditions, an inositol pyrophosphates (PP-InsPs) dependent negative regulator, the stand-alone SPX (SYG1/Pho81/XPR1) protein, binds to OsPHR2 and inactivates its transcriptional activity¹⁹^,²⁰^,²²^,²³ (Supplementary Fig. 1). Despite extensive studies on the rice PHR signalling pathway, how the SPX protein senses the Pi-level correlated PP-InsPs signal and transduces this signal into transcription repression remains largely unknown.

To reveal this underlying mechanism at molecular level, we sought to determine the structure of rice SPX2 in complex with rice PHR2 and InsP₆. We first constructed a PHR2 boundary PHR2^225-362 containing the MYB domain and the CC domain¹⁶^,²⁴^,²⁵. By co-expressing His-tagged PHR2^225-362 with SPX2 following Ni-NTA pull-down assay, we found that the interaction between PHR2^225-362 and SPX2 is InsP₆ dependent (Supplementary Fig. 2), in agreement with previous reports²²^,²⁶. Initial crystals of the of the SPX2/InsP₆/PHR2^225-362 ternary complex diffracted only to low resolutions. We next identified poorly conserved loop regions in rice SPX2 (residues 36-69 and 191-280; Supplementary Fig. 3). Deletion of non-conserved insertions represents one promising approach to improve protein stability and crystallizability²⁷. Based on this we expressed an engineered SPX^{21-202/∆47-59} construct fused to the C-terminus of the macro domain of human histone mH2A1.1^181-366 for carrier-driven crystallization²⁷. The crystal structure of the mH2A1.1^181-366 tagged SPX2^{1-202/∆47-59}/InsP₆/PHR2^225-362 ternary complex was subsequently determined at 3.1 Å resolution, using InsP₆ (phytic acid) as a commercially available surrogate for the bioactive PP-InsPs²²^,²⁵ (Fig. 1a, Supplementary Fig. 4, and Supplementary Table 1). In the complex structure, two InsP₆ molecules bind to an SPX2 dimer, and two copies of PHR2 wrap around this dimer. In order to check and assess if the mH2A1.1^181-366 fusion tag and the loop truncations may affect the SPX2 structure and its ability to bind PHR2, we performed in solution analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) analyses of the full-length SPX2^1-280/PHR2^225-362 complex and a SPX2^1-202/PHR2^225-362 complex in the presence of 1 mM InsP₆,respectively (Supplementary Figs. 5 and 6). We found that the full-length SPX2^1-280 binds PHR2^225-362 with 2:2 stoichiometry ratio, consistent with the complex crystal structure (Supplementary Fig. 5). In line with this, the SAXS profile derived from the crystal structure is consistent with that observed in solution (Supplementary Fig. 6). Taken together, the AUC and SAXS experiments corroborate the crystal structure of the ternary SPX2^{1-202/∆47-59}/InsP₆/PHR2^225-362 complex.

In the SPX2/InsP₆/PHR2 complex, the SPX2 dimer obtains a domain-swapped conformation, in which the N-terminal domain of one SPX2 protomer interacts with the C-terminal domain of the other SPX2 protomer, forming an intertwined dimer (Fig. 1b). Specifically, the domain swap involves helices α1 and α2 (NTD) from one protomer and helices α4 and α5 (CTD) from another protomer, which are bridged by two antiparallel extended helices α3 from these two protomers. We validated this dimeric conformation in solution by thiol-directed chemical crosslinking (Supplementary Fig. 7). Rice SPX2 is a stand-alone SPX protein, and a 3-dimensional structural homology search with the program DALI²⁸ revealed that its dimeric conformation has not been previously observed with other SPX domain structures ²²^,²⁹ (Figs. 1c, d). SPX domain-containing proteins for which ligand-bound structures are available, such as Chaetomium Thermophilum glycerophosphodiester phosphodiesterase 1 (SPX^CtGde1) or Vacuolar transporter chaperone 4 (SPX^CtVtc4), InsP₆ binds the monomeric SPX domain in a 1:1 stoichiometry ratio (Fig. 1c). In these previous structures, core helices α3 and α4 bridge helices α1, α2, α5 and α6, stabilizing a monomeric fold, and InsP₆ mainly interacts with helices α2 and α4. In contrast, the rice SPX2 adopts a domain-swapped dimer conformation and coordinates two InsP₆ molecules in a 2:2 stoichiometry ratio. The helix α2 of one protomer and the helix α3 of another protomer form the basic binding surface for InsP₆/ PP-InsPs.

To assess the recognition of inositol pyrophosphate signal by the domain-swapped rice SPX2 dimer, we performed extensive mutational analyses of the InsP₆/ PP-InsP binding site. We co-expressed 8×His-tagged PHR2^225-362 and untagged wild-type vs. mutant SPX2^1-280 and assessed their interaction in Ni-NTA pull-downs in vitro (see Methods). InsP₆ binds to a positively charged surface by a set of highly conserved residues in SPX proteins²² (Figs. 2a, b; Supplementary Fig. 3). The binding surface in rice SPX2 is formed by the basic residues K26, K29 and R31 from protomer A, and K143, K146, K147 and K150 from protomer B of the SPX2 dimer. The highly conserved Y25 and L28 also contribute the binding surface. In line with this, the Y25F, Y25A, L28A, K29A, or K143A/K147A mutations strongly reduced/abolished InsP₆ binding in vitro (Fig. 2c). Single amino-acid substitutions in the SPX basic binding surface had little effect on InsP₆ binding (Fig 2c). The area of the positively charged accessible surface for InsP₆ binding is larger than the shape of negatively charged InsP₆ (Fig. 2b), suggesting that, like previously shown for other SPX domains, PP-InsPs such as InsP₇ or InsP₈, may represent the bona fide Pi signalling molecule recognized/sensed by rice SPX2²²^,²⁶^,³⁰.

We next assessed the role of the domain-swapped SPX2 dimer in PHR2 binding. The intertwined dimer is mainly stabilized by hydrophobic and paired electrostatic interactions between the two anti-parallel helices α3. The hydrophobic network encompasses W18, F84, F87 and F88 from one protomer, and L129, L130, Y133 and N137 from another protomer (Fig. 2d). Substitutions of key residues in the network disrupted the PHR2 – SPX2 complex in vitro, whereas a substitution outside the hydrophobic network, R19A, had little impact on PHR2 – SPX2 complex association (Figs. 2d, e). The paired electrostatic interactions involve R105, E112 and E119 from one protomer, and E119, E112 and R105 from another protomer (Fig. 2d). Charge reversal mutation of these residues (R105E, E112R, and E119R) in the SPX2 dimer, that breaks paired electrostatic interactions, also abolished the binding of PHR2 (Figs. 2d, e). These residues contributing to SPX2 dimerization are well conserved in the stand-alone SPX proteins (Supplementary Fig. 3), supporting their importance in the dimerization of SPX proteins for the binding of PHR transcription factors.

We next assessed the interaction interface between SPX2 and PHR2 in vitro pull-down assays. Two copies of PHR2 are wrapped around the domain-swapped SPX2 dimer (Fig. 3a). The MYB domain and CC domain of PHR2 mainly interacts with the helix α3 of protomer A, and helices α3 and α5 of the protomer B in the SPX2 dimer, respectively. Residues R250, E257, H294, K297, Y298 and R302 in the MYB domain, and E92, E93, K100, E101 and E104 in the helix α3 of SPX2 protomer A, mediate the PHR2^MYB – SPX2 association. The interaction between the CC domain and SPX2 is stabilized by residues Q345, K346, H349, E353 and R356 in the CC domain and H124, E131, T172, Y176, E183 and D187 in the helices α3 and α5 of SPX2 protomer B (Fig. 3a). Consistent with this, mutations of these key interacting residues in the PHR2^MYB – SPX2 interface or in the PHR2^CC – SPX2 interface disrupted the association of SPX2 and PHR2 (Figs. 3b, c). We also validated these interactions by in vitro pull-down analyses using rice SPX4, a homolog of rice SPX2 (Supplementary Fig. 8).

We next tested if SPX – PHR complex interface mutations can modulate the function of the related PHR1 transcription factor in Arabidopsis. We expressed wildtype and point mutant versions of PHR1 from its native promoter in the previously characterized phr1 phl1 loss-of-function mutant¹⁴ and scored growth phenotypes of the T1 transformants (Supplementary Figs. 9a, b). We found that plants carrying mutations in the PHR^CC – SPX interface (AtPHR1^L331A; L352 in OsPHR2 and AtPHR1^L339A; L360 is OsPHR2) exhibited a growth phenotype similar to the parental phr1 phl1 line, suggesting that these mutations may disrupt the SPX – PHR complex and possibly AtPHR1 oligomerisation²⁵. We thus mutated additional polar residues in the PHR^CC – SPX complex interface that are located outside the AtPHR1 oligomerisation interface in the previously reported AtPHR1 CC structure²⁵. We found that the mutations of AtPHR1^Q324A (Q345 in OsPHR2), AtPHR1^E332A (E353 in OsPHR2) and AtPHR1^E342A (E363 in OsPHR2) resulted in severe dominant-negative growth phenotypes, that were reminiscent of the previously characterized AtPHR1^KHR mutant, which cannot bind SPX proteins and thus represents a constitutively active transcription factor²⁵ (Supplementary Figs. 9a, b). Taken together, these and previously characterized²⁵ PHR^CC – SPX interface mutations can release PHR transcription factors from the regulation by SPX domains and PP-InsPs, leading to autoactivation.

To gain insights into how rice SPX2 reduces the DNA binding ability of PHR2, we determined the structure of the PHR2 MYB domain in complex with P1BS motif at 2.7 Å resolution (Fig. 4a, and Supplementary Table 1). It turns out that two MYB molecules are coordinated into the major groove of the imperfect palindromic DNA mainly through helices α3. The P1BS recognition by MYB is mediated by the interaction between the residues K292, S293 and Q296 in MYB and the nucleotides G7, A9, A11, G4′, A6′ and A8′ in P1BS (Fig. 4b). Electrophoretic mobility shift assay (EMSA) results revealed that alanine substitution of theses residues attenuated the DNA binding ability of PHR2, and the charge reversal mutation of K292 abolished this interaction (Supplementary Fig. 10a). The structure of rice MYB/P1BS complex is consistent with classical binding models³¹, and is similar to the reported structure of the AtPHR1 MYB in complex with P1BS (Supplementary Fig. 10b)²⁴.

We next compared the PHR2^MYB – SPX2 and PHR2^MYB – DNA complex interfaces and isolated amino-acids that contribute to the formation of the PHR2^{MYB –} SPX2 complex, while being located outside the P1BS recognition site of the PHR2 MYB domain (Supplementary Figs. 9a, c). In vivo, mutation of AtPHR1^R229 (R250 in OsPHR2) or AtPHR1^H273/K276 (H294 and K297 in OsPHR2) to alanine again resulted in strong dominant growth phenotypes in the phr1 phl1 mutant background (Supplementary Figs. 9a, b), indicating that also the PHR2^MYB domain may contribute to PHR – SPX complex formation in vivo.

Structures of SPX2/InsP₆/PHR2 complex and MYB/DNA complex provide a framework for understanding how SPX2 senses the PP-InsP signal and transduces this signal into transcription inhibition. Superposing the two complex structures using the MYB domains as a reference, the position of SPX2 molecules heavily overlapped with the DNA position in the MYB/DNA complex, indicating that the binding of InsP₆-bound SPX2 to PHR2 would produce severe steric clashes and thus preventing DNA binding (Fig. 4c). It has been previously established that the CC domain of PHRs enables oligomerisation of the transcription factor critical for DNA binding¹⁷^,²⁵. In line with this, EMSA results showed that the DNA binding ability of PHR2^MYB-CC is stronger than PHR^MYB (Supplementary Fig. 10c). Mutation of K325, H328 and R335 at the surface of AtPHR1 CC domain disrupted its interaction with SPX receptors, and led to constitutive Pi starvation responses²⁵, whereas the mutation of R318 or R340 produced no effect²⁵. Mechanistically, our structure shows that these residues K346, H349 and R356 in OsPHR2 (corresponding to K325, H328 and R335 in AtPHR1) interact with OsSPX2, whereas R339 and R361 (corresponding to R318 and R340 in AtPHR1) do not contribute to the formation of the signalling complex (Fig. 4d, and Figs. 3a, c).

We have previously reported that the residues L319, I333 and L337 in AtPHR1 stabilize the CC oligomer required for DNA/promoter binding²⁵. Our complex structure now uncovers the mechanism by which rice SPX2 disassembles PHR2 CC oligomers and impairs DNA binding. Specifically, SPX2 blocks the assembly of PHR2^CC oligomers by exposing hydrophobic residues normally contributing to the stabilization of the coiled-coil structure (including L340, I354 and L358 that correspond to the previously characterized L319, I333 and L337 in AtPHR1)²⁵ (Fig. 4d). Collectively, the domain-swapped SPX2 dimer senses the InsP₆/ PP-InsP nutrient messenger to repress the PHR2 transcriptional activity by two distinct mechanisms: 1) The association of the InsP₆-bound SPX2 establishes severe steric clashes with the MYB domain, preventing DNA binding 2) Disruption of the CC domain oligomerisation motif in PHR2 attenuates its transcriptional activity (Figs. 4c, d).

Taken together, we propose a molecular model for the transcription inhibition of PHR2 by InsP₆/ PP-InsPs (Fig. 4e). Our structure clearly defines that in the case of PHR – SPX interactions, PP-InsPs do not act as "intermolecular glue" promoting the association of the signalling complex³². Instead, InsP₆ / PP-InsPs allosterically assemble a domain-swapped SPX2 dimer that targets two PHR2 monomers forming a 2:2:2 complex. Upon SPX2 dimer binding, the PHR2 CC domain can not longer oligomerise and the PHR2 MYB domain is inaccessible to DNA binding, leading to PHR2 inactivation. This unique molecular mechanism of plant phosphate homeostasis provides a framework for the rational engineering of crops with improved Pi use efficiency.

Molecular cloning. The codon-optimized complementary DNA of full length rice SPX2, SPX4 and PHR2 were synthesized. PHR2 was subcloned into a pET21b (Novagen) vector with a C-terminal 8×His tag. SPX4 was constructed with a 6×His tag and a caspase drICE protease cleavage site at the N-terminus, and was subcloned into a pET15D vector. SPX2 was cloned into a pBB75 vector without any tag. The site-specific mutations were introduced into PHR2 or SPX genes by overlap PCR. All the constructs were verified by DNA sequencing.

Protein expression and purification. For the preparation of SPX2/PHR2 complex, the SPX2 and PHR2, or the particular boundary and mutants, were co-expressed in E. coli strain BL21(DE3) using Lysogeny broth (LB) medium. The cells were induced with 0.2 mM isopropyl-b-D-thiogalactoside (IPTG) and 1 mM InsP₆ at 16 °C for 12 h. Harvested cells were lysed by a high-pressure cell disrupter in a buffer containing 25 mM Tris–HCl pH 8.0,150 mM NaCl, 1 mM InsP₆. Target protein was collected from the supernatant and purified over Ni²⁺ affinity resin and HiTrap Heparin column used in tandem. 1 mM InsP₆ was present during all the purification processes. The protein was further purified into homogeneity by gel-filtration chromatography (Superdex-200 Increase 10/300 GL, GE Healthcare) in a buffer containing 25 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM DTT and 1mM InsP₆. Target fractions were collected for biochemistry experiments and supplied with 10 mM InsP₆for crystallization.

PHR2 was expressed alone and purified similarly as the purification of SPX2/PHR2 complex, except there was no InsP₆ added.

SPX4 was expressed in E. coli strain BL21(DE3) using LB medium, and induced with 0.2 mM IPTG at 16 °C for 12 h. Harvested cells were lysed, and the target protein was purified over Ni²⁺ affinity resin. After removal of the His tag by drICE protease, target protein was further purified over Source 15Q and Superdex-200 Increase 10/300 columns used in tandem. The protein was finally prepared in a buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl and 1 mM InsP₆.

MYB domain of PHR2 (residues 180-313) was expressed in E. coli strain BL21(DE3) using LB medium, and induced with 0.2 mM IPTG at 16 °C for 16 h. Harvested cells were lysed, and the target protein was purified over Ni²⁺ affinity resin, Source 15Q, and Superdex-200 Increase 10/300 columns used in tandem. The protein was final prepared in a buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl₂, and 5 mM DTT. For crystallization trials, the PHR2 MYB was incubated with the P1BS (5′‐gCTTGAGGATATCCGA‐3′ and 5′‐cTCGGATATCCTCAAG‐3′) in a molar ratio of 1:1.5 at 4 °C for about 30 min.

Crystallization. Crystallization experiments were performed through hanging-drop vapour-diffusion methods by mixing the protein with an equal volume of reservoir solution at 18 °C. The mH2A1.1^181-366 tagged SPX2^{1-202/∆47-59}/InsP₆/PHR2^225-362 complex gave rise to best crystals under the condition of 8.4% PEG5000 MME, 6.5% Tacsimate pH 7.0, 0.15 M (NH4)₂SO₄, 0.1 M HEPES pH 6.8, 2.67% Pentaerythritol ethoxylate (3/4 EO/OH). Crystals were equilibrated in a cryoprotectant buffer containing 8%-12% PEG5000 MME, 5% Tacsimate pH 7.0, 0.1 M (NH4)₂SO₄, 0.1 M HEPES pH 7.0, 0.1 M KCl, 1.5 M L-Proline. By stepwise dehydration in the air, the crystal quality was improved. Finally, a best crystal from thousands of dehydrated crystals diffracted to 3.1 Å at Shanghai Synchrotron Radiation Facility beamline BL17U.

To crystalize the MYB domain of rice PHR2 (residues 180-313) in complex with DNA, the purified MYB was concentrated to about 200 μM and incubated with double-strand DNA (5′‐gCTTGAGGATATCCGA‐3′ and 5′‐cTCGGATATCCTCAAG‐3′). The crystal of MYB/DNA complex was grown in 13% (w/v) PEG 3350, 0.1 M sodium malonate pH 6.0. The crystals were flash frozen in liquid nitrogen using 25% glycerol as the cryoprotective buffer and diffracted to 2.7 Å.

Data collection and structure determination. X-ray diffraction datasets of mH2A1.1^181-366-tagged SPX2^{1-202/∆47-59}/InsP₆/PHR2^225-362 complex and MYB/DNA complex were collected at the Shanghai Synchrotron Radiation Facility (SSRF) on beamline BL17U or BL19U³³^,³⁴. The data were integrated and processed with the HKL2000 program suite or in XDS package³⁵. Further data processing was carried out using CCP4 suit³⁶. Crystal structures of the mH2A1.1^181-366-tagged SPX2^{1-202/∆47-59}/InsP₆/PHR2^225-362 complex and MYB/DNA complex were determined at resolutions of 3.1 Å, 2.7 Å, respectively. The structure of MYB/DNA was solved by molecular replacement using MYB domain of AtPHR1 (PDB ID 6J4R) as a search template. Using the resolved MYB domain and the human histone mH2A1.1 (PDB ID 1ZR3) as search the models, we determined the structure of mH2A1.1^181-366-tagged SPX2^{1-202/∆47-59}/InsP₆/PHR2^225-362 complex through molecular replacement by the program PHASER³⁷. All the structures were iteratively built with COOT³⁸ and refined using PHENIX program³⁹. Data collection and structure refinement statistics were summarized in Supplementary Table 1. All figures were generated using the PyMOL program (http://www.pymol.org/).

Analytical ultracentrifugation (AUC). The AUC experiment was performed in a Beckman Coulter XL-I analytical ultracentrifuge using two-channel centerpieces. The SPX2^1-280/PHR2^225-362 complex was prepared in a solution of 25 mM Tris-HCl pH8.0, 1mM InsP₆, and 150 mM NaCl. Data was collected via absorbance detection at 18 °C for protein at a concentration of 0.7 mg ml⁻¹ and rotor speed of 45,000 r.p.m. The SV-AUC data were globally analyzed using the SEDFIT program and fitted to a continuous c(s) distribution model to determine the molecular weight.

Small angle X-ray scattering (SAXS). SAXS data were collected at the BL19U2 beamline of the Shanghai Synchrotron Radiation Facility (SSRF) at room temperature. 30 μM SPX2^1-202/PHR2^225-362 complex was prepared in the buffer of 25 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM DTT and 1mM InsP₆ for SAXS measurement. For each measurement, 20 consecutive frames of 1-sec exposure were recorded and averaged, providing no difference between the first and the last frames. The background scattering was recorded for the matching buffer and was subtracted from the protein scattering data. The SAXS experiment was performed at room temperature. The data was visualized and analyzed using the software package ATSAS⁴⁰. For the calculation of the theoretical SAXS profile of SPX2^1-202/InsP₆/PHR2^225-362 complex, the invisible residues of the crystal structure were build using PyMOL and are refined by Xplor-NIH⁴¹. The theoretical SAXS profile was calculated using CRYSOL of software package ATSAS⁴⁰.

Thiol-directed chemical crosslinking. The complex of SPX2^1-280/PHR2^225-362, SPX2^1-280_K106C/PHR2^225-362 and SPX2^1-280_K106C/C182S /PHR2^225-362 was prepared in a buffer containing 25 mM Tris–HCl pH 8.0, 150 mM NaCl and 1mM InsP₆, respectively. About 30 mM complex protein was incubated with 200 mM M2M (1,2-ethanediyl bismethanethiosulfonate) at room temperature for 30 min. The reaction mixture was analyzed by SDS-PAGE and Coomassie Blue staining in the condition of 100 mM DTT, or not.

Co-expression coupled Ni-NTA pull-down. We have tried various expression systems, such as E. Coli., insects, or mammalian cells, to express the SPX2 alone, but no soluble SPX2 protein was obtained. Therefore, we applied a co-expression coupled Ni-NTA pull-down strategy to assess ligand binding and protein interactions (Supplementary Fig. 2). In this system, no tag SPX2 was co-expressed with 8×His-tagged PHR2, and then supernatant of the lysed cells were loaded on Ni-NTA beads. After washing unbound proteins, the His-tagged PHR2 and interacting SPX2 were co-eluted by imidazole.

Since the SPX2/PHR2 association is InsP₆ dependent (Supplementary Fig. 2)²²^,²⁶, the loss of InsP₆ binding ability will make SPX2 no longer interact with PHR2. Thus, the Ni-NTA pull-down assay of SPX2/PHR2 association by co-expressing His-tagged PHR2 with SPX2, or SPX2 mutants, in the presence of InsP₆, can be used to assess the recognition of InsP₆ by SPX2 (Fig. 2c). Similarly, we used this strategy assessed the role of SPX2 domain-swapped dimer in PHR2 recognition (Fig, 2e), and revealed keys residues for the interaction between SPX2 and PHR2 (Figs. 3b, c).

In vitro pull-down assay of SPX4 and PHR2. SPX4 and PHR2 was prepared in a buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl and 1 mM InsP₆, respectively. About 20 mM SPX4 was incubated with 20 mM PHR2 on ice for 30 minutes. Then the mixture was loaded onto Ni-NTA beads and incubated for 30 minutes. After extensively washing with a buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 15 mM imidazole and 1 mM InsP₆, the bound protein was eluted by a buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 250 mM imidazole and 1 mM InsP₆. The input protein and eluted fractions were analyzed by SDS-PAGE and Coomassie Blue staining.

Generation of constructs and transgenic lines. Binary constructs were generated using the Golden Gate system⁴². For Level I constructs, the full-length AtPHR1 coding sequence and the AtPHR1 promoter (pPHR1, 265bp) were cloned into pUC-BpiI via BpiI cut-ligation. Mutations targeting the AtPHR1 coding sequence were introduced by site-directed mutagenesis on the resulting Level I plasmid, as previously described⁴³ (primers are listed in Supplementary Table 2). Level I constructs were assembled in Level II constructs via BsaI cut-ligation (constructs are listed in Supplementary Table 2). All constructs were transformed into Agrobacterium tumefaciens strain pGV3101. Plants were transformed using the floral dip method⁴⁴. Transformants were identified by FastRed fluorescence with a Zeiss Axio Zoom.V16 stereomicroscope (mRFP filter) and a HXP200C illuminator.

Plant material and growth conditions. All experiments were done in Arabidopsis thaliana Colombia (Col-0) ecotype. Arabidopsis phr1 phl1 seeds¹⁴ were kindly provided by Dr. Yves Poirier (University of Lausanne, Switzerland). Plants were grown at 21°C, 60% humidity and in a 16/8 hour light/dark cycle.

Electrophoretic mobility shift assay (EMSA). FAM-labeled primers were used to generate the DNA fragment (F:5′-AAGCTTGAATATGCAATGGAATATGCTTAG-3′, R:5′-CTAAGCATATTCCATTGCATATTCAAGCTT-3′). The DNA fragment was annealed by heating to 95 °C for 5 min and gradually cooled to 25 °C. The FAM-labelled DNA (10 nM) was incubated with 0.3375, 0.45, 0.6, and 0.8 mM PHR2 proteins at 4 °C for 30 min, in a buffer of 25 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM DTT, 10% glycerol, and 200 ng ml⁻¹Heparin. The reactions were resolved on 8% native acrylamide gels (37.5:1 acrylamide:bis-acrylamide) in 0.5× Tris–Boric acid buffer at 150 V for about 3 h. Images of the gels were obtained using FLA5100(Typhoon, Fuji, Japan).

Data Availability

Atomic coordinates of SPX2/InsP₆/PHR2 and PHR2^MYB/DNA complex have been deposited in the Protein Data Bank (PDB) under accession number 7D3Y and 7D3T, respectively.

Acknowledgements

We thank staffs at the BL17U, BL19U and BL19U2 beamline of the NCPSS at Shanghai Synchrotron Radiation Facility for assistance with data collection, and research associate Dr. De-lin Zhang at the Center for Protein Research, Huazhong Agricultural University, for facilities support. We thank Prof. Lizhong Xiong for helpful discussions. The work has been supported by the National Key R&D Program of China (2018YFA0507700 to Z.L. ), and by the National Natural Science Foundation of China (32071226 to Z.L. ), by the Fundamental Research Funds for the Central Universities (Program No. 2662019PY004 to Z.L. ), and by the European Union’s Horizon 2020 research program under ERC consolidator grant agreement 818696 (INSPIRE) to M.H.. Zeyuan Guan also acknowledges the support of National Postdoctoral Program for Innovative Talents (BX2021108).

Author contributions

Z.L. and Z.Y.G. conceived the project and designed the experiments, Z.Y.G., Q.X.Z. and Z.F.Z. performed crystallization and resolved the structures, J.S., L.B. and M.H. designed and performed the genetic experiments, J.Q.Z., J.C., P.C., Q.W., K.P., D.L.Z., T.T.Z., J.J.Y. and P.Y. performed biochemistry experiments and analyzed data. Z.L. and M.H. wrote the manuscript with support from all authors.

Author information

Correspondence and requests for materials should be addressed to Z.L. ([email protected]). Z.Y.G., Q.X.Z. and Z.F.Z. contributed equally to this research.

Competing interests

The authors declare no competing interests.

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Mechanistic insights into the regulation of plant phosphate homeostasis by the rice SPX2 – PHR2 complex
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