DWARF 53 acts as a repressor of strigolactone signalling in rice

Abstract

Strigolactones (SLs) are a group of newly identified plant hormones that control plant shoot branching. SL signalling requires the hormone-dependent interaction of DWARF 14 (D14), a probable candidate SL receptor, with DWARF 3 (D3), an F-box component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. Here we report the characterization of a dominant SL-insensitive rice (Oryza sativa) mutant dwarf 53 (d53) and the cloning of D53, which encodes a substrate of the SCF(D3) ubiquitination complex and functions as a repressor of SL signalling. Treatments with GR24, a synthetic SL analogue, cause D53 degradation via the proteasome in a manner that requires D14 and the SCF(D3) ubiquitin ligase, whereas the dominant form of D53 is resistant to SL-mediated degradation. Moreover, D53 can interact with transcriptional co-repressors known as TOPLESS-RELATED PROTEINS. Our results suggest a model of SL signalling that involves SL-dependent degradation of the D53 repressor mediated by the D14-D3 complex.

Extended Data Figure 1. Characterization of the e9 mutant

a, Tiller number and plant height of wild-type, homozygous and heterozygous e9 plants at the heading stage. Values are means ±s.d. (n = 8). The double asterisks represent significant difference determined by the Student’s t-test at P <0.01. b, Kinetic comparison of tiller numbers between wild-type and e9 plants at different developmental stages. Values are means ±s.d. (n = 8). DAG, days after germination. Rice plants (a and b) were cultivated in the field in Beijing in the natural growing season. c, Four-week-old seedlings of wild-type, e9, d3 and d27 upon 1 μM GR24 treatment. Scale bars, 10 cm. Ten individual plants for each material were treated with GR24. d, The expression levels of the D10 gene revealed by qPCR in wild-type, e9, d3 and d27 mutant seedlings. Values are means ±s.d. (n = 3). The double asterisks represent significant difference determined by the Student’s t-test at P <0.01. e, The representative LC/MS–MS chromatograms for epi-5DS in root exudates of wild type and e9 before calibration against an internal standard.

Extended Data Figure 2. Cloning and confirmation of E9/D53

a, E9 was mapped in the interval between molecular markers Ds3 and K81114 on chromosome 11 using 142 recessive individual plants showing normal tillering phenotype from an F₂ population. Numbers under the markers indicate recombinants. b, E9/D53 mutation sites in e9/d53 in the coding region and its amino acid changes. c, Schematic diagram of D53:d53 constructs. d, Phenotypes of wild-type and D53:d53 transgenic plants at the mature stage. Scale bar, 20 cm. Nine independent transgenic lines showed the similar tillering and dwarf phenotypes. e, Comparison of tiller numbers between wild-type and D53:d53 transgenic plants at the mature stage. Values are means ±s.d. (n = 15). The double asterisks represent significant difference determined by the Student’s t-test at P <0.01. The plants were grown in the field in Beijing in the natural growing season.

Extended Data Figure 3. Phylogenetic tree of D53-like family proteins in rice and Arabidopsis

BlastP was done using the D53 protein sequence against all rice and Arabidopsis proteins at the MSU Rice Genome Annotation Project. In the BlastP result, rice proteins were filtered using cut-off E value <0.1 plus top query coverage >10% and Arabidopsis proteins were filtered using cut-off E value <0.0005. Multiple sequence alignment of the protein sequences was done using Clustalw2. Maximum-likelihood phylogenetic tree was drawn by MEGA 5.05 using default parameters with 100 times bootstrapping. Numbers above the branches represent bootstrap support based on 100 bootstrap replicates. Branch length represents substitutions per site.

Extended Data Figure 4. Alignment of D53 and D53-like proteins in rice and Arabidopsis

The graphic view of alignment was generated by BioEdit using Clustalw for multiple sequence alignment of protein sequences. Blue underline refers to the Double Clp-N domain, light green to atypical walker A motifs, dark green box to walker B motifs and red boxes to putative EAR motifs. D53, Os11g01330; D53-like, Os12g01360; AtD53-like 1, At1g07200; AtD53-like 2, At2g29970; AtD53-like 3, At2g40130; AtHSP101, At1g74310; OsHSP101, Os05g44340.

Extended Data Figure 5. D53 expression levels in d mutants and the mutation sites in other d mutants used in this study

a, Expression levels of D53 revealed by qPCR in wild-type, d3, d10, d14, d17 and d27 seedlings. Values are means with s.d. of three independent experiments. The double asterisks represent significant difference determined by the Student’s t-test at P <0.01. b, The mutation sites in other d mutants identified in this study.

Extended Data Figure 6. D3 and D14 protein levels are unaffected by GR24 treatment

a, Phenotypes of mature transgenic plants of D3-GFP and D14-GFP in the d3 or d14 background, respectively, showing that D3–GFP and D14–GFP can rescue the corresponding phenotypes of d3 and d14. Scale bar, 10 cm. Five independent transgenic lines were shown to have similar phenotypes. b, Comparison of the tiller number of transgenic plants of D3-GFP/d3 and D14-GFP/d14. Values are means ±s.d. (n = 5). The double asterisks represent significant difference determined by the Student’s t-test at P <0.01. c, D3–GFP abundance in d3 treated with 10 μM GR24 for 60 min. Protein level was analysed by immunoblotting using GFP antibody. d, The D14–GFP protein level in the d14 background as analysed by immunoblotting using GFP antibody. Transgenic plants of D14-GFP in the d14 background were treated with 10 μM GR24 for 60 min. Rice plants (T₂ generation) were grown in the field in Hainan province.

Extended Data Figure 7. Effects of GR24 and SL mimics on rice tillering

a, Chemical structures of GR24 and SL mimics. A, 4-[(2,5-dihydro-4-methyl-5-oxo-2-furanyl)oxy]benzonitrile; B, 5-(4-bromophenoxy)-3-methyl-2(5H)-furanone; C, 5-(4-iodophenoxy)-3-methyl-2(5H)-furanone. D, 3,3a,4,8b-tetrahydro-3-(hydroxymethylene)-2H-Indeno[1,2-b]furan-2-one (ABC rings of GR24); E, 5-hydroxy-3-methyl-2(5H)-furanone (D ring of GR24). b, Effects of 1 μM GR24 and SL mimics on tillering of 4-week-old seedlings. Values are means ±s.d. (n = 5).

Extended Data Figure 8. Phenotypes of transgenic plants of constitutively expressed D53–GFP in d3 and d14

a, Phenotypes of wild-type, d3 and Act:D53-GFP/d3 transgenic plants at the mature stage. Scale bar, 10 cm. Six independent transgenic lines showed similar phenotypes. b, Phenotypes of wild-type, d14 and Act:D53-GFP/d14 transgenic plants at the mature stage. Scale bar, 10 cm. Six independent transgenic plants showed similar phenotypes. c, Tiller numbers of Act: D53-GFP/d14 and Act:D53-GFP/d3 transgenic plants at the mature stage. Values are means ±s.d. (n =8). d, The D53–GFP abundance in d3 or d14 plants treated with 10 μM GR24 for 60 min. Protein levels were analysed by immunoblotting using GFP antibody. Rice plants (T₁ generation) were grown in the field in Hainan province.

Extended Data Figure 9. Phenotypes and confirmation of transgenic plants of D53-RNAi in d3 and d14

a, Phenotypes of wild-type, d3 and D53-RNAi/d3 transgenic plants. Scale bar, 10 cm. Four independent transgenic plants showed similar phenotypes. b, Phenotypes of wild-type, d14 and D53-RNAi/d14 transgenic plants at the mature stage. Scale bar, 10 cm. Five independent transgenic plants showed similar phenotypes. c, Tiller numbers of transgenic plants of D53-RNAi in d3 and d14 backgrounds at the mature stage. Values are means ±s.d. (n = 5). The double asterisks represent significant difference determined by the Student’s t-test at P <0.01. d, The D53 abundance of D53-RNAi in d3 or d14 transgenic plants. Protein levels were analysed by immunoblotting using D53 polyclonal antibodies. Rice plants (T₁ generation) were cultivated in the field in Hainan province. e, Sequence information of D53-RNAi constructs. Two complementary inverted D53 fragments are shown in black and the intron sequence between inverted DNA fragments is in red.

Extended Data Figure 10. Determination of D53 antibody specificity

Determination of D53 antibody specificity by D53–GFP transgenic calli. Left, anti-GFP (Roche), 1:10,000 dilution; right, anti-D53 (1:10,000). Horseradish-peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG antibodies were used as secondary antibodies to detect anti-D53 and anti-GFP.

Figure 1. D53 acts as a negative regulator in SL signalling

a, Phenotypes of e9 mutants. Scale bars, 20 cm. b, Tiller numbers of 4-week-old seedlings of wild-type, e9, d3 and d27 treated with or without 1 μM GR24. Values are means ±s.d. (n =10). c, Comparison of epi-5DS contents in wild-type and e9 root exudates. Values are means ±s.d. (n = 3), **P <0.01 (Student’s t-test). gfw⁻¹, per gram fresh weight. d, D53 transcript levels in various organs, including roots (R), shoot bases of seedlings (SB), axillary buds (AB), sheaths (SH), young leaves (L) and young panicles (P). Values are means with ±s.d. of three independent experiments. e, Subcellular localization of 35S:GFP (top), 35S:D53-GFP (middle) and 35S:d53-GFP (bottom) in rice protoplasts. Scale bars, 10 μm. f, D53 transcripts upon 20 μM GR24 treatment in wild-type seedlings revealed by quantitative (q)PCR. Values are means with ± s.d. of three independent experiments.

Figure 2. SL-induced D53 degradation by the ubiquitin proteasome system

a, Protein levels of D53 in seedlings of wild-type and d mutants detected by immunoblotting with anti-D53 antibodies. b, D53 protein levels in 3-week-old seedlings of the wild type (top) and d53 mutant (bottom) upon 10 μM GR24 treatment detected by immunoblotting with anti-D53 antibodies. c, Protein levels of D53–GFP in calli of Act:D53-GFP and Act:d53-GFP transgenic lines at different time points of GR24 (5 μM) treatment, as detected by immunoblotting with anti-GFP antibody. d, D53–GFP protein levels at different time points of GR24 treatment (10 μM) in the presence or absence of MG132 (50 μM), as detected by immunoblotting with anti-GFP antibody. e, Analysis of D53–GFP and d53–GFP ubiquitination in response to GR24 (10 μM) treatment. Top, immunoblot with anti-ubiquitin (Ubi) antibody. Bottom, immunoblot with anti-D53 antibodies. Asterisks indicate ubiquitinated D53–GFP proteins. f, D53–GFP protein levels in Act:D53-GFP transgenic calli treated with 10 μM GR24, KAR1 or SL mimics, as detected by immunoblotting with anti-GFP antibody. A, 4-[(2,5-dihydro-4-methyl-5-oxo-2-furanyl)oxy]benzonitrile; B, 5-(4-bromophenoxy)-3-methyl-2(5H)-furanone; C, 5-(4-iodophenoxy)-3-methyl-2(5H)-furanone; D, 3,3a,4,8b-tetrahydro-3-(hydroxymethylene)-2H-Indeno[1,2-b]furan-2-one (ABC rings of GR24); E, 5-hydroxy-3-methyl-2(5H)-furanone (D ring of GR24). g, D53 protein levels in calli of wild-type, d3, d14 and d27 upon 5 μM GR24 treatment at different time points, detected by immunoblotting with anti-D53 antibodies. h, Analysis of D53–GFP ubiquitination in d14 and d3 under GR24 (10 μM) treatment. Top, immunoblotting with a monoclonal anti-ubiquitin antibody. Bottom, immunoblotting with anti-D53 antibodies. Asterisks indicate ubiquitinated D53–GFP. Actin contents were used as loading controls in all the immunoblotting analyses. NP, Nipponbare.

Figure 3. Interactions among D3, D14 and D53

a, In vitro pull-down assay using maltose-binding protein (MBP)–D53, GST–D3 and Strep–D14–His recombinant proteins in the presence or absence of 10 μM GR24. b, Pull-down assays using recombinant GST–D53 or GST–d53 and lysates prepared from Act:D14-GFP/d14 calli in the absence or presence of 20 μM GR24. c, Pull-down assay using recombinant GST–D53 and lysates prepared fromAct:D14-GFP/d14 calli in the presence of GR24 at indicated concentrations. d, Pull-down assay using recombinant GST–D53 and lysates of Act:D14-GFP/d14 calli treated with 20 μM GR24 or SL mimics. e, D53 levels in the 10 μM-GR24-treated calli transformed with D14 mutated at the Ser-His-Asp catalytic triad sites, revealed by immunoblotting with anti-D53 antibodies. D14, Act:D14-GFP/d14; S147A, Act:D14(S147A)-GFP/d14; D268N, Act:D14(D268N)-GFP/d14; H297Y, Act:D14(H297Y)-GFP/d14. f, Pull-down assay using recombinant GST–D53 and lysates of the calli transformed with D14 mutated at its catalytic triad sites in the absence or presence of 20 μM GR24. g, Pull-down assay using recombinant GST–D3 and lysates of Act:D14-GFP/d14 calli in the absence or presence of 20 μM GR24. h, Pull-down assay using recombinant GST–D3 and lysates of transgenic calli transformed with D14 mutated at its catalytic triad sites in the absence or presence of 20 μM GR24. i, Pull-down assay using recombinant GST–D3 and His–Trx–D53 in the absence or presence of 5 μM GR24 or His–Sumo–D14 recombinant proteins. The D14 recombinant proteins in a were detected with anti-His antibody, GFP-fusion proteins in (b–d and f–h) were revealed with anti-GFP antibody, and His–Trx–D53 was detected with anti-D53 antibodies.

Figure 4. Interaction of D53 with TPR proteins

a, Mammalian two-hybrid assays showing interaction of D53 with TPR proteins. Values are means ±s.d. of three independent experiments. VP16, herpes simplex virus virion protein 16. b, Pull-down assay for GST–TPR2 and lysates of Act:D53-GFP calli in the absence or presence of 10 μM GR24. D53–GFP and GFP were detected with the monoclonal anti-GFP antibody.

Figure 5. A proposed model of D53 action

In the absence of SLs, D53 is stable and may recruit TPL/TPR proteins and repress downstream responses. In the presence of SLs, perception of SL leads to SCF^D3-mediated ubiquitination of D53 and its subsequent degradation by the proteasome system, which in turn releases the repression of downstream responses.