Abstract

The phytohormone auxin indole-3-acetic acid (IAA) regulates nearly all aspects of plant growth and development. Despite substantial progress in our understanding of auxin biology, delineating specific auxin response remains a major challenge. Auxin regulates transcriptional response via its receptors, TIR1 and AFB F-box proteins. Here we report an engineered, orthogonal auxin–TIR1 receptor pair, developed through a bump-and-hole strategy, that triggers auxin signaling without interfering with endogenous auxin or TIR1/AFBs. A synthetic, convex IAA (cvxIAA) hijacked the downstream auxin signaling in vivo both at the transcriptomic level and in specific developmental contexts, only in the presence of a complementary, concave TIR1 (ccvTIR1) receptor. Harnessing the cvxIAA–ccvTIR1 system, we provide conclusive evidence for the role of the TIR1-mediated pathway in auxin-induced seedling acid growth. The cvxIAA–ccvTIR1 system serves as a powerful tool for solving outstanding questions in auxin biology and for precise manipulation of auxin-mediated processes as a controllable switch.

  • Compound

    2-(5-(3-methoxyphenyl)-1H-indol-3-yl)acetic acid

  • Compound

    2-(5-(2-methoxyphenyl)-1H-indol-3-yl)acetic acid

  • Compound

    2-(5-(4-methoxyphenyl)-1H-indol-3-yl)acetic acid

  • Compound

    2-(5-(4-(trifluoromethyl)phenyl)-1H-indol-3-yl)acetic acid

  • Compound

    2-(5-([1,1'-biphenyl]-4-yl)-1H-indol-3-yl)acetic acid

  • Compound

    2-(5-phenyl-1H-indol-3-yl)acetic acid

  • Compound

    2-(5-(naphthalen-2-yl)-1H-indol-3-yl)acetic acid

  • Compound

    2-(naphthalen-1-yl)acetic acid

  • Compound

    2-(2,4-dichlorophenoxy)acetic acid

  • Compound

    2-(4-phenyl-1H-indol-3-yl)acetic acid

  • Compound

    methyl 2-(5-(2-methoxyphenyl)-1H-indol-3-yl)acetate

  • Compound

    methyl 2-(5-(3-methoxyphenyl)-1H-indol-3-yl)acetate

  • Compound

    methyl 2-(5-(4-methoxyphenyl)-1H-indol-3-yl)acetate

  • Compound

    methyl 2-(5-(naphthalen-2-yl)-1H-indol-3-yl)acetate

  • Compound

    methyl 2-(5-(4-(trifluoromethyl)phenyl)-1H-indol-3-yl)acetate

  • Compound

    methyl 2-(5-([1,1'-biphenyl]-4-yl)-1H-indol-3-yl)acetate

  • Compound

    2-(4-bromo-1H-indol-3-yl)-2-oxoacetic acid

  • Compound

    2-(4-bromo-1H-indol-3-yl)acetic acid

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Auxin: regulation, action, and interaction. Ann. Bot. 95, 707–735 (2005).

  2. 2.

    , & Auxin in action: signalling, transport and the control of plant growth and development. Nat. Rev. Mol. Cell Biol. 7, 847–859 (2006).

  3. 3.

    , & The F-box protein TIR1 is an auxin receptor. Nature 435, 441–445 (2005).

  4. 4.

    et al. Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana. Genes Dev. 13, 1678–1691 (1999).

  5. 5.

    & The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature 435, 446–451 (2005).

  6. 6.

    et al. Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446, 640–645 (2007).

  7. 7.

    , , , & Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Nature 414, 271–276 (2001).

  8. 8.

    et al. A combinatorial TIR1/AFB-Aux/IAA co-receptor system for differential sensing of auxin. Nat. Chem. Biol. 8, 477–485 (2012).

  9. 9.

    , , & Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates. Proc. Natl. Acad. Sci. USA 94, 3565–3570 (1997).

  10. 10.

    et al. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407, 395–401 (2000).

  11. 11.

    et al. A bump-and-hole approach to engineer controlled selectivity of BET bromodomain chemical probes. Science 346, 638–641 (2014).

  12. 12.

    et al. Auxin-mediated transcriptional system with a minimal set of components is critical for morphogenesis through the life cycle in Marchantia polymorpha. PLoS Genet. 11, e1005084 (2015).

  13. 13.

    , , & Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway. Curr. Biol. 20, 1907–1912 (2010).

  14. 14.

    et al. Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468, 400–405 (2010).

  15. 15.

    et al. Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426, 147–153 (2003).

  16. 16.

    et al. The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature 433, 39–44 (2005).

  17. 17.

    et al. Lateral root development in Arabidopsis: fifty shades of auxin. Trends Plant Sci. 18, 450–458 (2013).

  18. 18.

    , , & Lateral root formation is blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis. Plant J. 29, 153–168 (2002).

  19. 19.

    , & Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis. Plant Physiol. 159, 632–641 (2012).

  20. 20.

    Role of the plasma membrane H+-ATPase in auxin-induced elongation growth: historical and new aspects. J. Plant Res. 116, 483–505 (2003).

  21. 21.

    , , & Rapid auxin-induced cell expansion and gene expression: a four-decade-old question revisited. Plant Physiol. 152, 1183–1185 (2010).

  22. 22.

    et al. Complex regulation of the TIR1/AFB family of auxin receptors. Proc. Natl. Acad. Sci. USA 106, 22540–22545 (2009).

  23. 23.

    et al. Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis. Plant Physiol. 142, 542–552 (2006).

  24. 24.

    , & TIR1/AFB-Aux/IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls. eLife 5, e19048 (2016).

  25. 25.

    et al. The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion. Plant J. 70, 978–990 (2012).

  26. 26.

    et al. SAUR inhibition of PP2C-D phosphatases activates plasma membrane H+-ATPases to promote cell expansion in Arabidopsis. Plant Cell 26, 2129–2142 (2014).

  27. 27.

    & Enhancement of wall loosening and elongation by acid solutions. Plant Physiol. 46, 250–253 (1970).

  28. 28.

    et al. Constitutive expression of Arabidopsis SMALL AUXIN UP RNA19 (SAUR19) in tomato confers auxin-independent hypocotyl elongation. Plant Physiol. 173, 1453–1462 (2017).

  29. 29.

    , , , & An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6, 917–922 (2009).

  30. 30.

    , , & Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells. Proc. Natl. Acad. Sci. USA 109, E3350–E3357 (2012).

  31. 31.

    , , & The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans. Development 142, 4374–4384 (2015).

  32. 32.

    et al. Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell 169, 930–944 (2017).

  33. 33.

    , , & Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Rep. 15, 210–218 (2016).

  34. 34.

    et al. Mutations in the TIR1 auxin receptor that increase affinity for auxin/indole-3-acetic acid proteins result in auxin hypersensitivity. Plant Physiol. 162, 295–303 (2013).

  35. 35.

    et al. Untethering the TIR1 auxin receptor from the SCF complex increases its stability and inhibits auxin response. Nat. Plants 1, 14030 (2015).

  36. 36.

    & Plant synthetic biology for molecular engineering of signalling and development. Nat. Plants 2, 16010 (2016).

  37. 37.

    , , & Dose-response analysis using R. PLoS One 10, e0146021 (2015).

  38. 38.

    et al. Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115, 591–602 (2003).

  39. 39.

    et al. The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast grr1p. Genes Dev. 12, 198–207 (1998).

  40. 40.

    et al. New auxin analogs with growth-promoting effects in intact plants reveal a chemical strategy to improve hormone delivery. Proc. Natl. Acad. Sci. USA 105, 15190–15195 (2008).

  41. 41.

    , & Correlation of two-hybrid affinity data with in vitro measurements. Mol. Cell. Biol. 15, 5820–5829 (1995).

  42. 42.

    et al. Biochemical characterization of in vitro phosphorylation and dephosphorylation of the plasma membrane H+-ATPase. Plant Cell Physiol. 51, 1186–1196 (2010).

Download references

Acknowledgements

We thank M. Estelle (University of California, San Diego) for inspiring us and providing tir1-1 afb2-3 seeds; H. Fukaki (Kobe University) for slr-1 seeds; Yeast Genetics Resource Center and Y. Tsuchiya (Nagoya University) for Y2H vectors; Peptide/protein center at WPI-ITbM for biotinyl peptides; J. Bode for his visionary comments during the conceptualization of the project; J. Nemhauser and R. Cleland for commenting on the manuscript. K.U.T. dedicates this manuscript to R. Cleland, who proposed the acid growth theory in 1970, for his continued mentorship at the Univ. Washington. This work was funded by MEXT/JSPS KAKENHI (JP26291057, JP16H01237 and JP17H06476 to K.U.T.; JP16H01462, JP17H03695 and JP17KT0017 to N.U.; JP26440140 to K.T.; JP15H05956 to T.K.; JP17H06350 to S.H.; JP16H01472 and S1511023 to S.K.), Japan Science and Technology Agency (PRESTO, JPMJPR15Q9 to S.H.; ERATO, JPMJER1302 to K.I.), Howard Hughes Medical Institute (HHMI) and Gordon and Betty Moore Foundation (GBMF3035) to K.UT. S.H. is a JST PRESTO investigator, K.I. is a JST ERATO investigator and K.U.T. is an HHMI-GBMF Investigator and University Washington Endowed Distinguished Professor of Biology.

Author information

Author notes

    • Naoyuki Uchida
    •  & Koji Takahashi

    These authors contributed equally to this work.

Affiliations

  1. Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya, Japan.

    • Naoyuki Uchida
    • , Koji Takahashi
    • , Rie Iwasaki
    • , Hua Zhang
    • , Toshinori Kinoshita
    • , Kenichiro Itami
    • , Shinya Hagihara
    •  & Keiko U Torii
  2. Graduate School of Science, Nagoya University, Chikusa, Nagoya, Japan.

    • Naoyuki Uchida
    • , Koji Takahashi
    • , Ryotaro Yamada
    • , Masahiko Yoshimura
    • , Mika Nomoto
    • , Toshinori Kinoshita
    • , Kenichiro Itami
    • , Shinya Hagihara
    •  & Keiko U Torii
  3. Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.

    • Takaho A Endo
  4. Department of Bioresource and Environmental Sciences, Kyoto Sangyo University, Kyoto, Japan.

    • Seisuke Kimura
  5. Center for Gene Research, Nagoya University, Chikusa, Nagoya, Japan.

    • Yasuomi Tada
  6. PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan.

    • Shinya Hagihara
  7. Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA.

    • Keiko U Torii
  8. Department of Biology, University of Washington, Seattle, Washington, USA.

    • Keiko U Torii

Authors

  1. Search for Naoyuki Uchida in:

  2. Search for Koji Takahashi in:

  3. Search for Rie Iwasaki in:

  4. Search for Ryotaro Yamada in:

  5. Search for Masahiko Yoshimura in:

  6. Search for Takaho A Endo in:

  7. Search for Seisuke Kimura in:

  8. Search for Hua Zhang in:

  9. Search for Mika Nomoto in:

  10. Search for Yasuomi Tada in:

  11. Search for Toshinori Kinoshita in:

  12. Search for Kenichiro Itami in:

  13. Search for Shinya Hagihara in:

  14. Search for Keiko U Torii in:

Contributions

K.U.T. conceived the project; N.U., S.H., K.T. and K.U.T. designed research; N.U. and R.I. performed molecular cloning, yeast two-hybrid assays, and transgenic plants generation; N.U., R.I., and K.T. conducted qRT-PCR and phenotypic characterization; S.H., H.Z., R.Y., and M.Y. performed synthesis and NMR analyses of auxin analogs; K.T. performed biochemical analyses; S.K. performed next generation sequencing, and S.K., N.U. and T.A.E. performed bioinformatics; T.A.E. developed the Bobbin Dendrogram Plot Generator; M.N. and Y.T. provided reagents; N.U., S.H., K.T., T.K., K.I., and K.U.T. analyzed data; K.U.T. performed R-drc analysis; K.U.T., N.U., K.T., and S.H. wrote the manuscript; all authors read and approved the manuscript.

Competing interests

The cvxIAA–ccvTIR1 system reported here has been filed for a US provisional patent (No. 62/468642) in which N.U., R.I., K.I., S.H., and K.U.T. appear as inventors.

Corresponding authors

Correspondence to Shinya Hagihara or Keiko U Torii.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Tables 1–3, Supplementary Figures 1–14

  2. 2.

    Reporting Summary

  3. 3.

    Supplementary Note 1

    Synthetic procedures

Excel files

  1. 1.

    Supplementary Data Set 1

    Differentially expressed genes in wild type, 35S::TIR1 and 35S::ccvTIR1 by IAA and cvxIAA treatments

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nchembio.2555

Further reading