Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism

Nature volume 452, pages 755–758 (2008)Cite this article

Abstract

Pathogenic bacteria often use effector molecules to increase virulence. In most cases, the mode of action of effectors remains unknown. Strains of Pseudomonas syringae pv. syringae (Pss) secrete syringolin A (SylA), a product of a mixed non-ribosomal peptide/polyketide synthetase, in planta1. Here we identify SylA as a virulence factor because a SylA-negative mutant in Pss strain B728a obtained by gene disruption was markedly less virulent on its host, Phaseolus vulgaris (bean). We show that SylA irreversibly inhibits all three catalytic activities of eukaryotic proteasomes, thus adding proteasome inhibition to the repertoire of modes of action of virulence factors. The crystal structure of the yeast proteasome in complex with SylA revealed a novel mechanism of covalent binding to the catalytic subunits. Thus, SylA defines a new class of proteasome inhibitors that includes glidobactin A (GlbA), a structurally related compound from an unknown species of the order Burkholderiales2, for which we demonstrate a similar proteasome inhibition mechanism. As proteasome inhibitors are a promising class of anti-tumour agents, the discovery of a novel family of inhibitory natural products, which we refer to as syrbactins, may also have implications for the development of anti-cancer drugs3. Homologues of SylA and GlbA synthetase genes are found in some other pathogenic bacteria, including the human pathogen Burkholderia pseudomallei, the causative agent of melioidosis4. It is thus possible that these bacteria are capable of producing proteasome inhibitors of the syrbactin class.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Syringolin-negative mutant exhibits reduced virulence.
Figure 2: SylA inhibits the eukaryotic proteasome.
Figure 3: Structural basis for proteasome inhibition by syrbactins.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates have been deposited in the RCSB Protein Data Bank (http://www.rcsb.org/pdb) under the accession code 2ZCY (yeast 20S proteasome–syringolin A complex) and 3BDM (yeast 20S proteasome–glidobactin A complex).

References

  1. Wäspi, U., Blanc, D., Winkler, T., Ruedi, P. & Dudler, R. Syringolin, a novel peptide elicitor from Pseudomonas syringae pv. syringae that induces resistance to Pyricularia oryzae in rice. Mol. Plant Microbe Interact. 11, 727–733 (1998)

    Article  Google Scholar 

  2. Amrein, H. et al. Functional analysis of genes involved in the synthesis of syringolin A by Pseudomonas syringae pv. syringae B301D-R. Mol. Plant Microbe Interact. 17, 90–97 (2004)

    Article  CAS  Google Scholar 

  3. Borissenko, L. & Groll, M. 20S proteasome and its inhibitors: crystallographic knowledge for drug development. Chem. Rev. 107, 687–717 (2007)

    Article  CAS  Google Scholar 

  4. Schellenberg, B., Bigler, L. & Dudler, R. Identification of genes involved in the biosynthesis of the cytotoxic compound glidobactin from a soil bacterium. Environ. Microbiol. 9, 1640–1650 (2007)

    Article  CAS  Google Scholar 

  5. Hirano, S. S. & Upper, C. D. Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae – a pathogen, ice nucleus, and epiphyte. Microbiol. Mol. Biol. Rev. 64, 624–653 (2000)

    Article  CAS  Google Scholar 

  6. Michel, K., Abderhalden, O., Bruggmann, R. & Dudler, R. Transcriptional changes in powdery mildew infected wheat and Arabidopsis leaves undergoing syringolin-triggered hypersensitive cell death at infection sites. Plant Mol. Biol. 62, 561–578 (2006)

    Article  CAS  Google Scholar 

  7. Fleming, J. A. et al. Complementary whole-genome technologies reveal the cellular response to proteasome inhibition by PS-341. Proc. Natl Acad. Sci. USA 99, 1461–1466 (2002)

    Article  CAS  ADS  Google Scholar 

  8. Meiners, S. et al. Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of mammalian proteasomes. J. Biol. Chem. 278, 21517–21525 (2003)

    Article  CAS  Google Scholar 

  9. Colon-Carmona, A., You, R., Haimovitch-Gal, T. & Doerner, P. Spatio-temporal analysis of mitotic activity with a labile cyclin-GUS fusion protein. Plant J. 20, 503–508 (1999)

    Article  CAS  Google Scholar 

  10. Doerner, P., Jorgensen, J. E., You, R., Steppuhn, J. & Lamb, C. Control of root growth and development by cyclin expression. Nature 380, 520–523 (1996)

    Article  CAS  ADS  Google Scholar 

  11. King, R. W., Deshaies, R. J., Peters, J. M. & Kirschner, M. W. How proteolysis drives the cell cycle. Science 274, 1652–1659 (1996)

    Article  CAS  ADS  Google Scholar 

  12. Brukhin, V., Gheyselinck, J., Gagliardini, V., Genschik, P. & Grossniklaus, U. The RPN1 subunit of the 26S proteasome in Arabidopsis is essential for embryogenesis. Plant Cell 17, 2723–2737 (2005)

    Article  CAS  Google Scholar 

  13. Meng, L. H. et al. Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. Proc. Natl Acad. Sci. USA 96, 10403–10408 (1999)

    Article  CAS  ADS  Google Scholar 

  14. Groll, M. et al. Structure of 20S proteasome from yeast at 2.4 Å resolution. Nature 386, 463–471 (1997)

    Article  CAS  ADS  Google Scholar 

  15. Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  16. Oka, M. et al. Glidobactins A, B and C, new antitumor antibiotics. I. Production, isolation, chemical properties and biological activity. J. Antibiot. (Tokyo) 41, 1331–1337 (1988)

    Article  CAS  Google Scholar 

  17. Desveaux, D., Singer, A. U. & Dangl, J. L. Type III effector proteins: doppelgangers of bacterial virulence. Curr. Opin. Plant Biol. 9, 376–382 (2006)

    Article  CAS  Google Scholar 

  18. Galán, J. E. & Wolf-Watz, H. Protein delivery into eukaryotic cells by type III secretion machines. Nature 444, 567–573 (2006)

    Article  ADS  Google Scholar 

  19. Grant, S. R., Fisher, E. J., Chang, J. H., Mole, B. M. & Dangl, J. L. Subterfuge and manipulation: type III effector proteins of phytopathogenic bacteria. Annu. Rev. Microbiol. 60, 425–449 (2006)

    Article  CAS  Google Scholar 

  20. Coleman, C. S. et al. Syringolin A, a new plant elicitor from the phytopathogenic bacterium Pseudomonas syringae pv. syringae, inhibits the proliferation of neuroblastoma and ovarian cancer cells and induces apoptosis. Cell Prolif. 39, 599–609 (2006)

    Article  CAS  Google Scholar 

  21. Nomura, K. et al. A bacterial virulence protein suppresses host innate immunity to cause plant disease. Science 313, 220–223 (2006)

    Article  CAS  ADS  Google Scholar 

  22. Rosebrock, T. R. et al. A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity. Nature 448, 370–374 (2007)

    Article  CAS  ADS  Google Scholar 

  23. Dong, W. B., Nowara, D. & Schweizer, P. Protein polyubiquitination plays a role in basal host resistance of barley. Plant Cell 18, 3321–3331 (2006)

    Article  CAS  Google Scholar 

  24. Goritschnig, S., Zhang, Y. L. & Li, X. The ubiquitin pathway is required for innate immunity in Arabidopsis. Plant J. 49, 540–551 (2007)

    Article  CAS  Google Scholar 

  25. Adams, J. Proteasome inhibitors as new anticancer drugs. Curr. Opin. Oncol. 14, 628–634 (2002)

    Article  CAS  Google Scholar 

  26. Quinones, B., Dulla, G. & Lindow, S. E. Quorum sensing regulates exopolysaccharide production, motility, and virulence in Pseudomonas syringae. Mol. Plant Microbe Interact. 18, 682–693 (2005)

    Article  CAS  Google Scholar 

  27. Wäspi, U., Schweizer, P. & Dudler, R. Syringolin reprograms wheat to undergo hypersensitive cell death in a compatible interaction with powdery mildew. Plant Cell 13, 153–161 (2001)

    Article  Google Scholar 

  28. Fenteany, G. et al. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science 268, 726–731 (1995)

    Article  CAS  ADS  Google Scholar 

  29. Wallick, C. J. et al. Key role for p27(Kip1), retinoblastoma protein Rb, and MYCN in polyamine inhibitor-induced G(1) cell cycle arrest in MYCN-amplified human neuroblastoma cells. Oncogene 24, 5606–5618 (2005)

    Article  CAS  Google Scholar 

  30. Geerts, D. et al. Expression of PRA1 domain family, member 2 (PRAF2) in neuroblastoma: correlation with clinical features, cellular localization, and cerulenin-mediated apoptosis regulation. Clin. Cancer Res. 13, 6312–6319 (2007)

    Article  CAS  Google Scholar 

  31. Heiligtag, S. J., Bredehorst, R. & David, K. A. Key role of mitochondria in cerulenin-mediated apoptosis. Cell Death Differ. 9, 1017–1025 (2002)

    Article  CAS  Google Scholar 

  32. Rodrigues-Pousada, R. A. et al. The Arabidopsis 1-aminocyclopropane-1-carboxylate synthase gene-1 is expressed during early development. Plant Cell 5, 897–911 (1993)

    Article  CAS  Google Scholar 

  33. Groll, M. & Huber, R. Purification, crystallization and X-ray analysis of the yeast 20S proteasomes. Methods Enzymol. 398, 329–336 (2005)

    Article  CAS  Google Scholar 

  34. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  35. Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. A graphical user interface to the CCP4 program suite. Acta Crystallogr. D 59, 1131–1137 (2003)

    Article  Google Scholar 

  36. Turk, D. Improvement of a Programme for Molecular Graphics and Manipulation of Electron Densities and Its Application for Protein Structure Determination. PhD thesis, Technische Univ. München. (1992)

    Google Scholar 

Download references

Acknowledgements

We thank U. Grossniklaus and J. Celenza (Boston University) for the CycB1;1-GUS line. U. Grossniklaus and H. Gehring are thanked for reading the manuscript. We also thank D. Albert, H. Gehring, Z. Hasenkamp, R. Go, D. Koomoa, J. Molnar, A. Niewienda and C. Wallick for technical advice and assistance. We are grateful to L. Eberl for use of equipment. C.R.A was supported by a graduate student research assistantship from the Cell and Molecular Biology Graduate Program, University of Hawaii. Support by grants from the Swiss National Science Foundation to R.D. is acknowledged.

Author Contributions M.K., M.G., S.L., R.D. and A.S.B. designed experiments, analysed results and wrote the manuscript. R.D. constructed the sylA-negative mutant, T.K.P. performed the bean inoculation experiments, B.S. isolated SylA and GlbA, and performed in vitro proteasome and protease inhibition assays. M.G., M.K and R.H. performed and analysed crystallization experiments. C.R.A. performed the mammalian cell-based proteasome inhibition assays and immunoblot studies.

Author information

Author notes

  1. Michael Groll and Barbara Schellenberg: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Integrated Protein Science at the Department Chemie, Lehrstuhl für Biochemie, Technische Universität München, Lichtenbergstrasse 4, Garching D-85747, Germany,

    Michael Groll

  2. Zurich–Basel Plant Science Center, Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland,

    Barbara Schellenberg & Robert Dudler

  3. Cancer Research Center of Hawaii, University of Hawaii at Manoa, 1236 Lauhala Street, Honolulu, Hawaii 96813, USA,

    André S. Bachmann & Crystal R. Archer

  4. Cell and Molecular Biology Graduate Program, John A. Burns School of Medicine, University of Hawaii at Manoa, 651 Ilalo Street, Honolulu, Hawaii 96813, USA,

    André S. Bachmann & Crystal R. Archer

  5. Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany

    Robert Huber

  6. School of Biosciences, Cardiff University, Cardiff CF10 3US, UK

    Robert Huber

  7. Zentrum für medizinische Biotechnologie, Universität Duisburg-Essen, D-45117 Essen, Germany

    Robert Huber

  8. Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, California 94720-3102, USA,

    Tracy K. Powell & Steven Lindow

  9. Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany,

    Markus Kaiser

Authors
  1. Michael Groll
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Barbara Schellenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. André S. Bachmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Crystal R. Archer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Robert Huber
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tracy K. Powell
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Steven Lindow
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Markus Kaiser
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Robert Dudler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to André S. Bachmann, Markus Kaiser or Robert Dudler.

Supplementary information

The file contains Supplementary Figures 1 - 4 with Legends, Supplementary Table 1 and additional references.

The Supplementary Figure 1 shows results confirming that SylA inhibits proteasomes irreversibly. The Supplementary Figure 2 shows that SylA inhibits bean proteasomes in crude extracts of etiolated bean seedlings. The Supplementary Figure 3 exhibits root tips of Arabidopsis seedlings homozygous for a CyclinB1;1-uidA (GUS) reporter fusion gene stained for GUS activity, showing that SylA treatment leads to enhanced staining of dividing cells. This indicates that the fusion gene product, which is expressed only in the G2/M transition of the cell cycle and then is degraded by the proteasome, accumulates upon SylA treatment. The Supplementary Figure 4 shows the results of in vivo proteasome inhibition assays in cultured human cells using the known proteasome inhibitor epoxomicin and the apoptosis-inducing agent cerulenin as positive and negative control agents, respectively. The Supplementary Table 1 lists statistical parameters of crystallographic data collection and refinement. (PDF 484 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Groll, M., Schellenberg, B., Bachmann, A. et al. A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism. Nature 452, 755–758 (2008). https://doi.org/10.1038/nature06782

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature06782

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing