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.

S-nitrosylation of NADPH oxidase regulates cell death in plant immunity

This article has been updated

Abstract

Changes in redox status are a conspicuous feature of immune responses in a variety of eukaryotes1,2, but the associated signalling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide3,4 and a parallel accumulation of reactive oxygen intermediates, the latter generated by NADPH oxidases related to those responsible for the pathogen-activated respiratory burst in phagocytes5. Both nitric oxide and reactive oxygen intermediates have been implicated in controlling the hypersensitive response, a programmed execution of plant cells at sites of attempted infection3,5,6. However, the molecular mechanisms that underpin their function and coordinate their synthesis are unknown. Here we show genetic evidence that increases in cysteine thiols modified using nitric oxide, termed S-nitrosothiols, facilitate the hypersensitive response in the absence of the cell death agonist salicylic acid and the synthesis of reactive oxygen intermediates. Surprisingly, when concentrations of S-nitrosothiols were high, nitric oxide function also governed a negative feedback loop limiting the hypersensitive response, mediated by S-nitrosylation of the NADPH oxidase, AtRBOHD, at Cys 890, abolishing its ability to synthesize reactive oxygen intermediates. Accordingly, mutation of Cys 890 compromised S-nitrosothiol-mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: SNOs positively regulate cell death by hypersensitive response.
Figure 2: Increased SNO concentrations blunt NADPH oxidase activity and reduce ROI accumulation.
Figure 3: S -nitrosylation of AtRBOHD.
Figure 4: The AtRBOHD Cys890Ala mutant shows increased activity during the defence response, amplifying ROI accumulation and cell death development.

Change history

  • 13 October 2011

    The alignment of lane labelling was corrected in Fig. 3c.

References

  1. 1

    MacMicking, J. D. et al. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc. Natl Acad. Sci. USA 94, 5243–5248 (1997)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Tada, Y. et al. Plant immunity requires conformational changes of NPR1 via S-nitrosylation and thioredoxins. Science 321, 952–956 (2008)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Delledonne, M. Xia, Y. Dixon, R. A. & Lamb, C. J. Nitric oxide functions as signal in plant disease resistance. Nature 394, 585–588 (1998)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Durner, J., Wendehenne, D. & Klessig, D. F. Defense gene induction in tobacco by nitric oxide, cyclic GMP and cyclic ADP ribose. Proc. Natl Acad. Sci. USA 95, 10328–10333 (1998)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Torres, M. A., Dangl, J. L. & Jones, J. D. Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc. Natl Acad. Sci. USA 99, 517–522 (2002)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Delledonne, M., Zeier, J., Marocco, A. & Lamb, C. Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response. Proc. Natl Acad. Sci. USA 98, 13454–13459 (2001)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Leitner, M., Vandelle, E., Gaupels, F., Bellin, D. & Delledonne, M. NO signals in the haze: nitric oxide signalling in plant defence. Curr. Opin. Plant Biol. 12, 451–458 (2009)

    CAS  Article  Google Scholar 

  8. 8

    Feechan, A. et al. A central role for S-nitrosothiols in plant disease resistance. Proc. Natl Acad. Sci. USA 102, 8054–8059 (2005)

    ADS  CAS  Article  Google Scholar 

  9. 9

    He, Y. et al. Nitric oxide represses the Arabidopsis floral transition. Science 305, 1968–1971 (2004)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Grant, M. R. et al. Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance. Science 269, 843–846 (1995)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Gassmann, W., Hinsch, M. E. & Staskawicz, B. J. The Arabidopsis RPS4 bacterial-resistance gene is a member of the TIR-NBS-LRR family of disease resistance genes. Plant J. 20, 265–277 (1999)

    CAS  Article  Google Scholar 

  12. 12

    Liu, L. et al. Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock. Cell 116, 617–628 (2004)

    CAS  Article  Google Scholar 

  13. 13

    Shirasu, K., Nakajima, H., Rajasekhar, V. K., Dixon, R. A. & Lamb, C. J. Salicylic acid potentiates an agonist-dependent gain control that amplifies pathogen signals in the activation of defense mechanisms. Plant Cell 9, 261–270 (1997)

    CAS  Article  Google Scholar 

  14. 14

    Wildermuth, M. C., Dewdney, J., Wu, G. & Ausubel, F. M. Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414, 562–565 (2001)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Yu, I. C., Parker, J. & Bent, A. F. Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant. Proc. Natl Acad. Sci. USA 95, 7819–7824 (1998)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Holub, E. B. Beynon, J. L. & Crute, I. R. Phenotypic and genotypic characterization of interactions between isolates of Peronospora parasitica and accessions of Arabidopsis thaliana . Mol. Plant Microbe Interact. 7, 223–239 (1994)

    CAS  Article  Google Scholar 

  17. 17

    Keller, H. et al. Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance. Plant Cell 11, 223–236 (1999)

    CAS  Article  Google Scholar 

  18. 18

    Nawrath, C. & Metraux, J. P. Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation. Plant Cell 11, 1393–1404 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Wang, Y.-Q. et al. S-nitrosylation of AtSABP3 antagonises the expression of plant immunity. J. Biol. Chem. 284, 2131–2137 (2009)

    CAS  Article  Google Scholar 

  20. 20

    Romero-Puertas, M. C. et al. S-nitrosylation of peroxiredoxin II E promotes peroxynitrite-mediated tyrosine nitration. Plant Cell 19, 4120–4130 (2007)

    CAS  Article  Google Scholar 

  21. 21

    Lindermayr, C., Sell, S., Müller, B., Leister, D. & Durner, J. Redox regulation of the NPR1–TGA1 system of Arabidopsis thaliana by nitric oxide. Plant Cell 22, 2894–2907 (2010)

    CAS  Article  Google Scholar 

  22. 22

    Jaffrey, S. R., Erdjument-Bromge, H., Ferris, C. D., Tempst, P. & Snyder, S. H. Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nature Cell Biol. 3, 193–197 (2001)

    CAS  Article  Google Scholar 

  23. 23

    Selemidis, S., Dusting, G. J., Peshavariya, H., Kemp-Harper, B. K. & Drummond, G. R. Nitric oxide suppresses NADPH oxidase-dependent superoxide production by S-nitrosylation in human endothelial cells. Cardiovasc. Res. 75, 349–358 (2007)

    CAS  Article  Google Scholar 

  24. 24

    Ingelman, M., Bianchi, V. & Eklund, H. The three-dimensional structure of flavodoxin reductase from Escherichia coli at 1.7 Å resolution. J. Mol. Biol. 268, 147–157 (1997)

    CAS  Article  Google Scholar 

  25. 25

    Zhen, L., Yu, L. & Dinauer, M. C. Probing the role of the carboxyl terminus of the gp91 phox subunit of neutrophil flavocytochrome b 558 using site-directed mutagenesis. J. Biol. Chem. 273, 6575–6581 (1998)

    CAS  Article  Google Scholar 

  26. 26

    Matthews, J. R. et al. Inhibition of NF-κβ DNA binding by nitric oxide. Nucleic Acids Res. 24, 2236–2242 (1996)

    CAS  Article  Google Scholar 

  27. 27

    Mannick, J. B. et al. Fas-induced caspase denitrosylation. Science 284, 651–654 (1999)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Yun, B.-W. et al. Loss of actin cytoskeletal function and EDS1 activity, in combination, severely compromises non-host resistance in Arabidopsis against wheat powdery mildew. Plant J. 34, 768–777 (2003)

    CAS  Article  Google Scholar 

  29. 29

    Aboul-Soud, M. A. M., Cook, K. & Loake, G. J. Measurement of salicylic acid by a high-performance liquid chromatography procedure based on ion-exchange. Chromatographia 59, 129–133 (2004)

    CAS  Google Scholar 

  30. 30

    Liu, L. et al. Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock. Cell 116, 617–628 (2004)

    CAS  Article  Google Scholar 

  31. 31

    Foissner, I., Wendehenne, D., Langebartels, C. & Durner, J. In vivo imaging of an elicitor-induced nitric oxide burst in tobacco. Plant J. 23, 817–824 (2000)

    CAS  Article  Google Scholar 

  32. 32

    Whalen, M. C., Innes, R. W., Bent, A. F. & Staskawicz, B. J. Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean. Plant Cell 3, 49–59 (1991)

    CAS  Article  Google Scholar 

  33. 33

    Dellagi, A., Brisset, M.-N., Jean-Pierre Paulin, J.-P. & Expert, D. Dual role of desferrioxamine in Erwinia amylovora pathogenicity. Mol. Plant Microbe Interact. 11, 734–742 (1998)

    CAS  Article  Google Scholar 

  34. 34

    Liu, Q., Li, M., Leibham, D., Cortez, D. & Elledge, S. The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr. Biol. 8, 1300–1309 (1998)

    CAS  Article  Google Scholar 

  35. 35

    Sagi, M. & Fluhr, R. Superoxide production by plant homologues of the gp91phox NADPH oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection. Plant Physiol. 126, 1281–1290 (2001)

    CAS  Article  Google Scholar 

  36. 36

    Chen, Y. Y., Huang, Y. F., Khoo, K. H. & Meng, T. C. Mass spectrometry-based analyses for identifying and characterizing S-nitrosylation of protein tyrosine phosphatases. Methods 42, 243–249 (2007)

    CAS  Article  Google Scholar 

  37. 37

    Shen, A. L. &. Kasper, C. B. Differential contribution of NADPH-cytochrome P450 oxidoreductase FAD binding site residues to flavin binding and catalysis. J. Biol. Chem. 275, 41087–41091 (2000)

    CAS  Article  Google Scholar 

  38. 38

    Kelley, L. A. & Sternberg, M. J. Protein structure prediction on the Web: a case study using the Phyre server. Nature Protocols 4, 363–371 (2009)

    CAS  Article  Google Scholar 

  39. 39

    Guex, N. & Peitsch, M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997)

    CAS  Article  Google Scholar 

  40. 40

    Cole, C., Barber, J. D. & Barton, G. J. The Jpred 3 secondary structure prediction server. Nucleic Acids Res. 36, W197–W201 (2008)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We would like to acknowledge R. Innes and W. Gassmann for Pst DC3000 strains expressing either avrB or avrRps4, respectively. Arabidopsis transfer DNA insertion mutants were obtained from SAIL (Syngeneta) populations. We thank M. Tör for the H. arabidopsidis isolate Emwa1, and K. Kanchanawatee for the Drosophila cDNA clone and associated mutant. A.F. was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) CASE studentship. B.-W.Y. and E.K. were funded by BBSRC grant BB/D011809/1 awarded to G.J.L. J.W.M. received a grant from the Physical Sciences Research Council (EPSRC). M. Yu was the recipient of a Darwin Trust Scholarship. T.L.B. was supported by BBSRC and EPSRC grant BB/D019621/1. N.B.B.S. and M. Yin were funded by a Ministry of Education Malaysia scholarship and a Torrance Scholarship, respectively.

Author information

Affiliations

Authors

Contributions

G.J.L. designed the research and wrote the paper, and with B.-W.Y. planned experiments and analyses. B.-W.Y. conducted the majority of experiments. A.F., M. Yin, N.B.B.S., J.-G.K., J.W.M., M. Yu, E.K. and T.L.B. conducted experiments. S.H.S. generated and interrogated three-dimensional models. J.A.P. was the industrial supervisor of A.F. All authors, especially B.-W.Y. and S.S., commented on the manuscript.

Corresponding author

Correspondence to Gary J. Loake.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

The file contains Supplementary Figures 1-20 with legends. (PDF 2738 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yun, BW., Feechan, A., Yin, M. et al. S-nitrosylation of NADPH oxidase regulates cell death in plant immunity. Nature 478, 264–268 (2011). https://doi.org/10.1038/nature10427

Download citation

Further reading

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

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