Article | Published:

A type III effector ADP-ribosylates RNA-binding proteins and quells plant immunity

Nature volume 447, pages 284288 (17 May 2007) | Download Citation


The bacterial plant pathogen Pseudomonas syringae injects effector proteins into host cells through a type III protein secretion system to cause disease. The enzymatic activities of most of P. syringae effectors and their targets remain obscure. Here we show that the type III effector HopU1 is a mono-ADP-ribosyltransferase (ADP-RT). HopU1 suppresses plant innate immunity in a manner dependent on its ADP-RT active site. The HopU1 substrates in Arabidopsis thaliana extracts were RNA-binding proteins that possess RNA-recognition motifs (RRMs). A. thaliana knockout lines defective in the glycine-rich RNA-binding protein GRP7 (also known as AtGRP7), a HopU1 substrate, were more susceptible than wild-type plants to P. syringae. The ADP-ribosylation of GRP7 by HopU1 required two arginines within the RRM, indicating that this modification may interfere with GRP7’s ability to bind RNA. Our results suggest a pathogenic strategy where the ADP-ribosylation of RNA-binding proteins quells host immunity by affecting RNA metabolism and the plant defence transcriptome.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    , & Type III protein secretion mechanism in mammalian and plant pathogens. Biochim. Biophys. Acta 1694, 181–206 (2004)

  2. 2.

    & Protein delivery into eukaryotic cells by type III secretion machines. Nature 444, 567–573 (2006)

  3. 3.

    & The bacterial injection kit: type III secretion systems. Ann. Med. 37, 234–249 (2005)

  4. 4.

    , & Bacterial elicitation and evasion of plant innate immunity. Nature Rev. Mol. Cell Biol. 7, 601–611 (2006)

  5. 5.

    & Disabling surveillance: bacterial type III secretion system effectors that suppress innate immunity. Cell. Microbiol. 6, 1027–1040 (2004)

  6. 6.

    New insights to the function of phytopathogenic bacterial type III effectors in plants. Annu. Rev. Plant Biol. 56, 509–531 (2005)

  7. 7.

    & Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4. Cell 112, 369–377 (2003)

  8. 8.

    , , , & Arabidopsis RIN4 is a target of the type III virulence effector AvrRpt2 and modulates RPS2-mediated resistance. Cell 112, 379–389 (2003)

  9. 9.

    et al. Cleavage of Arabidopsis PBS1 by a bacterial type III effector. Science 301, 1230–1233 (2003)

  10. 10.

    , , & HopPtoN is a Pseudomonas syringae Hrp (type III secretion system) cysteine protease effector that suppresses pathogen-induced necrosis associated with both compatible and incompatible plant interactions. Mol. Microbiol. 54, 353–365 (2004)

  11. 11.

    et al. A translocated protein tyrosine phosphatase of Pseudomonas syringae pv. tomato DC3000 modulates plant defence response to infection. Mol. Microbiol. 49, 389–400 (2003)

  12. 12.

    , , , & The Pseudomonas syringae type III-secreted protein HopPtoD2 possesses protein tyrosine phosphatase activity and suppresses programmed cell death in plants. Mol. Microbiol. 49, 377–387 (2003)

  13. 13.

    , , & A bacterial inhibitor of host programmed cell death defenses is an E3 ubiquitin ligase. Science 311, 222–226 (2006)

  14. 14.

    , , , & Subterfuge and manipulation: Type III effector proteins of phytopathogenic bacteria. Annu. Rev. Microbiol. 60, 425–449 (2006)

  15. 15.

    et al. Closing the circle on the discovery of genes encoding Hrp regulon members and type III secretion system effectors in the genomes of three model Pseudomonas syringae strains. Mol. Plant Microbe Interact. 19, 1151–1158 (2006)

  16. 16.

    et al. A functional screen for the type III (Hrp) secretome of the plant pathogen Pseudomonas syringae. Science 295, 1722–1726 (2002)

  17. 17.

    et al. Genomewide identification of proteins secreted by the Hrp type III protein secretion system of Pseudomonas syringae pv. tomato DC3000. Proc. Natl Acad. Sci. USA 99, 7652–7657 (2002)

  18. 18.

    , , & Stealth and mimicry by deadly bacterial toxins. Trends Biochem. Sci. 31, 123–133 (2006)

  19. 19.

    & Pseudomonas aeruginosa ExoS and ExoT. Rev. Physiol. Biochem. Pharmacol. 152, 79–92 (2004)

  20. 20.

    & Functional aspects of protein mono-ADP-ribosylation. EMBO J. 22, 1953–1958 (2003)

  21. 21.

    et al. Identification of Pseudomonas syringae type III effectors that suppress programmed cell death in plants and yeast. Plant J. 37, 554–565 (2004)

  22. 22.

    , , & Innate immunity in plants and animals: striking similarities and obvious differences. Immunol. Rev. 198, 249–266 (2004)

  23. 23.

    , & Pathogen recognition and innate immunity. Cell 124, 783–801 (2006)

  24. 24.

    & The plant immune system. Nature 444, 323–329 (2006)

  25. 25.

    , & A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana. Plant J. 18, 277–284 (1999)

  26. 26.

    , , & Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean. Plant Cell 3, 49–59 (1991)

  27. 27.

    , & Three types of nuclear genes encoding chloroplast RNA-binding proteins (cp29, cp31 and cp33) are present in Arabidopsis thaliana: presence of cp31 in chloroplasts and its homologue in nuclei/cytoplasms. Plant Mol. Biol. 27, 529–539 (1995)

  28. 28.

    & Two cDNAs from Arabidopsis thaliana encode putative RNA binding proteins containing glycine-rich domains. Plant Mol. Biol. 21, 695–699 (1993)

  29. 29.

    & Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615–621 (1994)

  30. 30.

    , , , & Identification of molecular contacts between the U1 A small nuclear ribonucleoprotein and U1 RNA. EMBO J. 10, 3447–3456 (1991)

  31. 31.

    et al. A gene induced by the plant hormone abscisic acid in response to water stress encodes a glycine-rich protein. Nature 334, 262–264 (1988)

  32. 32.

    et al. A glycine-rich RNA-binding protein gene is differentially expressed during acute hypersensitive response following Tobacco Mosaic Virus infection in tobacco. Plant Mol. Biol. 37, 571–576 (1998)

  33. 33.

    , , & AtGRP7, a nuclear RNA-binding protein as a component of a circadian-regulated negative feedback loop in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 94, 8515–8520 (1997)

  34. 34.

    , , , & The circadian clock regulated RNA-binding protein AtGRP7 autoregulates its expression by influencing alternative splicing of its own pre-mRNA. Plant J. 33, 361–371 (2003)

  35. 35.

    , & The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J. 272, 2118–2131 (2005)

  36. 36.

    et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657 (2003)

  37. 37.

    et al. Shigella effector IpaH9.8 binds to a splicing factor U2AF35 to modulate host immune responses. Biochem. Biophys. Res. Commun. 333, 531–539 (2005)

  38. 38.

    et al. Pseudomonas syringae type III chaperones ShcO1, ShcS1, and ShcS2 facilitate translocation of their cognate effectors and can substitute for each other in the secretion of HopO1-1. J. Bacteriol. 187, 4257–4269 (2005)

  39. 39.

    , & In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris Ser. III 316, 1194–1199 (1993)

  40. 40.

    & Identification of SpyA, a novel ADP-ribosyltransferase of Streptococcus pyogenes. Mol. Microbiol. 54, 89–98 (2004)

  41. 41.

    & Pseudomonas aeruginosa ExoT ADP-ribosylates CT10 regulator of kinase (Crk) proteins. J. Biol. Chem. 278, 32794–32800 (2003)

  42. 42.

    & The ARTT motif and a unified structural understanding of substrate recognition in ADP-ribosylating bacterial toxins and eukaryotic ADP-ribosyltransferases. Int. J. Med. Microbiol. 291, 523–529 (2002)

Download references


We thank the members of the Alfano laboratory for many fruitful discussions, Y. Zhou and C. Elowsky for technical assistance with confocal microscopy, T. Clemente and S. Sato for constructing transgenic plants, G. Li and C. Bryan for assistance in the identification of the A. thaliana grp7 mutants, A. Collmer for reviewing the manuscript, P. Seitz for assistance in plasmid constructions, and J. T. Barbieri for help initiating the ADP-RT assays in our laboratory. We are grateful to the Ohio State University Arabidopsis Biological Resource Center, the Salk Institute Genomic Analysis Laboratory, and the Arabidopsis research community for providing the Arabidopsis SALK lines used in this study. This research was supported by grants from the National Science Foundation and the National Institutes of Health, and funds from the Plant Science Initiative at the University of Nebraska to J.R.A, and a grant from the German Research Council to D.S.

Author Contributions Z.Q.F. constructed the DC3000 ΔhopU1 mutant, made the transgenic HopU1–HA-expressing A. thaliana plants, and performed the experiments in Figs 1a, b; 2a, b; 3; 4a, d and Supplementary Figs 1, 2, 4, 5b–d and 6d; M.G. identified the homozygous A. thaliana grp7 mutant plants, cloned the HopU1–His substrate complementary DNAs, and performed the experiments in Figs 2c, d and 5 and Supplementary Figs 3, 4, 6a–c and 7; B.-r.J. performed the experiments in Figs 1c and 4c and Supplementary Fig. 5a, d; and F.T. provided technical support for several experiments. T.E.E. helped direct the identification of the HopU1–His substrates; R.L.C. performed the mass spectrometry and peptide database searches; D.S. provided the anti-GRP antibody, plasmids pGRP7-Gly and pGRP7-RRM, and insights on RNA-binding proteins; J.R.A. helped design the experimental plan, designed Supplementary Fig. 8, and was the primary writer of the paper. All of the authors discussed the results and commented on the paper.

Author information

Author notes

    • Zheng Qing Fu
    •  & Ming Guo

    These authors contributed equally to this work.


  1. Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68588-0660, USA

    • Zheng Qing Fu
    • , Ming Guo
    • , Byeong-ryool Jeong
    • , Fang Tian
    •  & James R. Alfano
  2. School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588-0118, USA

    • Fang Tian
    •  & Thomas E. Elthon
  3. Department of Agronomy and Horticulture, University of Nebraska, Lincoln, Nebraska 68588-0915, USA

    • Thomas E. Elthon
  4. Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA

    • Ronald L. Cerny
  5. Molecular Cell Physiology, University of Bielefeld, 33501 Bielefeld, Germany

    • Dorothee Staiger


  1. Search for Zheng Qing Fu in:

  2. Search for Ming Guo in:

  3. Search for Byeong-ryool Jeong in:

  4. Search for Fang Tian in:

  5. Search for Thomas E. Elthon in:

  6. Search for Ronald L. Cerny in:

  7. Search for Dorothee Staiger in:

  8. Search for James R. Alfano in:

Competing interests

Reprints and permissions information is available at The authors declare no competing financial interests.

Corresponding author

Correspondence to James R. Alfano.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Methods, Supplementary Tables 1-3, Supplementary Figures 1-8 with Legends, Supplementary Notes and additional references. Supplementary Methods section describes materials and procedures. The Supplementary Tables contain a list of the identified HopU1 substrates (Supplementary Table 1), bacterial strains and plasmids used in this study (Supplementary Table 2), and the nucleotide sequences of the primers used in this study. The Supplementary Figures show that HopU1 is injected into plant cells by the type III secretion system of P. syringae and that a hopU1 mutant is reduced in virulence (Supplementary Fig. 1); that HopU1 suppresses ion leakage in plants (Supplementary Fig. 2); that HopU1-expressing plants are altered in the AvrRpt2-dependent HR (Supplementary Fig. 3); representative two-dimensional PAGE gels that led to the identification of HopU1 substrates (Supplementary Fig. 4); localization of HopU1 and HopU1 substrates in plant cells (Supplementary Fig. 5); data showing that an A. thaliana Col-0 knock-out mutant is homozygous for the T-DNA insert in the AtGRP7 locus (Supplementary Fig. 6); Similar data for an independent Atgrp7 mutant and pathogenicity-related phenotypes (Supplementary Fig. 7); and a proposed model of suppression of plant innate immunity by HopU1.

About this article

Publication history





Further reading


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.