1887

Abstract

is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects more than 70 effector proteins into host plant cells via the Hrp type III secretion system to cause a successful infection. However, the function of these effectors in plant cells, especially in the suppression of plant immunity, remains largely unknown. In this study, we characterized two effectors, RipAW and RipAR, which share homology with the IpaH family of effectors from animal and plant pathogenic bacteria, that have a novel E3 ubiquitin ligase (NEL) domain. Recombinant RipAW and RipAR show E3 ubiquitin ligase activity . RipAW and RipAR localized to the cytoplasm of plant cells and significantly suppressed pattern-triggered immunity (PTI) responses such as the production of reactive oxygen species and the expression of defence-related genes when expressed in leaves of . Mutation in the conserved cysteine residue in the NEL domain of RipAW completely abolished the E3 ubiquitin ligase activity and the ability to suppress PTI responses in plant leaves. These results indicate that RipAW suppresses plant PTI responses through the E3 ubiquitin ligase activity. Unlike other members of the IpaH family of effectors, RipAW and RipAR had no leucine-rich repeat motifs in their amino acid sequences. A conserved C-terminal region of RipAW is indispensable for PTI suppression. Transgenic plants expressing RipAW and RipAR showed increased disease susceptibility, suggesting that RipAW and RipAR contribute to bacterial virulence in plants.

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2017-07-01
2024-04-19
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References

  1. Han Z, Sun Y, Chai J. Structural insight into the activation of plant receptor kinases. Curr Opin Plant Biol 2014; 20:55–63 [View Article][PubMed]
    [Google Scholar]
  2. Couto D, Zipfel C. Regulation of pattern recognition receptor signalling in plants. Nat Rev Immunol 2016; 16:537–552 [View Article][PubMed]
    [Google Scholar]
  3. Chisholm ST, Coaker G, Day B, Staskawicz BJ. Host-microbe interactions: shaping the evolution of the plant immune response. Cell 2006; 124:803–814 [View Article][PubMed]
    [Google Scholar]
  4. Dodds PN, Rathjen JP. Plant immunity: towards an integrated view of plant-pathogen interactions. Nat Rev Genet 2010; 11:539–548 [View Article][PubMed]
    [Google Scholar]
  5. Hueck CJ. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 1998; 62:379–433[PubMed]
    [Google Scholar]
  6. Galán JE, Lara-Tejero M, Marlovits TC, Wagner S. Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells. Annu Rev Microbiol 2014; 68:415–438 [View Article][PubMed]
    [Google Scholar]
  7. Poueymiro M, Genin S. Secreted proteins from Ralstonia solanacearum: a hundred tricks to kill a plant. Curr Opin Microbiol 2009; 12:44–52 [View Article][PubMed]
    [Google Scholar]
  8. Mukaihara T, Tamura N, Iwabuchi M. Genome-wide identification of a large repertoire of Ralstonia solanacearum type III effector proteins by a new functional screen. Mol Plant Microbe Interact 2010; 23:251–262 [View Article][PubMed]
    [Google Scholar]
  9. Peeters N, Carrère S, Anisimova M, Plener L, Cazalé AC et al. Repertoire, unified nomenclature and evolution of the type III effector gene set in the Ralstonia solanacearum species complex. BMC Genomics 2013; 14:859 [View Article][PubMed]
    [Google Scholar]
  10. Feng F, Zhou JM. Plant-bacterial pathogen interactions mediated by type III effectors. Curr Opin Plant Biol 2012; 15:469–476 [View Article][PubMed]
    [Google Scholar]
  11. Hicks SW, Galán JE. Exploitation of eukaryotic subcellular targeting mechanisms by bacterial effectors. Nat Rev Microbiol 2013; 11:316–326 [View Article][PubMed]
    [Google Scholar]
  12. Büttner D. Behind the lines–actions of bacterial type III effector proteins in plant cells. FEMS Microbiol Rev 2016; 40:894–937 [View Article][PubMed]
    [Google Scholar]
  13. Hochstrasser M. Ubiquitin-dependent protein degradation. Annu Rev Genet 1996; 30:405–439 [View Article][PubMed]
    [Google Scholar]
  14. Pickart CM. Mechanisms underlying ubiquitination. Annu Rev Biochem 2001; 70:503–533 [View Article][PubMed]
    [Google Scholar]
  15. Hershko A, Heller H, Elias S, Ciechanover A. Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J Biol Chem 1983; 258:8206–8214[PubMed]
    [Google Scholar]
  16. Maculins T, Fiskin E, Bhogaraju S, Dikic I. Bacteria-host relationship: ubiquitin ligases as weapons of invasion. Cell Res 2016; 26:499–510 [View Article][PubMed]
    [Google Scholar]
  17. Zhang Y, Higashide WM, Mccormick BA, Chen J, Zhou D. The inflammation-associated Salmonella SopA is a HECT-like E3 ubiquitin ligase. Mol Microbiol 2006; 62:786–793 [View Article][PubMed]
    [Google Scholar]
  18. Göhre V, Spallek T, Häweker H, Mersmann S, Mentzel T et al. Plant pattern-recognition receptor FLS2 is directed for degradation by the bacterial ubiquitin ligase AvrPtoB. Curr Biol 2008; 18:1824–1832 [View Article][PubMed]
    [Google Scholar]
  19. Gimenez-Ibanez S, Hann DR, Ntoukakis V, Petutschnig E, Lipka V et al. AvrPtoB targets the LysM receptor kinase CERK1 to promote bacterial virulence on plants. Curr Biol 2009; 19:423–429 [View Article][PubMed]
    [Google Scholar]
  20. Rohde JR, Breitkreutz A, Chenal A, Sansonetti PJ, Parsot C. Type III secretion effectors of the IpaH family are E3 ubiquitin ligases. Cell Host Microbe 2007; 1:77–83 [View Article][PubMed]
    [Google Scholar]
  21. Quezada CM, Hicks SW, Galán JE, Stebbins CE. A family of Salmonella virulence factors functions as a distinct class of autoregulated E3 ubiquitin ligases. Proc Natl Acad Sci USA 2009; 106:4864–4869 [View Article][PubMed]
    [Google Scholar]
  22. Zhu Y, Li H, Hu L, Wang J, Zhou Y et al. Structure of a Shigella effector reveals a new class of ubiquitin ligases. Nat Struct Mol Biol 2008; 15:1302–1308 [View Article][PubMed]
    [Google Scholar]
  23. Singer AU, Rohde JR, Lam R, Skarina T, Kagan O et al. Structure of the Shigella T3SS effector IpaH defines a new class of E3 ubiquitin ligases. Nat Struct Mol Biol 2008; 15:1293–1301 [View Article][PubMed]
    [Google Scholar]
  24. Ashida H, Nakano H, Sasakawa C. Shigella IpaH0722 E3 ubiquitin ligase effector targets TRAF2 to inhibit PKC-NF-κB activity in invaded epithelial cells. PLoS Pathog 2013; 9:e1003409 [View Article][PubMed]
    [Google Scholar]
  25. de Jong MF, Liu Z, Chen D, Alto NM. Shigella flexneri suppresses NF-κB activation by inhibiting linear ubiquitin chain ligation. Nat Microbiol 2016; 1:16084 [View Article][PubMed]
    [Google Scholar]
  26. Bernal-Bayard J, Ramos-Morales F. Salmonella type III secretion effector SlrP is an E3 ubiquitin ligase for mammalian thioredoxin. J Biol Chem 2009; 284:27587–27595 [View Article][PubMed]
    [Google Scholar]
  27. Xin DW, Liao S, Xie ZP, Hann DR, Steinle L et al. Functional analysis of NopM, a novel E3 ubiquitin ligase (NEL) domain effector of Rhizobium sp. strain NGR234. PLoS Pathog 2012; 8:e1002707 [View Article][PubMed]
    [Google Scholar]
  28. Haraga A, Miller SI. A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1. Cell Microbiol 2006; 8:837–846 [View Article][PubMed]
    [Google Scholar]
  29. Bhavsar AP, Brown NF, Stoepel J, Wiermer M, Martin DD et al. The Salmonella type III effector SspH2 specifically exploits the NLR co-chaperone activity of SGT1 to subvert immunity. PLoS Pathog 2013; 9:e1003518 [View Article][PubMed]
    [Google Scholar]
  30. Chou YC, Keszei AF, Rohde JR, Tyers M, Sicheri F. Conserved structural mechanisms for autoinhibition in IpaH ubiquitin ligases. J Biol Chem 2012; 287:268–275 [View Article][PubMed]
    [Google Scholar]
  31. Keszei AF, Tang X, Mccormick C, Zeqiraj E, Rohde JR et al. Structure of an SspH1-PKN1 complex reveals the basis for host substrate recognition and mechanism of activation for a bacterial E3 ubiquitin ligase. Mol Cell Biol 2014; 34:362–373 [View Article][PubMed]
    [Google Scholar]
  32. Ashida H, Sasakawa C. Shigella IpaH family effectors as a versatile model for studying pathogenic bacteria. Front Cell Infect Microbiol 2016; 5:100 [View Article][PubMed]
    [Google Scholar]
  33. Miao EA, Scherer CA, Tsolis RM, Kingsley RA, Adams LG et al. Salmonella typhimurium leucine-rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems. Mol Microbiol 1999; 34:850–864 [View Article][PubMed]
    [Google Scholar]
  34. Fernandez-Prada CM, Hoover DL, Tall BD, Hartman AB, Kopelowitz J et al. Shigella flexneri IpaH7.8 facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages. Infect Immun 2000; 68:3608–3619 [View Article][PubMed]
    [Google Scholar]
  35. Ashida H, Toyotome T, Nagai T, Sasakawa C. Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors. Mol Microbiol 2007; 63:680–693 [View Article][PubMed]
    [Google Scholar]
  36. Wang F, Jiang Z, Li Y, He X, Zhao J et al. Shigella flexneri T3SS effector IpaH4.5 modulates the host inflammatory response via interaction with NF-κB p65 protein. Cell Microbiol 2013; 15:474–485 [View Article][PubMed]
    [Google Scholar]
  37. Mukaihara T, Tamura N, Murata Y, Iwabuchi M. Genetic screening of Hrp type III-related pathogenicity genes controlled by the HrpB transcriptional activator in Ralstonia solanacearum. Mol Microbiol 2004; 54:863–875 [View Article][PubMed]
    [Google Scholar]
  38. Nakano M, Nishihara M, Yoshioka H, Takahashi H, Sawasaki T et al. Suppression of DS1 phosphatidic acid phosphatase confirms resistance to Ralstonia solanacearum in Nicotiana benthamiana. PLoS One 2013; 8:e75124 [View Article][PubMed]
    [Google Scholar]
  39. Mukaihara T, Hatanaka T, Nakano M, Oda K. Ralstonia solanacearum type III effector RipAY is a glutathione-degrading enzyme that is activated by plant cytosolic thioredoxins and suppresses plant immunity. MBio 2016; 7:e00359-16 [View Article][PubMed]
    [Google Scholar]
  40. King EO, Ward MK, Raney DE. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med 1954; 44:301–307[PubMed]
    [Google Scholar]
  41. Ohtsubo N, Mitsuhara I, Koga M, Seo S, Ohashi Y. Ethylene promotes the necrotic lesion formation and basic PR gene expression in TMV-infected tobacco. Plant Cell Physiol 1999; 40:808–817 [View Article]
    [Google Scholar]
  42. Markwell J, Osterman JC, Mitchell JL. Calibration of the Minolta SPAD-502 leaf chlorophyll meter. Photosynth Res 1995; 46:467–472 [View Article][PubMed]
    [Google Scholar]
  43. Nishinaka Y, Aramaki Y, Yoshida H, Masuya H, Sugawara T et al. A new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells. Biochem Biophys Res Commun 1993; 193:554–559 [View Article][PubMed]
    [Google Scholar]
  44. Flury P, Klauser D, Schulze B, Boller T, Bartels S. The anticipation of danger: microbe-associated molecular pattern perception enhances AtPep-triggered oxidative burst. Plant Physiol 2013; 161:2023–2035 [View Article][PubMed]
    [Google Scholar]
  45. Aoyama T, Chua NH. A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 1997; 11:605–612 [View Article][PubMed]
    [Google Scholar]
  46. Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 1998; 16:735–743 [View Article][PubMed]
    [Google Scholar]
  47. Lorrain S, Vailleau F, Balagué C, Roby D. Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants?. Trends Plant Sci 2003; 8:263–271 [View Article][PubMed]
    [Google Scholar]
  48. Gimenez-Ibanez S, Boter M, Fernández-Barbero G, Chini A, Rathjen JP et al. The bacterial effector HopX1 targets JAZ transcriptional repressors to activate jasmonate signaling and promote infection in Arabidopsis. PLoS Biol 2014; 12:e1001792 [View Article][PubMed]
    [Google Scholar]
  49. Teper D, Burstein D, Salomon D, Gershovitz M, Pupko T et al. Identification of novel Xanthomonas euvesicatoria type III effector proteins by a machine-learning approach. Mol Plant Pathol 2016; 17:398–411 [View Article][PubMed]
    [Google Scholar]
  50. Heese A, Hann DR, Gimenez-Ibanez S, Jones AM, He K et al. The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc Natl Acad Sci USA 2007; 104:12217–12222 [View Article][PubMed]
    [Google Scholar]
  51. Nguyen HP, Chakravarthy S, Velásquez AC, Mclane HL, Zeng L et al. Methods to study PAMP-triggered immunity using tomato and Nicotiana benthamiana. Mol Plant Microbe Interact 2010; 23:991–999 [View Article][PubMed]
    [Google Scholar]
  52. Segonzac C, Feike D, Gimenez-Ibanez S, Hann DR, Zipfel C et al. Hierarchy and roles of pathogen-associated molecular pattern-induced responses in Nicotiana benthamiana. Plant Physiol 2011; 156:687–699 [View Article][PubMed]
    [Google Scholar]
  53. Lebrun-Garcia A, Ouaked F, Chiltz A, Pugin A. Activation of MAPK homologues by elicitors in tobacco cells. Plant J 1998; 15:773–781 [View Article][PubMed]
    [Google Scholar]
  54. He P, Shan L, Lin NC, Martin GB, Kemmerling B et al. Specific bacterial suppressors of MAMP signaling upstream of MAPKKK in Arabidopsis innate immunity. Cell 2006; 125:563–575 [View Article][PubMed]
    [Google Scholar]
  55. de Vries JS, Andriotis VM, Wu AJ, Rathjen JP. Tomato Pto encodes a functional N-myristoylation motif that is required for signal transduction in Nicotiana benthamiana. Plant J 2006; 45:31–45 [View Article][PubMed]
    [Google Scholar]
  56. Hwang IS, Kim NH, Choi DS, Hwang BK. Overexpression of Xanthomonas campestris pv. vesicatoria effector AvrBsT in Arabidopsis triggers plant cell death, disease and defense responses. Planta 2012; 236:1191–1204 [View Article][PubMed]
    [Google Scholar]
  57. Priller JP, Reid S, Konein P, Dietrich P, Sonnewald S. The Xanthomonas campestris pv. vesicatoria type-3 effector XopB inhibits plant defence responses by interfering with ROS production. PLoS One 2016; 11:e0159107 [View Article][PubMed]
    [Google Scholar]
  58. Liu L, Wang Y, Cui F, Fang A, Wang S et al. The type III effector AvrXccB in Xanthomonas campestris pv. campestris targets putative methyltransferases and suppresses innate immunity in Arabidopsis. Mol Plant Pathol 2016 doi:10.1111/mpp.12435 [View Article][PubMed]
    [Google Scholar]
  59. Fabro G, Steinbrenner J, Coates M, Ishaque N, Baxter L et al. Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity. PLoS Pathog 2011; 7:e1002348 [View Article][PubMed]
    [Google Scholar]
  60. Li Q, Zhang M, Shen D, Liu T, Chen Y et al. A Phytophthora sojae effector PsCRN63 forms homo-/hetero-dimers to suppress plant immunity via an inverted association manner. Sci Rep 2016; 6:26951 [View Article][PubMed]
    [Google Scholar]
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