Skip to main content
Book cover

Type 3 Secretion Systems pp 141–153Cite as

Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants

Part of the Methods in Molecular Biology book series (MIMB,volume 1531)

Abstract

Animal and plant pathogenic bacteria use type III secretion systems to translocate proteinaceous effectors to subvert innate immunity of their host organisms. Type III secretion/effector systems are a crucial pathogenicity factor in many bacterial pathogens of plants and animals. Pseudomonas syringae pv. tomato (Pst) DC3000 injects a total of 36 protein effectors that target a variety of host proteins. Studies of a subset of Pst DC3000 effectors demonstrated that bacterial effectors, once inside the host cell, are localized to different subcellular compartments, including plasma membrane, cytoplasm, mitochondria, chloroplast, and Trans-Golgi network, to carry out their virulence functions. Identifying the subcellular localization of bacterial effector proteins in host cells could provide substantial clues to understanding the molecular and cellular basis of the virulence activities of effector proteins. In this chapter, we present methods for transient or stable expression of bacterial effector proteins in tobacco and/or Arabidopsis thaliana for live cell imaging as well as confirming the subcellular localization in plants using fluorescent organelle markers or chemical treatment.

Key words

  • Plant pathogen
  • Bacterial pathogenesis
  • Type III secretion
  • Plant immunity
  • Tobacco
  • Arabidopsis thaliana
  • Agrobacterium
  • Confocal microscopy

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

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-6649-3_12
  • Chapter length: 13 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   109.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-6649-3
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   139.00
Price excludes VAT (USA)
Hardcover Book
USD   169.99
Price excludes VAT (USA)
Learn about institutional subscriptions
Fig. 1
Fig. 2

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Whalen MC, Innes RW, Bent AF, Staskawicz BJ (1991) Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean. Plant Cell 3(1):49–59. doi:10.1105/tpc.3.1.49

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wei HL, Chakravarthy S, Mathieu J, Helmann TC, Stodghill P, Swingle B, Martin GB, Collmer A (2015) Pseudomonas syringae pv. tomato DC3000 Type III secretion effector polymutants reveal an interplay between HopAD1 and AvrPtoB. Cell Host Microbe 17(6):752–762. doi:10.1016/j.chom.2015.05.007

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  3. Xin XF, Nomura K, Ding X, Chen X, Wang K, Aung K, Uribe F, Rosa B, Yao J, Chen J, He SY (2015) Pseudomonas syringae effector avirulence protein E localizes to the host plasma membrane and down-regulates the expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 gene required for antibacterial immunity in Arabidopsis. Plant Physiol 169(1):793–802. doi:10.1104/pp.15.00547

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  4. Nomura K, Mecey C, Lee YN, Imboden LA, Chang JH, He SY (2011) Effector-triggered immunity blocks pathogen degradation of an immunity-associated vesicle traffic regulator in Arabidopsis. Proc Natl Acad Sci U S A 108(26):10774–10779. doi:10.1073/pnas.1103338108

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  5. Block A, Toruno TY, Elowsky CG, Zhang C, Steinbrenner J, Beynon J, Alfano JR (2014) The Pseudomonas syringae type III effector HopD1 suppresses effector-triggered immunity, localizes to the endoplasmic reticulum, and targets the Arabidopsis transcription factor NTL9. New Phytol 201(4):1358–1370. doi:10.1111/nph.12626

    CrossRef  CAS  PubMed  Google Scholar 

  6. Jelenska J, Yao N, Vinatzer BA, Wright CM, Brodsky JL, Greenberg JT (2007) A J domain virulence effector of Pseudomonas syringae remodels host chloroplasts and suppresses defenses. Curr Biol 17(6):499–508. doi:10.1016/j.cub.2007.02.028

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rodriguez-Herva JJ, Gonzalez-Melendi P, Cuartas-Lanza R, Antunez-Lamas M, Rio-Alvarez I, Li Z, Lopez-Torrejon G, Diaz I, Del Pozo JC, Chakravarthy S, Collmer A, Rodriguez-Palenzuela P, Lopez-Solanilla E (2012) A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses. Cell Microbiol 14(5):669–681. doi:10.1111/j.1462-5822.2012.01749.x

    CrossRef  CAS  PubMed  Google Scholar 

  8. Li G, Froehlich JE, Elowsky C, Msanne J, Ostosh AC, Zhang C, Awada T, Alfano JR (2014) Distinct Pseudomonas type-III effectors use a cleavable transit peptide to target chloroplasts. Plant J 77(2):310–321. doi:10.1111/tpj.12396

    CrossRef  CAS  PubMed  Google Scholar 

  9. Block A, Guo M, Li G, Elowsky C, Clemente TE, Alfano JR (2010) The Pseudomonas syringae type III effector HopG1 targets mitochondria, alters plant development and suppresses plant innate immunity. Cell Microbiol 12(3):318–330. doi:10.1111/j.1462-5822.2009.01396.x

    CrossRef  CAS  PubMed  Google Scholar 

  10. Fu ZQ, Guo M, Jeong BR, Tian F, Elthon TE, Cerny RL, Staiger D, Alfano JR (2007) A type III effector ADP-ribosylates RNA-binding proteins and quells plant immunity. Nature 447(7142):284–288. doi:10.1038/nature05737

    CrossRef  CAS  PubMed  Google Scholar 

  11. Giska F, Lichocka M, Piechocki M, Dadlez M, Schmelzer E, Hennig J, Krzymowska M (2013) Phosphorylation of HopQ1, a type III effector from Pseudomonas syringae, creates a binding site for host 14-3-3 proteins. Plant Physiol 161(4):2049–2061. doi:10.1104/pp.112.209023

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  12. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909. doi:10.1038/nmeth819

    CrossRef  CAS  PubMed  Google Scholar 

  13. Runions J, Hawes C, Kurup S (2007) Fluorescent protein fusions for protein localization in plants. Methods Mol Biol 390:239–255. doi:10.1007/978-1-59745-466-7_16

    CrossRef  CAS  PubMed  Google Scholar 

  14. Lee LY, Gelvin SB (2008) T-DNA binary vectors and systems. Plant Physiol 146(2):325–332. doi:10.1104/pp.107.113001

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  15. Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS (2006) Gateway-compatible vectors for plant functional genomics and proteomics. Plant J 45(4):616–629. doi:10.1111/j.1365-313X.2005.02617.x

    CrossRef  CAS  PubMed  Google Scholar 

  16. Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K, Jinbo T, Kimura T (2007) Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104(1):34–41. doi:10.1263/jbb.104.34

    CrossRef  CAS  PubMed  Google Scholar 

  17. Reumann S, Quan S, Aung K, Yang P, Manandhar-Shrestha K, Holbrook D, Linka N, Switzenberg R, Wilkerson CG, Weber AP, Olsen LJ, Hu J (2009) In-depth proteome analysis of Arabidopsis leaf peroxisomes combined with in vivo subcellular targeting verification indicates novel metabolic and regulatory functions of peroxisomes. Plant Physiol 150(1):125–143. doi:10.1104/pp.109.137703

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zordan RE, Beliveau BJ, Trow JA, Craig NL, Cormack BP (2015) Avoiding the ends: internal epitope tagging of proteins using transposon Tn7. Genetics 200(1):47–58. doi:10.1534/genetics.114.169482

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sauer M, Friml J (2010) Immunolocalization of proteins in plants. Methods Mol Biol 655:253–263. doi:10.1007/978-1-60761-765-5_17

    CrossRef  CAS  PubMed  Google Scholar 

  20. Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51(6):1126–1136. doi:10.1111/j.1365-313X.2007.03212.x

    CrossRef  CAS  PubMed  Google Scholar 

  21. Geldner N, Denervaud-Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59(1):169–178. doi:10.1111/j.1365-313X.2009.03851.x

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  22. Collings DA (2013) Subcellular localization of transiently expressed fluorescent fusion proteins. Methods Mol Biol 1069:227–258. doi:10.1007/978-1-62703-613-9_16

    CrossRef  CAS  PubMed  Google Scholar 

Download references

Acknowledgement

This work was supported by funding from the National Institutes of Health R01 GM109928; the Chemical Sciences, Geosciences, Department of Energy DE–FG02–91ER20021 (support of research infrastructure); U.S. Department of Agriculture/National Institute of Food and Agriculture AFRI-004412; and the Gordon and Betty Moore Foundation GBMF3037. SYH is a Howard Hughes Medical Institute-Gordon Betty Moore Foundation Investigator.

Author information

Authors and Affiliations

  1. Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824, USA

    Kyaw Aung, Xiufang Xin, Christy Mecey & Sheng Yang He

  2. Department of Plant Biology, Michigan State University, East Lansing, MI, 48824, USA

    Sheng Yang He

  3. Howard Hughes Medical Institute, Michigan State University, East Lansing, MI, 48824, USA

    Kyaw Aung & Sheng Yang He

Authors
  1. Kyaw Aung
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiufang Xin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christy Mecey
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sheng Yang He
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sheng Yang He .

Editor information

Editors and Affiliations

  1. Department of Biomedical Sciences, School of Medicine and Health Sciences University of North Dakota, Grand Forks, North Dakota, USA

    Matthew L. Nilles

  2. Department of Biomedical Sciences, School of Medicine and Health Sciences University of North Dakota, Grand Forks, North Dakota, USA

    Danielle L. Jessen Condry

Rights and permissions

Reprints and Permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Aung, K., Xin, X., Mecey, C., He, S.Y. (2017). Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants. In: Nilles, M., Condry, D. (eds) Type 3 Secretion Systems. Methods in Molecular Biology, vol 1531. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6649-3_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6649-3_12

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6647-9

  • Online ISBN: 978-1-4939-6649-3

  • eBook Packages: Springer Protocols