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

  • Kyaw Aung
  • Xiufang Xin
  • Christy Mecey
  • Sheng Yang He
Part of the Methods in Molecular Biology book series (MIMB, volume 1531)


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 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.


  1. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 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 CrossRefPubMedGoogle Scholar
  6. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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 CrossRefPubMedGoogle Scholar
  8. 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 CrossRefPubMedGoogle Scholar
  9. 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 CrossRefPubMedGoogle Scholar
  10. 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 CrossRefPubMedGoogle Scholar
  11. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909. doi: 10.1038/nmeth819 CrossRefPubMedGoogle Scholar
  13. 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 CrossRefPubMedGoogle Scholar
  14. 14.
    Lee LY, Gelvin SB (2008) T-DNA binary vectors and systems. Plant Physiol 146(2):325–332. doi: 10.1104/pp.107.113001 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 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 CrossRefPubMedGoogle Scholar
  16. 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 CrossRefPubMedGoogle Scholar
  17. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 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 CrossRefPubMedGoogle Scholar
  20. 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 CrossRefPubMedGoogle Scholar
  21. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 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 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Kyaw Aung
    • 1
    • 3
  • Xiufang Xin
    • 1
  • Christy Mecey
    • 1
  • Sheng Yang He
    • 1
    • 2
    • 3
  1. 1.Department of Energy Plant Research LaboratoryMichigan State UniversityEast LansingUSA
  2. 2.Department of Plant BiologyMichigan State UniversityEast LansingUSA
  3. 3.Howard Hughes Medical InstituteMichigan State UniversityEast LansingUSA

Personalised recommendations