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Identification of Lipid-Binding Effectors

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Plant Fungal Pathogens

Part of the book series: Methods in Molecular Biology ((MIMB,volume 835))

Abstract

In recent years, the functional roles of effectors from a wide variety of fungal and oomycete pathogens have begun to emerge. As a product of this work, the importance of effector-lipid interactions has been made apparent. Phospholipids are not only important signaling molecules, but they also play important roles in the trafficking of endosomes and the localization of proteins. Characterizing effector-lipid interactions can provide novel information regarding the functions of effectors relevant to their cellular and subcellular targeting and their potential effects on host signaling and vesicle trafficking. We present here two techniques that can be used to screen for and validate protein-lipid interactions without the need to access highly specialized machinery. We describe in detail how to perform lipid filter and liposome-binding assays and provide suggestions for troubleshooting potential problems with these assays.

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References

  1. Kooijman EE et al (2003) Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid. Traffic 4: 162–174.

    Article  PubMed  CAS  Google Scholar 

  2. Athenstaedt K, Daum G (1999) Phosphatidic acid, a key intermediate in lipid metabolism. Eur J Biochem 266: 1–16.

    Article  PubMed  CAS  Google Scholar 

  3. Delon C et al (2004) Sphingosine kinase 1 is an intracellular effector of phosphatidic acid. J Biol Chem 279: 44763–44774.

    Article  PubMed  CAS  Google Scholar 

  4. Young BP et al (2010) Phosphatidic acid is a pH biosensor that links membrane biogenesis to metabolism. Science 329: 1085–1088.

    Article  PubMed  CAS  Google Scholar 

  5. Anthony RG et al (2006) The Arabidopsis proteinkinase PTI1-2 is activated by convergent phosphatidic acid and oxidative stress signaling pathways downstream of PDK1 and OXI1.

    Google Scholar 

  6. Park J et al (2004) Phosphatidic acid induces leaf cell death in Arabidopsis by activating the rho-related small G protein GTPase-mediated pathway of reactive oxygen species generation. Plant Physiol 134: 129–136.

    Article  PubMed  CAS  Google Scholar 

  7. Daleke DL (2003) Regulation of transbilayer plasma membrane phospholipid asymmetry. J Lipid Research 44: 233–242.

    Article  CAS  Google Scholar 

  8. An X et al (2004) Phosphatidylserine binding sites in erythroid spectrin: location and implications for membrane stability. Biochemistry 43: 310–315.

    Article  PubMed  CAS  Google Scholar 

  9. Sahu SK et al (2007) Phospholipid scramblases: an overview. Arch Biochem Biophys 462: 103–114.

    Article  PubMed  CAS  Google Scholar 

  10. Zwaal RF et al (2005) Surface exposure of phosphatidylserine in pathological cells. Cell Mol Life Sci 62: 971–988.

    Article  PubMed  CAS  Google Scholar 

  11. Vermeet JEM et al (2009) Mapping phosphatidylinositol 4-phosphate dynamics in living plant cells. Plant J 57: 356–372.

    Article  Google Scholar 

  12. Garofalo RS et al (2003) Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta. J Clinic Invest 112: 197–208.

    CAS  Google Scholar 

  13. Franke TF et al (2003) PI3K/Akt and apoptosis: size matters. Oncogene, 22: 8983–8998.

    Article  PubMed  CAS  Google Scholar 

  14. Godi A et al (2004) FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P. Nat Cell Biol 6: 393–404.

    Article  PubMed  CAS  Google Scholar 

  15. D’Angelo et al (2008) The multiple roles of PtdIns(4)P- not just the precursor of PtdIns(4,5)P2. J Cell Sci 121: 1955–1963.

    Article  PubMed  Google Scholar 

  16. Wurmser AE, Emr SD (1998) Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires luminal vacuolar hydrolase activities. EMBO Journal 17: 4930–4942.

    Article  PubMed  CAS  Google Scholar 

  17. Zoncu R et al (2009) A Phosphoinositide switch controls the maturation and signaling properties of APPL endosomes. Cell 136: 1110–1121.

    Article  PubMed  CAS  Google Scholar 

  18. Kale SD et al (2010) External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells. Cell 142: 284–295.

    Article  PubMed  CAS  Google Scholar 

  19. Stahelin RV (2009) Lipid binding domains: more than simple lipid effectors. J Lipid Res 50: 299–304.

    Article  Google Scholar 

  20. Colon-Gonzalez F et al (2006) C1 domains exposed: From diacylglycerol binding to protein-protein interactions. BBA- Mol Cell Biol L 1761: 827–837.

    CAS  Google Scholar 

  21. Bolsover SR et al (2003) Role of Ca2+/phospha-tidylserine binding region of the C2 domain in the translocation of protein kinase Cα to the plasma membrane. J Biol Chem 278: 10282–10290.

    Article  PubMed  CAS  Google Scholar 

  22. Craig KL, Harley CB (1996) Phosphorylation of human pleckstrin on Ser-113 and Ser-117 by protein kinase C. Biochem J 314: 937–942.

    PubMed  CAS  Google Scholar 

  23. Dowler S et al (2000) Identification of pleckstrin-homology-domain containing proteins with novel phosphoinositide binding specificities. Biochem J 351: 19–31.

    Article  PubMed  CAS  Google Scholar 

  24. Gaullier JM et al (2004) FYVE finger bind PtdIns(3)p. Nat Cell Biol 5: 393–404.

    Google Scholar 

  25. Lee SA et al (2006) Molecular Mechanism of Membrane Docking by the Vam7p PX Domain J Biol Chem 281: 37091–37101.

    Article  PubMed  CAS  Google Scholar 

  26. McLaughlin S et al (2005) Reversible - through calmodulin - electrostatic interactions between basic residues on proteins and acidic lipids in the plasma membrane. Biochem Soc Symp 72: 189–198.

    PubMed  CAS  Google Scholar 

  27. Ghosh S et al (1996) Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. J Biol Chem 271: 8472–8480.

    Article  PubMed  CAS  Google Scholar 

  28. Grange M et al (2000) The cAMP-specific phosphodiesterase PDE4D3 is regulated by phosphatidic acid binding. J Biol Chem: 275, 33379–33387.

    Article  CAS  Google Scholar 

  29. Dou D et al (2008) RXLR-mediated entry of Phytophthora sojae effector Avr1b into soybean cells does not require pathogen encoded machinery. Plant Cell 20: 1930–1947.

    Article  PubMed  CAS  Google Scholar 

  30. Rafiqi M et al (2010) Internalization of flax rust avirulence proteins into flax and tobacco cells can occur in the absence of the pathogen. Plant Cell 22: 2017–2032.

    Article  PubMed  CAS  Google Scholar 

  31. Whisson SC et al (2007) A translocation signal for delivery of oomycete effector proteins into host plant cells. Nature 450: 115–118.

    Article  PubMed  CAS  Google Scholar 

  32. Jiang RHY et al (2008) RXLR effector reservoir in two Phytophthora species is dominated by a single rapidly evolving superfamily with more than 700 members. Proc Natl Acad Sci USA 12: 4874–4879.

    Article  Google Scholar 

  33. Dou D et al (2008) Conserved C-terminal motifs required for avirulence and suppression of cell death by Phytophthora sojae effector Avr1b. Plant Cell 20: 1118–1133.

    Article  PubMed  CAS  Google Scholar 

  34. Bos JI et al (2007) The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana. Plant Journal 48: 165–76.

    Article  Google Scholar 

  35. Sohn KH et al (2007) The Downy mildew effector proteins ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana. Plant Cell 19: 4077–4090.

    Article  PubMed  CAS  Google Scholar 

  36. Lafont F et al (2004) Bacterial subversion of lipid rafts. Curr Opin Microbiol 7: 4–10.

    Article  PubMed  CAS  Google Scholar 

  37. Murata-Kamiya N et al (2010) Helicobacter pylori Exploits Host Membrane Phosphatidylserine for Delivery, Localization, and Pathophysiological Action of the CagA Oncoprotein. Cell Host Microbe 7: 338–339.

    Article  Google Scholar 

  38. Rüter C et al (2010) A newly identified bacterial cell-penetrating peptide that reduces the transcription of pro-inflammatory cytokines. J Cell Sci 123: 2190–2198.

    Article  PubMed  Google Scholar 

  39. Schoebel, S et al (2010) High-affinity binding of phosphatidylinositol 4-phosphate by Legionella pneumophila DrrA. EMBO reports 11: 598–604.

    Article  PubMed  CAS  Google Scholar 

  40. Weber SS et al (2006) Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole. PLoS Pathog 2(5): e46. doi:10.1371/journal.ppat.0020046.

    Article  PubMed  Google Scholar 

  41. Brombacher E et al (2009) Rab1 guanine nucleotide exchange factor SidM is a major PtdIns(4)P-binding effector protein of Legionella pneumophila. J Biol Chem 284: 4846–4856.

    Article  PubMed  CAS  Google Scholar 

  42. Tawk L et al (2010) Phosphatidylinositol 3-phosphate, an essential lipid in Plasmodium, localises to the food vacuole membrane and the apicoplast. Eukaryotic Cell doi:10.1128/EC.00124-10.

  43. Lee SW et al (2005) PilT is required for PI(3,4,5)P3-mediated crosstalk between Neisseria gonorrhoeae and epithelial cells. Cell Microbiol 7: 1271–1284.

    Article  PubMed  CAS  Google Scholar 

  44. Narayan K, Lemmon MA (2006) Determining selectivity of phosphoinositide-binding domains. Methods 39: 122–133.

    Article  PubMed  CAS  Google Scholar 

  45. Echelon Biosciences Inc. (2005). Technical Data Sheet. Q&A P-6000 Rev: 1 (08/08/05). http://www.echelon-inc.com/corp/p-6000%-20qa.pdf.

  46. Ju H et al (2009) Membrane insertion of the FYVE domain is modulated by pH. Proteins 76: 852–860.

    Article  Google Scholar 

  47. Silvius JR (1982) Thermotropic phase transitions of pure lipids in model membranes and their modification by membrane proteins. In: Jost PC, Griffith OH (eds) Lipid-protein interactions, vol 2. Wiley, New York.

    Google Scholar 

  48. Diraviyam K et al (2003) Computer modeling of the membrane interaction of FYVE Domains. J Mol Biol 328: 721–736.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

    Shiv D. Kale & Brett M. Tyler

Authors
  1. Shiv D. Kale
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  2. Brett M. Tyler
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Corresponding author

Correspondence to Shiv D. Kale .

Editor information

Editors and Affiliations

  1. Northern Crops Science Laboratory, USDA-ARS, North 18th Street 1307, Fargo, 58105-5677, North Dakota, USA

    Melvin D. Bolton

  2. Lab. Phytopathology, Wageningen University, Droevendaalsesteeg 1, Wageningen, 6708 PB, Netherlands

    Bart P.H.J. Thomma

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Kale, S.D., Tyler, B.M. (2012). Identification of Lipid-Binding Effectors. In: Bolton, M., Thomma, B. (eds) Plant Fungal Pathogens. Methods in Molecular Biology, vol 835. Humana Press. https://doi.org/10.1007/978-1-61779-501-5_24

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  • DOI: https://doi.org/10.1007/978-1-61779-501-5_24

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-500-8

  • Online ISBN: 978-1-61779-501-5

  • eBook Packages: Springer Protocols

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