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22/05/2014

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  1. The identification of lipopolysaccharide (LPS)-binding proteins in Arabidopsis thaliana plasma membranes. 22/05/2014 Name: Mr. Cornelius Sipho VilakaziSupervisor: Dr. LizellePiaterCo-supervisor: Prof. Ian Dubery

  2. Hypothesis Lipopolysaccharide (LPS) from the outer membrane of Gram- negative bacteria binds to plasma membrane localized protein receptor(s) in plants.

  3. Background

  4. ∫ Plant Immunity∫ Pre-formed defenses∫ Inducible defense responses Figure 1: Plant innate immunity active defense mechanisms. (Jones and Dangl, 2006; Muthamilarasan and Prasad, 2013; Zhang et al., 2013; Klemptneret al., 2014)

  5. Lipopolysaccharide (LPS) is a M/PAMP therefore a potent inducer of innate immunity. Figure 2: General structure of LPS (taken from Erridgeet al., 2002). • (Erridgeet al., 2002; Silipoet al., 2010)

  6. Why is the plant plasma membrane an interesting target for LPS investigations? Figure 3: Membrane-associated pattern recognition receptors (PRRs) can perceive microbial patterns (P/MAMP) from different microbes (taken from Mazzotta and Kemmerling, 2011).

  7. Affinity chromatography (2) (1) (3) • (Pierce Biotechnology, RESYN Biosciences; Hyglos GmbH)

  8. Research aims The objective of the study was to capture, identify and characterize LPS-interacting proteins from Arabidopsis thaliana plasma membranes (PM) in order to elucidate the LPS receptor/receptor complex leading to the activation of host plant defense responses.

  9. Materials and methods

  10. Extraction and purification of bacterial LPS

  11. Preparation of plant material Arabidopsis thaliana (Columbia ecotype) were grown in soil under a 16 h/ 8 h light-dark cycle in a green house.

  12. Plasma membrane isolation

  13. Affinity chromatography

  14. Results and discussion

  15. Extraction and purification of the bacterial lipopolysaccharides Table 1: Summary of the characterization of LPS from Burkholderia cepacia. • (Coventry and Dubery, 2001)

  16. Extraction and purification of the bacterial lipopolysaccharides kDaA B Mature O-antigen, core oligosaccharide attached with lipid A 150 100 70 50 40 30 20 15 Core oligosaccharide attached with lipid A Free lipid A Figure 4: SDS-PAGE analysis of B. cepacia LPS samples. Underivatized LPS sample (A) . Biotinylated LPS sample (B).

  17. Plasma membrane isolation kDa1 2 3 150 100 70 50 40 30 20 15 10 (A) (B) 1 - Homogenate (HG) 2 - Microsomal fraction (MCF) 3 - Plasma membrane (PM) • Figure 5:Sucrose density gradient for the isolation of the PM fraction (A). Comparison by SDS-PAGE of the HG, MCF and PM proteins isolated from A. thaliana leaves (B).

  18. Plasma membrane H+-ATPase activity determination Figure 6: H+-ATPase activity of the plasma membrane fractions and vanadate inhibition of the enzyme.

  19. Affinity chromatography (A) (B) • Figure 7: Elution curves of non-specifically bound and LPS-interacting proteins. Fractions were collected subsequent to affinity chromatography using endotoxin removing columns (A) and streptavidin magnetic microspheres (B).

  20. SDS-PAGE analysis of eluted fractions kDa1 2 3 4 5 150 100 70 50 40 30 20 15 10 1 & 2 - Membrane fractions 3 – NaCl fraction 4 & 5 - 1% SDS fractions (LPS-interacting proteins) • Figure 8: SDS-PAGE analysis of eluted fractions following the chromatographic experiment with polymixin B basedendotoxin removing columns.

  21. SDS-PAGE analysis of eluted fractions kDa1 2 3 4 5 6 7 150 100 70 50 40 30 20 15 10 1 – PM fraction 2 – PM supernatant 3 – Membrane fraction 4 – NaCl fraction 5 – 100 µl/ml LPS 6-7 – 1% SDS fractions (LPS-interacting proteins) • Figure 9: SDS-PAGE analysis of eluted fractions from the magnetic polymeric microsphere affinity-capture procedure.

  22. Table 2: List of LPS-interacting proteins after in situ digestion of bands from sample bound fractions.

  23. Table 2 : (Continued)

  24. Conclusion The novel affinity-capture strategy for the enrichment of LPS-interacting proteins proved to be effective in specifically binding proteins involved in plant defense responses . The identification of MAMP receptors will lead to a better understanding of pathogen perception in plants and may lead to the development of new and innovative ways to control plant diseases. (Giangrande et al., 2013)

  25. References Abas, L. and Luschnig, C. (2010). Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation. Analytical Biochemistry, 401: 217-227. Coventry, H.S. and Dubery, I.A. (2001). Lipopolysaccharides from Burkholderia cepacia contribute to an enhanced defensive capacity and the induction of pathogenesis-related proteins in Nicotiana tabacum. Physiological and Molecular Plant Pathology, 58: 149-158. Erridge, C., Bennett-Guerrero, E. and Poxton, I.R. (2002). Structure and function of lipopolysaccharides. Microbes and Infection, 4: 837-851. Giannini, L., Ruiz-Christin, J. and Briskin, D. (1988). A small scale procedure for the isolation of transport competent vesicles from plant tissues. Analytical Biochemistry, 174: 561-567. Giangrande, C., Colarusso, L., Lanzetta, R., Molinaro, A., Pucci, P. and Amoresano, A. (2013). Innate immunity probed by lipopolysaccharides affinity strategy and proteomics. Analytical Bioanalytical , 174: 561-567. Jones, J.D.G. and Dangl, J.L. (2006). The plant immune system. Nature, 444: 323-329. Klemptner, R.L., Sherwood, J.S., Tugizimana, F., Dubery, I.A. And Piater., L.A. (2014). Ergosterol, an orphan fungal microbe-associated molecular pattern (MAMP). Molecular Plant Pathology, 1: 1-15. Ligaba, A., Yamaguchi, M., Shen, H., Sasaki, T., Yamamoto, Y., and Matsumoto, H. (2004). Phosphorous deficiency enhances plasma membrane H+-ATPase activity and citrate exudation in greater purple lupin (Lupinuspilosus). Functional Plant Biology, 31: 1075-1083. Mazzotta, S. and Kemmerling, B. (2011). Pattern recognition in plant innate immunity. Journal of Plant Pathology, 93: 7-17. Muthamilarasan, M. and Prasad, M. (2013). Plant innate immunity: An updated insight into defense mechanism. Journal of Biosciences, 38: 1-17. Silipo, A., Erbs, G., Shinya, T., Dow, J.M., Parrilli, M., Lanzetta, R., Shibuya, N., Newman, M-A. and Molinaro, A. (2010). Glycoconjugates as elicitors or suppressors of plant innate immunity. Glycobiology, 20: 406-419. Zhang, J. and Zhou, J-M. (2010). Plant immunity triggered by microbial molecular signals. Molecular Plant, 3: 783-793.

  26. Muchas gracias