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
Hypothesis Lipopolysaccharide (LPS) from the outer membrane of Gram- negative bacteria binds to plasma membrane localized protein receptor(s) in plants.
∫ 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)
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)
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).
Affinity chromatography (2) (1) (3) • (Pierce Biotechnology, RESYN Biosciences; Hyglos GmbH)
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.
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.
Extraction and purification of the bacterial lipopolysaccharides Table 1: Summary of the characterization of LPS from Burkholderia cepacia. • (Coventry and Dubery, 2001)
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).
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).
Plasma membrane H+-ATPase activity determination Figure 6: H+-ATPase activity of the plasma membrane fractions and vanadate inhibition of the enzyme.
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).
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.
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.
Table 2: List of LPS-interacting proteins after in situ digestion of bands from sample bound fractions.
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)
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