The identification of lipopolysaccharide (LPS)-binding proteins in Arabidopsis thaliana plasma membranes. 22/05/2014 Name: Mr. Cornelius Sipho Vilakazi Supervisor: Dr. Lizelle Piater Co-supervisor: Prof. Ian Dubery Lipopolysaccharide (LPS) from the outer membrane of Gramnegative 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; Klemptner et al., 2014) Lipopolysaccharide (LPS) is a M/PAMP therefore a potent inducer of innate immunity. Figure 2: General structure of LPS (taken from Erridge et al., 2002). (Erridge et al., 2002; Silipo et 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) Endotoxin removing columns MagResyn ™ Strepavidin (3) EndoTrap® HD Protein identification (Pierce Biotechnology, RESYN Biosciences; Hyglos GmbH) Research aims The objective of the study was to capture, identify and characterize LPSinteracting 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. Materials and methods PREPARATION OF CULTURES LPS EXTRACTION • Endophytic strain of Burkholderia cepacia (ASP B 2D) was cultivated in Nutrient broth (Merck, RSA) liquid medium and incubated at 28oC on a continuous rotary shaker for 10-14 days. • LPS was extracted from freeze dried bacterial cell walls using an adaptation of the phenol-water method where the LPS fractionates into the water phase at 65 oC. • For further purification, the extract was digested with RNase, DNase, Proteinase K LPS PURIFICATION (Sigma, USA), dialyzed and lyophilized. • The 2-keto-3-deoxyoctonate (KDO), carbohydrate as well as the protein content of the LPS was determined. LPS CHARACTERIZATION • Purified LPS was further analyzed on 10% SDS-PAGE gels containing 4M urea. Arabidopsis thaliana (Columbia ecotype) were grown in soil under a 16 h/ 8 h light-dark cycle in a green house. Plasma membrane isolation PM ISOLATION • The plasma membrane was isolated according to a small scale procedure as described by Giannini et al. (1988) as well as by Abas and Luschin (2010). PM ISOLATION • Approximately 20 g of leaf tissue was homogenized and centrifugation was employed to isolate the microsomal fraction from the homogenate. PM ISOLATION • The micosomal fraction was then layered onto a 25/38% sucrose density gradient and centrifuged at 13 000 xg for 1 h. H+-ATPase assay • The plasma membrane H+-ATPase activity was measured following a method by Ligaba et al. (2004) and Giannini et al. (1988). Affinity chromatography • 1 mg/ml LPS in 10mM Tris-Cl pH 7.5 was bound to Endotoxin removing affinity columns. LPS Immobilization PM addition Elimination of non-specificity Elution of target proteins Analysis • 600 pmol biotinylated LPS was bound to 10 µl of streptavidin microspheres. • 1 mg/ml of plasma membrane was added to each affinity-capture method and incubated for 2 h at RT. • Non-specifically bound proteins were then washed off using 10mM Tris-Cl, 0.1-0.2 M NaCl and 100 µg/ml LPS. • LPS-interacting proteins were eluted out using 1% SDS. • SDS-PAGE, band excision and MALDI-TOF-MS analysis. Results and discussion 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 kDa A B 150 100 Mature O-antigen, core oligosaccharide attached with lipid A 70 50 40 30 Core oligosaccharide attached with lipid A 20 15 Free lipid A . Figure 4: SDS-PAGE analysis of B. cepacia LPS samples Underivatized LPS sample (A) . Biotinylated LPS sample (B). Plasma membrane isolation kDa 1 2 3 150 (A) (B) 100 70 50 40 1 - Homogenate (HG) 2 - Microsomal fraction (MCF) 3 - Plasma membrane (PM) 30 20 15 10 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 2.5 nmol Pi/min/mg 2 PM H+-ATPase activity (- Inhibitor) PM H+-ATPase activity (+ Inhibitor) 1.5 1 0.5 0 0 10 20 30 40 50 60 Time (min) Figure 6: H+-ATPase activity of the plasma membrane fractions and vanadate inhibition of the enzyme. Affinity chromatography (A) (B) 1.2 1.2 1 Membrane fraction 0.1 M NaCl fractions 0.8 1% SDS fractions 0.6 0.4 Absorbance (280nm) Absorbance (280nm) 1 Membrane fraction 0.2 M NaCl fractions 0.8 1% SDS fractions 0.6 0.4 0.2 0.2 0 0 0 5 10 15 Fraction number 20 25 0 5 10 15 Fraction number 20 25 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 kDa 1 2 3 4 5 150 100 70 50 40 1 & 2 - Membrane fractions 3 – NaCl fraction 4 & 5 - 1% SDS fractions (LPS-interacting proteins) 30 20 15 10 Figure 8: SDS-PAGE analysis of eluted fractions following the chromatographic experiment with polymixin B based endotoxin removing columns. SDS-PAGE analysis of eluted fractions kDa 1 2 3 4 5 6 7 150 100 70 50 40 30 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) 20 15 10 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. Table 2 : (Continued) 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) Abas, L. and Luschnig, C. (2010). 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