Abstract
Lipopolysaccharide (LPS) from Gram-negative bacteria is recognized as a microbe-associated molecular pattern (MAMP) and not only induces an innate immune response in plants, but also stimulates the development of characteristic defense responses. However, identification and characterization of a cell surface LPS-receptor/binding site, as described in mammals, remains elusive in plants. As an amphiphilic, macromolecular lipoglycan, intact LPS potentially contains three MAMP-active regions, represented by the O-polysaccharide chain, the core and the lipid A. Binding site studies with intact labeled LPS were conducted in Arabidopsis thaliana protoplasts and quantified using flow cytometry fluorescence changes. Quantum dots (Qdots), which allow non-covalent, hydrophobic labeling were used as a novel strategy in this study and compared to covalent, hydrophilic labeling with Alexa 488. Affinity for LPS-binding sites was clearly demonstrated by concentration-, temperature-, and time-dependent increases in protoplast fluorescence following treatment with the labeled LPS. Moreover, this induced fluorescence increase was convincingly reduced following pre-treatment with excess unlabeled LPS, thereby indicating reversibility of LPS binding. Inhibition of the binding process is also reported using endo- and exocytosis inhibitors. Here, we present evidence for the anticipated presence of LPS-specific binding sites in Arabidopsis protoplasts, and furthermore propose Qdots as a more sensitive LPS-labeling strategy in comparison to the conventional Alexa 488 hydrazide label for binding studies.
Highlights
Lipopolysaccharides (LPSs) are complex lipoglycans found in the outer membrane of Gramnegative bacteria and is generally composed of three regions namely the fatty acid lipid A disaccharide, a core region of short oligosaccharide chains and an O-antigen region of polysaccharides (Madala et al, 2011, 2012)
LPS binds to a LPS binding protein (LBP) to form a LPS–LBP complex which is translocated to myeloid differentiation 2 (MD2) with the presence/absence of its co-receptor, a glycosylphosphatidylinositol (GPI)-linked protein, CD14 (Triantafilou et al, 2001; Sasaki and White, 2008)
2 g A. thaliana leaves were cut into 1–2 mm strips and the protocol according to Yoo et al (2007) was followed using enzymatic digestion with a solution containing cellulase “Onosuka”R10, macerozyme R10 (Yakult Pharmaceutical Industry, Tokyo, Japan) and pectinase
Summary
Lipopolysaccharides (LPSs) are complex lipoglycans found in the outer membrane of Gramnegative bacteria and is generally composed of three regions namely the fatty acid lipid A disaccharide, a core region of short oligosaccharide chains and an O-antigen region of polysaccharides (Madala et al, 2011, 2012). Systemic acquired resistance (SAR) is known to be triggered by LPS elicitation through in the expression of PR genes in upper leaves upon lower leaf treatment (Coventry and Dubery, 2001; Zeidler et al, 2004; Mishina and Zeier, 2007) Such defense components lead to heightened plant sensitivity to subsequent stimuli and microbe sensing, termed a primed state (Newman et al, 2007; Sanabria et al, 2008; Madala et al, 2012). Since most LPS labels, including Qdots, are fluorophores, flow cytometry can be used for quantification of cell–ligand binding interactions through the analysis of optical properties such as fluorescence and light scatter at a specific emission wavelength This technique, requires single cells, and the production of protoplasts from higher plant tissues (Galbraith, 1994; Doležel et al, 2007; Yoo et al, 2007). Inhibitor studies suggest that the binding site-ligand interaction may be subject to binding site recycling via endo- and exocytosis processes similar to that reported for MAMP receptors (Zipfel, 2014)
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