Comparative conventional- and quantum dot-labeling strategies for LPS binding site detection in Arabidopsis thaliana mesophyll protoplasts

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.

Alternative splicing of the receptor‐like kinase Nt‐Sd‐RLK in tobacco cells responding to lipopolysaccharides: suggestive of a role in pathogen surveillance and perception?

“…Our previous work has shown that LPS from B. cepacia initiate characteristic MAMP responses typically mediated by pattern‐recognition receptors (PRRs) in plants . Nt‐Sd‐RLK was first identified as a differentially expressed, LPS‐responsive transcript .…”

Identification of Candidate Ergosterol-Responsive Proteins Associated with the Plasma Membrane of Arabidopsis thaliana

“…Both light chains (CLCs) and clathrin heavy chains (CHCs), including CHC1 and CLC3, were identified in the non-enriched PM-associated fraction (Table 1 and Table 2). In this regard, Mgcina et al [41] also speculated that the binding-site of lipopolysaccharide (LPS) as a bacterial MAMP to A. thaliana protoplasts is internalized into the cell by endocytosis, thus leading to the reduced level of receptors on the surface.…”

Plasma Membrane-Associated Proteins Identified in Arabidopsis Wild Type, lbr2-2 and bak1-4 Mutants Treated with LPSs from Pseudomonas syringae and Xanthomonas campestris

“…SA regulates endocytosis by interfering with clathrin-mediated protein trafficking but did not affect flg22-induced endocytosis of the FLS2 receptor during pathogen responses in Arabidopsis [143]. Mgcina et al [144] proposed that LPS binding might involve ligandinduced receptor endocytosis and recycling in Arabidopsis. Endocytosis of PM-localised PRR for recycling or degradation involving transport and trafficking proteins is part of the regulation of ligand-induced defence signalling [63,121].…”