Conventional Alexa 488 Hydrazide in Quantum dot labelling of conjugates for lipopolysaccharide binding studies in <i>Arabidopsis thaliana</i> protoplast

Abstract

Conventional Alexa 488 Hydrazide in Quantum dot labelling of conjugates for lipopolysaccharide binding studies in Arabidopsis thaliana protoplast Lipopolysaccharide (LPS) is a complex lipoglycan that is found in the outer membrane of Gram-negative bacteria and composed of three regions namely the fatty acid Lipid A, a core region of short oligosaccharide chains and an O-antigen region of polysaccharide. When LPS is used as a MAMP/PAMP, it not only induces an innate immune response in plants but also stimulates the development of defense responses such as the immediate release of ROS/ROI, PR gene expression and activation of the hypersensitive response (HR), resulting in stronger subsequent pathogen interactions. The identification and characterization of the elusive LPS receptor or receptor complex in plants is thus of importance, since understanding the mechanism of perception and specific signal transduction pathways will clarify, and lead to the advancement of, basal resistance in plants in order to decrease plant crop losses due to pathogen attack. In mammals, LPS binds to LBP (LPS binding protein) to form a LPS-LBP complex which is translocated to MD2 with the presence or absence of its co-receptor, a glycosylphosphatidylinositol (GPI)-linked protein, CD14. The interaction occurs on the host membrane and triggers an inflammatory defense response through the signaling cascade activated by the interaction with Toll-like receptor 4 (TLR4). A similar LPS-receptor interaction is however, unknown in plants. To address this, biological binding studies with regard to concentration, incubation time and temperature, affinity, specificity and saturation were conducted using LPS labeled with Alexa 488 hydrazide. Although such labeling does not affect the biological activity in mammalian studies, the same cannot necessarily be said for plant systems. Thus, quantum dots, which allow non-covalent hydrophobic labelling of LPS, were further employed in binding studies. The conjugation to LPS was confirmed by transmission electron microscopy and results illustrated higher fluorescence values as compared to Alexa-LPS fluorescence analysis. Furthermore, inhibition of the process is also reported using Wortmannin and Brefeldin A as suitable endocytosis inhibitors.