Proteomic profiling of cellular targets of lipopolysaccharide-induced signalling in Nicotiana tabacum BY-2 cells.

Plants constantly monitor for pathogen challenge and utilize a diverse array of adaptive defense mechanisms, including differential protein regulation, during pathogen attack. A proteomic analysis of Nicotiana tabacum BY-2 cells was performed in order to investigate the dynamic changes following perception of bacterial lipopolysaccharides. A multiplexed proteome analysis, employing two-dimensional difference-in-gel-electrophoresis with CyDye DIGE fluors, as well as Ruthenium II tris (bathophenanthroline disulfonate) fluorescence staining and Pro-Q Diamond phosphoprotein-specific gel staining, monitored over 1500 proteins and resulted in the identification of 88 differentially regulated proteins and phosphoproteins responsive to LPS(B.cep.)-elicitation. Functional clustering of the proteins both at the level of their abundance and phosphorylation status, revealed 9 proteins involved in transport, ion homeostasis and signal transduction. A large number of responsive proteins were found to be involved in metabolism- and energy-related processes (36), representing various metabolic pathways. Another abundant category corresponded to proteins classified as molecular chaperones and involved in protein destination/targeting (12). Other categories of proteins found to be LPS(B.cep.)-responsive and differentially regulated include cell structure- and cytoskeletal rearrangement proteins (8) and proteins involved in transcription and translation as well as degradation (11). The results indicate that LPS(B.cep.) induces metabolic reprogramming and changes in cellular activities supporting protein synthesis, -folding, vesicle trafficking and secretion; accompanied by changes to the cytoskeleton and proteosome function. Many of the identified proteins are known to be interconnected at various levels through a complex web of activation/deactivation, complex formation, protein-protein interactions, and chaperoning reactions. The presented data offers novel insights and further evidence for the biochemical action of LPS(B.cep.) as a resistance elicitor, a pathogen-associated molecular pattern molecule and triggering agent of defense responses associated with innate immunity.

The O-chain structure from the LPS of the endophytic bacterium Burkholderia cepacia strain ASP B 2D.

The O-chain polysaccharide of the lipopolysaccharide from the endophytic bacterium Burkholderia cepacia strain was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy and shown to be the following: -->2)-beta-D-Ribf-(1-->6)-alpha-D-Glcp-(1-->.

Protein phosphorylation in Nicotiana tabacum cells in response to perception of lipopolysaccharides from Burkholderia cepacia.

Bacterial LPS have the ability to act as modulators of the innate immune response in plants. Complex and largely unresolved perception systems exist for LPS on the plant cell surfaces that lead to the activation of multiple intracellular defense signaling pathways. The aim of the present study was to investigate the perception mechanism of cultured Nicotiana tabacum cells towards LPS from Burkholderia cepacia (LPS(B.cep.)), with regard to the role of protein phosphorylation during signal perception-related responses to gain a better understanding of the chemosensory perception of LPS elicitor signals in plant cells. In vivo labeling of protein phosphorylation events during signal transduction indicated the rapid phosphorylation of several proteins with the hyperphosphorylation of two proteins of 28 and 2 kDa, respectively. Significant differences and de novo LPS-induced phosphorylation were also observed with two-dimensional analysis. The protein kinase inhibitor, staurosporine, totally inhibited the extracellular alkalinization response induced by LPS(B.cep.), while the oxidative burst was only partially inhibited by staurosporine. Inhibition of protein phosphatase activity by calyculin A intensified the LPS(B.cep.) responses. The results indicate that perception- and signal transduction responses during LPS(B.cep.) elicitation of tobacco cells require a balance between the actions of certain protein kinases and protein phosphatases.

Early perception responses of Nicotiana tabacum cells in response to lipopolysaccharides from Burkholderia cepacia.

Lipopolysaccharides (LPS) are cell surface components of Gram-negative bacteria and, as microbe-/pathogen-associated molecular patterns, have diverse roles in plant-microbe interactions, e.g. LPS are able to promote plant disease tolerance through activation of induced or acquired resistance. However, little is known about the mechanisms of signal perception and transduction in response to elicitation by these bio-active lipoglycans. The present study focused on the involvement of LPS isolated from the outer cell wall of the Gram-negative bacterium Burkholderia cepacia (strain ASP B 2D) in the molecular mechanisms and components involved in signal perception and transduction and defense-associated responses in suspension-cultured tobacco (Nicotiana tabacum L.) cells. The purified LPS(B.cep.) was found to trigger a rapid influx of Ca2+ into the cytoplasm of aequorin-transformed tobacco cells. An oxidative burst, concomitant with the production of reactive oxygen and nitrogen species was measured by chemiluminescence and fluorescence. These early perception responses were accompanied by K+/H+ exchange and alkalinization of the extracellular medium. Through the use of various inhibitors of the oxidative burst reaction, as well as scavengers of produced radicals, the biochemical basis of the cellular response to LPS(B.cep.) elicitation was dissected, elucidated and compared to that induced by a yeast elicitor. These results suggest that LPS(B.cep.) interacts with tobacco cells in a manner different from the response elicited by yeast elicitor.

Fluorescence microplate assay for the detection of oxidative burst products in tobacco cell suspensions using 2',7'-dichlorofluorescein.

The technique of 2',7'-dihydrodichlorofluorescin diacetate (H2DCF-DA)-derived fluorescence was applied to measurements of the oxidative burst reaction in plant cell suspension cultures in an automatic fluorometric multiwell microplate assay. The developed procedure was found to be versatile and effective for the determination of the oxidative burst reaction in plant cell cultures. Using this assay, cumulative production of reactive oxygen intermediates may be monitored and recorded non-destructively on a real-time scale for a large number of samples at frequent and continual time intervals for time course experiments. Through the use of various inhibitors and inducers or elicitors of the oxidative burst in combination with H2DCF-DA, this assay aids in the dissection of the signal transduction pathways and the determination of the origins of the oxidative burst in plant cells.