Abstract
Plants respond to pathogen attack by inducing a coordinated resistance strategy, which
results in the expression of defense gene products. When a plant-pathogen interaction
results in disease establishment, parasite colonization is caused by a delayed plant defense
response, not due the absence of any response. Thus, the speed and intensity of the plant
response and intracellular signalling determines the outcome of a plant-pathogen interaction.
The acceleration of plant responses by the application of resistance inducers could provide a
commercially, biologically and environmentally feasible alternative to existing pathogen
control methods.
Lipopolysaccharides are amphipathic lipoglycans that are attached to the outer bacterial
membrane by a lipidic entity inserted into the bacterial phospholipid monolayer, with the
saccharidic part oriented towards the exterior. The general structure of this compound is
comprised of an anchor named lipid A associated with a core polysaccharide, which bears an
O-antigen domain.
LPS has been described as one of the pathogen-associated molecular patterns (PAMPs)
capable of eliciting the activation of the plant innate immune system. LPS present in the outer
membranes of plant growth-promoting rhizobacteria (PBPR) are major determinants of
induced systemic resistance (ISR). In addition, LPS may function as an activator of systemic
acquired resistance (SAR), providing non-specific immunization against later infection.
Evidence suggests that LPS may advance plant disease resistance using the mechanism of ISR
or SAR through its application to plants as a sensitizing agent, priming them to respond more
effectively to subsequent pathogen attack.
Phosphorylation plays a major role during the plant defense response, exemplified by its
phosphorylation of transcription factors, required for the expression of defense-related
genes. One of the most extensively documented phosphorylation responses is that of MAP
kinase activation by phosphorylation in response to elicitation by race-specific and non-racespecific
elicitors in various plant species.Proteins that undergo differential phosphorylation as a result of elicitation could be
components of signal transduction pathways which connect pathogen perception with
defense responses. Thus the identification of protein kinases, protein phosphatases and their
substrates is essential in the elucidation of plant defense responses.
The hypothesis behind this dissertation is that LPS elicitation results in alterations in the
phosphorylation profile of Arabidopsis thaliana proteins.
In this study, LPS was extracted from the cell walls of Burkholderia cepacia, a bacterial
endophyte, and characterized by SDS-PAGE. The exposure of Arabidopsis callus culture cells
to LPS resulted in distinctive changes in the phosphoprotein profile of the cells. Radioactive
phosphorous labelling of proteins provided evidence that phosphorylation occurs in
Arabidopsis following LPS perception, as part of a defense response related to LPS elicitation.
Further investigation of differential protein phosphorylation via immunoblotting with antiphosphotyrosine
antibodies revealed that tyrosine phosphorylation of Arabidopsis proteins
occurs in response to LPS.
One of the tyrosine-phosphorylated proteins was found to be a 42 kDa kinase, activated
in response to LPS elicitation. The identity of the kinase as a mitogen-activated protein
(MAP) kinase was confirmed by immunoblotting with anti-active MAP kinase antibodies. In
addition, an assay of MAP kinase activity demonstrated the ability of the LPS-responsive MAP
kinase to phosphorylate the ERK-MAP kinase substrate Elk1.
In terms of the global phosphoproteome of Arabidopsis in response to LPS,
phosphopeptides were purified from a crude protein digest by immobilized metal affinity
chromatography and analyzed by liquid chromatography-tandem mass spectrometry (LCMS/
MS). While LC indicated both quantitative and qualitative differences resulting from LPS
elicitation, no peptides could be positively identified as phosphopeptides by MS analysis. This
work can however be repeated with further precautions to prevent the loss of phosphate
groups prior to analysis.
The results obtained in this study indicate that LPS causes specific alterations in Arabidopsis
protein phosphorylation as a post-translational modification in response to the perception of
LPS during a plant-pathogen interaction, proving the original hypothesis.
Prof. I.A. Dubery