Identification and characterisation of mitogen activated protein kinases in leaf tissue of Nicotiana tabacum in response to elicitation by Lipopolysaccharides.

Lipopolysaccharides from Gram-negative bacteria are amphipathic, tripartite molecules consisting of a hydrophobic lipid A portion, a core hetero-oligosaccharide and a repetitive hydrophilic O-antigen polysaccharide region. Through cell : cell interactions, plants can come into contact with LPS originating from root-associated rhizobacteria, bacterial endophytes as well as bacterial pathogens. Biologically active LPS molecules have been shown to act as determinants of bacterial virulence but also as determinants of induced systemic resistance (ISR) and activators of the phenotypically related systemic acquired resistance (SAR), characterised by accelerated and enhanced defence responses. LPS as a ¡¥pathogen associated molecular pattern, PAMP¡¦ molecule, has the ability to activate the innate mammalian immunity system and to act as an immunomodulator of immune ¡V and inflammatory systems via the conserved lipid A region. It is thus believed that LPS is able to promote plant disease resistance through activation of ISR and/or SAR; however here, the O-antigen region is also implicated to play a pivotal role in the signal perception and transduction in response to elicitation by this bio-active lipoglycan. LPS was isolated from the cell walls of the endophyte, Burkholderia cepacia, characterised by denaturing electrophoresis and compared to the equivalent from the pathogen Ralstonia solanacearum. When dissolved in the presence of Ca2+ and Mg2+, the LPS attained its biologically active micellar state through complex formation. The former LPS strongly induced the activation of two MAPKs following treatment of Nicotiana tabacum cv Samsun leaves, while comparative inductions with the R. solanacearum counterpart were extremely weak and might be ascribed to it lacking an extensive O-antigen region. No previous reports on LPS-responsive MAP kinases in plant tissues exist in the literature. The time- and dose dependent activation of the two kinases were therefore investigated and their physico-chemical properties compared. A novel 32 kDa MAP kinase was transiently activated in response to exposure to LPS with optimal activation at 7 min post-elicitation with 100 ƒÝg.ml-1 LPS. Its identity as an ERK (extracellular signal-related) MAPK was confirmed by immunodetection with a pTEpY-specific (anti-active) MAPK antibody, tyrosine-phosphorylated association of activation and inhibition of activation by U0126, an inhibitor of upstream MAPKKs. The kinase did not utilise casein, histone or myelin basic protein as substrates and no endogenous substrate could be identified. The activated MAP kinase exhibited a pI of 6.3, but two charge isomers of 32 kDa respectively were found upon two-dimensional electrophoresis. Although loss of the dual-phosphorylated epitope during purification attempts prevented extensive purification, 30% ammonium sulphate fractionation significantly (33 fold) enriched the MAPK. A second, distinct, 30 kDa MAP kinase was transiently activated in response to 125 ƒÝg.ml-1 LPS at 40 min post-elicitation, and its identity as a p38 MAPK, to date not yet found in plants, was confirmed by immunodetection with a pTGpY-specific (anti-active) MAPK antibody, tyrosine-phosphorylation associated with activation and inhibition of activation by SB203580, a direct inhibitor of p38 MAPKs. The kinase did not utilise casein, histone or MBP as substrates and no endogenous substrate could be identified. The kinase displayed a pI of 6.0, but two charge isomers of 30 kDa respectively were found following two-dimensional electrophoresis. Loss of the dual-phosphorylated epitope again prevented significant purification, but the protein was found to be significantly (83 fold) enriched by 30% ammonium sulphate fractionation. Although LPS has been reported to be capable of altering Ca2+ permeability and perturbation of Ca2+ homeostasis across plasma membranes, Ca2+ did not appear to potentiate or reduce the activation of either the 30 or the 32 kDa kinases. To date other MAP kinases have been shown to act either independently or upstream from reactive oxygen intermediates (ROI) produced during the oxidative burst. It was found that peroxide and concomitant ROI is either not generated in leaf tissue in response to LPS elicitation, or if generated, do not trigger the activation of the two kinases. The identification and partial characterisation of these two novel tobacco MAPKs in the signal perception and transduction response to LPS, significantly contributes to understanding the biochemical basis of the mechanism of action of LPS as a ¡¥resistance elicitor¡¦ involved in the triggering of effective plant defence responses and contributes towards relating the activation of mammalian innate immunity to similar responses in plants.Prof. I.A. Duber