Identification of a lipopolysaccharide responsive erk-like MAP kinase in tobacco leaf tissue

Lipopolysaccharides (LPS)are indispensable cell surface components of Gram-negative bacteria and have diverse roles in plant-microbe interactions. Treatment of Nicotiana tabacum with the LPS of an endophytic strain of Burkholderia cepacia results in an enhanced defensive capacity in the tissue. In this study the rapid and transient phosphorylation of an extracellular signal-regulated (ERK)-like mitogen-activated protein (MAP)kinase in response to LPS from B. cepacia (LPS B.cep. ) elicitation is reported. Based on in-gel kinase assays it was found that this 43-kDa LPS B.cep. -responsive kinase is optimally activated following 7 min elicitation with 100 ì g/mL LPS. Its identity as an ERK MAPK was supported by tyrosinephosphorylated association with induction, immunodetection with pTEpY-specific MAPK antibodies and inhibition of phosphorylation by U0126, an upstream MAPKK inhibitor. The kinase utilized myelin basic protein, but not casein or histone, as substrate. Ca 2+ did not modulate the phosphorylation, nor did wounding. To date, other MAP kinases have been shown to act either independently or upstream from reactive oxygen intermediates produced during the oxidative burst. It was found that hydrogen peroxide is either not generated in leaf tissue in response to LPS elicitation or, if generated, does not trigger the phosphorylation of the kinase. Physicochemical characterization of the ERK-like MAPK indicated a molecular mass of 43 kDa and a pI of 6.3; two-dimensional gel analysis indicated two charge isomers. This is the first demonstration of such an LPS-responsive MAP kinase phosphorylation in plants.