Initial signalling processes induced by elicitors of ectomycorrhiza-forming fungi in spruce cells can also be triggered by G-protein-activating mastoparan and protein phosphatase-inhibiting cantharidin

The first responses in spruce [Picea abies (L.) Karst.] cells induced by el icitors (N-acetylglucosamine oligomers) from ectomycorrhizal fungi have bee n described as follows. efflux of Cl- and K+, influx of Ca2+, extracellular alkalinization, phosphorylation of a 63-kDa protein (pp63), dephosphorylat ion of a 65-kDa protein (pp65) and synthesis of H2O2 (Salzer et al. 1996, P lanta 198. 118-126). In order to obtain new insights into the triggering me chanism and the sequence of these rapid responses we used compounds which a re known to activate or block specific steps within an elicitor-induced sig nal transduction cascade in plant cells. Comparable to elicitors the two pr otein phosphatase inhibitors, cantharidin and calyculin A, as well as masto paran, an activator of trimeric G-proteins, were able to induce the release of Cl- and K+ from spruce cells and the alkalinization of the medium. Half -maximal activation of the alkalinization occurred at 133 nM calyculin A, 2 .3 mu M cantharidin and 1.6 mu M mastoparan. The structural analogue of mas toparan, Mas 17, which has no G-protein-stimulating properties, was unable to trigger the above-mentioned reactions. In addition, cantharidin and caly culin A induced an increased synthesis of H2O2 in spruce cells which was pr olonged in comparison to the elicitor-induced transient formation of H2O2 A lso, the cantharidin-induced release of K+ was more pronounced and longer l asting than that caused by elicitors from the ectomycorrhizal fungus Hebelo ma crustulini-forme (Bull. ex Fries.) and N-acetylglucosamine oligomers. Fu rthermore, cantharidin, calyculin A and mastoparan induced the phosphorylat ion of pp63. Remarkably, the protein kinase inhibitor, staurosporine, inhib ited all the rapid responses described above, no matter whether they were t riggered by fungal elicitors or by the protein phosphatase inhibitors. Thes e results indicate that in the initial signalling events in spruce cells, e ssential protein phosphorylations occur either as an (auto) phosphorylation of a membrane-bound receptor kinase prior to the activation of a G-protein or (and) immediately downstream of the activated G-protein in a phosphoryl ation cascade and are the basic requirements for the ion fluxes following d ownstream.