Phosphoproteins involved in the signal transduction of cryptogein, an elicitor of defense reactions in tobacco.

We previously reported that the signal transduction of cryptogein, an elicitor of defense reactions in Nicotiana tabacum cells, involves upstream protein phosphorylation. In the present study, induction of these early physiological events was further investigated with inhibitors of protein phosphatase (PP), okadaic acid, and calyculin A. Calyculin A mimicked the effects of cryptogein, inducing an influx of calcium, an extracellular alkalinization, and the production of active oxygen species (AOS), suggesting that during cryptogein signal transduction the balance between specific protein kinase (PK) and PP activities was modified. To identify the phosphorylated proteins that could be involved early in the elicitor signaling pathway, we analyzed by 2-D electrophoresis the in vivo phosphorylation status of proteins after cryptogein, staurosporine, and calyculin A treatments of tobacco cells (5 min). Of about 100 phospho-labeled polypeptides, 19 showed increased 32P incorporation after 5 min of cryptogein treatment. Phosphorylation of 12 of the 19 polypeptides depended upon calcium influx. Staurosporine inhibited the phosphorylations induced by cryptogein whereas calyculin A activated the phosphorylation of 18 of these polypeptides. This study highlighted the role of PKs and/or constitutive active PPs whose activation and inhibition, respectively, resulted in an increased phosphorylation of proteins that may be involved in cryptogein signal transduction. Identification of the phosphoproteins is in progress and will increase our knowledge of signal transduction pathways implicated in plant defense responses.

Involvement of plasma membrane proteins in plant defense responses. Analysis of the cryptogein signal transduction in tobacco.

Cryptogein, a 98 amino acid protein secreted by the fungus Phytophthora cryptogea, induces a hypersensitive response and systemic acquired resistance in tobacco plants (Nicotiana tabacum var Xanthi). The mode of action of cryptogein has been studied using tobacco cell suspensions. The recognition of this elicitor by a plasma membrane receptor leads to a cascade of events including protein phosphorylation, calcium influx, potassium and chloride effluxes, plasma membrane depolarization, activation of a NADPH oxidase responsible for active oxygen species (AOS) production and cytosol acidification, activation of the pentose phosphate pathway, and activation of two mitogen-activated protein kinase (MAPK) homologues. The organization of the cryptogein responses reveals that the earliest steps of the signal transduction pathway involve plasma membrane activities. Their activation generates a complex network of second messengers which triggers the specific physiological responses. This study may contribute to our understanding of plant signaling processes because elicitors and a variety of signals including hormones, Nod factors, light, gravity and stresses share some common transduction elements and pathways.

Activation of MAPK homologues by elicitors in tobacco cells.

Elicitors of plant defence reactions (such as cryptogein, an elicitin produced by Phytophthora cryptogea, or oligogalacturonides (OGs)), induced in tobacco cell suspensions (Nicotiana tabacum var Xanthi) a rapid and transient activation of two protein kinases (PKs) with apparent molecular masses of 50 and 46 kDa, respectively. These PKs activated and phosphorylated at tyrosine residues, phosphorylated myelin basic protein (MBP) at serine/threonine residues. Both are recognized by anti-MAPK antibodies. The two MBP kinases possessed the same kinetics of activation, and their activation depended, to the same extent, on different exogenously applied compounds (staurosporine, lanthanum, EGTA). We demonstrate here that the activation of the MBP kinases is calcium dependent and sensitive to staurosporine, a protein kinase inhibitor which annihilates all known responses of tobacco cells to cryptogein. The activation of MBP kinases appeared to be independent of the production of active oxygen species (AOS) and insensitive to calyculin A, a protein phosphatase type 1 and 2A inhibitor. The activation of MAPKs is discussed in relation to the early responses induced by cryptogein.