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 elici tor of defense reactions in Nicotiana tabacum cells, involves upstream prot ein phosphorylation, In the present study, induction of these early physiol ogical events was further investigated with inhibitors of protein phosphata se (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 protei ns that could be involved early in the elicitor signaling pathway, we analy zed by 2-D electrophoresis the in vivo phosphorylation status of proteins a fter cryptogein, staurosporine, and calyculin A treatments of tobacco cells (5 min). Of about 100 phospho-labeled polypeptides, 19 showed increased P- 32 incorporation after 5 min of cryptogein treatment. Phosphorylation of 12 of the 19 polypeptides depended upon calcium influx. Staurosporine inhibit ed the phosphorylations induced by cryptogein whereas calyculin A activated the phosphorylation of 18 of these polypeptides, This study highlighted th e role of PKs and/or constitutive active PPs whose activation and inhibitio n, respectively, resulted in an increased phosphorylation of proteins that may be involved in cryptogein signal transduction, Identification of the ph osphoproteins is in progress and will increase our knowledge of signal tran sduction pathways implicated in plant defense responses.