Mechanism Of Apoptosis In Tobacco Suspension Cells Induced By Phytophthora Elicitor PB90

PB90, a novel protein elicitor with a molecular mass of 90 kDa purified from culture filtrate of the Oomycete Phytophthora boehmeriae, an important plant pathogen that causes serious boll rot of cotton in P.R. China, could induce hypersensitive cell death (HCD) and systemic acquired resistance in non-host plants. In this study, we elucidate that PB90-induced HCD shares some features of the apoptotic cell death process in animals and investigate the signaling events that mediate HCD.We observed cell death in suspension-cultured cells of Nicotiana tabacum BY-2 with PB90 treatment using Trypan blue staining method. And this cell death could be suppressed by Cycloheximide, an inhibitor of proteins synthesis, which implies that PB90-induced cell death was an active cell death process requiring new protein synthesis. DAPI staining revealed that PB90 induced rapid chromatin condensation, margination, apoptotic bodies formation and DNA laddering, further TUNEL assay also observed the specific breakage of 3'-OH ends. All of the above common morphological characteristics indicated that PB90 induced apoptosis in suspension cultures of tobacco.During the process of PB90-induced apoptosis in tobacco suspension cells, we observered NO burst after 5 min treated with 1 nmol/LPB90 by DAF-2DA staining method. The kinetics of NO burst within 8 h treatment was biphasic: first phase peaked at 0.5 h and second peaked at 6 h post-treatment. The culture medium of tobacco cells alkalization is another early event induced by PB90. Then, we detected that PB90-induced ascrobate (ASC) and glutathione (GSH) metabolisms alteration and cytochrome c (Cyt c) released from mitochondria to cytosol after treatment with PB90. Moreover, the results showed PB90 induced tobacco bax inhibitor gene (TBI) and PR genes (PR1a, PR1b, PR1c) expression.The application of EGTA (a Ca²⁺ specific chelator), LaCl₃ (an inhibitor of Ca²⁺ channel), L-NAME (an inhibitor of NOS), DPI (an inhibitor of mammalian neutrophil plasma membrane NADPH oxidase) and Cycloheximide (CHX, an inhibitor of proteins synthesis) suppressed PB90-induced redox state aleration, TBI and PR genes expression in tobacco cells, and also suppressed PB90-induced tobacco cell death at different levels. Taken together,the result indicated that culture medium alkalization, NO burst, Cyt c reselseand TBI gene expression were involved in the PB90-induced apoptotic cell death transduction pathway; NO, H₂O₂ and Ca²⁺ played important roles in PB90-induced apoptosis and defense response activation, suggesting that the singal transduction pathways of apoptosis and defense response are overlapping.