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Induction of Hypersensitive Cell Death by a Fungal Protein in Cultures of Tobacco Cells

Treatment of suspension-cultured tobacco (Nicotiana tabacum cv. Xanthi) cells (line XD6S) with fungal proteinaceous elicitors, namely, xylanase (EC 3.2.1.8) from Trichoderma viride (TvX) and xylanase from T. reesei (TrX), induced shrinkage of the cytoplasm, condensation of the nucleus, and, finally, cell death, which were accompanied by typical defense responses that included an oxidative burst and expression of defense genes. A Ca²⁺ channel blocker, Gd³⁺, inhibited the typical response of XD6S cells to TvX, which resembled the hypersensitive reaction (HR). These results suggested that the influx of Ca²⁺ ions plays an important role as a secondary signal. The HR was not observed in TvX-treated tobacco cells (line BY-2) derived from cv. Bright Yellow 2. This result suggests that key features of cultivar-specific interaction can be observed in cultures of tobacco cells. Xylanase from Bacillus circulans (BcX) and B. subtilis (BsX), which has enzymatic properties similar to those of TvX but an amino acid sequence different from that of TvX, did not induce the HR-like response in XD6S cells. These results suggest that the elicitor action of TvX is not due to its ability to hydrolyze cell walls but requires the TvX-specific recognition factors in plant cells. Thus, TvX-induced cell death was not due to some general toxic effect, but seems to be mediated by the activation of a specific cellular signal-transduction cascade that converges with a pathway that activates the intracellular cell death program.