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Rapid, Cf-9- and Avr9-Dependent Production of Active Oxygen Species in Tobacco Suspension Cultures

The tomato Cf-9 gene confers resistance to races of Cla-dosporium fulvum expressing the corresponding avirulence gene Avr9. The availability of transgenic tobacco lines carrying Cf-9, and the well-characterized 28 amino acid Avr9 elicitor, make this an excellent system to study resistance gene function. In this paper we establish tobacco suspension cultures derived from transgenic tobacco plants containing Cf-9, as well as from plants without Cf-9. Cultures derived from Cf9 tobacco produce active oxygen species (AOS) within 5 min of treatment with pure or synthetic Avr9. This enabled us to perform biochemical and pharmacological analysis in cell culture of the very earliest events in resistance gene function. In addition to AOS production, an increase in oxygen uptake was detected in the Avr9-treated Cf9 cells. Both phenomena were inhibited by low concentrations of diphenyleneiodonium (DPI). Additional pharmacological inhibitor studies suggest that uptake of calcium, activation of protein kinases, and probably phospholipase A₂ (PLA₂) activity are intermediates in the Cf-9- and Avr9-dependent signaling pathway that leads to AOS production. Interestingly, those defense responses did not result in plant cell death.