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

Abstract

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.

Induction of defense responses in tobacco by the protein Nep1 from Fusarium oxysporum

“…The sensitivity of tobacco cells to Nep1 is similar to, if not greater than, their sensitivity to xylanase [19] and cryptogein [24]. Similar specific activity levels have been demonstrated for the Avr9 elicitor in Cf-9 tomato [42], the Pell 3 pectate lyase/elicitor in tobacco [43], and the host/cultivar specific toxin Tox A in wheat [7] when variations in experimental systems are taken into account.…”

TherolBGene Suppresses Reactive Oxygen Species in Transformed Plant Cells through the Sustained Activation of Antioxidant Defense

“…The production of ROS in callus cultures was measured by the luminolbased luminometric method according to Piedras et al (1998). The control and rolB-transformed calli were harvested at 21 and 28 d of cultivation and analyzed using a RF-1501 instrument (Shimadzu EUROPA GmbH).…”

Nicotiana benthamiana gp91phox Homologs NbrbohA and NbrbohB Participate in H2O2 Accumulation and Resistance to Phytophthora infestans

“…However, the oxidative burst has been shown to be controlled through phosphorylation activation by its upstream kinase (Levine et al, 1994;Miura et al, 1995;Piedras et al, 1998) and dephosphorylation inactivation by its negative regulator, phosphatase. Treatment of cultured rice cells with the phosphatase inhibitor calyculin A without elicitor rapidly stimulates a diphenylene iodonium-sensitive oxidative burst (Kawasaki et al, 1999), suggesting that a small portion of its upstream kinase is active.…”

“…Incubation of potato tuber slices with an incompatible race of Phytophthora infestans , the potato late blight pathogen, or with a hyphal wall elicitor stimulated NADPH-dependent O 2 Ϫ -generating activity, which was located predominantly in the plasma membrane fraction (Doke, 1985;Doke and Miura, 1995). Diphenylene iodonium, an inhibitor of the phagocyte NADPH oxidase, blocks the oxidative burst in plant cells (Levine et al, 1994;Auh and Murphy, 1995;Piedras et al, 1998). Homologs of gp91 phox (respiratory burst oxidase homolog [ rboh ]), which is the catalytic subunit of phagocyte NADPH oxidase, were isolated from several plants (Groom et al, 1996;Keller et al, 1998;Torres et al, 1998;Amicucci et al, 1999).…”

“…However, no homologs of the p22 phox , p47 phox , or p67 phox regulators of the phagocyte NADPH oxidase were found in the Arabidopsis genome (Dangl and Jones, 2001). Calcium influx into the cytoplasm (Chandra and Low, 1997;Piedras et al, 1998) and changes in protein Functional Analysis of rboh Gene by VIGS 707 phosphorylation (Kauss and Jeblick, 1995;Miura et al, 1995) are implicated in the activation process of the oxidative burst. To address the function of rboh genes in plant defense, we used virus-induced gene silencing (VIGS) in Nicotiana benthamiana as an efficient reverse genetics approach (Baulcombe, 1999).…”