Involvement of Plasma Membrane Calcium Influx in Bacterial Induction of the K⁺/H⁺ and Hypersensitive Responses in Tobacco

Abstract

An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K+/H+ response characterized by specific plasma membrane K+ efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension gested by Keppler et al. (15,16). Bacterial induction of the K+/H+ response is determined by genes within a 31 kb DNA fragment that also controls HR induction (13).We are interested in the mechanism by which the K+/H+ response and active oxygen production are activated during the HR. Rapid stimulation of plasma membrane ion channel, antiporter, and oxidase activities by transmembrane signal transduction pathways has been demonstrated in numerous animal cell systems (10, 22). In addition, phytoalexin synthesis in plants, a phenomenon often associated with the HR, may be triggered by a signaling pathway (20, 30). We therefore postulated that such a pathway is involved in induction of the K+/H+ and hypersensitive responses in tobacco by P. syringae pathovars. In this paper, we show that increased plasmalemma Ca2" influx is associated with and required for these host responses. MATERIALS AND METHODS ChemicalsThe HR3 is a rapid but localized necrotic response of plant cells to microbial invasion and is associated with disease resistance (18). Although its biochemical basis is not known, changes in membrane potential (24), ion fluxes (3, 11), lipid peroxidation (16, 17), and membrane-associated NADPHdependent oxidase activity (6) suggest plasma membrane involvement. In tobacco, initiation of the HR to Pseudomonas syringae pathovars pisi and syringae is marked by active oxygen production (15, 16), K+ efflux, intracellular acidification, and a net uptake of H+ from the extracellular medium (3,4). The K+/H+ response was initially described as a K+ efflux/H+ influx exchange (3), but H+ influx is unproven. Inhibition of this response and HR-associated cell death by increased extracellular pH and ATPase inhibitors (1, 27

Electrophysiological Characterization of the Arabidopsis avrRpt2-Specific Hypersensitive Response in the Absence of Other Bacterial Signals

“…The ion fluxes during HR are likely to be complex; simultaneous transient extracellular alkalinization, Ca 21 uptake, and K 1 efflux are characteristic of bacterial HR in suspension cells (Atkinson et al, 1985(Atkinson et al, , 1990. Because all these ion fluxes are down the chemical gradient, they would occur as a direct consequence of membrane depolarization caused by membrane damage or H 1 -ATPase down-regulation, and do not necessarily constitute a specific signal.…”

Measurement of Ca2+ Fluxes during Elicitation of the Oxidative Burst in Aequorin-transformed Tobacco Cells

“…Indeed, several groups have reported that extracellular EGTA can inhibit the mobilization of selected disease resistance mechanisms (52,53). Other workers have shown that introduction of exogenous Ca 2ϩ into resting cells can initiate a defense response in the absence of the usually required elicitors (16,54).…”

“…Other workers have shown that introduction of exogenous Ca 2ϩ into resting cells can initiate a defense response in the absence of the usually required elicitors (16,54). Still other laboratories have demonstrated that Ca 2ϩ channel blockers such as La 3ϩ can prevent expression of a normal pathogen resistance mechanism when conditions are otherwise designed to stimulate its induction (52,54). Nevertheless, it has never been determined wherein Ca 2ϩ actually exerts control over processes leading to disease resistance.…”

The generation of oxygen radicals during host plant responses to infection

“…H 2 0 2 has been implicated in signal transduction during insulin action in animal systems [67,81]. Significantly, plasma membrane calcium influx, another recognized component of mammalian cell signal-transduction [122], has been observed as an early event in the bacteria-induced hypersensitive reaction of tobacco suspension cells [4].…”