The Hypersensitive Reaction of Tobacco to Pseudomonas syringae pv. pisi

Abstract

Net electrolyte efflux from suspension-cultured tobacco cells undergoing the hypersensitive reaction to Pseudomonas syringae pv. pisi resulted from a specific efflux of K which was accompanied by an equimolar net influx of H+. These fluxes began 60 to 90 minutes after inoculation of tobacco cells with bacteria, reached maximum rates of 6 to 9 micromoles per gram fresh weight tobacco cells per hour within 2.5 to 3 hours, and dropped below 4 micromoles per gram per hour within 5 hours. Tobacco cells lost approximately 35% of total K' during this period, and average cellular pH declined by approximately 0.75 pH unit. These events were accompanied by a 30% decrease in cellular ATP. K+ and H' fluxes were inhibited by the protonophore (p-trifluoromethoxy)carbonyl cyanide phenylhydrazone and by increasing the K' concentration of the external solution. Tobacco leaf discs inoculated with the bacterium also exhibited a specific net K+ efflux and H+ influx. These results suggest that induction of the hypersensitive reaction in tobacco proceeds through the activation of a passive plasmalemma K+/H+ exchange mechanism. It is hypothesized that activation of this exchange is a major contributing factor in hypersensitive plant cell death.The hypersensitive reaction is characterized by the rapid death ofindividual plant cells which come into contact with pathogenic organisms, and is generally associated with disease resistance of the whole plant to the pathogen (1 1, 13, 14). The capacity to express the HR3 appears to be universal among highet plants and can be triggered by bacterial, fungal, viral, and nematode pathogens. Despite its close association with resistance, the HR and its role in pathogen localization are not well understood. It has even been argued that hypersensitivity is a consequence rather than a cause ofdisease resistance (1 1 for 16 to 20 h before each experiment. Bacteria were suspended in standard assay medium (0.175 M mannitol, 0.5 mm K2SO4, 0.5 mm CaCl2, 5 mm Mes adjusted to pH 6.0 with Tris), washed twice by centrifugation, and resuspended in this medium to an inoculum density of 8 x 109 viable bacteria/ml unless otherwise stated. Suspension-cultured tobacco cells were derived from Nicotiana tabacum var Hicks (10) and maintained as previously described (3). Cells were collected by filtration from logarithmically growing cultures, washed with 10 ml assay medium/g fresh weight tobacco, and resuspended in 28.5 ml assay medium/g tobacco. Cell suspensions were incubated in 50-ml beakers at 27°C on a reciprocal shaker at 150 oscillations/min for approximately 1 h. This preincubation period was necessary for consistent induction of the HR. Tobacco cell suspensions were then inoculated with 1.5 ml bacterial suspension/g tobacco. This

Effects of Extracellular pH on UV-Induced K+ Efflux from Cultured Rose Cells

“…1 B): in respiring cells, the H+ on the outside would be supplied to a great extent by the hydration of CO2 and dissociation of H2CO3 outside the cell. The second mechanism would be consistent with the conclusions of Atkinson et al (2) regarding the K+/H+ counterflux induced in tobacco cells treated with P. syringae. In both hypothetical mechanisms, the result is a net efflux of K+ (cultured rose cells and tobacco cells both lose about 30% of the available soluble K+), alkalinization of the exterior of the cells, and acidification of the interior.…”

“…Cells were irradiated with UV-C for 2 Exposure to the modified atmosphere began 15 min before treating with UV in order to allow equilibration. All beakers were exposed to normal atmosphere during UV treatment, after which they were returned to the modified atmosphere.…”

“…Ion leakage as a stress response has also been shown to be caused by other factors such as herbicides (14), chilling (7), ozone (5), heavy metals (8), and certain plant pathogens (2). However, while most of these stress factors cause a generalized leakage of a variety of solutes, UV at low fluences causes a specific K+ leakage from cultured rose cells (12).…”

“…However, while most of these stress factors cause a generalized leakage of a variety of solutes, UV at low fluences causes a specific K+ leakage from cultured rose cells (12). Treatment of suspension-cultured tobacco cells with Pseudomonas syringae, a bacterial plant pathogen, can also cause a specific leakage of K+ (2).…”

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.…”