1.2.31
DOES HYDROGEN PEROXIDE ACT AS A TOXIN IN THE HYPERSENSITIVE RESPONSE?

H LU and VJ HIGGINS

Department of Botany, University of Toronto, Toronto, Canada M5S 3B2

Background and objectives
An oxidative burst appears to be a general feature of the hypersensitive response (HR) of plants to avirulent pathogens. Possible roles of active oxygen species, particularly hydrogen peroxide, in the plant defence response include a direct toxic effect on the pathogen and/or the host, cross-linking of host cell-wall components, and signalling to activate other host defences. Our objective was to compare the in planta levels of hydrogen peroxide occurring during the HR with the levels required to show toxicity to host cells and to a fungal pathogen.

Materials and methods
Races of Cladosporium fulvum virulent or avirulent on tomato plants with the Cf5 or Cf9 genes for resistance were used to produce intercellular fluids (IFs) with or without AVR5 or AVR9. Leaves of Cf9 plants and cell suspensions prepared from Cf5 plants were used to test the ability of IFs, or hydrogen peroxide, to cause necrosis (by a rating system) and cell death (by an Evans Blue assay [1]) and to estimate the amount of hydrogen peroxide accumulating after elicitation using the probe 21,71-dichlorofluorescin diacetate [2].

Results and conclusions
The concentration of hydrogen peroxide accumulating in Cf9 leaf tissue 2.5 h after injection with IF containing AVR9 (at a concentration causing total necrosis within 12 h) was estimated to be >10 mM [2]. In contrast, injection of hydrogen peroxide at even 100 mM caused an insignificant degree of necrosis. Assays with cell suspensions, confirmed the low toxicity of hydrogen peroxide to tomato cells but, as expected, with the addition of Fe2+ with hydrogen peroxide (or with IF) enhanced toxicity (e.g. at 10 mM hydrogen peroxide and 1 mM Fe2+) cells retained about three times more Evans Blue than cells treated only with hydrogen peroxide. Germination and germ-tube growth of conidia of C. fulvum were significantly retarded by 4-5 mM hydrogen peroxide, and at higher concentrations death of germ tubes was observed (EDSO=22 mM). The addition of Fe2+ with hydrogen peroxide had little effect on fungal growth or viability. In conclusion, the amount of hydrogen peroxide accumulating during an elicitor-induced response in leaves appears to be sufficient to affect fungal colonization but not to directly affect viability of host cells unless the Fe2+ status in the apoplast is in some way altered by the elicitor.

References
1. Turner JG, Novacky A, 1974. Phytopathology 64, 885-890.
2. Lu H, Higgins VJ, 1998. Physiological and Molecular Plant Pathology (in press).