Elicitor stimulation of the defense response in cultured plant cells monitored by fluorescent dyes

Addition of fungal elicitors to plant cells in suspension is known to stimulate biochemical changes in the plant cell leading to production of defense compounds. In this paper we demonstrate that introduction of elicitors from the pathogenic fungus Verticillium dahliae to cultured cotton, tobacco, or soybean cells leads to a rapid, dramatic change in the fluorescence of several membrane-associated potentiometric or pH-sensitive dyes. The fluorescence transitions occur abruptly following a brief (0 to 10 min) lag period in apparently most cells of the suspension simultaneously. Furthermore, both the length of the lag period and the rate of the subsequent fluorescence change were shown to be highly dependent on elicitor concentration. When the crude elicitor extract was separated by gel filtration chromatography into several active fractions, the ability of each fraction to stimulate phytoalexin production in the cotton cell suspension was found to correlate directly with the rate of the fluorescence decrease in the fluorescence assay. Because the assay is rapid, simple to perform, quantitative, and reproducible, it represents an attractive alternative to the more cumbersome and perhaps less quantitative elicitor assays currently in use. The fact that membrane-potential-sensitive dyes of different structure respond to elicitation of plant cells similarly further suggests, but does not prove, that asymmetric ion fluxes into or out of the plant cell are involved in the initial events of elicitor signal transduction.