Changes in Tobacco Cell Membrane Composition and Structure Caused by Cercosporin

Abstract

Cercosporin, a toxin produced by Cercospora species, rapidly kills plant cells in the Light. Previous work has shown that cercosporin treatment causes products of lipid peroxidation to be released. We have found that the unsaturated acyl chains of lipids in tobacco (Niotiana tabacum) cell membranes are destroyed when cells are treated with cercosporin. Concomitant with this change in composition is a change in structure of the membranes as detected by two different fatty acid spin labels, 2-(3-carboxypropyl)4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl (denoted 1112,31) and 2-(1'-carboxytetradecyl)-2-ethyl-4,4-dmethyl-3-oxazolidinyloxyl (denoted 111,141). Cercosporin causes the membranes to become more rigid at all temperatures tested and increases the membrane phase transformation temperature from 12.70C to 20.80C.Cercosporin, a toxic compound from Cercospora species, affects both hosts and non-hosts of the producing fungi. Cercosporin was first isolated in 1957 by Kuyama and Tamura (14) from C. kikuchii, a soybean pathogen, and has since been isolated from a large number of Cercospora species (1,8,16) and from Cercosporainfected plants (8,14). The structure of cercosporin was determined independently by Lousberg et al. (15) and Yamazaki and Ogawa (25).Cercosporin is a photosensitizing agent capable of generating both singlet oxygen and superoxide during irradiation (M. Daub and R. Hangarter, unpublished). Plant cells, bacteria, and mice are killed rapidly by cercosporin in the presence of light (5,26). Cercosporin causes electrolytes to leak from plant tissues (6, 17), destroys plant protoplasts (6), and causes peroxidation of plant membrane lipids both in vitro (3) and in vivo (6). These data suggest that cercosporin acts directly on membranes in situ.We report here that cercosporin causes a large increase in the ratio of saturated to unsaturated fatty acids extracted from membranes of toxin-treated cells. Concomitant with this change is a decreased membrane fluidity and the possible appearance of a phase separation in the membranes at the growth temperature of the cells. Electrolyte leakage and cell death may be accounted for by these perturbations of membrane composition and structure. MATERIALS AND METHODS Cercosporin. Cercosporin was isolated and purified from cultures of Cercospora nicotianae as previously described (5). Stock solutions were prepared in acetone and stored at -20°C in the dark. Control cultures were either treated with the same concentration of acetone or treated with cercosporin but incubated in the dark. Final acetone concentrations in both treated and control cultures did not exceed 1% (v/v).Host Material. All experiments were conducted with Nicotiana tabacum cv. 'Wisconsin 38' cell culture line NT575. Liquid suspension cultures were maintained as previously described (5).Protoplast Isolation. Protoplasts were isolated from NT575 suspension cultures 2 d after subculture (early log phase). The cells were preplasmolyzed in 0.6 M mannitol for 2 h followed by centrifugation. Packed ...

The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis

“…These reactive oxygen species cause damage to various cellular components including lipids, proteins and nucleic acids (Daub and Ehrenshaft, 2000). Cercosporin has been shown to produce both 1 O 2 and O ÁÀ 2 , causing peroxidation of cell membrane lipids and consequently electrolyte leakage in plants (Daub, 1982a;Daub and Briggs, 1983;Daub and Hangarter, 1983). Due to the production of reactive oxygen species, cercosporin has universal toxicity not only to host plants, but also to fungi, bacteria and mice (Daub, 1982b;Ito, 1981;Yamazaki et al, 1975).…”

Functional characterization of three genes encoding putative oxidoreductases required for cercosporin toxin biosynthesis in the fungus Cercospora nicotianae

“…Cercosporin is capable of breaking down different cellular components such as lipids, proteins and nucleic acids, depending on its localization in cells. During pathogenesis in host plants, cercosporin has been shown to damage cell membranes by causing lipid peroxidation, electrolyte leakage and eventually cell death (Daub, 1982;Daub & Briggs, 1983).…”

Hypericin and Its Photodynamic Action

“…, 1975). This compound is also photodynamically active in damaging plant cell membranes (Marcri and Vianello, 1979; Daub, 1982a,b; Daub and Briggs, 1983). Singlet oxygen has been implicated as the oxidant in the'photodynamic action of cercosporin (Dobrowolski and Foote, 1983).…”