Effects of Helminthosporium maydis Race T Toxin on Electron Transport in Susceptible Corn Mitochondria and Prevention of Toxin Actions by Dicyclohexylcarbodiimide

Abstract

The effect of Helminthosporium maydis race T toxin on electron transport in susceptible cytoplasmic male-sterile Texas corn (Zea mays L.) mitochondria was investigated, using dichlorophenol indophenol and ferricyanide as electron acceptors. Succinate-dependent electron transport was stimulated by the toxin, consistent with the well described increase in membrane permeability induced by the toxin. Malate-dependent electron transport was inhibited. This inhibition of electron transport increased as a function of time of exposure to the toxin. Mitochondria from normal-fertile (N) com were not affected by the toxin. Both the inhibition of electron transport and the increase in ion permeabil-ity, such as dissipation of membrane potential and Ca2+ gradients, induced by the toxin in T corn was prevented by N,N'-dicyclohex-ylcarbodiimide, a hydrophobic carbodiimide. A water-soluble car-bodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, was ineffective in preventing dissipation of membrane potential by the toxin. These results suggest that the various toxin actions are mediated via interaction of the toxin with one target site, most probably a 13 kilodalton polypeptide unique to T mitochondria. N,N'-dicyclohexylcarbodiimide may confer protection by modifying an amino acid residue in a hydrophobic portion of the target site. Helminthosporium maydis race T toxin specifically targets mitochondria of the Texas (T) line ofcytoplasmic male-sterile corn (Zea mays L.), while having no effect on normal-fertile corn or other types of cytoplasmic male-sterile corn. HmT3 toxin sensitivity and cytoplasmic male-sterility are tightly linked and are probably the result of the same recombination event in the mitochondrial DNA (8-10). The molecular mechanism of toxin action is unknown. One primary action ' of HmT toxin is to increase the permeability of the inner mitochondrial membrane to ions. Supporting this thesis is evidence that HmT toxin: (a) stimulates respiration with succinate and NADH as substrates (6), (b) stimulates mito-chondrial ATPase activity (2, 16), (c) dissipates mitochondrial membrane potential (3, 16), and (d) increases membrane permeability to calcium (15) and protons (20). However, these studies provide no clues regarding the site of toxin action or the nature of the toxin target site. HmT toxin also inhibits malate-driven respiration as measured by oxygen consumption (6). This inhibition may be the result of a direct interaction of the toxin with a target site in the NADH-ubiquinone oxidoreductase (complex I) or due to leakage of a cofactor, NAD. Malate-driven respiration, unlike succinate-driven respiration, requires a soluble cofactor, NAD. NAD has been shown to leak slowly from toxin-treated mitochondria (3, 22). Thus, it is possible that inhibition of malate-dependent respiration is caused by toxin-induced increases in mitochondrial membrane permeability and subsequent NAD leakage. However, in one case, HmT toxin inhibition of malate-dependent electron transport was shown to be separate from the action ofthe toxin on membrane perme-ability (6, 30), suggesting that the toxin could possibly interact with two target sites. To understand the nature and the location of the toxin target site(s), we have investigated the effect of HmT toxin on electron transport activities by functionally dissecting the electron transport chain using ferricyanide and DCPIP as electron acceptors. The rationale was that assaying for accep-tor reduction would be a more direct measurement ofelectron transport from complex I or II than assaying for 02 reduction. We also studied the effect of DCCD on toxin-induced increase in membrane permeability and inhibition of electron transport to determine if the different activites are the result of toxin interaction with one or more target sites. Bouthyette et al. (4) reported that DCCD prevents toxin-induced absorb-ance changes attributed to mitochondrial swelling. We found that malate-dependent ferricyanide and DCPIP reduction is inhibited by HmT toxin and that DCCD protected against the toxin effects on three different activities (electron transport , Ca2+ gradients, and membrane potential). A preliminary report of some of these findings has been made (17). After our studies were completed, Dewey et al. (10) reported that the 13 kD polypeptide, unique to T mitochondria, bound 1296