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

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) corn were not affected by the toxin. Both the inhibition of electron transport and the increase in ion permeability, such as dissipation of membrane potential and Ca²⁺ gradients, induced by the toxin in T corn was prevented by N,N′-dicyclohexylcarbodiimide, a hydrophobic carbodiimide. A water-soluble carbodiimide, 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 Induces Leakage of NAD from T Cytoplasm Corn Mitochondria

The mechanism by which Helminthosporium maydis race T toxin inhibits respiration dependent on NAD⁺-linked substrates in T cytoplasm corn mitochondria was investigated. The toxin did not cause leakage of the soluble matrix enzyme malate dehydrogenase from the mitochondria or inhibit malate dehydrogenase or isocitrate dehydrogenase directly. The toxin did increase the permeability of the inner membranes of T cytoplasm, but not N cytoplasm, mitochondria to NAD⁺. Added NAD⁺ partially or fully restored toxin-inhibited electron transport in T cytoplasm mitochondria. Thiamin pyrophosphate had a similar effect when malate was the substrate. It was concluded that the inhibition of respiration of NAD⁺-linked substrates by the toxin is due to depletion of the intramitochondrial pool of NAD⁺ and other coenzymes.     

Evaluation of a Chemical Method for Assay of Helminthosporium maydis Race T Toxin

A chemical test reported by Karr et al. (Plant Physiol. 55:727) to assay for host-specific toxin produced by Helminthosporium maydis race T was evaluated. Preparations from culture filtrates of both race T isolates, containing host-specific toxin, and similar preparations from race O isolates, containing no detectable host-specific toxin, gave positive reactions in the chemical assay. Also, preparations containing active or inactive toxin gave equal responses in this test. The procedure does not provide a reliable method for assaying H. maydis race T toxin.     

Reversible Effects of Toxin from Helminthosporium maydis Race T on Oxidative Phosphorylation by Mitochondria from Maize

Host-selective toxin from Helminthosporium maydis race T inhibited oxidative phosphorylation (AT³²P formation) and stimulated ATPase activity by mitochondria from male-sterile (T) but not from normal (N) cytoplasm maize (Zea mays L.). Toxin increased the rate of NADH oxidation, but succinate oxidation was slightly, and malate-pyruvate oxidation was strongly inhibited as the associated ATP formation was abolished. There was a 1-minute lag before toxin gave maximal stimulation of NADH oxidation; the responses to 2,4-dinitrophenol and valinomycin were immediate. There was also a delay in the effect of toxin on ATP formation. T mitochondria were more sensitive than were N mitochondria to uncoupling by nigericin plus K⁺; there was no evidence, however, that the action of toxin is related to that of nigericin or other ionophores. With NADH as the substrate, the degree of uncoupling increased with increases in toxin concentration up to a saturating level; kinetics of the response suggested reversibility. T mitochondria exposed to toxin for 5 minutes regained normal rates of respiration and of ATP formation when they were washed with toxin-free medium, showing that the uncoupling effect is reversible. Evidently HM-T toxin does not bind firmly to its site(s) of action, in contrast to reports for another hostselective toxin.     

Reduction of Adenosine Triphosphate Levels in Susceptible Maize Mesophyll Protoplasts by Helminthosporium maydis Race T Toxin

Helminthosporium maydis race T (HMT) toxin caused a reduction in the steady-state ATP levels when leaf mesophyll protoplasts isolated from maize containing Texas male-sterile (T) but not male-fertile (N) cytoplasm were incubated in the dark. At a toxin concentration 10 times the mean effectived dose for inhibition of root growth, the ATP levels began to fall in 30 to 90 seconds, fell by 50% in about 4 minutes, and reached 23% of the original levels in 100 minutes. This is faster than any previously observed response of whole cells or tissues to HMT toxin. In protoplasts incubated in the light, ATP levels were 25% higher than in the dark and were either unaffected or only slightly diminished by toxin. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport, overcame the effect of light on both toxin-treated and control protoplasts. Oligomycin, an inhibitor of mitochondrial ATP synthesis, mimicked the effects of toxin in the dark, in the light, and in the light plus DCMU, but it was not specific for T cytoplasm. During the first 24 hours of culture, ATP levels in control protoplasts increased in both the light and dark. In the dark, ATP was not detectable after 24-hour incubation in the presence of toxin, whereas in the light a substantial amount of ATP remained. Our results are compatible with the hypothesis that mitochondria in vivo are inhibited by HMT toxin. Other responses of cells and tissues to toxin can be explained in terms of reduced ATP levels.     

Cytoplasm-specific Effects of Helminthosporium maydis Race T Toxin on Survival of Corn Mesophyll Protoplasts

High yields of mesophyll protoplasts were obtained from leaves of corn (Zea mays L., inbred W64A). Many protoplasts survived a week in the dark in a simple osmoticum. Culture filtrate from Helminthosporium maydis race T at dilutions of 1:10,000 to 1:20,000 destroyed protoplasts with Texas male-sterile (T) cytoplasm. Substantial damage to protoplasts with nonmale-sterile (N) cytoplasm occurred only at a 1:20 dilution. High concentrations of partially purified H. maydis race T (HMT) toxin (32.5-130 μg dry weight/ml) did not reduce survival of protoplasts with N cytoplasm or C or S male-sterile cytoplasms after 6 days of exposure. Protoplasts with T or TRf (fertility restored) cytoplasm collapsed within 1 to 3 days after treatment with 0.13 μg of HMT toxin/ml, which was one-fifth the level causing 50% inhibition of T cytoplasm seedling root growth. Protoplasts with T cytoplasm which were washed after 30 minutes or more of exposure to HMT toxin also collapsed within a few days. Cultured W64A T protoplasts and freshly isolated protoplasts from inbreds C103 and Mo17 with T cytoplasm were less sensitive to HMT toxin than freshly isolated W64A T protoplasts. Toxin-treated protoplasts survived longer in the light than in the dark. The sensitivity and specificity of the system described will facilitate physiological, ultrastructural, and genetic studies of toxin action.     

Influence of Helminthosporium maydis, Race T, Toxin on Potassium Uptake in Maize Roots

The effect of a toxin extract of Helminthosporium maydis, race T on K⁺ (⁸⁶Rb) uptake by excised root segments of normal (N) and Texas cytoplasmic male-sterile (T) versions of corn inbred W64A was investigated. The uptake of K⁺ was inhibited in both N and T roots by the toxin. This was true for both basal (freshly excised) and augmented (pretreated with aeration) K⁺ uptake. Augmented uptake was more toxin-sensitive than basal uptake (irrespective of cytoplasm type), and the augmented uptake in T roots was seven to eight times more toxin-sensitive than in N roots.     

Dissipation of the Membrane Potential in Susceptible Corn Mitochondria by the Toxin of Helminthosporium maydis, Race T, and Toxin Analogs

We have tested directly the effect of Helminthosporium maydis T (Hmt) toxin and various analogs on the membrane potential formed in mitochondria isolated from a Texas (T) cytoplasmic male-sterile and a normal (N) corn. ATP, malate or succinate generated a membrane potential (negative inside) as monitored by the absorbance change of a cationic dye, safranine. The relative membrane potential (Δψ) could also be detected indirectly as ⁴⁵Ca²⁺ uptake. Hmt toxin added to T mitochondria dissipated the steady state Δψ similar to addition of a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Toxin analogs (Cpd XIII: C₄₁H₆₈O₁₂ and Cpd IV: C₂₅H₄₄O₆), reduced native toxin (RT2C: C₄₁H₈₄O₁₃) and Pm toxin (band A: C₃₃H₆₀O₈, produced by the fungus, Phyllosticta maydis) were effective in dissipating Δψ and decreasing Ca²⁺ uptake with the following order: Pm (100) » HmT (23-30) > Cpd XIII (11-25) » RT2C (0-4−1.8) > Cpd IV (0.2−1.0). In contrast, the toxins and analogs had no effect on Δψ formed in N mitochondria. The striking similarities of the HmT toxin (band 1: C₄₁H₆₈O₁₃) and Cpd XIII on T mitochondrial activities provide strong evidence supporting the correctness of the polyketol structure assigned to the native toxin. Since the Δψ in energized mitochondria is caused mainly by the electrogenic extrusion of H⁺, the results support the idea that HmT toxin increases membrane permeability of T mitochondria to H⁺. The host specificity of the toxin suggests that an interaction with unique target site(s) on the inner mitochondrial membrane of T corn causes H⁺ leakage.     

Electrophoretic Comparison of Mitochondrial Polypeptides from Maize Lines Susceptible and Resistant to Helminthosporium maydis Race T

The mitochondrial polypeptides from maize lines susceptible and resistant to Helminthosporium maydis were studied by single- and two-dimensional gel electrophoresis. Approximately 120 polypeptides were detected by two-dimensional gel electrophoresis. However, it was not possible to detect qualitative differences between the mitochondrial polypeptides of the two inbreds. These observations are discussed with reference to the putative mechanism of action of the pathotoxin.     

Loss of Sensitivity to Helminthosporium maydis Race T Toxin during Aging of Mitochondria Isolated from Texas Cytoplasm Corn

Helminthosporium maydis Race T toxin caused the expected changes in freshly isolated mitochondria from T cytoplasm corn, namely complete uncoupling of oxidative phosphorylation, pronounced stimulation of succinate and NADH respiration, complete inhibition of malate respiration, and increased mitochondrial swelling. In contrast, identical toxin treatments of the mitochondria after 12 hours aging on ice resulted in partial uncoupling, much lower stimulation of succinate and NADH respiration, no inhibition of malate respiration, and no mitochondrial swelling. Almost all of the toxin sensitivity was lost by 6 hours aging. At this stage, the mitochondria were 208× and 66× less sensitive to toxin-induced changes in coupling of malate respiration and state 4 malate respiration rates, respectively. Loss of toxin sensitivity did not occur when the mitochondria were aged under nitrogen or in the presence of 5 millimolar dithiothreitol. This suggested that the aging effect was due to oxidation, possibly of sulfhydryl groups in one or more mitochondrial membrane proteins.     

Effects of Purified Helminthosporium maydis Race T Toxin on the Structure and Function of Corn Mitochondria and Protoplasts

A toxin preparation from Helminthosporium maydis Race T containing several closely related molecules with apparently identical biological activities was highly active against mitochondria and protoplasts from Texas male-sterile (T) cytoplasm corn (T mitochondria and T protoplasts, respectively) but had no effect on their male-fertile (N) cytoplasm counterparts. The toxin preparation caused multiple changes in isolated T mitochondria, including uncoupling of oxidative phosphorylation, stimulation of succinate and NADH respiration, inhibition of malate respiration, increased swelling, loss of matrix density, and unfolding of the inner membrane. Only 6 to 7 nanograms toxin per milligram mitochondrial protein (1.8 nanogram per milliliter) were required to fully uncouple oxidative phosphorylation and to completely inhibit malate respiration in isolated T mitochondria. Similar low concentrations of toxin caused collapse of T protoplasts after several days of culture. Severe ultrastructural damage to mitochondria in T protoplasts was observed within 20 minutes; no changes in other cellular components were observed at this time. These observations on the cytoplasmic specificity, multiple effects, and high activity of the toxin at the mitochondrial and cellular levels highlight its biological significance and potential usefulness in determining the molecular basis of southern corn leaf blight disease.     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

Induction of a Sensitive Response to Helminthosporium maydis Race T Toxin in Resistant Mitochondria of Corn (Zea mays L.) by Removal of the Outer Mitochondrial Membrane

Mitochondria isolated from Texas cytoplasmically male sterile (Tms) and normal (N) versions of corn (Zea mays L.) exhibit differential sensitivity to toxin(s) produced by Helminthosporium maydis race T, the causal organism of southern corn leaf blight. Malate dehydrogenase was inhibited by toxin(s) in intact Tms mitochondria but was unaffected in N mitochondria. Removal or rupture of the outer mitochondrial membrane resulted in retention of sensitivity of malate dehy-drogenase in Tms mitochondria to toxin(s), and induction of a sensitive response in normally toxin-insensitive N mitochondria. This suggests that a permeability difference in the respective outer membranes of N and Tms mitochondria may affect the passage of toxin(s) to a mitochondrial site of action. Mitochondrial bioassays indicate that more toxin was bound by Tms mitochondria than by N mitochondria; the greatest toxin binding was associated with the inner membrane of Tms mitochondria.     

Helminthosporium maydis T Toxin Increased Membrane Permeability to Ca in Susceptible Corn Mitochondria

Though Helminthosporium maydis race T (HmT) toxin decreased active Ca(2+) uptake into mitochondria isolated from susceptible (T) but not resistant (N) corn (Kimber, Sze, 1984 Plant Physiol 74: 804-809 the mode of toxin action is not understood. This study shows that HmT toxin or A23187 (a Ca(2+) ionophore) dissipated a Ca(2+) gradient in T mitochondria. However, HmT toxin had no effect on Ca(2+) gradients in N mitochondria or microsomal vesicles from T or N corn. The results suggest that HmT toxin increased membrane permeability to Ca(2+) in mitochondria of T corn specifically.     

Comparison of Activities of the Host-Specific Toxin of Helminthosporium maydis, Race T, and a Synthetic C(41) Analog

It previously had been proposed that the host-selective toxin of Helminthosporium maydis race T consists of a series of unusual linear (C₃₅ to C₄₅)polyketols, of equal toxicity on a weight or molar (10⁻⁸−10⁻⁹) basis. Previous laboratory synthesis of T-toxin analogs was limited to shorter (C₁₅ to C₂₆) versions which possessed the requisite specificity for susceptible corn (Zea mays) but were less toxic on a weight or molar (10⁻⁶−10⁻⁷) basis. In the present study, a C₄₁ analog with four β-ketol units spaced by CH₂ bridges as in native toxin has been synthesized. On a weight or molar basis, it is as effective as native toxin or its purified components in stimulating NADH oxidation of mitochondria from susceptible corn, thus providing firm evidence for the correctness of the proposed structures of T-toxin. Additional support derives from the observation that C₂₄ and C₂₆ analogs with -(CH₂)₄- and -(CH₂)₆- bridges between ketol groups are not as effective in stimulating NADH oxidation as are C₂₃ and C₂₅ analogs with the -(CH₂)₃- and -(CH₂)₅- bridges of native T-toxin.

It was calculated that a single molecule of the C₄₁ analog is at least 300 times more effective in stimulating mitochondrial oxidation than a molecule of the C₂₃ or C₂₅ analogs. This emphasizes the importance of chain length for toxicity, perhaps through perturbation of membrane functions of mitochondria and/or chloroplasts.

     

Isolation and Partial Characterization of Four Host-specific Toxins of Helminthosporium maydis (Race T)

Helminthosporium maydis, race T, produces four host-specific toxins in culture. These have been designated toxins I, II, III, and IV. A method for isolation and purification of the four toxins is presented, and the criteria of purity of preparations of toxins I, II, and III are given. Toxins I and II are chemically similar and yield the same molecular ion when subjected to mass spectrometry, while toxin III appears to be a glycoside of a compound related to toxins I and II. Toxins I, II, and III can be biologically derived from ¹⁴C-mevalonic acid or ¹⁴C-acetate, permitting preparation of ¹⁴C-labeled toxins. Some chemical, spectral, and chromatographic properties of toxins I, II, and III are presented, and these data are discussed relative to the possible structure of the three compounds. In addition, four host-specific toxins have been isolated from corn infected with H. maydis (race T). These toxins are recovered in the same fractions as toxins I, II, III, and IV using the isolation procedure described here. Three of the toxins isolated from infected corn cannot be distinguished from toxins I, II, and III on the basis of infrared spectra or chromatographic mobility.     

Inhibition of Dark CO(2) Fixation and Photosynthesis in Leaf Discs of Corn Susceptible to the Host-specific Toxin Produced by Helminthosporium maydis, Race T

The host-specific toxin produced by Helminthosporium maydis, race T, causes 50% inhibition of dark fixation of ¹⁴CO₂ by leaf discs of susceptible (Texas male sterile) corn when it is diluted to approximately 1/10,000 of the volume of the original fungus culture filtrate. Dilutions of 1/10 or less are required for equivalent inhibition of discs prepared from resistant (N) corn. Root growth and photosynthesis were considerably less sensitive (dilution values 1/3000 and 1/1200, respectively), as was leakage of ¹⁴C induced by toxin from preloaded discs. Based on literature values for dilutions causing ion leakage or inhibition of mitochondrial oxidation, toxin dilutions several orders of magnitude greater bring about inhibition of dark CO₂ fixation. Preincubation of discs in light increased sensitivity of dark fixation to toxin and an effect of light on symptom development was shown. Phosphoenolypruvate carboxylase activity in extracts of roots or leaves was not affected by toxin nor was the enzyme level altered in excised leaves treated with toxin. Inhibition of dark fixation of CO₂ provides a bioassaay for race T toxin which is both reliable and rapid.     

A Comparison of Purified Host Specific Toxin from Helminthosporium maydis, Race T, and Its Acetate Derivative on Oxidation by Mitochondria from Susceptible and Resistant Plants

NADH or succinate oxidation and malate oxidation were differentially affected in mitochondria from both susceptible and resistant corn by a purified and chemically characterized preparation of host-specific toxin from Bipolaris (Helminthosporium) maydis, race T. NADH and succinate oxidation by susceptible T corn mitochondria were stimulated 50 to 200% with apparent uncoupling from the cytochrome chain at approximately 10(-9)m toxin (5 to 20 ng/ml). Significant inhibition of malate oxidation was observed at slightly higher toxin concentrations, but oxidation was still coupled to ADP utilization. Inhibition of malate oxidation also was observed in N corn (resistant) and soybean mitochondria at approximately 1,000-fold greater concentrations, but stimulation of NADH and succinate oxidation was not found at any toxin concentration tested.A fully acetylated toxin derivative at approximately 1 microgram per milliliter also caused stimulation of NADH or succinate oxidation in T corn mitochondria, but not those of N corn or soybean mitochondria at 100 micrograms per milliliter. Malate oxidation was inhibited to the same extent by toxin acetate with mitochondria from T corn, N corn, and soybean. The blocking of hydroxyl groups in race T toxin by acetyl functions eliminated selectivity toward malate oxidation only. The data suggest that inhibition of malate oxidation is either a separate or secondary effect of selective action of toxin on T corn mitochondria, perhaps by interference with transport in or out of the matrix. Sensitivity of T, but not N, corn mitochondria to purified toxin decays within minutes after pellets are suspended in aqueous osmotica, with no obvious change in mitochondrial integrity. The action of race T toxin seems to involve a labile process, such as ion gradient(s), or an unstable structural conformation of T corn mitochondria.     

Influence of Helminthosporium maydis, Race T, Toxin on Potassium Uptake in Maize Roots: II. Sensitivity of Development of the Augmented Uptake Potential to Toxin and Inhibitors of Protein Synthesis

Basal K⁺ uptake in the root midzone region (cm 2 + 3 + 4) of N and T cytoplasmic versions of each of four maize inbreds was equally sensitive to the toxin(s) of Helminthosporium maydis, race T. Basal K⁺ uptake in the root apex (0-1 cm) and augmented K⁺ uptake in the root midzone were more toxin-sensitive in inbreds W64A(T) and Mo17(T) than in inbreds W64A(N) and Mo17(N). This differential response of N and T cytoplasms to toxins was not found for corresponding cytoplasms of inbreds WF9 and B37.