Inhibition of Dark CO<sub>2</sub> Fixation and Photosynthesis in Leaf Discs of Corn Susceptible to the Host-specific Toxin Produced by <i>Helminthosporium maydis</i>, Race T

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The effect of purified host-specific toxin from Bipolaris (Helminthosporium) maydis, race T, on dark or light-dependent CO2 fixation was studied with thin (1 x 8 mm) corn (Zea mays L.) leaf slices supplied H14C03-. At 5 to 30 nanograms per milliliter (z5 nanomolar), toxin significantly inhibited (20 to 40%) dark CO2 fixation in susceptible (T) corn slices after either dark or light preincubations of 10-20 minutes. The same concentrations were effective to the same degree on photosynthesis, but the effect differed in that significant inhibition occurred after 25 minutes and only with light preincubation. Light preincubation without toxin did not shorten the time required for inhibition of photosynthesis after addition of toxin. Once photosynthetic inhibition was entrained, it was not reversed by subsequent periods of darkness. The results suggest the possibility that race T toxin affects two separate metabolic sites, and the data are discussed in view of currently held concepts of toxin action in susceptible tissue.observation suggested that leaf damage and necrosis may be separate from disturbance of mitochondrial functions.There are few studies of the effects of race T toxin on chloroplastic functions. Arntzen et al. (1), using crude toxin preparations taken up by excised leaves, ascribed a rapid inhibition of photosynthesis and transpiration to secondary consequences of stomatal closure. Bhullar et al. (6), using floating 5-mm leaf discs and gaseous "CO2 at approximately ambient concentrations, reported inhibition of dark CO2 fixation and photosynthesis which, from results of studies with compounds known to affect stomata, were not readily attributable to stomatal closure. With this experimental system, several hours, rather than minutes (6), of preincubation with toxin were required for significant effects. Preincubation in light enhanced toxicity.Because of the required long incubation periods, in contrast to rapid effects on mitochondria (20) and, more recently (19), on ion leakage, the studies with leaf discs (6) are ambiguous because of potential permeation problems through the epidermis by toxin of unknown purity. With the availability of purified toxin, we have reinvestigated the effect of toxin on leaf metabolism using thin (I mm) leaf sections (21) in order to improve permeation.It is widely assumed (3,4,8,9,14,17,26) that the biochemical effects of the host-specific pathotoxin from Bipolaris (Helminthosporium) maydis, race T, during disease progression in susceptible corn are primarily the result of its action on mitochondria (17 Longitudinal leaf-strips (6-10 cm x 8 mm) were excised from the basal half of fully expanded, fourth true leaves of plants 18 to 23 days after sowing. It was necessary to include the main vein in the leaf strip in order to obtain thin leaf sections (1 mm) at right angle to the leaf axis. Laminar tissue without the main vein did not section satisfactorily with the apparatus and procedures described previously (21).In initial experiments, the handling of thin sections...

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confidence: 99%

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confidence: 99%

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confidence: 99%

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A Differential Effect of Race T Toxin on Dark and Photosynthetic CO₂ Fixation by Thin Leaf Slices from Susceptible Corn

Daly¹,

Barna²

1980

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“…Mitochondria isolated from susceptible tissue are very sensitive to HM-T toxin (14) whereas mitochondria from resistant maize will tolerate at least 1000 times higher concentrations. The mitochondrion appears to contain a major primary site for action of toxin, although sites in addition to the mitochondrial site are not entirely eliminated (1,3).…”

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confidence: 99%

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

Bednarski¹,

Izawa²,

Scheffer³

1977

Host-selective toxin from Helminthosporium maydis race T inhibited oxidative phosphorylation (AT32P formation) and stimulated ATPase activity by mitochondrin 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 saturatng 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 flnnly to its site(s) of action, in contrast to reports for another hostselective toxin.Maize (Zea mays L.) with an extranuclear or cytoplasmic gene for male sterility (Texas male sterile cytoplasm, Tmsc or T) is highly susceptible to Helminthosporium maydis race T. Susceptible maize is also highly sensitive to a low mol wt substance (HM-T toxin) released by the causal fungus. Resistant (N cytoplasm, or N) maize and other plant species are resistant to the fungus and insensitive to the toxin. Mitochondria isolated from susceptible tissue are very sensitive to HM-T toxin (14) whereas mitochondria from resistant maize will tolerate at least 1000 times higher concentrations. The mitochondrion appears to contain a major primary site for action of toxin, although sites in addition to the mitochondrial site are not entirely eliminated (1, 3).Other known host-selective toxins do not affect isolated organelles (20), and sensitivities to the other toxins are determined by nuclear genes rather than by cytoplasmic factors.Mitochondria isolated from susceptible (T) maize seedlings are known to swell at an accelerated rate (9) and to lose respira-I Supported by National Science Foundation Grants GB-24962 and GB-37959. tory control after exposure to HM-T toxin (14), indicating an uncoupling effect. We report here further studies that confirm and extend previous work on the effects of HM-T toxin on the mitochondrial site. Included are data on effects of toxin on AT32P formation and on ATP hydrolysis by isolated mitochondria.A major objective of this work was to obtain some understanding of the nature of the relationship between HM-T toxin and its site of action in the cell. Our results indicate a relationship of HM-T toxin with its site of action which differs radically from that suggested by Str...

“…However, the length of the lesions was highly variable and could not adequately be adapted for assaying the toxin. Bhullar et al (1) and Wheeler (18) have drawn attention to this variability in reports which use the lesion length bioassay. Two alternative assays based on conductivity increases in leachates oftoxin-treated leaf tissues were developed and are exemplified in Figures 1 and 2.…”

Section: Resultsmentioning

confidence: 99%

Eyespot Disease of Sugarcane

Larkin¹,

Scowcroft²

1981

Hebninthosporium sacchari produces a toxin which is responsible for the symptoms of eyespot disease In Sacchanw oficinarwn A rapid and highly repeatable bioassay based on increase in conductivity of tissue leachates showed that the interaction of toxin with sugarcane obeys MichaelisMenten hyperbolic saturation kinetics. There was no evidence for positive or negative cooperation interaction. Resistant (Table I). T2 medium contains serinol as an activator for toxin production (7).Sugarcane cell suspension cultures were initiated from leaf base callus in CSV(3-5) medium (Table I) Helminthosporium sacchari is the causal organism of eyespot disease in sugarcane (Saccharum officinarum) and occurs in all growing areas of the world. This pathogen produces a host-specific toxin(s) responsible for the disease symptoms (9). The degree of sensitivity of plants to the toxin correlates well with their susceptibility to the pathogen, and the toxin is thus an attractive agent for the selective recovery of resistant variants from sugar cane tissue cultures. This report examines the induction of toxin and characterizes its interaction with sugarcane leaf cells. This work required the development of a rapid bioassay for toxin sensitivity. The kinetics of interaction are consistent with a plasmalemmalocated binding protein, as suggested by Strobel and co-workers (12,13,(15)(16)(17). The binding appears to be noncooperative. Steiner and Strobel (10) proposed that the major toxin fraction is 2-hydroxycyclopropyl-Ci-D-galactopyranoside. However, they have not confirmed this structure by synthesis. The sensitivity to the toxin appears to be affected by the temperature history of the leaf. Brief leaf pretreatments at temperatures either higher or lower than the growth temperature induce tolerance to the toxin. This report also demonstrates that, when the fungus is grown together with sugarcane suspension cultures from susceptible or resistant cultivars, the production of toxin is increased up to 1,000 times the level in pure fungal cultures grown in the presence of serinol (2-amino-1,3-propanediol) (7). (Table I). Fresh subcultures of Q99 and Q1O1 in CSV(3-5) and Q47 in CSV were inoculated with H. sacchari and cultured at 27 C in a gyratory shaker at 80 rpm.Pure cultures of H. sacchari were also established in both CSV(3-5) and CSV. Toxin was extracted by the usual procedure after 12 or 13 days. TOXIN ACTIVITY BIOASSAYSHalf-leaf Method. Leaf pieces (15 cm long) of comparable developmental stage were divided along the central vein with a scalpel. One-half was used as the control and placed in a tube with 1 ml distilled H20 and the other half was placed in I ml of toxin solution. Such pairs of half-leaves were subjected to vacuum of 93 kPa for 10 min and then left overnight (16 h) in the dark at 25 C. The lower portion in direct contact with the solution was discarded, and the remainder was rinsed three times with distilled H20, cut into 1-cm pieces, and incubated at 25 C in 10 ml distilled H20. The conductivity (jLnho em-l)...