Nitrite reduction is inhibited by anaerobiosis in barley aleurone layers (5) and wheat roots (18), whereas the reduction of nitrate to nitrite by nitrate reductase is enhanced (4). Nitrite accumulates under such conditions and the anaerobic production of nitrite has been used to assay nitrate reductase in situ in a variety of plant tissues (4, 11-13, 17, 22, 24, 25). The reaction was found to be linear for more than 1 hr and dependent upon the addition of exogenous nitrate (4,13,24

Susceptible corn tissues exposed to the host-specific toxin of Helminthosporiuna carbonum race 1 reduced more nitrate to nitrite than did control tissues, as measured by an in vivo method. There were no differences in nitrate reductase activities extracted from treated and control tissues and assayed by an in vitro method. Toxin-treated susceptible roots (15,16). This "host-specific toxin" is required by the fungus for pathogenicity and for initial colonization of susceptible plant tissues (1, 16). Among its described physiological effects on susceptible tissues, HC toxin increases electrolyte leakage, respiration, dark fixation of CO2, and the incorporation of uridine and amino acids. Most of these effects are not evident unless tissues are exposed to toxin for 8 to 12 hr (9, 17); they could be secondary results of an initial toxic effect.The original aim of this work was to determine whether or not toxin has a direct effect on protein synthesis. An inducible enzyme, nitrate reductase, was chosen to study this question. Zielke and Filner (23) MATERIALS AND METHODSPlants. H. carbonum-resistant (Prl X K61) or susceptible (Pr X K61) corn hybrids were used in all experiments. Roots were produced by two methods. (a) Seeds (250) were incubated 8 to 12 hr in water, then were washed and placed embryo side down on a cheesecloth surface in a beaker above 4 liters of 0.2 mm CaCl2. A second layer of cheesecloth was placed over the seeds, the beaker was covered with plastic film, and the solution was aerated. Primary roots were used 4 days later when they were 8 to 12 cm long. (b) Seeds (150) were placed embryo side down on an agar medium (100 ml of 0.9% agar in a 15-cm Petri dish) containing White's solution (20) minus nitrate. After 60 to 84 hr of incubation, the germinated seeds had 4-to 8-cm roots which were used as experimental tissues.Toxin Preparation. Toxin was isolated from H. carbonum race 1 by previously described procedures (15, 21). Culture filtrates were concentrated, deproteinized by methanol precipitation, and extracted with chloroform. The chloroform solution was taken to dryness in vacuo, and the residue was dissolved in ethanol, mixed with 20 volumes of diethyl ether, and stored at 5 C for 24 hr. The precipitate was discarded, and the solution was taken to dryness. A second ethanol-ether extract was taken to dryness, dissolved in water, and passed through a BioGel P-2 column. Fractions with host-specific toxicity were dried, and the residues were stored under nitrogen over CaCl2 at -20 C. This preparation caused 50% inhibition of susceptible corn roots at 0.2 ug/ml, and of resistant roots at 20 ug/ml, when assayed by the seedling root growth method (16). Thus, the toxin preparation was active at 0.295 ,uM (mol wt = 679), which is as active as the best crystalline preparations (14, 15). Reaction mixtures were assayed before and after each experiment; there were no losses in host-specific toxicity.Nitrate Reductase Assays. Nitrate reductase activity was determined in vivo by the method of Ferr...

Susceptible corn roots exposed to the host-selective toxin of Helminthosporium carbonum took up and retained more NO3-, Na+, Cl-, 3-o-methylglucose, and leucine than did control roots. Stimulatory effects on uptake were more pronounced with freshly cut roots than with roots that were washed and aged.Solutes were accumulated against a concentration gradient, and toxin-treated tissues developed a steeper gradient than did control tissues. Toxin affected both the low and high affinity uptake systems for Na+ and C1. Toxin did not affect uptake of Na2_, K+, Ca'+, phosphate ion (H2PO4-and HP04-), S04-, and glutamic acid. No toxin-induced leakage of any solute tested was detected within 5 to 6 hr after initial exposure to toxin. The data suggest that toxin from H. carbonum does not cause the general plasma membrane derangement caused by other host-selective toxins. Instead, H. carbonum toxin may cause specific changes in characteristics of the plasmalemma, which result in increased uptake of certain solutes.Helminthosporium carbonum Ullstrup race 1 produces a toxic compound which selectively affects the corn genotypes that are hosts of the fungus. Susceptible genotypes are those with a homozygous recessive factor at the locus conditioning disease reaction. Several lines of evidence show that the toxin is required for pathogenicity. All visible and known biochemical symptoms of infection are reproduced by the toxin (26). Thus, an understanding of disease and disease resistance should be possible from studies of toxic action. Metabolites with selective toxicity are now known from nine plant-infecting fungi, including Helminthosporium victoriae, Helminthosporium maydis race T, Periconia circinata, Alternaria kikuchiana, and Phyllosticta maydis (26,29 (28). Likewise, the apparent effect on nitrate reductase is secondary, because the increase in activity was traced to toxin-stimulated uptake of substrate, not to increased synthesis of the enzyme (31). Some of the other stimulatory effects of HC toxin may be secondary to initial toxic lesions. Metabolic activity induced by toxin at damaging concentrations is followed some hours later by decreased activity, cell leakiness, and death.The purpose of this work was to determine effects of toxin on uptake and retention of representative solutes in addition to nitrate. Nitrite, C1-, SO,-, Na+, K+, Ca2+, phosphate ion (H2PO-and HP042-), 3-o-methylglucose, leucine, and glutamic acid were selected for further study. HC toxin is selective for susceptible corn in its effects on NO3-uptake, and the increase in uptake is the earliest effect of toxin reported to date (31). An abstract describing some of the work has been published (30).Plant cells are thought to have at least two uptake systems for each of several ions (2, 12). Mechanism 1 of ion transport has a high affinity for ions and functions when ion concentrations are below 0.5 mM; this system is generally thought to be located in the plasmalemma, and it accumulates ions against a concentration gradient. Ion transport mech...

Leaf protoplasts from resistant (N, C, and S cytoplasm) and susceptible (T cytoplasm) corn inbred W182B (Zea mays L.) exhibited a differential response after exposure to Helminthosporium maydis race T toxin. The volume of untreated protoplasts increased twofold during the first 24 h after isolation and by 48–72 h, most protoplasts exhibited an extensive network of cytoplasmic strands and the chloroplasts were distributed throughout the cytoplasm by cyclosis. However, susceptible protoplasts exposed to ≥2 μg toxin/ml failed to increase in volume and cytoplasmic streaming was rarely observed. By 72 h there was extensive degradation and collapse of susceptible protoplasts exposed to toxin. Resistant protoplasts exposed to up to 100 μg toxin/ml exhibited a twofold volume increase and were indistinguishable from untreated protoplasts. Susceptible protoplasts exposed to 0.1 μg toxin/ml also appeared unaffected, but at 1.0 μg toxin/ml an intermediate response was observed. The differential response to H. maydis race T toxin of protoplasts from resistant and susceptible corn correlates well with those effects observed in intact plant tissues and may serve to explain further the mode of action of the toxin on susceptible corn cells.