STU‘aBEES Gfi {HE MS 0% ACTION OF THE GSLSF’EUHC TGXWS ERQM HELMINYHOSPORIUM VEC'EQERLAE AND PERE‘CGNEA CERCENATA Ehesis for The Degree of PM). MlCHlGAN SIATE UNWERSITY .EOHN MacGREGOR GARDNER 1971 This is to certify that the thesis entitled Studies on the Sites of Action of the Host-Specific Toxins from He Zminthosporium Victoriae and Perioonia Circinata presented by John MacGregor Gardner has been accepted towards fulfillment of the requirements for Doctoral degree in Botany and Plant Pathology Major professor Date April 28, 1971 0-7639 ., . 12.61;? “‘3 MiChlgEIYI :2 Law University ABSTRACT STUDIES ON THE SITES OF ACTION OF THE HOST-SPECIFIC TOXINS FROM HEIMNTHOSPORIUM VICTORIAE AND PERICONIA CIRCINATA By John MacGregor Gardner Pathogenicity of several plant-infecting fungi requires the pro- duction of certain compounds specifically toxic to host plants. This study is a further examination of the hypothesis that Helminthosporium victoriae (RV) and Periconia circinata (PC) toxins cause initial biochemical lesions in the plasma membranes of the susceptible plant cell. A related hypothesis is that resistant plants are not affected because they lack toiin receptor sites. Experiments described herein were focused on the biochemical factors which contribute to suscept- ibility to toxins. A convenient assay for toxins was employed, based on the toxins' ability to disrupt permeability barriers and to induce electrolyte loss from plant cells. Twelve hr pretreatment of susceptible tissues with cycloheximide (CH) (2-5 ug/ml) gave 70 to 90% protection against both HV and PC- toxins. CH protection of oat tissues against HV-toxin was clearly reversible when CH was removed. CH did not protect oat tissues against electrolyte loss induced by nonspecific toxic compounds. Other inhibitors gave relatively little protection against either toxin in comparable experiments. It is possible that cycloheximide protects by inhibiting synthesis and turnover of receptor proteins. When oat tissues were pretreated for 30 min with N-ethyl- maleimide (NEM) (2 mM), 2,4-dinitrof1uorobenzene (DNFB) (2 mM), John MacGregor Gardner iodoacetate (2 mM), or arsenite (4 mM), all of which are sulfhydryl- binding compounds, there was 70 to 90% protection against toxin- induced loss of electrolytes. Significant protection was evident within one min after exposure to NEM or DNFB. Beta-mercaptoethanol reversed the protective effects of DNFB and arsenite. None of these reagents gave protection of susceptible sorghum tissue against PC- toxin, nor did they protect against loss of electrolytes induced by nonspecific toxic compounds. The protective compounds did not act via competitive effects with toxin. NEM did not react with the toxin molecule, as determined by a photometric assay. HV-toxin breakdown products (TBP) partially countered NEM pro- tection, as measured by a reduction in the protective effects of NEM. TBP also partially countered toxin activity, as determined by the electrolyte loss assay. The possibility that ‘TBP was competing with NEM for the same sites was tested. Both TBP and toxin reduced 14C- NEM labelling of cell-free, membrane enriched fractions from suscepti- ble but not resistant oats. This indicates that HV-toxin and TBP may Cmnpete with NEM for sulfhydryl groups associated with toxin receptor Sites. Other possible explanations have not been eliminated. Certain other pretreatments with protein-binding reagents, pro- nase, and detergents, gave no protection against either toxin. Pre- treatment of sorghum tissue with phospholipase D reduced subsequent losses of electrolytes caused by PC-toxin. Pretreatments with uranyl 8alts, which bind to membranes, gave partial protection against both toKins; however, uranyl salts also gave partial protection against 1088 of electrolytes induced by nonspecific toxic compounds. Carbonyl- bil'lding reagents were confirmed as protectants against HV—toxin, but John MacGregor Gardner not against PC-toxin. HV and PC-toxins caused changes in Single cell electrOpotentials, but under most conditions these came later than the rapid efflux of materials from cells into distilled water. No effects of toxins on isolated organelles have been demonstrated. Both HV and PC-toxins were inactivated by treatment with dry methanol-RC1, a specific reagent for carboxyl groups. The esterified, inactivated toxins gave some protection against active toxin in electrolyte loss assays; it is possible that esterified toxin com- petes with active toxin for receptor sites. Carboxyl groups are therefore required for both HV and PC-toxin activity. The data support the hypothesis of toxin receptors in plasma membranes of susceptible cells. Data with cycloheximide suggest that toxin receptors are proteins; data with certain sulfhydryl- binding reagents suggest that sulfhydryl groups are involved. More conclusive studies will require techniques for isolation of plant cell membranes. STUDIES ON THE SITES OF ACTION OF THE HOST—SPECIFIC TOXINS FROM HELMINTHOSPORIUM VICTORIAE AND PERICONIA CIRCINATA By John MacGregor Gardner A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1971 DEDICATION TO FINE PARENTS K. S . GO. RO.F..LS. P.M.L. ii ACKNOWLEDGMENTS I sincerely thank my thesis advisor, Dr. Robert P. Scheffer, for his valuable guidance, encouragement, and interest during the course of this study. His dedicated review of the manuscript has been greatly appreciated. I also wish to thank the other members of my thesis committee, Drs. R. S. Bandurski, A. H. Ellingboe, H. H. Murakishi, and J. E. Verner for reviewing the manuscript. The study on single cell electropotentials was possible through the kindness of Dr. Noe Higinbotham of washington State University. This study would not have been possible without the patience, optimism, and interest shown by my wife, Joy. In more subtle ways, Scot and Wendy provided their own inspiration. Finally, a word of thanks to my typist, Mrs. Helen Curro, for her excellent work. iii TABLE OF CONTENTS Eggs LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . vii LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . x LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . xiii PART I. EXPERIMENTS WITH HV-TOXIN INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . 2 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . 5 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . 15 Plants and Fungus Cultures . . . . . . . . . . . . . . . . 15 Preparation of Toxin and Toxin Breakdown Products (TBP) . . 15 Toxin Assays . . . . . . . . . . . . . . . . . . . . . . . 17 Determination of Potassium and Sodium in Extracts and Leachates from Tissues . . . . . . . . . . . . . . . . . . 18 Electropotentials of Single Plant Cells . . . . . . . . . . 19 Isolation of Nuclei and Determination of Protein Synthesis. 20 Preparation and Labelling of (Presumed) Membrane Fragments. 22 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Effects of Tbxin on Ion Fluxes and Electropotentials of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Toxin-induced electrolyte efflux from cells . . . . . 24 Effect of toxin on electr0potentials in single cells of oat coleoptiles . . . . . . . . . . . . . . . . . . 29 Counteracting Effects of Various Substances on Toxin Action . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Carbonyl reagents . . . . . . . . . . . . . . . . . . 37 iv Uranyl salts . . . . . . Pretreatment with cycloheximide and other inhibitors Pretreatment with sulfhydryl-binding reagents Competition between toxin breakdown products (TBP) and toxin . . . . . . . . Competition between esterified, inactive toxin and active toxin . Experiments with Cell Particles and Nuclei Interactions of a particulate fraction from cells with 14C-NEM, toxin, and toxin breakdown products (TBP) . . . . . . . . . . . . . . . Effect of toxin on uptake and incorporation of amino acids by isolated nuclei . . . DISCUSSION . SUMMARY . . . LITERATURE CITED . . . . . . . . . . . . . . . PART II. EXPERIMENTS WITH PC-TOXIN INTRODUCTION . LITERATURE REVIEW . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . Host Plants and FUngus Cultures Purification and Isolation of PC-Toxin Toxin Assays . . . . . Amylase Production by Aleurone Cells Preparation of Mitochondria . Respiratory Measurements . . . . . . . . . Measurement of Electropotentials in Single Cells Incorporation of Amino Acids by Nuclei RESULTS 40 47 56 73 77 79 79 84 86 95 96 105 108 111 111 111 112 113 114 115 116 116 117 Effects of PC-Toxin on Membrane Function . . . . . . . . . 117 Effects of PC-Toxin on Metabolism and Subcellular Organelles . . . . . . . . . . . . . . . . . . . . . . . . 124 Effects of Inhibitors on Susceptibility of Sorghum to PC-Toxin . . . . . . . . . . . . . . . . . . . . . . . . 128 Uptake and Recovery of PC-Toxin, and Its Action on Resting Seeds . . . . . . . . . . . . . . . . . . . . . . . 134 Effects of Chemically Altering the Toxin Molecule . . . . . 139 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . 143 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . 148 vi LIST OF TABLES Part I Temperature Coefficients (Q10) of HV-Toxin-Induced Electrolyte Efflux . . . . . . . . . . Effect of HV-Toxin on Pbtassium.and Sodium Contents of 53293 Coleoptile Cells . . . - . . - . - Effect of 2-Mercaptoethanol (ME) Treatments on HV- Toxin-Induced Efflux of Electrolytes . Effect of Uranyl Nitrate Pretreatments on HV-Toxin- Induced Efflux of Electrolytes . . . . . . . . . . . Effect of various Rinse Treatments on Uranyl (U02) Nitrate Protection Against HV-Toxin . . . . . . Effect of Uranyl Nitrate on Leakage of Electrolytes Induced by mCl-CCP and Methanol. . . . . . . Effect of Cycloheximide (CH) Pretreatments on HV- Toxin-Induced Electrolyte Efflux from Susceptible Oat Tissue . . . . . . . . . . . . . . . . . . . Effect of Metabolic Inhibitors on HV-Toxin-Induced Loss of Electrolytes from Susceptible Oat Tissue - - Dinitrofluorobenzene (DNFB) Protection against HV- Toxin-Induced Loss of Electrolytes, and Reversal of the Protective Effect by Beta-mercaptoethanol (ME) . vii 3O 38 42 44 45 46 49 55 62 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Arsenite Protection against HV-Toxin-Induced Loss of Electrolytes, and Reversal of the Protective Effect by Beta-mercaptoethanol (ME) . . Photometric Assay for a Possible Interaction between HV-Toxin Breakdown Products (TBP) and NEM N-ethylmaleimide (NEM) Protection against HV-Toxin- Induced Loss of Electrolytes, and Countereffects of Toxin Breakdown Products (TBP) . . . . N-ethylmaleimide (NEM) Protection against HV-Toxin- Induced Loss of Electrolytes, and Countereffects of Toxin Breakdown Products (TBP) . . . . . . Countereffects of Esterified (Inactive) Toxin (ET) on HV-Toxin-Induced Loss of Electrolytes . . Effect of HV-Toxin Breakdown Products (TBP) on Labelling of Particulate Fractions with 14C-NEM . . Effect of HV-Toxin and HVeToxin Breakdown Products (TBP) on the Labelling of a Particulate Fraction with 14C-NEM . . . . . . . . . . . . . . . . Labelling of Bovine Serum Albumin (BSA) with 14C-NEM in the Presence of HV-Toxin Breakdown Products (TBP) and HV-Toxin . . . . . . . . . . . . . . . . Effect of HV-Toxin on Incorporation of 14C-Lysine into Protein of Nuclei Isolated from Susceptible Oat Leaves . . . . . . . . . . . . . . . . . . Part II The Effect of Cycloheximide (CH) on PC-Toxin Sensitivity of Susceptible Sorghum Tissue . viii 63 69 71 72 78 81 82 83 85 129 20. 21. 22. 23. 24. 25. Effect of Phospholipase D (PL-D) Pretreatment on Susceptibility to PC-Toxin . . . . . . . Recovery of PC-Toxin from Cuttings and Seeds as Determined by Seedling Bioassay . . . . . . . . Recovery of PC-Toxin from Leaf Tissues of Resistant and Susceptible Seedlings Following Exposure of Intact Roots to Toxin . . . . . . . . . . . . Germination of Resistant and Susceptible Sorghum Seeds Exposed to PC-Toxin 3 Weeks to 30 Months Previously . . . . . . . . . . . . . . . . . . Inactivation of PC-Toxin by Esterification, and Reversal of the Inactivation at pH 7.0 . . . . . . Comparative Characteristics of HV and PC-Toxins . ix 133 135 137 138 140 144 LIST OF FIGURES Part I The effect of HV-toxin concentration on efflux of electrolytes from leaf tissue. . . . . . . . . . . . Hill plot of HV-toxin-induced efflux of electrolytes. Initial effect of HV-toxin on potassium efflux from oat coleoptile tissue. . . . . . . . . . . . . . . . . The effects of HV-toxin (16 ug/ml) on membrane electropotentials in 5 single cells of susceptible oat coleoptiles. . . . . . . . . . . . . . . . . . . . The effect of HV-toxin (16 ug/ml) on membrane electropotentials in 4 single cells of susceptible or resistant oat coleoptiles. . . . . . . . . . . Effect of m-ClCCP on membrane electropotentials in 4 single cells of oat coleoptiles. . . . . . . . . Effect of HC-toxin (5 ug/ml) on membrane electro- potentials in 5 single cells of susceptible corn coleoptiles. . . . . . . . . . . . . . . . . . . . . . WOlff plots showing effects of inhibitors on HV-toxin- induced loss of electrolytes. . . . . . . Effect of semicarbazide-HCl ($32) on HV-toxin- induced efflux of electrolytes as a function of toxin concentration. . . . . . . . . . . . . . . . . . . . . 25 27 28 32 34 35 36 39 41 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Time required for development of cycloheximide (CH) protection against HV-toxin-induced efflux of electrolytes. . . . . . . . . . . . . . . . . . . . . . 50 Cycloheximide (CH) protection against HV-toxin- induced efflux of electrolytes and reversal of the protective effect. . . . . . . . . . . . . . . . . 52 Effect of HV-toxin concentration on cycloheximide (CH) protection against HV-toxin-induced efflux of electro- lytes. . . . . . . . . . . . . . . . . . . . . . . . . 53 Effect of brief pretreatments with sulfhydryl binding reagents on HV-toxin-induced loss of electrolytes. . . 59 Effect of N-ethylmaleimide (NEM) pretreatment on HV-toxin-induced electrolyte efflux from susceptible oat leaf tissue. . . . . . . . . . . . . . . . . . . . 60 Protection against HV-toxin-induced lossof electro- lytes by N-ethylmaleimide (NEM) at several concentra- tions. . . . . . . . . . . . . . . . . . . . . . . . . 65 Time required for development of protective effects of N-ethylmaleimide (NEM) and dinitrofluorobenzene (DNFB) against HV-toxin-induced loss of electrolytes. . 67 Countereffects of toxin breakdown products (TBP) on HV-toxin-induced loss of electrolytes for oat leaves. . 74 Chromatography of HV-toxin breakdown products on a Sephadex G-15 column (1.5 x 88 cm). . . . . . . . . . . 76 Part II Time course of electrolyte efflux induced by PC-toxin. 118 xi The effects of PC-toxin at several concentrations on loss of electrolytes from sorghum leaves. . . . . . . . . . . . 119 Hill plot of data from Fig. 20. . . . . . . . . . . . . . 121 Loss of Na and K from PC-toxin-treated sorghum coleoptiles. . . . . . . . . . . . . . . . . . . . . . . . 122 The effect of PC-toxin (80 ug/ml) and m-ClCCP (20 uM) on membrane electropotentials in 3 Single cells of susceptible sorghum coleoptiles. . . . . . . . . . . . . . 123 Effects of PC-toxin (Tox) and cycloheximide (CH) on gibberellic acid (GA) induction of a-amylase in sorghum aleurone cells. . . . . . . . . . . . . . . . . . 126 Succinoxidase activity of isolated mitochondria, with and without PC-toxin. . . . . . . . . . . . . . . . . . . 127 Development of cycloheximide (CH) insensitivity to PC-toxin by sorghum tissue. . . . . . . . . . . . . . . . 131 Counteracting effects of esterified toxin (ET) on toxin induced loss of electrolytes from sorghum leaves. . . . . . . . . . . . . . . . . . . . . . . . . . 142 xii LIST OF ABBREVIATIONS ATP - Adenosine 5'-triphosphate CoA - Coenzyme A CH - Cycloheximide m-ClCCP - Carbonyl cyanide m-chlorophenylhydrazone EDTA - Ethylenediamine tetraacetic acid HC - Helminthosporium carbonum HV - Helminthosporium victoriae NAD - Nicotinamide adenosine diphosphate NEM - N-ethylmaleimide PC - Periconia circinata POP - 2,5-diphenyloxazole POPOP - 1,4-bis 2-(5-phenyloxazolyl) benzene TCA - Trichloroacetic acid TPP - Thiamine pyrophosphate Tris - Tris (hydroxylmethyl) aminomethane xiii Part I Experiments with Helminthosporium Victoriae Toxin INTRODUCTION Host-specific toxins are pathogen-produced determinants of disease which affect the same plants as does the pathogen. The toxins also reproduce the disease symptoms caused by the pathogen. At least eight host-specific toxins are now recognized (75,79,84). In addi- tion, several non-specific toxins are known (53); the roles of non- specific toxins in disease development have seldom been evaluated. The mechanisms by which pathogens attack their hosts may differ in many respects, but parasitism and pathogenicity must have chemical bases. A primary biochemical lesion, or interaction, is therefore a logical prerequisite for parasite invasion. The initial or primary chemical lesion in the host is of interest in understanding both the action of toxin and the mechanism of pathogenicity. HV-toxin is produced by the fungus Helminthosporium Victoriae Meehan and Murphy, the causal agent of Victoria blight of cats (Aygga sativa L1). Toxic activity was first described in 1947 (48) after'H. Victoriae had removed the popular cultivars of cats from production in the Midwest. The toxin principle was purified and partially char- acterized by Pringle and Braun (56,57). The toxin is produced by germinating spores and appears to be necessary for initial coloniza- tion of the fungus in susceptible host tissue (50). Toxin is highly Specific for susceptible oats; resistant oats and other species will tolerate > 400,000 times higher concentrations than will susceptible 3 cats (39). Susceptibility or resistance to toxin is controlled by one dominant allele in the host. Therefore, this decrease may be de- fined both chemically and genetically. The initial site of action for HV-toxin is believed to be in the plasma membrane (75). The rapid effects of toxin on electrolyte loss from tissues, and the destruction of cell wall-free protoplasts, were considered as evidence for an early lesion in the plasma membrane. Effects of toxin on apparent free space and plasmolytic ability fur- ther confirm the drastic effect on the membrane (75). Much evidence would indicate the toxin-induced symptoms such as increased respira- tion (72,62), decreased uptake and incorporation of various solutes (68), and damage to cellular organelles (45), are all secondary and result from initial effects of toxin on the plasma membrane. If the hypothesis of initial action on the membrane is correct, this toxin is the only known substance which can selectively destroy the plasma membrane of a specific organism. Previous workers (73) postulated that the plasma membrane of the susceptible cell has a distinct receptor site for toxin. They also postulated that the resistant cell lacks a receptor site, or that the receptor site has lost its affinity for toxin. There is no direct evidence for these hypotheses, but several observations seem to offer support (68,73). The standard toxin bioassay based on root growth inhibition proved to be impractical for many of my experiments. .An assay based on toxin- induced efflux of electrolytes from susceptible cells, originally sug- gested by Wheeler and Black (91), was therefore developed. This assay proved to have many advantages over the seedling root growth assay. 4 The aim of my work was to examine the receptor site hypothesis. First, I have used the counteracting effects of various substances to develop some information on the general nature of hypothetical receptor sites. I have then attempted to devise a technique to iso- late the receptor sites, based on an indirect chemical assay for the receptor components. Such an ig‘yitgg assay should help to locate the receptor in a subcellular fraction. This approach, if success- ful, would be a major step in understanding toxin action. A covalent labelling of the receptor site was a major concern in my approach to identification of toxin receptor sites. Many substances were tested for ability to alter the sensitivity of susceptible cells to toxin, in the hope of finding a clue to the nature of the receptor site. Cycloheximide, which primarily inhibits protein synthesis, was found to decrease toxin susceptibility. N- ethylmaleimide (NEM), which reacts covalently with sulfhydryl groups in protein, was found to have strong protective properties against toxin. The role of protein and sulfhydryl groups in membranes is well documented (22,49,64,77,83,85). The idea that receptor sites are proteins gradually evolved. Further experiments were designed to show the specificity of the NEM effects, and possible applications to isolation and characterization of receptdr sites for toxin. The data in this thesis are consistent with the original receptor site hypothesis outlined by Scheffer and Pringle (73). The data pre- sented should aid and stimulate more definitive investigations on the toxin-receptor site interactions. LITERATURE REVIEW Much of the literature on host-specific toxins is discussed in several recent reviews (53,74,75). HV-toxin was first character- ized by Pringle and Braun (57) as a low molecular weight substance G< 2000) which breaks down under mild alkaline conditions to give two ninhydrin-positive components. One, named victoxinine, was de- scribed as a tricyclic secondary amine (CI7H29NO) with a molecular weight of 264. The other moiety, a peptide, had an empirical ratio of 1 leucine:1 valine:1 g1ycine:l glutamic acid:l aspartic acid. The peptide was thought to account for specificity, although it has not been characterized further. Victoxinine has a non-specific activity about 0.0001 as much as the complete toxin. The linkage between peptide and Victoxinine apparently involves the single nitrogen of Victoxinine and the terminal amino nitrogen of the peptide. The complete toxin molecule does not react with ninhydrin. Recently, Victoxinine was characterized as a sesquiterpene having properties of both secondary and tertiary amines (unpublished data from.R. B. Pringle). The first physiological work with HV toxin was concerned with the increase in respiration occurring in diseased and toxin-treated sus- ceptible oats (93). 'More definitive data on this phenomenon came from Scheffer and Pringle (72), who demonstrated that toxin has no effect on oxygen uptake by isolated mitochondria. Also, respiratory 6 responses are readily abolished by rinsing tissue in distilled water (1). It is therefore safe to conclude that increased respiration in tissues is an indirect consequence of toxin damage. Wheeler and Black (91) reported that efflux of electrolytes from susceptible tissues increased as early as 15 min after toxin treat- ment. Reapiration increases were detected 10 to 20 min after toxin treatment (72). Wheeler and Black found that toxin-treated tissues took up less electrolytes than control tissues 10 min after toxin treatment, and thereafter toxin-treated tissues lost more electro- lytes than controls. These data were clarified by Scheffer and Samaddar (75) who detected toxin-induced electrolyte efflux as early as 2 min after exposure to toxin. Wheeler and Luke prophetically stated in a 1963 review (93) that the primary effect of toxin was most likely on cellular permeability and that all other changes were probably indirect effects. Five years later, Sammadar and Scheffer (68) showed by several criteria that toxin had a very rapid effect on the plasma membrane. Extra- polation of the electrolyte efflux curve to zero time would suggest that the plasma membrane effects are almost immediate (75). However, it is still possible that the initial effect may occur elsewhere, and that changes in the plasma membrane are secondary. Final conclusions should be withheld until effects on isolated plasma membranes are demonstrated. Toxin action does not appear to require metabolic energy and ap- parently involves a physical interaction with receptors not present or nonfunctional in resistant tissues (73). The hypothesis is that receptors are present at the cell surface, but so far there is no 7 conclusive supporting evidence. There are some observations that seem to fit the hypothesis. First, breakdown products of toxin, but not victoxinine, reduced toxin activity in a seedling bioassay. Second, bisulfite and other carbonyl-binding compounds also reduced toxicity, perhaps by affecting the hypothetical receptor sites (70, 73). An alternative was suggested by Romanko (62), who postulated that resistance is based on ability to inactivate toxin. Romanko's hypo- thesis was based on apparent recovery of toxin from treated suscep- tible cuttings, and lack of recovery from resistant cuttings. This implied that resistant tissues had toxin sensitive sites, but that toxin was inactivated too rapidly for the sites to be affected. Romanko's results could not be repeated (73,90). Theoretically, tox- in would have to be inactivated immediately to account for resistance. All existing data would argue against this poSsibility. Recently, still another hypothesis was preposed by Wheeler and Pirone (94) to account for resistance to toxin. They observed that when bean cuttings were given moderate doses of toxin for 36 hr, the leaves of this toxin-insensitive plant were resistant to tobacco mosaic and alfalfa mosaic viruses. The authors suggested that toxin stimulates a repair mechanism.in the plant membrane. Even if this hypothesis were true, we would still need to know why the susceptible cell membrane is affected, while the resistant membrane is stimulated to repair. Direct proof for the toxin receptor site requires experiments to show that specific sites are affected in susceptible cells, and that these sites in resistant cells are missing, nonfunctional, or have low 8 affinity for toxin. Attempts to detect adsorption of toxin to tis- sues by assaying removal of toxin from solution were unsuccessful (73). This leads to two possibilities: that toxin is not bound or inactivated on the cell surface or within the cell; or, that the assays for toxin are not sensitive enough to detect removal of small quantities of toxin. Very few molecules of toxin may be required to disrupt the membrane. Several attempts have been made to alter susceptibility to toxin by chemical treatments. One report (16) indicates that calcium at a high concentration (0.1 M) will protect oat cuttings from toxin, but only when toxin is mixed with the calcium solution. This was inter- preted as a membrane stabilizing effect. However, calcium.is known to inhibit transpiration, and the protective effect could very well be an artifact. Similarly, reports of synergistic effects of cyto- kinins on toxin action (46) were later shown to be artifacts (43). Cytokinins increase transpiration and therefore more toxin was taken up by intact cuttings. ‘Uranyl ions were reported to protect cells against toxin when added to toxin solutions (27,70). However, binding of uranyl ion by toxin in solution was demonstrated (70). To circumvent mixing of toxin and uranyl ion, cuttings were pretreated for 12 hr with uranyl nitrate (27). The pretreatment also protected against toxin, but there is still a possibility of uranyl ions accumulating in tissues and ac- counting for toxin inactivation. Hanchey nevertheless interpreted her data to indicate that toxin acts on the cell surface, since it is known that uranyl ion does not penetrate the plasma membrane of yeast cells (65). Uranyl ion has a strong affinity for phosphoryl and 9 carboxyl groups (66), and has some affinity for sulfhydryl groups (60). The damage to the plasma membrane by toxin appears to be general and massive, since leakage of potassium, phosphate, sugars, and amino acids increased after exposure of cells to toxin (8). The rate of electrolyte efflux was said to be unaffected by oxygen tension and was characterized by a low temperature coefficient (Q10 1.2). Electron microscope studies of toxic effects have given ambiguous results. Derangement of nuclear membranes, endoplasmic reticula, chloroplasts and mitochondria have been observed (45), but these ob- servations were made after long exposures to toxin. The plasma mem- brane appeared to be intact, but smoother than in untreated cells; the bi-layer appearance was retained for 24 hr after toxin exposure. Other studies have shown that definite changes in membrane charac- teristics are not apparent in electron microScope pictures (28). The long toxin treatment periods used in these studies make results difficult to interpret. The genetics of susceptibility and resistance to toxin is especi- ally interesting since susceptibility to HV-toxin is linked to re- sistance to several strains of crown rust (Puccinia coronata) (58). Susceptibility to toxin in oats is dominant over resistance, and the reaction is controlled by the Vb locus. Other genes have been found in certain oat selections which may modify the reaction (44). It is possible that resistance to.§. Victoriae and susceptibility toug. coronata are not controlled by the same gene pair, but are only linked very closely. The qualitative ability of H. Victoriae to pro- duce HV-toxin also is controlled by a single gene, as shown by mating 10 experiments and genetic analysis (71). A major question is whether or not the primary gene product of the 22 allele in susceptible plants is the toxin receptor. If the toxin receptor is established as being located in the plasma membrane, then the final understanding of the mechanism of toxin ac- tion will result on a basic knowledge of the structure and function of the plasma membrane. Therefore, certain information related to the plasma membrane is included here. Progress in this general field is summarized in several recent reviews (10,38,47). Danielli and Davson's original hypothesis that the membrane is basically a protein-lipid-protein sandwich has survived 30 yr of criticism; only recently have solid objections been raised, based on data from work with electron microscOpy, infrared spectroscopy, cir- cular dichroism, and other techniques. Recent data have emphasized that protein-protein interactions are major determinants of membrane structure (38). Even though the role of lipid in membrane structure has been de-emphasized, there is good evidence to show that hydro- phobic lipid-protein interactions determine some of the structural characteristics of proteins in membranes (88). The most striking demonstration of the importance of structural proteins in membranes was provided by Fleisher ggflgl (21). In their experiments, 95% of the lipid was extracted from mitochondrial mem- branes without altering their bilayer appearance. Nevertheless, phospholipids do play an important role in electron transport and succinate oxidation (26). Furthermore, membranes of the gas vacuoles from Microcystis aeruginosa are composed entirely of protein, and apparently only a single protein moiety is involved (33). 11 Structural proteins from.many membrane types bind hydrophobically to lipid (35,38). Such proteins are heterogeneous, resistant to pronase, and soluble only in detergent or non-aqueous solvent sys- tems. Alkaline butanol is very effective in extracting protein from membranes (3,38). Spectroscopic studies on plasma membranes from human erythrocytes (88) have shown that membrane proteins are, to a great extent, globular with a high helical content. These studies suggest that the architecture of membrane proteins is dependent on lipid-protein and/or lipid sensitive protein-protein interactions. Opposing the bilayer lipid-protein hypothesis is the view that membranes are composed basically of repeating subunits, each unit composed of independent sets of catalytic and structural proteins (26). Electron microscopy in part has supported this hypothesis, but there may be artifacts in the techniques (10). Many so-called transport proteins for various sugars and amino acids have been described (54). The techniques involved in identi- fying and isolating transport proteins have potential application to studies on toxin receptors. Therefore, some of the more important experimental procedures, especially those concerned with sulfhydryl functions, will be described in some detail. Stein (83) was the first to develop a technique for isolating a membrane transport protein. Previous work (15) had shown that fluorodinitrobenzene (FDNB) inhibited the glucose transport system in red blood cells. Therefore, Stein labelled cells induced for glucose transport with 14C-FD'NB, and cells not induced for glucose transport with 3H-FDNB. The cell cultures were then mixed, homogenized, and the protein fraction chromatographed on DEAE cellulose. The protein 12 fractions with the highest 14C/3H ratio were presumed to be specific for, or at least associated with, the induction of glucose trans- port. Kolber and Stein (37) tried another double labelling technique with the galactoside transport system, also inducible by substrate. In this technique, induced cultures of E..ggli were labelled with 3H- phenylalanine and non-induced cultures with 14C-phenylalanine. The same type of fractionation method used with the glucose transport sys- tem was successful in showing 3 proteins with high 3H/14C ratios. Two of these proteins were galactosidase and transacetylase; the other was presumably a soluble protein associated with galactoside trans- port, although more definitive work by Fox and Kennedy (22) has cast some doubt on the significance of this protein. Fox and Kennedy developed a labelling approach using N-ethylmaleimide (NEM), a co- valent sulfhydryl reagent. With thiodigalactoside, a galactose ana- logue, they protected a specific binding protein against inactivation by NEM. Using induced and non-induced cultures of £3,221i, they were able to show that induced cultures had a binding protein. They were able to block 23% of the reactive sulfhydryl groups in the membrane fraction from NEM labelling by adding thiodigalactoside to the NEM solution. The binding protein, called M protein, was extracted with detergent from membrane fractions and therefore appeared to be a lipo- protein. ATPase is another well-studied membrane protein. NEM completely inhibited this enzyme (86). ATP can partially protect against NEM l4 inactivation, and against C-NEM labelling of red blood cell ghosts (86). However, ATP blocked many sulfhydryl groups not related to l3 ATPase activity. The author suggested that ATP could have induced conformational changes in the membrane and therefore was affecting nonspecific sulfhydryl groups. ‘Membrane ATPase is probably a lipo- protein, since attempts to solubilize it have not been successful (47). Sulfhydryl groups are important for other membrane functions. Glu- cagon and epinephrine binding to the rat liver plasma membrane is in- hibited by parachloromercuribenzoate (PCMB) and 5',5' dithiobisnitro- benzoic acid (DTNB) (85). These studies demonstrated that a hormone binding protein was different from adenyl cyclase, which mediates the hormonal action by producing cyclic 3',5' adenosine monophosphate. Similarly, the acetylcholine receptor was shown to involve a distinct acetylcholine receptor protein in the membrane of the eel electroplax (13). Experiments with equilibrium dialysis demonstrate that the receptor protein is blocked by PCMB (51). Reversible disulfide link- ages are involved in the regulation of both the enzyme and the re- ceptor protein (49). The evidence is strong that sulfhydryl groups are important in mem- brane function. However, Benson (6) pointed out that many membrane types have very low content of sulfhydryl groups (e.g., the red blood cell membrane has 1.4% cysteine). This implies the near absence of disulfide cross-linkages in membrane protein and a freedom of config- urational alteration. I The environment of the sulfhydryl group usually determines what reagent will react with it (2,12). For example, iodoacetate and arsenite do not inhibit the glucose transport system, whereas NEM, dinitrofluorobenzene (DNFB), and several mercurials are effective in- hibitors (15). NEM (9 mM) gave maximal inhibition of glucose transport 14 in red blood cells after 30 min of exposure. The red blood cell membrane was not penetrated to any extent by three mercurials: PCMB, parachloromercuriphenylsulfonate (PUMPS), and chlormerodrin. This made it possible to locate the glucose transport system on the outer surface of the membrane (87). The same approach was used by Pardee and Watanabe (55) to locate the sulfate binding protein on the memr brane of Salmonella typhimurium; in this case, diazonium salts were used to react with histidine and tyrosine groups in protein. Two inherently different types of membrane proteins, soluble and insoluble, have been described. Insoluble proteins are firmly bound to lipid and their isolation requires organic solvents and detergents. Soluble membrane proteins have been found mostly in bacteria. They are released by an osmotic shock technique described by Heppel (29). PUrification of membranes by density gradient centrifugation is done routinely for animal and bacteria cells,but apparently diffi- culties with plant cells have been encountered. It is evident that biochemical markers will be necessary for plant cell membrane work. MATERIALS AND METHODS Plants and Fungus Cultures: Oat cvs. Park, Rodney and Clinton were used in most experiments. Park oat has the dominant Vb.allele for susceptibility to H. Victoriae and to its toxin; Rodney and Clinton are resistant. Seedlings were grown in the laboratory at 22 C in vermiculite plus White's nutrient solution (95). Light (100- 200 ft. candles) was furnished by Gro-lux fluorescent tubes (Sylvania). Unless otherwise stated, the first true leaf above the primary leaf was used as a source of leaf tissue for experiments. For toxin production, highly virulent strains of H. Victoriae were grown in one liter Roux bottles, each containing 200 m1 modified Fries no. 3 basal mediuma Liquid medium was seeded with small pieces of mycelium from potato dextrose agar slant cultures. The still cul- tures were incubated for 3 weeks at 22 C. Preparation of Toxin and Toxin Breakdown Products (TBP): Toxin was isolated from culture filtrates by the method of Pringle and Braun (56). Filtrates were concentrated ig;gaggg, equal parts of methanol were added, and the precipitate was discarded. After meth- anol was removed by evaporation lg Egggg, the filtrate was extracted 3 times with n-butanol. The butanol extracts were combined and con- centrated in 23222, and an equal volume of methanol was added to the concentrate. This solution was then passed through an alumina column. 15 l6 Methanol and aqueous methanol, respectively, were run through the column. The toxin, which was adsorbed to the alumina, was eluted with 1% acetic acid. Unless otherwise mentioned, the eluate from alumina was used for experiments. This preparation completely in- hibited root growth of susceptible plants at 0.001 ug/ml. For some experiments, toxin was further purified by gel filtration (39) using Biogel P-2 (200-400 mesh) or Sephadex G-10 or G-15. Unless otherwise mentioned, columns were 1.5 cm.x 25 cm. The toxin at this stage of purity could not be stored for very long without gradual loss of activity. Toxin was broken down by the following method. The active frac- tions from gel filtration were pooled, and NaOH was added to bring the pH to 11.5. The solution was held at room temperature for one week, when the solution was readjusted to pH 7.0 to 8.0 with HCl. The preparation was evaporated to dryness in 13232, and repeatedly dried with acetone to yield a white friable powder. This preparation was called toxin breakdown product (TBP). A small amount of residual. activity remained, but this was reduced still further after storage at room temperature. Descending chromatography of TBP on Whatman no. 1 paper using propanol : acetic acid : water (RAW, 200 : 3 : 100 v/v) resulted in a major ninhydrin positive Spot at Rf 0.65 - 0.70. This was the Rf reported for peptide in breakdown products of toxin (57). A ninhydrin positive spot at Rf 0.80 - 0.90 was seen occasionally; this was presumed to be Victoxinine or a rearrangement thereof. The latter interpretation was favored since the spot was not reactive with iodoplatinate reagent (59), whereas authentic Victoxinine gave an R f of 0.84 - 0.89 and was strongly reactive with iodoplatinate. 0n l7 silica gel thin layer plates (Brinkman.MN silica gel N-HR/UV254)’ using the same solvent system, authentic Victoxinine gave an Rf of 0.70 - 0.75, whereas TBP preparations gave a ninhydrin positive spot of Rf 0.65 - 0.70. A much better separation was obtained when butanol : acetic acid : water (BAW, 8 : 2 : 2 v/v) was used. In this system, authentic Victoxinine had an R of 0.67 - 0.72, whereas the f TBP preparation gave a major ninhydrin positive spot with Rf of 0.52 - 0.56. Victoxinine was not detected in TBP with the iodo- platinate reagent. lgxin Assays: A seedling root growth bioassay (73) for toxin was used. Hulled oat seeds were germinated for 24 hr between moist fil- ter paper. Serial dilutions of toxin in White's solution or in water were prepared, and 5 m1 of each dilution wasplaced in each of two 60 x 15 mm Petri dishes. Five seedlings were placed in each dish, and root growth was measured after 48 to 72 hr incubation time. The highest dilution which restricted root growth to 1 cm or less was considered the dilution end-point. Roots of control plants were approximately 6 cm long. A toxin conductance assay was deve10ped, modified from.methods of Wheeler and Black (91). Toxin-treated or control tissue samples (0.53 - 1.0g) were enclosed in cheesecloth, rinsed thoroughly in distilled water, and suspended in 50 ml toxin solution in 125 m1 flasks which were placed on a reciprocal shaker (70 - 100 strokes/ min). After toxin treatment, tissue samples were washed 4 to 5 times in 100 ml of distilled water over a 10 min period, then suspended in 50 ml distilled water (conductance approximately 1 nmho) and shaken 18 (70 - 100 strokes/min) for up to 6 hr. Conductivity of the ambient solution was measured at intervals with a model RC 1631 Industrial Instruments conductivity bridge, using a dip type electrode with a constant of 1.0 for solutions below 200,000 ohms and a constant of 0.1 for solutions above 200,000 ohms. Specific conductivity was ex- pressed as reciprocal ohms by the following equation: Kc Ia =IE; (Ls = specific conductance, Kc = cell constant, and Rm = resistance at 22 C). Oat leaf tissue, from plants 5 to 15 days old, was used. In all experiments, conductance values obtained from.water control samples were subtracted from the values of toxin-treated samples to give toxin-induced electrolyte efflux. In experiments with inhibi- tion of toxin action being estimated, the term "percent protection" was used to show the reduction in toxin-induced electrolyte loss. The following equation was used to calculate percent protection: . . _ (EEEOS I-TD - (“mhos I) A protection = l (umhos T) _ (umhos W) x 100 (umhos I-T, I, T, or W’8 conductivity of leachates from inhibitor plus toxin, inhibitor, toxin, or water-treated tissues, respectively). MEasurement of Sodium and Potassium Contents in Extracts and leachates from Tissues: HCl was added to leachates from toxin-treated and control tissues to bring HCl concentration to 0.2N. .All solu- tions were held at 3 C prior to analysis. To obtain tissue extracts, 100 mg coleoptile tissue was extracted in boiling water for 10 to 15 min and the final volume was adjusted to 10 m1. This solution was filtered through Whatman ash free filter paper. A Jarrel-Ash "Dial l9 Atom”.Atomic absorption photometer or a Coleman flame photometer was used to measure the ion content of both leachates and tissue ex- tracts. Standard solutions of sodium and potassium were used to obtain estimates of ion concentration. Electropotentials of Single Plant Cells: These experiments were conducted in the laboratory of Dr. Noe Higinbotham of the Department of Botany, Washington State University. Methods of measuring elec- tropotentials in coleoptile cells of oat, corn and sorghum were essentially those used by Etherton and Higinbotham (19). The changes in potential between the cytoplasm.and bathing solutions were mea- sured with microelectrodes prepared with an automatic electrode puller. Cell microelectrodes were filled with electrolyte by boiling in 3MZKC1 for 15 min; tip diameters were approximately 0.5u. Refer- ence electrodes were filled with 3M KCl in agar; tip diameters were approximately 10 - 20p. The electrodes were connected to a measuring circuit with.AgeAgCl wires. The measuring circuit consisted of a Keithly electrometer with preamplifier connected to a Heath variable time recorder. Coleoptile sections were cut into 1 cm sections 3 to 6 hr before use and floated on a 1X nutrient solution (a salt solution lmM with respect to sodium, potassium, and calcium) (30) before they were mounted in a perfusion chamber. The perfusion chamber was continu- ally flushed with the 1X nutrient solution. Coleoptile sections were ‘mounted so that a cut surface could be observed microscopically. The cell microelectrode was then inserted into a cell and the reference microelectrode was placed in the bathing solution. This made it 20 possible to determine the resting potential across the plasma mem- brane. Isolation of Nuclei and Determination of Protein Synthesis: Nuclei were isolated using standard methods developed for plants (7,20). Oat seedlings were grown in the dark for 5 to 6 days, then leaf tissue was cut into 2 - 4 cm.sections, washed with distilled water and floated on White's solution at 3 C for 0.5 to 1 hr. The tissue was chopped rather than ground to eliminate shear forces which injure nuclei. The leaf sections were placed in a 250 m1 beaker with a poly- ethylene-covered bottom. Two ml/g tissue of the following buffer was used: sucrose, 0.5M; MgClz, 5mm; CaCl 5mm; EDTA, SmM; BSA, 0.2%; 2: mercaptoethanol, 10 mM. Tissue was chopped to a fine mince with single edge razor blades held firmly with long-nose pliers. Total chopping time was no longer than 10 min. The mince was filtered through 4 layers of cheesecloth followed by filtration through one layer Miracloth. The filtrate was centrifuged at 350g for 10 min and the pellet was resuspended in approximately 1 ml chopping buffer. The suspension was layered on a 1.2 M sucrose solution buffered with Tris-H01 (0.05M, pH 7.4). After centrifugation at 450g for 15 min, the pellet was resuspended in 1 m1 chapping buffer and once more centrifuged through a 1.2 M sucrose solution. The final pellet was resuspended in a buffer containing Tris-H01 (0.08M, pH 7.4), sucrose (0.6M) and chloramphenicol (300 ug/ml). Many nuclei were evident by microscopic examination. Toxin in 0.05M Tris-HCI (pH 7.4) or Tris buffer alone was added to aliquots of the nuclear suspension and held on ice 10 to 15 min 21 before addition of other materials. At 0 time, after the tubes had equilibrated for 5 min at 32 C in a water bath, radioactive sub- strate was added. This was either lysine 14C (uniformly labelled, 269 mm/mM) or leucine 14C (carboxyl labelled, 55.3 mc/mM). The final activity was from 0.5 to 1.0 uc/ml. Further additions were required for experiments with an ATP regenerating system.as follows: Tris- .ATP, 0.1mM; phosphocreatine (Na salt), 2mM; and creatine kinase, 100 ug/ml. Total volume of the reaction mixture was 0.9 m1. Duplicate tubes were incubated at 32 C, and the reaction was terminated by the addition of 40% TCA (1 ml). The 40% TCA step was followed by the addition of 5 ml cold 20% TCA containing, in some cases, approxi- mately 10 mg/ml D,L lysine-HCI. Samples were chilled, centrifuged at 10,000g for 30 min, and pellets were redissolved in 0.2N HCl. The tubes were incubated at 90 C for 30 min to degrade amino acyl RNA. The tubes were then cooled, and an equal volume of 40% TCA was added. After centrifugation, the pellets were washed once in 20% TCA followed by two washings in ethanol:ether (1:1). Precipitates were redispersed in 20% TCA, filtered on Millipore filters (type PHWP, 0.3a, 47mm) and washed with 50 m1 cold 10% TCA. The filter pad was dried with 20 ml ethyl ether and placed in scintillation vials at 50 C for several hours. Scintillation solution (10 ml, containing 43 PPO and 0.1g POPOP per liter of toluene) was added to each vial and vials were counted in a Packard Tri-Carb scintillation counter with approximately 40% efficiency. In each experiment a sample of the nuclear suspension was frozen and later assayed for protein by the modified Folin-Ciocalteau method (42) using BSA as the standard. The calculation of the number of MMoles amino acid incorporated into 22 protein was as follows: cpm incorporated/eff. x (dpm/uc)-1 x S.A..1 x mg protein.1 = umoles incorporated per mg protein (eff. = counter efficiency; dpm/uc = 2.2 x 106; S.A. = specific activity in uc/uM) Preparatign and Labelling of (Presumed) Mambrane Fragments: Oat seedlings were grown in the dark for 5 to 6 days and the leaves were cut, sectioned into pieces 2-3 cm long, rinsed with distilled water, and placed in White's solution at 3 C for 30 min. Tissue was then placed in a buffer solution in a 250 m1 beaker with a polyethylene- covered bottom. Approximately 2 ml buffer was used per g tissue. The buffer contained sucrose, 0.5M; Tris-HCl, 0.05M (pH 7.4); KCl, lOmM; CaCl 2mM; BSA, 0.1%; and beta-mercapto- 2mM; MgCl 2mM; EDTA-Na 2’ 2’ 2’ ethanol, 20mm. Tissue was chopped rapidly with single edge razor blades held with long-nose pliers, and the brei was filtered through 2 layers of cheesecloth and 1 layer Miracloth. The filtrate was cen- trifuged for 10 min at 12,000g and the pellet discarded. The superna- tant was centrifuged for 1 hr at 50,000g, or (in some cases) for 40 min at 30,000g. The two methods gave comparable results. The pellets were resuspended in lM.sucrose buffered with 0.1M Tris-HC1, pH 7.2; or in 0.1M K-phosphate buffer, pH 6.7. This preparation was clarified at 2000g for 5 min. The supernatant was called the particulate frac- tion and is believed to contain plasma membrane fragments (41). The particulate fraction was incubated with NEM-14C, with and without toxin or its breakdown products (TBP). .A 0.3 ml aliquot of the particulate preparation was mixed with 0.3 ml buffer (Tris-H01, 23 pH 7.4 or K-phosphate, pH 6.7; 0.05M), TBP (final conc. of TBP, 500 ug/ml) in buffer, or toxin (final conc. 16 ug/ml) in buffer. NEM-14C (New England Nuclear Corp., 2.9 mc/mM) was diluted with NEM to give a final concentration of 0.1 - 0.03mM (activity 0.1 - 0.5 uc/ml) in the reaction mixture. After 10 min, 0.3 ml of the NEM solution was added. .After incubation for 15 min, 1 m1 200mm NEM was added, and the reaction was terminated by adding an equal volume of 40% TCA; Triplicate tubes were used for each sample. The solution was diluted by adding 5 ml 10% TCA containing excess NEM, and the mixtures were centrifuged at 10,000g for 30 min. The pellet was washed again with 20% TCA, followed by l or 2 washes each with ethanol and ethanol:ether (1:1 v/v). The pellets were resuspended in 10% TCA, filtered on Millipore filters (pore size 0.3g or 0.45u) and washed with 60 ml 10% TCA. The filter pads were washed with 20 ml ethyl ether, placed in scintillation vials, dried for several hr at 50 C, and then 10 ml scintillation solution (toluene, 1 liter; PPO, 4g; POPOP, 100mg) was added. Preparations were counted with a Packard Tri-Carb scintillation counter with approximately 40% efficiency; samples were counted long enough to be within 1% reliability. In many experiments, control tubes terminated at 0 time were used to determdne adsorption of radioactivity; these values, plus the back- ground counts, were subtracted from the other sample counts. In a few experiments, protein in the particulate fraction was estimated by the modified Folin-Ciocalteau reaction (42). All experiments described were repeated one or more times with essentially the same results. RESULTS Effects of Toxin on Ion Fluxes and Electropotentials of Cells: Toxin-induced electrolyte efflux from cells: HV-toxin-treated tissues lose more organic and inorganic substances than do untreated control tissues (8). Within 2 min after toxin exposure, an increase in loss of total electrolytes over the control can be detected (75). Electrolyte efflux has been suggested as a possible toxin assay system (91), but there are not enough data to evaluate such an assay. Therefore, the relationship between toxin concentration and electro- lyte efflux was examined and characteristics of toxin-induced efflux were determined. 1 When toxin concentrations were plotted against conductance of leaching solutions (in umho), hyperbolic curves resulted (Figure 1, top). These resemble a plot of substrate concentration versus enzyme activity. Plots based on log toxin concentration were also shmilar to enzyme plots (Figure 1, top). This relationship suggested that data could be plotted reciprocally; therefore, a modified Lineweaver- Burk (WOlff) plot is shown, which yields a straight line and an esti- mation of the Km, or an estimation of the affinity of the toxin for its receptor (Figure 1, bottom). Km values were approximately 1 HM, assuming a molecular weight for toxin of about 1000. This use of re- ciprocal plots was helpful for checking on the competitive or non- competitive nature of a toxin inhibitor, as will be examined in a 24 25 , A_.IH_.._ 2.6 mg/ml two or more peptide positive inactivates increase decrease none not tested increase no effect decrease no effect no effect no effect decrease decrease 40 to 80% 145 gene locus; therefore, one gene product, presumably a receptor site, has been proposed. The data from experiments with cycloheximide can be interpreted according to the toxin receptor hypothesis. Cycloheximide protection against toxin could mean that the receptor is a protein with a 6 to 12 hr turnover time. However, other possible explanations have not been ruled out. Even the data with phospholipase D pretreatments are not counter to the receptor hypothesis. Phospholipase D protection against toxin-induced electrolyte loss could mean that the sensitive component is a lipoprotein, or is closely associated with a lipopro- tein. The apparent competition of esterified PC-toxin with active toxin is also consistent with the hypothesis of a receptor site. Each mole- cule of PC-toxin contains two glutamic acid residues and four aspartic acid residues; some or all of these may be esterified by methanol- HCl. The latter treatment is quite specific for carboxyl groups (5,32). The reduction of toxicity by esterified toxin could be ex- plained if carboxyl groups are necessary for toxin activity, but not required for stereospecific adsorption of toxin to receptor sites. A toxin analog might be useful in future studies of toxin. The identification of a receptor site for PC-toxin need not util- ize a reagent that binds with a specific group. Another approach, based on the incorporation of amino acids into the protein in ques- tion, would appear to be a more direct way to identify receptor pro- teins. This double labelling procedure was used by Kolber and Stein (9) to isolate membrane proteins associated with galactoside trans- port. This type of experiment would require that sorghum cultivars 146 be isogenic, except for the single gene controlling susceptibility. The susceptible cultivar would be labelled with 14C-amino acid and the resistant cultivar with 3H-amino acid. The tissues would be homo- genized, the homogenates mixed, and then proteins would be fraction- ated. A protein with a high 14C/3H ratio should be the specific gene product for susceptibility. PC-toxin is similar to HV-toxin in many respects. Resistance and susceptibility to each is based on single gene differences, and prob- ably is based on lack of receptor sites. The mechanism Of toxin ac- tion, in terms of interaction with receptors, could differ in each case. However, the available data are in accord with the hypothesis of receptor sites in plasma membranes. SUMMARY The earliest observed response of susceptible tissues to PC-toxin was increased loss of electrolytes. A toxin assay based on the abil- ity to induce loss of electrolytes was developed; the assay was shown to be practical, and was similar in nature to the assay used for HV- toxin. PC and HV-toxins are similar in many other respects. The data suggest that susceptibility and resistance to PC-toxin are con- stitutive characteristics which do not depend on active metabolism, Increased respiration and decreased amino acid incorporation are apparently secondary effects in toxic action. 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