Host-parasite relations in a mycoparasitic system : alterations in membrane permeability and lipid composition in Choanephora cucurbitarum infected by Piptocephalis virginiana /

examined in Choanephora cucurbita rum during the early stages of infection by Piptocephalis virginiana » There was a small but consistent increase in the leakage of electrolytes, amino acids and sugars as a result of infection. These low levels of differential leakage in infected tissues are explained on the basis of the nature of this obligate, biotrophic, mycoparasitic system. Quantitative analysis of the twenty six amino acids and amino compounds detected in the leacheates — showed similar profiles in infected and control host and no new species of amino acids or amino compounds were detected in either infected or control host leacheates. Comparatively high amounts of aspartic acid, glutamic acid and alanine were found in the leacheates of host and infected host . Analyses of the sugars comprising the leacheates of infected and control host showed the presence of eight sugars, among which glucose was found in significant amounts (50-53%) ' The nutritional implication of this preferential leakage is discussed. No significant difference was observed in the leacheates of infected host sugar profiles compared with that of the control host. Profiles of the internal pool sugars of infected and control host did not reflect that obtained from the leacheate data, perhaps owing to leakage of sugars in a selective manner . Membrane lipid analyses yielded higher levels of lipid in infected host compared with the control, both at the 24 h and 36 h analyses. In addition, preliminary investigations of phosphorous-32 incorporation and turnover in phospholipids showed higher levels of 32p incorporation and turnover in infected host compared with the control. No apparent difference was noted in the profiles of the neutral lipid classes and the polar lipid classes of the membrane lipids as determined by one and two dimensional thin-layer chromatography respectively. However, a small but consistently higher degree of unsaturation was detected in the fatty acids of infected tissue compared with the control. Also, '^''-^^''^^'-'-^'^^c acid, a polyunsaturated fatty acid previously reported to show a direct correlation during the early stages of infection and the degree of parasitism of P. virginiana on C. cucurbitarum , was found in higher amounts in infected host membrane lipids compared with that of the control host. The implications of these membrane lipid alterations are discussed with particular reference to the small but consistently higher leakage of electrolytes, amino acids and sugars observed during infection in this study.

The effects of cultural conditions and parasitism on the lipid composition of choanephora cucubitarum (Berk. Rav.) thaxter /|nJohn M. Deven. -- 260 St. Catharines [Ont. : s. n.],

The fatty acid composition of the total, neutral, sterol, free fatty acid and polar-lipid fractions in the mycelium of Choanephora cucurbitarum was determined. The major fatty acids in all lipid fractions were palmitic, oleic, linoleic and y-linolenic acid. Different lipid fractions did not show any particular preference for any individual fatty acid; however, the degree of unsaturation was different in various lipid fractions. Addition of glutamic acid to the malt-yeast extract medium resulted in the biosynthesis of a number of long-chain fatty acids beyond y-linolenic acid. These fatty acids, e.g. C22~1' C24:0 and C26=Q were never observed to be present in the fungus when grown on a malt-yeast extract medium without glutamic acid. Furthermore, thin-layer chromatographic analysis showed a larger and denser spot of diphosphatidyl glycerol from the mycelium grown on the glutamic acid medium than from the control mycelium. Various cultural conditions such as temperature, age, pH, light and carbon:nitrogen ratio in the growth medium used in this study did not alter the qualitative profile of fatty acids normally present in the organism. Neither did these conditions stimulate the production of further long-chain fatty acids (C20 - C26) beyond y-linolenic acid as observed in growth media containing glutamic acid. These cultural conditions influenced the degree of unsaturation, this being due mainly to changes in the concentration of y-linolenic acid. The fatty acid pattern of the lipid fractions though the same qualitatively, differed quantitatively due to the variation in the y-linolenic acid content under different cultural conditions. The degree of unsaturation of various lipid fractions decreased with increases in temperature, light intensity and pH, but within each treatment the same pattern of decreasing degree of unsaturation with increasing age was observed. The cultural conditions, used in this study, are also known to influence the degree and rate of development of the parasite, Piptocephalis virginiana. A direct correlation was observed between the levels of y-linolenic acid in C. cucurbitarum during the early stages of growth (24 h) and the degree of parasitism of P. virginiana. The amount of y-linolenic acid present in the host mycelium was found to be unrelated to either the dry weight of the mycelium or to the total lipid contents. K. virginiana is confined to host species which produce y-linolenic acid in their mycelium. The lipid profile of the host, C. cucurbitarum, did not show a significant qualitative or quantitative change in the lipid profile as a result of infection by the parasite, P. virginiana,e However, an increase in the total lipid was observed in the infected host mycelium. The significance of these results is discussed.

Investigations on the production of long chain fatty acids in Choanephora cucurbitarum (Berk.

The fatty acid composition of the total cellular lipids of Choanephora cucurbitarum incubated for 96 hrs on either glucose-ammonium sulfate or malt-weast extract media was determined. The major fatty acids were palmitic, palmitoleic, stearic and linoleic acids. The saturated fatty acid possessing the longest acyl chain was stearate (C 18:0). The presence of glutamic acid (2.0 x 10-1% or 1.36 x la-2M) in either of the above growth media resulted in increase in percent of 1f-linolenic acid, decrease in percent of linoleic ~iCid and appearance of a new series of fatty acid> C ~8 e.g. C ",,,,'V' C2k:O, C26,O. The addition of glutamic acid had no effect on the lipid yield but slightly decreased the degree of unsaturation. Compounds which duplicated the effect of glutamic acid were acetate, malate, citrate, succinate, 0( -ketoglutarate, prOline, -y -aminobutyric acid and glucose (3%) but not aspartic acid or alanine. ~o correlation was found between glutamic acid pool concentration and the presence in the growth medium of those compounds which stimulate long chain fatty acid production. Four hours of incubation with 27 JJ 1-1 glutamate supported the production of long chain fatty acids. This stimulation is inhibited if 272 .u M isophthalic acid is added with 27 AJ M glutamate. But, long chain fatty acids were detected when 27 JJ M eX -ketoglutarate is also present in the incubation mixture. Five hours of incubation with 100 ,Mg/ml of cycloheximide resulted in over 9CY/o inhibition of cytoplasmic :protein synthesise Glutamate (27 .uM) enhanced the synthesis of long chain fatty acids under these conditions. These findings are discussed in an attempt to provide a plausible explanation COmmon to compounds that support the production of long chain fatty acids.

Does Gamma-aminobutyric acid function as a plant resistance mechanism against phytophagous insect activity? /

Gamma-aminobutyric acid (GAB A) is a ubiquitous non-protein amino acid synthesized via the decarboxylation of L-glutamate in a reaction catalyzed by the cytosolic enzyme L-glutamate decarboxylase (GAD). In animals it functions as an inhibitory neurotransmitter. In plants it accumulates rapidly in response to various stresses, but its function remains unclear. The hypothesis that GABA accumulation in leaf tissue may function as a plant resistance mechanism against phytophagous insect activity was investigated. GABA accumulation in response to mechanical stimulation, mechanical damage and insect activity was demonstrated. In wt tobacco (Nicotiana tabacum cv Samsun), mechanical stimulation or damage caused GABA to accumulate within 2 min from mean levels of 14 to 37 and 1~9 nmol g-l fresh weight (FW), respectively. In the transgenic tobacco strain CaMVGAD27c overexpressing Petunia GAD, the same treatments caused GABA to accumulate from 12 to 59 and 279 nmol g-l FW, respectively. In the transgenic tobacco strain CaMVGADilC 11 overexpressing Petunia GAD lacking an autoinhibitory domain, mechanical stimulation or damage caused GABA to accumulate from 180 to 309 and 630 nmol g-l FW, respectively. Ambulatory activity by tobacco budworm (TBW) larvae (Heliothis virescens) on leaves of CaMVGAD27c tobacco caused GABA to accumulate from 28 to 80 nmol g-l FW within 5 min. Ambulatory and leaf-rolling activity by oblique banded leaf roller (OBLR) larvae (Choristoneura rosaceana cv Harris) on wt soybean leaves (Glycine max cv Harovinton) caused GABA to accumulate from 60 to 1123 nmol g-l FW within 20 min. Increased GABA levels in leaf tissue were shown to affect phytophagous preference in TBW larvae presented with wt and transgenic tobacco leaves. When presented with leaves of Samsun wt and CaMVGAD27c plants, TBW larvae consumed more wt leaf tissue (640 ± 501 S.D. mm2 ) than transgenic leaf tissue (278 ± 338 S.D. mm2 ) nine times out of ten. When presented with leaves of Samsun wt and CaMVGAD~C11 plants, TBW larvae consumed more transgenic leaf tissue (1219 ± 1009 S.D. mm2 ) than wt leaf tissue (28 ± 31 S.D. mm2 ) ten times out of ten. These results indicate that: (1) ambulatory activity of insect larvae on leaves results in increased GABA levels, (2) transgenic tobacco leaves with increased capacity for GABA synthesis deter feeding, and (3) transgenic tobacco leaves with constitutively higher GABA levels stimulate feeding.

L-Glutamate uptake, decarboxylation to -aminobutyric acid and GABA efflux in isolated Asparagus sprengeri mesophyll cells

Addition of L-glutamate caused alkalinization of the medium surrounding Asparagus spreng.ri mesophyll cells. This suggests a H+/L-glutmate symport uptake system for L-glutamate. However stoichiometries of H+/L-glutamate symport into Asparagus cells were much higher than those in other plant systems. Medium alkalinization may also result from a metabolic decarboxylation process. Since L-glutmate is decarboxylated to r-amino butyric acid (SABA) in this system, the origin of medium alkalinization was reconsidered. Suspensions of mechanically isolated and photosyntheically competent Asparagus sprengeri mesophyll cells were used to investigate the H+/L-glutamate symport system, SABA production, GABA transport, and the origin of L-glutamate dependent medium alkalinization. The major results obtained are summarized as follows: 1. L-Glutamate and GABA were the second or third most abundant amino acids in these cells. Cellular concentrations of L-glutamate were 1.09 mM and 1.31 mM in the light and dark, respectively. Those of SABA were 1.23 mM and 1.17 mM in the light and dark, respectively. 2. Asparagine was the most abundant amino acid in xylem sap and comprised 54 to 68 1. of the amino acid pool on a molar basis. GABA was the second most abundant amino acid and represented 10 to 11 1. of the amino acid pool. L-Slutamate was a minor component. 3. A 10 minute incubation with 1 mM L-glutamate increased the production of GABA in the medium by 2,743 7. and 2,241 7. in the light and dark, respectively. 4. L-Glutamate entered the cells prior to decarboxylation. 5. There was no evidence for a H+/GABA symport process • 6. GABA was produced by loss of carbon-1 of L-glutamate. 7. The specific activity of newly synthesized labeled GABA suggests that it is not equilibrated with a storage pool of GABA. 8. The mechanism of GABA efflux appears to be a passive process. 9. The evidence indicates that the origin of L-glutamate dependent medium alkalinization is a H+/L-glutamate symport not an extracellular decarboxylation. The possible role of GABA production in regulating cytoplasmic pH and L-glutamate levels during rapid electrogenic H+/L-glutamate symport is discussed.

Rapid [Gamma]-amino acid synthesis and the inhibition of the growth and development of oblique banded leaf-roller larvae

The hypothesis that rapid y-aminobutyric acid (GABA) accumulation is a plant defense against phytophagous insects was investigated. Simulation of mechanical damage resulting from phytophagous insect activity increased soybean (Glycine max L.) leaf GABA 10- to 25-fold within 1 to 4 min. Pulverizing leaf tissue resulted in a value of 2. 15 (±O. 11 SE) ~mol GABA per gram fresh weight. Increasing the GABA levels in a synthetic diet from 1.6 to 2.6 Jlffiol GABA per gram fresh weight reduced the growth rates, developmental rates, total biomass (50% reduction), and survival rates (30% reduction) of cultured Oblique banded leaf-roller (OBLR) (Choristonellra rosacealla Harris) larvae. In field experiments OBLR larvae were found predominantly on young terminal leaves which have a reduced capacity to produce GABA in response to mechanical damage. Glutamate decarboxylase (GAD) is a cytosolic enzyme which catalyses the decarboxylation of L-Glu to GABA. GAD is a calmodulin binding enzyme whose activity is stimulated dramatically by increased cytosolic H+ or Ca2 + ion concentrations. Phytophagous insect activity will disrupt the cellular compartmentation of H+ and Ca2 +, activate GAD and subsequent GABA accumulation. In animals GABA is a major inhibitory neurotransmitter. The possible mechanisms resulting in GABA inhibited growth and development of insects are discussed.