1. BTOCHElYlirftl INTERACTIONS INVOLVING PARASITTE Clavicepx purpurea A thesis presented in fulfilment of the requirements for the degree of Ph.D. of the University of London by JULIA WILLINGALE April 1983 Department of Biochemistry, Imperial College of Science and Technology, London, SU7. 2. BIOCHEMICAL INTERACTIONS INVOLVING PARASTIC CJavicens PURPUREA ABSTRACT Artificial inoculation of wheat, infected with the bunt fungus Tilletia caries with Claviceps purpurea strain 12/2 [ALK~] resulted in a dual infection of the host ovary. Spatial interaction between the two pathogens during concurrent occupation of the ovary were studied by immunofluorescent microscopy, based on antibody raised against C. purpurea glucanase. Experiments on the specificity of this antibody showed no cross reaction with the mycelium of T. caries grown in axenic culture. Using this apparent specificity the investigations revealed penetration through the integument wall at the base of the ovary. Within 5 days, T. caries teliospores, at the base of the ovary, had been displaced in an acropetal direction by sporing,sphacelial tissue of C, purpurea. Establishment of the sphacelium was marked by copious honeydew exudation. At the fungal-fungal interface teliospores were completely surrounded by Claviceps hyphae. Above and below this interface, free teliospores and homogeneous sphacelial tissue were evident. As growth and differentiation to sclerotial tissue occurred, the bunted region became pushed to the tip of the developing ergot, forming a cap of black teliospores. During parasitic development on rye, a strain of Claviceps purpurea produced the primary pathway precursor dimethylaliyltryptophan (DMAT) but no tetracyclic ergoline alkaloids. Incubation of fresh sclerotial tissue with radioactively labelled primary pathway precursors demonstrated the biosynthesis of DMAT during the incubation period from tryptophan and mevalonate. There was no chromatographic evidence of N-methyl DMAT the next intermediate in ergoline biosynthesis. This was confirmed by a more sensitive approach in which it was found that 14C-methyl methionine was not incorporated into any indolic 3. metabolite. Thus the biosynthetic block was located beyond DFIAT at the step DMAT to N-methyl DMAT. Further inv/estigation into the operation of later steps in the ergoline pathway were explored by incubating parasitic tissue with 1^C-agroclauine, prepared biosynthetically by fermentation of C. fusiformis, and lysergic acid. Addition of the alkaloid intermediates did not result in production of the ergoline alkaloids usually formed by C. purpurea, but both promoted the biosynthesis of a single product, novel in C. purpurea, identified as lysergic acid amide. This strain of C. purpurea therefore has potential for exploring the control of the tryptophan prenylation step and provides new evidence concerning the role of lysergic acid amide in the biosynthesis of the cyclic tripeptide ergot alkaloids. Preliminary experiments suggest that at physiological concentrations phosphate does not inhibit the prenylation step. To compare the relative toxicity of alkaloid-free ergot and ergot rich in ergotoxine, young lean and obese mice were fed diets containing ergot. Animals on diets containing alkaloid-free ergot maintained a normal growth rate when a 10% ergot diet was given. In contrast mice consuming ergot rich in alkaloid became anorexic, and further administration of a diet containing 3% ergot was lethal. A group of mycotoxins which are structually related to the ergot alkaloids are the indole containing tremorgens, the most potent of which is verruculogen. TR-2 a structually related derivative of verruculogen was successfully incorporatec into verruculogen and fumitremorgen B by submerged culture of P. raistrickii . The finding that this tremorgenic alkaloid can be readily prenylated and therefore act as an intermediate in the biosynthesis of verruculogen is of some ecological interest when considering the possible role of soil-borne fungi in " staggers" syndromes. There are undoubtedly idiopathic syndromes amoungst livestock which occur due to synergistic effects of exo- mycotoxins. Tremorgenic species of Aspergillus and Penicillium commonly mould grain and grain products. Poor storage could easily have resulted in the proliferation of these ubiquitous, saprophytic moulds on ergotised grain, the consumption of which might have resulted in dominant expression of the neurological toxicity rather than the vasopressor type charactoristic of ergot alkaloids. Therefore a possible role for tremorgenic mycotoxins in convulsive ergotism, the cause of which is still in doubt, is discussed. 4. TABLE OF CONTENTS PAGE Abstract 2 Table of Contents 4 Index of Figures 7 Index of Tables 9 Index of Plates 10 Acknowlegements 11 1. INTRODUCTION 12 1.1 Ergot fungi as plant pathogens 12 1.1.1 Life cycle 12 1.1.2 Plant disease aetiology 14 1.1.3 Interaction between Claviceps 15 ptirpureaand the common bunt fungus Tilletia caries. 1.1.4 The role of fungal 8-1-3, 18 glucanases in plant pathogenicity 1.2 Ergot alkaloids 23 1.2.1 Chemistry 23 1.2.2 Biosynthesis 29 1.2.3 Regulation and control of alkaloid 37 biosynthesis 113.Toxicity 40 1.3.1 Ergotism and Nan 40 1.3.2 Toxicity in livestock 42 1.4.Tremorgenic mycotoxins 45 2. NATERIALS AND METHODS 51 2.1.Growth and maintenance'of fungi 51 2.1.1 Ergot 51 2.1.2 Penicillium spp. 52 2.2.General analytical methods 53 2.2.1 Detection and isolation of ergot 53 alkaloids 2.2.2 Layer chromatography (TLC and PLC) 54 5. 2.2.3 High performance liquid 55 chromatography (HPLC) 2.2.4 Centrifugation 55 2.2.5 Detection and assay of 56 biosynthetically radiolabelled metabolites 2.3 Axenic culture of the common bunt 57 fungus Tilletia caries 2.4 3-glucanase activity of wheat ovary 65 tissue infected with C. purpurea strain 12/2 and T, caries 2.5 3-glucanase activity of T• caries 69 grown in axenic culture 2^6 Immunofluorescent technique to 70 study spatial interaction between C. pupurea and T. caries during concurrent parasitism of the host ovary 2.7 Detection and promotion of ergot 77 alkaloid biosynthesis in C. purpurea strain 12/2 [ALK~] 2.8 Biosynthesis of verruculogen 82 2.8.1 Incorporation of radiolabelled TR-2 82 into verruculogen and fumitremorgen B by P. raistrickii 2.8.2 Incorporation of ll*C-proline into 88 verruculogen related metabolites of P. simplicissimum 2.9 Toxicity of ergot sclerotia gg 2.9.1 Preliminary investigation into the '90 effect on growth of young lean mice and young obese mice, resulting from the ingestion of sclerotial tissue of C. purpurea 2.9.2 Statistical analysis 93 3. RESULTS 94 3.1 Growth of T. caries in axenic culture 94 3.2 3-glucanase activity during the early 102 stage of ovary infection 3.3 3-glucanase activity of T. caries in 102 axenic culture 3.4 Spatial interaction between C, purpurea 107 and T. caries during concurrent parasitism of wheat ovary tissue 3.5 Biosynthesis 119 3.6.1 Incorporation of radiolabelled 14C-TR-2 into verruculogen and fumitremorgen B by P. raistrickii 3.6.2 Application of the fluorimetric assay to fumitremorgen B 3.6.3 Biosynthetically-radiolabelled, verruculogen related,metabolites of P. simplicissimum 3.7 Toxicity 4. DISCUSSION 4.1 8-glucanase activity of T. caries 4.2 Interaction between C, purpurea and T. caries in wheat ovary tissue 4.3 Promotion of alkaloid biosynthesis in a Q\LK J C. purpurea mutant 4.4 Biosynthesis of verruculogen 4.5 Toxicity APPENDIX I Technique to reduce unwanted background fluorescence in fluorescence microscopy APPENDIX II Preparation of plant tissue for fluorescence microscopy APPENDIX III Statistical analysis for the difference between two sample means 5. REFERENCES INDEX OF FIGURES 'PAGE Fig. 1 Life cycle of ergot 13 Fig. 2 Life cycle of bunt Tilletia caries 17 Fig. 3 The tetracyclic ergoline ring system 23 Fig. 4 Biosynthetic origin of the tetracyclic 32 ergoline ring Fig. 5 Relationships between clavine alkaloids 35 Fig. 6 Chemical structure of tremorgenic 47 mycotoxins of the paspalinine group Fig. 7 Structure of verruculogen 48 Fig. 8 Chemical structure of the fumitremorgens 49 Fig. 9 Chemical structure of TR-2 49 Fig.ID Four populations of bunt teliospores 58 obtained by centrifugation Fig.11 Diagramatic representation of longitudinal 67 section through a bunt infected ovary at "anthesis" Fig.12 Elution of IgG fractions of 3-1,3 glucanase 72 antiserum fr.Dma DEAE-cellulose column Fig.13 Resolution of C-verruculogen and llfC- 85 fumitremorgen B by HPLC Fig.14 HPLC extract of fumitremorgen B left in 87 aqueous solution for 6h; repurified by HPLC Fig.15 Resolution of fumitremorgen B by HPLC 87 Fig.16 3-glucanase activity of control and 165 infected ovaries with increasing age Fig.17 3-glucanase activity of control, 104 "healthy bunted", and infected bunted ovaries with increasing age of infection Fig.18 Time course of hydrolysis of S. sorghi 106 glucan by dialysed extracts of T. caries tissue from axenic culture Fig.19 Diagramatic representation of a dual ^11 infected wheat ovary, 6 days after inoculation with C, purpurea 8. Fig.20 Diagramatic representation of a 113 longitudinal section through a dual infected ovary, 10 days after inoculation with C, purpurea Fig.21 Diagram representing sections of dual 123 infected ovaries stained with (3-1,3 glucanase antiserum, indicating the areas of the sections illustrated in Plate 8 and 9 Fig.22 Incubation of fresh parasitic tissue 126 of C. purpurea strain 12/2 [ALK"] with C-methyl methionine. TLC analysi s Fig.23 Incubation of fresh parasitic tissue 130 of C, purpurea strain 12/2 [ALK~] with 1^C-tryptophan in the presence of O.OIM 0.1M and 1.01*1 phosphate Fig.24 Excitation and emission spectra for 136 acid-treated verruculogen and fumitremorgen B Fig.25 Growth curves for 3 groups of obese mice 140 A & B over the 50 day experimental period Fig.26 Growth curves for 3 groups of lean mice 142 A & B over the 50 day experimental period Fig.27 Limitation of, and potential for, ergot 158 alkaloid biosynthesis in a mutant strain of C.