|
|
|
Project Number: WVA00360 MANIPULATION OF ERGOT ALKALOID PRODUCTION IN THE ENDOPHYTE OF TALL FESCUE Investigators: Panaccione,
D. Termination Date: 09/30/2002 Progress Report: Research focused on two strategies for manipulating the accumulation of toxic ergopeptines in fungi associated with grasses. One strategy is to prevent ergopeptine production by using transformation-mediated gene disruption to block a specific step in the ergopeptine biosynthesis pathway. A cloned ergopeptine biosynthesis gene is necessary for the execution of this strategy. A peptide synthetase gene (Cp605) that catalyzes a critical step in the pathway has been cloned from the pathogenic fungus Claviceps purpurea; characterization of this gene continued. DNA sequence analysis indicated that peptide synthetase gene Cp605 has the ability to incorporate several amino acids into a small peptide. Genes homologous to Cp605 were detected in the agriculturally important grass endophytes Neotyprodium coenophialum and Neotyphodium sp. Lp1, and isolation of these genes based on their hybridization with the C. purpurea gene was initiated. Copies of this gene from the grass endophytes will be used to construct ergopeptine-deficient strains of these fungi. An alternative strategy for manipulating the accumulation of ergopeptines in grass-associated fungi is to metabolize the ergopeptines as they are made. Execution of this strategy will require the discovery of ergopeptine-metabolizing microorganisms, followed by the isolation of genes for ergopeptine-metabolizing enzymes, and, finally, the expression of these genes in the endophytic fungi. Two different fungi with the ability to metabolize ergotamine (an important ergopeptine) were identified while screening large numbers of microorganisms associated with grasses. Ergotamine and other ergopeptines are normally detected and quantified by the characteristic fluorescence that results from the interaction of their multicyclic ring component with ultraviolet radiation. Metabolism of ergotamine by the two fungi is indicated by a loss of fluorescence after ergotamine is incubated with the fungi. The lack of fluorescence in the product of metabolism indicates an opening or other oxidation of the ring component but also makes identification of the metabolite(s) difficult. Strategies based on ultraviolet absorbance will be pursued to find and isolate the products of metabolism. Publications: Annis, S.L., and D.G. Panaccione. 1998. Presence of peptide synthetase gene transcripts and accumulation of ergopeptines in Claviceps purpurea and Neotyphodium coenophialum. Canadia Journal of Microbiology 44:80-86. Panaccione, D.G., J. Wang, C.L. Schardl, S.L. Annis, and P. Damrongkool. 1998. Association of a Claviceps purpurea peptide synthetase gene with ergopeptine biosynthesis. Phytopathology 88:S131 (abstract). Wang, J., D.G. Panaccione, and C.L. Schardl. 1998. Ergot alkaloid biosynthesis genes in Claviceps and Neotyphodium species. Phytopathology 88:S133 (abstract).
|