Curt Genet (1992)22:399 406 Current Genetics 9 Springer-Verlag 1992 Transformation of Acremonium coenophialum, a protective fungal symbiont of the grass Festuca arundinacea Huei-Fung Tsai, Malcolm R. Siegel, and Christopher L. Schardl Department of Plant Pathology, S-305 Agriculture Science Center-North, University of Kentucky, Lexington, KY 40546-0091, USA Received March 19, 1992 Summary. Acremonium coenophialum is a mutualistic my- cosymbiont and natural agent of biological protection of the widely distributed grass Festuca arundinacea (tall fes- cue). An electroporative transformation system was de- veloped for A. coenophialum. Segments of DNA 5' to the /~-tubulin gene (tub2) of the closely related ascomycete Epichlod typhina, fused to the Escherichia coli hph gene encoding hygromycin B phosphotransferase, conferred hygromycin resistance when introduced into A. coenophialum by electroporation. The incorporation of the Emericella nidulans trpC terminator greatly increased protoplast germination on selective medium and im- proved transformation efficiencies 30-200 % depending on the plasmid construct. Plasmid pCSN43, which incor- porates the trpC controlling elements for hph expression, was also used to transform A. coenophialum. Southern blot analysis of ten pCSN43 transformants indicated the possibility of random integration of this vector into the genome. Key words: Acremonium coenophialum - EpichloO" typhina - fl-tubulin gene - Electroporative transformation Introduction Acremonium coenophialum is a seedborne, endophytic fungal symbiont of the grass Festuca arundinacea (tall rescue). The mycosymbiont is an anamorphic (asexual) strain of Epichlob typhina (Clavicipitaceae) (White and Morgan-Jones 1987; Schardl et al. 1991) and one of a group of seedborne symbionts commonly found in spe- cies of Festuca (fescues), Lolium (ryegrasses), and other C3 grasses (Bacon and De Battista 1990). The life cycle of A. coenophialurn is strictly tied to that of its host maternal lineage because the mycosymbiont has no natural means of infection or dissemination other than through seed transmission. Therefore, it constitutes a heritable compo- nent of the grass-fungus symbiotum (Siegel and Schardl 1991), and some important genetic and physiological Correspondence to. C. L. Schardl characteristics of this symbiotum depend on the my- cosymbiont or on interactions between the mycosym- biont and its host. Among these characteristics are nu- merous fitness benefits. A. coenophialum has been shown to provide its host with enhanced growth and competi- tiveness (De Battista et al. 1990; Hilt et al. 1990, i991), enhanced drought tolerance (Arachevaleta et al. 1989), greater seed set (Rice et al. 1990), enhanced resistance to diseases caused by Rhizoctonia zeae (Gwinn and Gavin 1992) and Puccinia coronata (Ford and Kirkpatrick 1989), and a substantial degree of protection against ne- matode (West et al. 1988; Kimmons et al. 1990), insect (Clay 1988; Bacon and De Battista 1990; Dahlman et al. 1991) and mammalian herbivores (Siegel etal. 1987; Stuedemann and Hoveland 1988). Because of these char- acteristics, A. coenophialum is critical for the fitness of tall rescue in a range of natural environments, and the plant and fungus exist in an interdependent, mutualistic associ- ation (Clay 1990). Tall fescue is widely distributed in North America, where it is grown on approximately 14 million ha. It is an important forage grass for livestock and is also used as a turf and conservation grass. A high percentage of tall rescue is symbiotic with A. coenophialum (Shelby and Dalrymple 1987). The economic benefits of this grass- fungus association are partially offset by 'fescue toxico- sis,' the manifestation of anti-mammalian activity which results in seasonal, sometimes severe, detrimental effects on grazing livestock (Raisbeck et al. 1991). Within the United States, the cost of fescue toxicosis affecting cattle alone is estimated to total more than 600 million dollars per year (Hoveland 1992). Because of the natural role of A. coenophialum in biological protection (Clay 1990), the approach of permanently removing the mycosymbiont, though technically feasible, may be impractical in most circumstances (Siegel and Schardl 1991). Among the al- ternative approaches are remedial treatment of the live- stock, identification and use of related mycosymbionts with less anti-mammalian activity, or genetic alteration of the fungus to eliminate or reduce its ability to produce the toxic factors (Siegel 1992).