Abstract Experiments were designed to clone and iden- tify genes of the fungal phytopathogen Colletotrichum gloeosporioides expressed at high levels during growth on the compatible host Stylosanthes guianensis when com- pared with expression in axenic culture. A cDNA clone (pCgGS) that hybridised preferentially to a cDNA probe prepared from infected leaves was isolated by the differen- tial screening of a cDNA library from a nitrogen-starved axenic culture of C. gloeosporioides. The DNA sequence of pCgGS is highly homologous to genes for glutamine synthetase (GS) in other organisms. pCgGS contained all of the conserved regions assigned as catalytic domains in GS enzymes. Comparison with genomic sequences indi- cated that in C. gloeosporioides the GS gene is present as a single copy with three introns. To our knowledge this is the first report of the cloning of a GS from a filamentous fungus. A second clone (pCgRL1) was also isolated and represented a partial cDNA of the 25s rRNA of C. gloeo- sporioides. Because pCgRL1 did not hybridise to plant rRNA under high-stringency hybridisation conditions, it was used as a reference to quantify the expression of fun- gal GS mRNA during pathogenesis in S. guianensis com- pared to fungal growth in axenic culture. The results indi- cated that elevated expression of GS occurred during path- ogenesis of C. gloeosporioides on S. guianensis, particu- larly at early stages of infection where expression was about six-times higher than during growth in rich culture media. This work also demonstrates that fungal-specific 25s rRNA fragments, such as pCgRL1, have considerable utility as a reference for quantifying pathogen gene expres- sion in infected plants. Key words Expression quantification · Glutamine synthetase · Pathogenesis · Nitrogen metabolism Introduction The ability of a fungal pathogen to infect a host plant de- pends on its capacity to obtain adequate nutrients while avoiding or negating any defense responses. Genes that are either induced or highly expressed in the pathogen during the infection process may play a direct role in pathogene- sis, and hence contribute to events conferring compatibil- ity in host-pathogen interactions (Pieterse et al. 1993; Haw- thorne et al. 1994; Schäfer 1994). Pathogen genes induced in planta may produce products required for pathogenesis processes that suppress the host defences, re-direct nutri- ent supplies to the pathogen, and control essential adapta- tions to the nutritional environment encountered by the pathogen. Identification of such fungal genes and the elu- cidation of the regulatory processes necessary for their ex- pression during successful pathogenesis may reveal new information that can be targeted for the development of durable strategies of disease management (Valent and Chumley 1991; Schäfer 1994; Oliver and Osbourne 1995). Anthracnose caused by Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. is an important disease of many tropical plants and restricts the utilisation of the tropical forage legume Stylosanthes guianensis and other Stylo- santhes spp. in Australia, South-East Asia, Africa and South America (Manners et al. 1992; Lenne 1994). The in- fection processes of C. gloeosporioides on S. guianensis have been well studied histologically (Trevorrow et al. 1988; Ogle et al. 1990). In compatible interactions of bio- type B of C. gloeosporioides on S. guianensis, conidia ger- minate within 12 h to give melanized appressoria, followed by penetration of the cuticle by means of a penetration peg within 24 h after inoculation. After infecting two to four adjacent epidermal cells, hyphae begin to spread intra- and inter-cellularly into the mesophyll. Collapse of the meso- Curr Genet (1997) 31: 447 – 454 © Springer-Verlag 1997 Received: 14 November 1996 / 22 January 1997 Sally-Anne Stephenson · Jonathan R. Green John M. Manners · Donald J. Maclean Cloning and characterisation of glutamine synthetase from Colletotrichum gloeosporioides and demonstration of elevated expression during pathogenesis on Stylosanthes guianensis ORIGINAL PAPER S.-A. Stephenson · J. M. Manners () · D. J. Maclean Cooperative Research Centre for Tropical Plant Pathology, John Hines Building, The University of Queensland, Brisbane 4072, Australia J. R. Green School of Biological Sciences, The University of Birmingham, P.O. Box 363, Birmingham B15 2TT, UK Communicated by P. de Wit