CLONING AND CHARACTERIZATION OF GLUTAMINE-SYNTHETASE FROM COLLETOTRICHUM-GLOEOSPORIOIDES AND DEMONSTRATION OF ELEVATED EXPRESSION DURING PATHOGENESIS ON STYLOSANTHES-GUIANENSIS

Experiments were designed to clone and identify genes of the fungal ph ytopathogen Colletotrichum gloeosporioides expressed at high levels du ring growth on the compatible host Stylosanthes guianensis when compar ed with expression in axenic culture. A cDNA clone (pCgGS) that hybrid ised preferentially to a cDNA probe prepared from infected leaves was isolated by the differential screening of a cDNA library from a nitrog en-starved axenic culture of C. gloeosporioides. The DNA sequence of p CgGS is highly homologous to genes for glutamine synthetase (GS) in ot her organisms. pCgGS contained all of the conserved regions assigned a s catalytic domains in GS enzymes. Comparison with genomic sequences i ndicated 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. gloeosporioides, Because pCgRL1 did not hybridise to plant rRNA und er high-stringency hybridisation conditions, it was used as a referenc e to quantify the expression of fungal GS mRNA during pathogenesis in S. guianensis compared to fungal growth in axenic culture. The results indicated that elevated expression of GS occurred during pathogenesis of C. gloeosporioines on S. guianensis, particularly at early stages of infection where expression was about six-times higher than during g rowth in rich culture media. This work also demonstrates that fungal-s pecific 25s rRNA fragments, such as pCgRL1, have considerable utility as a reference for quantifying pathogen gene expression in infected pl ants.