A SECRETED ASPARTIC PROTEINASE FROM GLOMERELLA-CINGULATA - PURIFICATION OF THE ENZYME AND MOLECULAR-CLONING OF THE CDNA

A secreted aspartic proteinase from Glomerella cingulata (GcSAP) was p urified to homogeneity by ion exchange chromatography, The enzyme has an M-r of 36 000 as estimated by SDS-PAGE, optimal activity from ph 3. 5 to ph 4.0 and is inhibited by pepstatin. The N-terminal sequence, 23 residues long, was used to design a gene-specific primer, This was us ed in 3' RACE (rapid amplification of cDNA ends) PCR to amplify a 1.2 kb fragment of the gcsap cDNA, A second gene-specific primer was desig ned and used in 5' RACE PCR to clone the 5' region, This yielded a 600 bp DNA fragment and completed the open reading frame. the gcsap open reading frame encodes a protein with a 78 residue prepro-sequence typi cal of other fungal secreted aspartic proteinases. Based on the deduce d sequence, the mature enzyme contains 329 amino acids and shows appro ximately 40 % identity to other fungal aspartic proteinases. Subsequen t cloning and sequencing Of gcsap fragments obtained from PCR with gen omic DNA revealed a 73 bp intron beginning at nt 728, Southern analyse s at medium and high stringency indicated that C. cingulata possesses one gene for the secreted aspartic proteinase, and Northern blots indi cated that gene expression was induced by exogenous protein and repres sed by ammonium salts, GcSAP isa putative pathogenicity factor of G. c ingulata, and it will now be possible to create SAP(-) mutants and ass ess the role GcsAP plays in pathogenicity.