Characterization of SNP1, a cell wall-degrading trypsin, produced during infection by Stagonospora nodorum

Stagonospora (= Septoria) nodorum when grown in liquid culture with wheat c ell walls as the sole carbon and nitrogen source secretes numerous extracel lular depolymerases, including a rapidly produced, alkaline, trypsinlike pr otease (SNP1). The enzyme was purified 417-fold by cation exchange chromato graphy and has a molecular mass of 25 kDa on sodium dodecyl sulfate gels, p i 8.7, and pH optimum of 8.5. It cleaved peptide bonds on the carboxyl side of lysine or arginine, was strongly inhibited by the trypsin inhibitors ap rotinin and leupeptin and weakly by phenylnethylsulfonyl fluoride, and its activity was stimulated by calcium. SNP1 has the characteristic, conserved, fungal, trypsin N terminus. Polymerase chain reaction (PCR) primers based on this sequence and the conserved trypsin active site were used to amplify a DNA fragment that facilitated isolation of the corresponding genomic clo ne from a lambda library of S. nodorum. The full-length sequence confirmed its identity as a trypsin-like protease containing the N-terminal sequence of the previously purified enzyme. Infected leaf tissue contained a proteas e, not present in controls, that coeluted with the fungal trypsin from cati on exchange, and had properties (pI and inhibitor characteristics) similar to those of the fungal trypsin. SNP1 expression in planta was detected by N orthern (RNA) blotting, reverse transcription PCR, and green fluorescent pr otein confocal microscopy. SNP1 released hydroxyproline from wheat cell wal ls. The release of hydroxyproline, together with its early expression in pl anta, suggests that SNP1 participates in the degradation of host cell walls during infection.