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Characterization of SNP1, a Cell Wall-Degrading Trypsin, Produced During Infection by Stagonospora nodorum

May 2000 , Volume 13 , Number  5
Pages  538 - 550

A. J. Carlile , 1 L. V. Bindschedler , 1 A. M. Bailey , 2 P. Bowyer , 3 J. M. Clarkson , 1 and R. M. Cooper 1

1Microbial Pathogenicity Group, Department of Biology and Biochemistry, University of Bath, Claverton Down, BA2 7AY, U.K.; 2School of Biological Sciences, University of Bristol, Woodland Rd., Bristol, U.K.; 3Cell Biology Department, IACR Long Ashton Research Station, Bristol BS18 9AF, U.K.

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Accepted 25 January 2000.

Stagonospora (= Septoria) nodorum when grown in liquid culture with wheat cell walls as the sole carbon and nitrogen source secretes numerous extracellular depoly-merases, including a rapidly produced, alkaline, trypsin-like protease (SNP1). The enzyme was purified 417-fold by cation exchange chromatography and has a molecular mass of 25 kDa on sodium dodecyl sulfate gels, pI 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 aprotinin and leupeptin and weakly by phenylmethylsulfonyl 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 clone 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 protease, not present in controls, that coeluted with the fungal trypsin from cation exchange, and had properties (pI and inhibitor characteristics) similar to those of the fungal trypsin. SNP1 expression in planta was detected by Northern (RNA) blotting, reverse transcription PCR, and green fluorescent protein confocal microscopy. SNP1 released hydroxyproline from wheat cell walls. The release of hydroxyproline, together with its early expression in planta, suggests that SNP1 participates in the degradation of host cell walls during infection.

Additional keywords: cell wall proteins, plant pathogen, virulence.

© 2000 The American Phytopathological Society