Characterization of SNP1, a cell wall-degrading trypsin, produced during infection by Stagonospora nodorum.
Stagonospora (= Septoria) nodorum when grown in liquid culture with wheat cell walls as the sole carbon and nitrogen source secretes numerous extracellular depolymerases, 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.
Carlile
A J
AJ
Microbial Pathogenicity Group, Department of Biology and Biochemistry, University of Bath, Claverton Down, UK.
Bindschedler
L V
LV
Bailey
A M
AM
Bowyer
P
P
Clarkson
J M
JM
Cooper
R M
RM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002473:Cell Wall / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D003904:Mitosporic Fungi / Q000472:pathogenicity*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014908:Triticum / Q000382:microbiology*
D018076:DNA, Complementary; C409688:SNP1 protein, Phaeosphaeria nodorum; D012697:Serine Endopeptidases