Characterization of SNP1, a cell wall-degrading trypsin, produced during infection by Stagonospora nodorum
Characterization of SNP1, a cell wall-degrading trypsin, produced during infection by Stagonospora nodorum
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DOI：
10.1094/mpmi.2000.13.5.538
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发表时间：
2000-05-01
影响因子：
3.5
通讯作者：
Cooper, RM
中科院分区：
文献类型：
--
作者：
Carlile, AJ;Bindschedler, LV;Cooper, RM
当在以小麦细胞壁作为唯一碳源和氮源的液体培养物中生长时，Stagonospora（=壳针孢）nodorum分泌许多细胞外解聚酶，包括快速产生的碱性胰蛋白酶样蛋白酶（SNP 1）。该酶通过阳离子交换色谱法纯化417倍，在十二烷基硫酸钠凝胶上分子量为25 kDa，pi为8.7，最适pH为8.5。它裂解的赖氨酸或精氨酸的羧基侧的肽键，强烈抑制胰蛋白酶抑制剂抑肽酶和亮抑酶肽和弱的苯甲基磺酰氟，其活性被钙刺激。SNP 1具有特征性的、保守的、真菌的胰蛋白酶N末端。基于该序列和保守的胰蛋白酶活性位点的聚合酶链反应（PCR）引物用于扩增DNA片段，该DNA片段有助于从S.结节病。全长序列证实了其作为含有先前纯化的酶的N-末端序列的胰蛋白酶样蛋白酶的身份。感染的叶组织中含有蛋白酶，不存在于控制，与真菌胰蛋白酶从阳离子交换共洗脱，并具有类似的真菌胰蛋白酶的属性（PI和抑制剂特性）。通过北方（RNA）印迹、反转录PCR和绿色荧光蛋白共聚焦显微镜检测植物中SNP 1的表达。SNP 1从小麦细胞壁释放羟脯氨酸。羟脯氨酸的释放及其在植物中的早期表达表明，SNP 1参与了感染过程中宿主细胞壁的降解。
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, trypsinlike 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 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 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.