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首页> 外文期刊>Molecular Plant-Microbe Interactions >Characterization of SNP1, a Cell Wall-Degrading Trypsin, Produced During Infection by Stagonospora nodorum
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Characterization of SNP1, a Cell Wall-Degrading Trypsin, Produced During Infection by Stagonospora nodorum

机译：SNP1的表征，一种细胞壁降解的胰蛋白酶，由No. Stagonospora nodorum感染过程中产生

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摘要

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.
机译：在液体培养中以小麦细胞壁作为唯一碳源和氮源生长的Stagonospora（= Septoria）瘤菌会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（SNP1）。该酶通过阳离子交换色谱纯化417倍，在十二烷基硫酸钠凝胶上的分子量为25 kDa，pI为8.7，最适pH为8.5。它裂解赖氨酸或精氨酸羧基侧的肽键，被胰蛋白酶抑制剂抑肽酶和亮肽素强烈抑制，并被苯甲基磺酰氟弱抑制，其活性被钙刺激。 SNP1具有保守，真菌，胰蛋白酶N末端的特征。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）引物被用于扩增DNA片段，该DNA片段促进了从No. S. nodorum的λ文库中分离相应的基因组克隆。全长序列证实其与包含先前纯化的酶的N-末端序列的胰蛋白酶样蛋白酶相同。受感染的叶片组织含有一种蛋白酶，该蛋白酶不存在于对照中，并与阳离子交换的真菌胰蛋白酶共洗脱，并且具有与真菌胰蛋白酶相似的特性（pI和抑制剂特性）。通过Northern（RNA）印迹，逆转录PCR和绿色荧光蛋白共聚焦显微镜检测了植物中SNP1的表达。 SNP1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，SNP1在感染过程中参与了宿主细胞壁的降解。

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