Martínez M, López-Solanilla E, Rodríguez-Palenzuela P, Carbonero P, Díaz I
Laboratorio de Bioquímica y Biología Molecular, Departamento de Biotecnología-UPM, ETSI Agrónomos, 28040 Madrid, Spain.
Mol Plant Microbe Interact. 2003 Oct;16(10):876-83. doi: 10.1094/MPMI.2003.16.10.876.
The recombinant barley cystatin Hv-CPI inhibited the growth of three phytopathogenic fungi (Botrytis cinerea, Colletotrichum graminicola, and Plectosphaerella cucumerina) and the saprotrophic fungus Trichoderma viride. Several mutants of barley cystatin were generated by polymerase chain reaction approaches and both their antifungal and their cysteine-proteinase inhibitory properties investigated. Point mutants R38-->G, Q63-->L, and Q63-->P diminished their capacity for inhibiting papain and cathepsin B, retaining their antifungal properties. However, mutant C68-->G was more active for papain and cathepsin B than the wild type. These results indicate that in addition to the consensus cystatin-reactive site, Q63-V64-V65-A66-G67, the A37-R38-F39-A40-V41 region, common to all cereal cystatins, and the C68 residue are important for barley cystatin activity. On the other hand, the K92-->P mutant is inactive as a fungicide, but still retains measurable inhibitory activity for papain and cathepsin B. Against B. cinerea, the antifungal effect of Hv-CPI and of its derived mutants does not always correlate with their activities as proteinase inhibitors, because the Q63-->P mutant is inactive as a cystatin, while still inhibiting fungal growth, and the K92-->P mutant shows the reciprocal effects. These data indicate that inhibition of plant-pathogenic fungi by barley cystatin is not associated with its cysteine-proteinase inhibitory activity. Moreover, these results are corroborated by the absence of inhibition of intra- and extramycelia-proteinase activities by barley cystatin and by other well-known inhibitors of cysteine-proteinase activity in the fungal zymograms of B. cinerea.
重组大麦半胱氨酸蛋白酶抑制剂Hv-CPI抑制了三种植物病原真菌（灰葡萄孢菌、禾谷炭疽菌和黄瓜盘长孢）以及腐生真菌绿色木霉的生长。通过聚合酶链反应方法产生了几种大麦半胱氨酸蛋白酶抑制剂的突变体，并对它们的抗真菌特性和半胱氨酸蛋白酶抑制特性进行了研究。点突变体R38→G、Q63→L和Q63→P降低了它们抑制木瓜蛋白酶和组织蛋白酶B的能力，但保留了它们的抗真菌特性。然而，突变体C68→G对木瓜蛋白酶和组织蛋白酶B的活性比野生型更高。这些结果表明，除了共有半胱氨酸蛋白酶抑制剂反应位点Q63-V64-V65-A66-G67外，所有谷物半胱氨酸蛋白酶抑制剂共有的A37-R38-F39-A40-V41区域以及C68残基对大麦半胱氨酸蛋白酶抑制剂的活性很重要。另一方面，K92→P突变体作为杀菌剂没有活性，但仍保留对木瓜蛋白酶和组织蛋白酶B的可测量抑制活性。对于灰葡萄孢菌，Hv-CPI及其衍生突变体的抗真菌作用并不总是与其作为蛋白酶抑制剂的活性相关，因为Q63→P突变体作为半胱氨酸蛋白酶抑制剂没有活性，但仍能抑制真菌生长，而K92→P突变体则表现出相反的效果。这些数据表明，大麦半胱氨酸蛋白酶抑制剂对植物病原真菌的抑制作用与其半胱氨酸蛋白酶抑制活性无关。此外，在灰葡萄孢菌的真菌酶谱中，大麦半胱氨酸蛋白酶抑制剂以及其他著名的半胱氨酸蛋白酶活性抑制剂对菌丝内和菌丝外蛋白酶活性均无抑制作用，这证实了这些结果。
