Laboratorio de Bioquímica y Biología Molecular, Departamento de Biotecnología-UPM, ETSI Agrónomos, 28040 Madrid, Spain
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 anti-fungal 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.