Inhibition of Plant-Pathogenic Fungi by the Barley Cystatin Hv-CPI (Gene Icy) Is Not Associated with Its Cysteine-Proteinase Inhibitory Properties

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 cysteineproteinase inhibitory properties investigated. Point mutants R(^38)-G, Q(^63)-L, and Q(^63)-P diminished their capacity for inhibiting papain and cathepsin B, retaining their antifungal properties. However, mutant C(^68)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, Q(^63)-V(^64)-V(^65)-A(^66)-G(^67), the A(^37)-R(^38)-F(^39)-A(^40)V(^41) region, common to all cereal cystatins, and the C(^68) residue are important for barley cystatin activity. On the other hand, the K(^92)-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 Q(^63)-P mutant is inactive as a cystatin, while still inhibiting fungal growth, and the K(^92)-P mutant shows the reciprocal effects. These data indicate that inhibition of plant-pathogenic fungi by barley cystatin is not associated with its cysteineproteinase 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.