In vitro and in vivo antifungal properties of cysteine proteinase inhibitor from green kiwifruit.

BACKGROUND: Higher plants possess several mechanisms of defense against plant pathogens. Proteins actively synthesized in response to those stresses are called defense-related proteins which, among others, include certain protease inhibitors. It is of particular relevance to investigate plant natural defense mechanisms for pathogen control which include cystatins-specific inhibitors of cysteine proteases. RESULTS: In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. Immuno-tissue print results indicated that CPI is most abundant in the outer layer of pericarp, near the peel, and the inner most part of the pulp-sites where it could act as a natural barrier against pathogens entering the fruit. The purified protein (15 µmol L(-1)) showed antifungal activity against two phytopathogenic fungi (Alternaria radicina and Botrytis cinerea) by inhibiting fungal spore germination. In vivo, CPI (10 µmol L(-1)) was able to prevent artificial infection of apple and carrot with spore suspension of B. cinerea and A. radicina, respectively. It also exerted activity on both intracellular and fermentation fluid proteinases. CONCLUSION: Identification and characterization of plant defense molecules is the first step towards creation of improved methods for pathogen control based on naturally occurring molecules.

Cysteine proteinase inhibitor Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy.

Kiwifruit has become a frequent cause of fruit allergy in the recent years. The molecular basis of type I hypersensitivity to kiwifruit is attributed to 11 IUIS allergens, with Act d 1, Act d 2 and Act d 5 characterized in extenso. Evaluation of the allergenic properties of Act d 4, a cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa) was performed in this study. Identity of the purified glycoprotein was determined by Edman degradation and by mass fingerprint whereby more than 90% of the primary structure of the mature kiwifruit cystatin was confirmed. Using MALDI TOF analysis, molecular masses of 10902.5 and 11055.2 Da were detected for Act d 4, respectively. Positive skin prick reactivity with Act d 4 was induced in three kiwifruit allergic patients, as well as the upregulation of CD63 and CD203c molecules in the basophile activation assay. The IgE reactivity was detected in dot blot analysis while Western blot analysis was negative using sera from six kiwifruit patients, suggesting the presence of conformational IgE epitopes on the Act d 4 molecule. As activator of effector cells in type I hypersensitivity Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy.