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JXB Advance Access originally published online on May 16, 2005
Journal of Experimental Botany 2005 56(417):1821-1829; doi:10.1093/jxb/eri172
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Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.

RESEARCH PAPER

The strawberry gene Cyf1 encodes a phytocystatin with antifungal properties

Manuel Martinez *, Zamira Abraham *, Marina Gambardella * {dagger}, Mercedes Echaide, Pilar Carbonero and Isabel Diaz{ddagger}

Laboratorio de Bioquímica y Biología Molecular, Departamento de Biotecnología-UPM, ETSI Agrónomos, Ciudad Universitaria s/n, E-28040 Madrid, Spain

{ddagger} To whom correspondence should be addressed. Fax: +34 9 1336 5695. E-mail: Isabel{at}bit.etsia.upm.es

An EST, encoding a strawberry phytocystatin (PhyCys) obtained from a developing fruit of Fragariaxananassa cv. Elsanta has been characterized. The corresponding gene (Cyf1) had three introns interrupting its ORF that codes for a protein (FaCPI-1) of 235 amino acid residues with a putative signal peptide of 29 residues and an estimated molecular mass for the mature protein of 23.1 kDa. This protein contains, besides a C-terminal extension, several motifs conserved in all members of the PhyCys superfamily: (i) a GG and LARFAV-like motifs towards the N-terminal part of the protein; (ii) the reactive site QVVAG, and (iii) a conserved PW, downstream of the reactive site. Northern blot and in situ hybridization analyses indicated that the Cyf1 gene was expressed in fully expanded leaves, in roots and in achenes, but not in the receptacle (pseudocarp) during fruit development. The recombinant FaCPI-1 protein expressed in E. coli efficiently inhibited papain (Ki 1.9x10–9 M) and less so cathepsin H (Ki 4.7x10–7 M) and cathepsin B (Ki 3.3x10–6 M), and was a good inhibitor of the in vitro growth of phytopathogenic fungi Botrytis cinerea (EC50: 1.90 µM) and Fusarium oxysporum (EC50: 2.28 µM).

Key words: Antifungal activity, cathepsin B and H, cysteine proteinase inhibitor, intron–exon structure, papain, strawberry cystatin


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