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
Laboratorio de Bioquímica y Biología Molecular, Departamento de Biotecnología-UPM, ETSI Agrónomos, Ciudad Universitaria s/n, E-28040 Madrid, Spain
To whom correspondence should be addressed. Fax: +34 9 1336 5695. E-mail: Isabel{at}bit.etsia.upm.es
Received 10 December 2004; Accepted 30 March 2005
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Abstract |
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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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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Cystatins are a group of proteins specifically inhibiting cysteine-proteinases that have been identified in vertebrates, invertebrates, and plants. Those from plants, referred to as phytocystatins (PhyCys), comprise more than 80 members (Pfam databank; Bateman et al., 2002
), which cluster in a major evolutionary tree branch of the cystatin superfamily of proteins (Margis et al., 1998). In addition to the reactive site motif QXVXG and a glycine residue near the N-terminal part of the protein, and to the A/PW residues located in the second half of the molecule, common to all cystatins, the PhyCys present a consensus LARFAV-like sequence upstream of the reactive site and contain neither disulphide bonds nor putative glycosylation sites. Most PhyCys are small proteins ranging from 12–16 kDa. However, a group of them with a molecular weight of 
23 kDa, containing a carboxy-terminal extension, has been described (Lim et al., 1996; Misaka et al., 1996; Shyu et al., 2004). Several 85 kDa multicystatins, with eight cystatin domains, have been reported in tomato and potato (Waldrom et al., 1993; Wu and Haard, 2004).
Several cystatin isoforms and cystatin-encoding genes, with different spatial and temporal expression patterns and different inhibitory activities towards different cysteine-proteinases have been characterized in maize, rice, wheat, sunflower, and kiwi (Kondo et al., 1990; Abe et al., 1992; Kouzuma et al., 1996; Kuroda et al., 2001; Rassam and Laing, 2004). Recently, a comparative phylogenetic analysis has been done with the annotated cystatin genes from rice and arabidopsis and from seven different barley cystatin genes whose ORFs were derived from ESTs (Martinez et al., 2005a). This study has identified, according to the gene structure, three groups of cystatin genes by considering the intron–exon structure of their ORFs. The first and second groups encode proteins with a molecular mass of about 12–16 kDa and include genes without introns or with one intron, located between the motifs LARFAV and QXVXG. A third group of genes encoding 23 kDa proteins have three introns interrupting their ORFs and C-terminal extension sequences.
PhyCys have been implicated in two main functions: (i) in the endogenous regulation of protein turnover during seed development and germination (Arai et al., 2002; Corre-Menguy et al., 2002; Martinez et al., 2005b) and in programmed cell death (Solomon et al., 1999; Belenghi et al., 2003), and (ii) in defence against pathogens and pests. The first function is supported by the inhibition of cysteine proteinases from rice and wheat seeds that efficiently hydrolyse the storage proteins (Arai et al., 2002; Corre-Menguy et al., 2002). The defence role is sustained by in vitro data on inhibition against insect gut proteinases and on bioassays against pests (Kuroda et al., 1996; Pernas et al., 1998, 2000b; Haq et al., 2004), as well as by the enhanced resistance obtained against insects, nematodes, slugs, and potyviruses in transgenic plants over-expressing PhyCys genes (Vain et al., 1998; Gutierrez-Campos et al., 1999; Walker et al., 1999; Bouchard et al., 2003). The induction of some PhyCys by wounding and methyl-jasmonate (Zhao et al., 1996; Pernas et al., 2000b; the authors' unpublished results) further supports its putative role in defence. Moreover, antifungal and antimite activities have also been described for certain PhyCys (Pernas et al., 1999; Siqueira-Junior et al., 2002; Soares-Costa et al., 2002; Martinez et al., 2003). Although the mode of action of these PhyCys against phytopathogenic fungi has not been yet established, Martinez et al. (2003) have demonstrated by site-directed mutagenesis studies that the inhibition of Botrytis cinerea by the barley cystatin HvCPI is not associated with its cysteine-proteinase inhibitory properties.
The strawberry fruit crop is of great economic importance worldwide and its edible part is a false fruit originating mainly from the expansion of the flower base (receptacle) or pseudocarp where the real fruits (achenes) are attached. In recent years, several groups have reported the characterization and expression analyses of genes associated with various aspects of strawberry achene and receptacle maturation, including fruit flavour, either on a single gene basis or on a large-scale basis using microarrays (Moyano et al., 1998; Aharoni and O'Connell, 2002; Aharoni et al., 2004; Castillejo et al., 2004). Modification of important nutritional or physical properties of the strawberry fruit have also recently been described through over-expression or antisense expression of certain genes (Jimenez-Bermudez et al., 2002; Agius et al., 2003). However, the characterization of defence-related genes in strawberry is rather limited.
In this paper, the molecular and functional characterization is reported of an EST from the developing fruit of Fragariaxananassa cv. Elsanta, encoding the first cystatin (FaCPI-1) described in this crop. The cystatin gene (Cyf1) that has three introns interrupting its ORF, is expressed in leaves and roots and in the seeds of the achenes, but not in the receptacle. The recombinant protein expressed in E. coli is a good inhibitor of papain and other cysteine proteinases and has antifungal properties against two important plant pathogens Botrytis cinerea and Fusarium oxysporum.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Sequence analysis of the Cyf1 gene encoding a cystatin from strawberry
The D8 EST was obtained from the strawberry EST collection derived from the ripe fruit cDNA library constructed in Fragariaxananassa cv. Elsanta (Aharoni and O'Connell, 2002
) at Plant Research International (PRI B.V.), Wageningen, The Netherlands. Nucleotide sequences were determined on both strands using vector-specific primers and the automated sequencer (ABI PRISM TM 3100, Applied Biosystems).
To determine if the ORF was interrupted by introns, total DNA was extracted from leaves of Fragariaxananassa cv. Elsanta, following the procedure described by Taylor and Powell (1982) and a PCR amplification was carried out. The forward primer Fa-CYS1: 5'-GATCCATGGCCACCCTCGGCGGAATC-3' was used, which incorporated a NcoI restriction site (underlined) upstream of the GCC codon (bold) corresponding to the initial alanine of the mature protein. As the reverse primer, Fa-CYS2 was used: 5'-TCAAAGCTTTCAGTGCTCCACCTCCATC-3', containing a HindIII site (underlined) upstream of the complementary sequence of the stop codon (bold), as indicated in Fig. 1. The amplification product was first cloned in pGEM-T easy vector (Promega) and then sequenced on both strands and a comparison was done between the sequence of this fragment and the sequence of the cDNA clone (D8 EST). Alignments of protein sequences with the Clustal W program (Thompson et al., 1994) were performed at the DNA Data Bank of Japan (http://www.ddbj.njg.ac.jp). To explore the presence of the putative signal peptide, the SignalP v.3.0 (http://www.cbs.dtu.dk/services/SignalP) program was used (Bendtsen et al., 2004). Bootstrapping analysis with a PHYLIP format tree output was carried out after the Neighbor–Joining method and the phylogenetic tree was obtained with the TREEVIEW (v.1.6.6) software (Page, 1996).
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Northern blot analysis
Achene and receptacle tissues derived from medium size ripe-stage strawberry fruits (partially pigmented), newly expanded leaves, and secondary roots from adult plants of the domesticated strawberry Fragariaxananassa cv. Elsanta, were used for RNA extraction and purification by the phenol/chloroform method, followed by precipitation with 3 M LiCl (Lagrimini et al., 1987
). Total denatured RNA was electrophoresed in 1.2% agarose gels containing 7% formaldehyde, and blotted onto Hybond N+ membranes (Amersham). Hybridization and washings were done under stringent conditions, following standard procedures (Sambrook and Russell, 2001), with a specific strawberry cystatin probe corresponding to a 242 bp fragment, spanning from position 611 to 855 in the 3' end of the cDNA (Fig. 1). This probe was amplified by PCR using as forward and reverse primers, FaCYS3: 5'-CTACTCAAACTGAAGAGG-3' and FaCYS4: 5'-CAAGGAAAGCTGATACTG-3', respectively, both indicated in Fig. 1.
mRNA in situ hybridization analysis
Medium-sized partially pigmented turning fruits obtained from strawberry (Fragariaxananassa cv. Elsanta) were collected and fixed in FAE solution (ethanol:acetic acid:formaldehyde:water, 50:5:3.5:41.5 by vol.) for 2 h at room temperature, dehydrated, and embedded in paraffin and sectioned to 8 µm. After de-waxing in histoclear and rehydration, sections were treated with 0.2 M HCl, neutralized, and incubated with 1 µg ml–1 proteinase K as described by Ferrandiz et al. (1999). Finally, tissue sections were dehydrated in an ethanol dilution series and dried under vacuum before applying the hybridization solution (100 µg ml–1 tRNA, 6x SSC, 3% SDS, and 50% formamide), containing approximately 100 ng µl antisense or sense DIG-labelled specific Cyf1 probe, previously described for northern blot analysis. Hybridization was performed overnight at 52 °C, followed by two washes in 2x SSC and 50% formamide for 90 min at the same temperature. Antibody incubation and colour detection were carried out according to the manufacturer's instructions (Boehringer).
Expression and purification of a recombinant strawberry cystatin from E. coli
A 624 bp fragment, spanning the ORF of the mature strawberry cystatin protein without the signal peptide (positions 93–716 in Fig. 1), was amplified by PCR. The oligonucleotides used as primers were previously described: forward primer Fa-CYS1 and reverse primer Fa-CYS2. The amplified DNA fragment was inserted in frame into the fusion expression vector pRSETB (Invitrogen) and introduced into E. coli BL21 (DE3) pLysS. Bacterial over-expressing cells were harvested after 2 h of induction with IPTG (isopropyl ß-D-thiogalactopyranoside). The fusion protein with the N-terminal histidine tag was purified to homogeneity, using a His-Band resin and elution conditions from the Ni+-column, following the manufacturer's instructions (Novagen). Control of the purification process was carried out by separation of proteins by SDS–PAGE (Laemmli, 1970).
Cysteine-proteinase inhibitory activity
Inhibitory activity of the recombinant FaCPI-1 protein purified from E. coli was tested against papain (Sigma; EC 3.4.22.2
[EC]
), cathepsin B (Calbiochem; EC 3.4.22.1
[EC]
), and cathepsin H (Calbiochem; EC 3.4.22.16
[EC]
), essentially as described by Gaddour et al. (2001), using BANA (N-benzoyl-DL-arginine-ß-naphthylamide) as substrate. Protein concentration was quantified by the Bio-Rad kit with bovine albumin as standard and the Ki values were determined from Dixon plots (1/V versus [I]).
Fungal growth inhibitory assays
Phytopathogenic fungal strains from the laboratory collection, Botrytis cinerea and Fusarium oxysporum, were the gift of Dr A Molina and Dr P Rodriguez-Palenzuela (Biotechnology Department, Universidad Politécnica de Madrid, Spain). Bacteria were grown on potato-dextrose agar medium at 28 °C. The in vitro inhibition assays were performed as described by Martinez et al. (2003). Approximately 104 spores of each fungal strain were incubated in 100 µl of one-third potato dextrose broth at 28 °C for 48 h in the absence and presence of different concentrations of the recombinant strawberry cystatin, FaCPI-1. The incubation was carried out in sterile microtitre plates and fungal growth was monitored by measuring absorbance at 492 nm and by microscopic observations. Results were expressed as the percentage of growth in the absence of the inhibitory agent.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Molecular characterization of the Cyf1 gene encoding the FaCPI-1 cystatin
The search for a cystatin gene in Fragariaxananassa was done by analysing the EST collection from PRI B.V. (The Netherlands). An EST (D8), putatively encoding a cystatin protein, was sequenced in both strands and the sequence analysis indicated that this clone represented a new cystatin gene, Cyf1 (Cystatin gene from Fragaria). The SignalP software predicted a signal peptide of 29 residues, which suggests a subcellular location of the mature protein to the endoplasmic reticulum (Womack et al., 2000
). Although the sequence contains an ORF of 858 nt (an estimated 26.39 kDa protein), taking into account the putative signal peptide, this will correspond to a mature cystatin of 206 amino acid residues with a molecular mass of 23.1 kDa (Fig. 1). The mature protein shared with other PhyCys those motives involved in the three-point inhibitor–proteinase interaction: two glycines (Gly4-Gly5), the putative reactive domain QXVXG (Q49-V50-V51-A52-G53), and the A/PW motif (P79-W80). In addition, the FaCPI-1 protein contained the LARFAV-like sequence (L22-G23-R24-F25-A26-V27) in the N-terminal part of the molecule, common to all PhyCys, and an extension at its C-terminal end (Fig. 1a). Although this extension (residues 115–206) has certain similarities to 12–16 kDa PhyCys and most of the conserved cystatin motifs are still recognizable (see dotted lines in Fig. 1a: GG; AANHAV; QEVVH), the overall similarity between the two halves of this protein is very low, which suggests that the C-extension is a degenerated part of a dimeric cystatin that probably originated by gene duplication followed by divergent evolution. Another level of complexity derives from the fact that the strawberry (Fragariaxananassa) is an octoploid and thus four allelic variants of the Cyf1 gene could be expected to occur. Allelic evolution has not been explored, since this was outside the scope of this work.
The comparison between the cDNA sequence and the PCR fragment amplified from the genomic DNA corresponding to the same ORF, has determined that the ORF of the Cyf1 gene is interrupted by three introns (Fig. 1). Exon 1 codified for the first 61 amino acid residues, spanning the signal peptide and the first 32 residues of the mature protein, including the motifs G4-G5 and L22-G23-R24-F25-A26-V27. Exon 2 codifies amino acid residues 33–113, containing the reactive site Q49-V50-V51-A52-G53 and the P79-W80 motif. The C-terminal extension of 93 amino acid residues begins with the G114-G115 motif in exon 3 and is interrupted by intron 3. All three introns, flanked by typical GT/AG boundaries, are essentially similar in number and position to those found in the soya cystatin gene, the only PhyCys of this class whose genomic clone has been characterized (Misaka et al., 1996). The first intron (87 nt) was located between the LGRFAV and the reactive site (QVVAG) motives, as in other PhyCys genes, even those with just one intron (Kondo et al., 1991; Waldron et al., 1993; Abe et al., 1996; Martinez et al., 2005a). Intron 2, of 79 nt, was positioned between Gln113 and Gly114, which corresponds to the positions Asp152 and Gly153 in soyacystatin; in both species this intron is located at the beginning of the C-terminal extension. The third intron of 451 nt, is situated between residues Glu162 and Val163, corresponding to identical residues in positions 200 and 201 in the soyacystatin (Misaka et al., 1996). As expected, little homology in length and sequence of the three introns between the two genes from Fragariaxananassa and Glycine max, was observed. The three introns plus the C-terminal extension present in the strawberry cystatin gene justifies the integration of Cyf1, as well as the soyacystatin gene into the third PhyCys group, as discussed by Martinez et al. (2005a).
The phylogenetic dendrogram (Fig. 2) based on the deduced sequence comparisons of the mature PhyCys proteins of 
23 kDa, found in the data banks, clearly indicated that the strawberry FaCPI-1 was closely related to the apple (Malus domestica) cystatin, the only PhyCys characterized from a cultivated fruit crop, belonging also to the Rosaceae family. Both cystatins share 79% identical amino acid residues in their C-terminal tails. This percentage is also quite high when comparisons are made with cystatins from Lotus corniculatus (71%), Glycine max (69%), and Oryza sativa (69%), but falls to 47%, when this comparison is made with the corresponding C-tail of that from Lycopersicum esculentum.
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Expression analysis of the strawberry Cyf1 gene
The Cyf1 gene expression was examined by northern blot analysis among the major strawberry organs: (i) expanded leaves, (ii) secondary roots of adult plants, and (iii) receptacles and achenes of medium-sized immature fruits. As hybridization probe, a 242 bp fragment (nucleotides 520 to 762; see Fig. 1a) of the Cyf1 cDNA clone was used, checking previously that a single band appeared in Southern blot analysis (data not shown). The strawberry cystatin transcripts were abundant in achenes and in expanded leaves (Fig. 3a). Upon long exposures of the membranes, the mRNA could be also detected in the RNA sample from secondary roots of adult plants while still undetectable in the receptacle or pseudocarp (data not shown).
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To localize more precisely the spatial expression of the Cyf1 gene, mRNA in situ hybridization studies were done. Medium-sized ripe-stage turning fruits of strawberry were analysed. A clear signal with the antisense probe was only detected (Fig. 3b) in the immature seeds, as observed in the longitudinal sections of the achenes (Fig. 3b, A, C). Within the seed, only the embryo cells were expressing the Cyf1 mRNA; no signal could be detected in the one cell layer endosperm or in the testa, or in the pericarp (Perkins-Veazie, 1995
). No specific signal above background was detected when sections were hybridized with the sense probe, used as negative control (Fig. 3b, B, D). The Icy gene, encoding barley cystatin HvCPI, was found to be ubiquitously expressed and within the seed its transcripts were detected not only in the embryo but also throughout the endosperm (Gaddour et al., 2001).
To date, most of the PhyCys have been purified from seeds and their genes are abundantly expressed in cDNA libraries from developing seeds (Misaka et al., 1996; Gaddour et al., 2001; Corre-Menguy et al., 2002), although they have also been detected in vegetative tissues, including roots and leaves (Lim et al., 1996; Pernas et al., 2000a; Gaddour et al., 2001). Regarding the strawberry Cyf1 gene, these data show it is almost ubiquitously expressed, with the exception of the pseudocarp, and this expression is particularly intense in leaves and embryos.
Proteinase inhibitory activity and antifungal properties of the recombinant strawberry FaCPI-1
The recombinant FaCPI-1 expressed in E. coli as a fusion protein with a histidine tail was purified as shown in Fig. 4a. As expected, incubation with IPTG induced the expression of the recombinant cystatin that was particularly relevant in the supernatant of the bacterial lysate (lane 2), compared with the non-induced control (lane 1). The recombinant protein was purified to homogeneity by affinity chromatography to a Ni2+ column and finally eluted from it (lane 4). Proteins not retained in the column are shown in lane 3 (Fig. 4a).
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The recombinant FaCPI-1 cystatin was assayed against several cysteine proteinases. The FaCPI-1 inhibited papain and cathepsin H and to a lesser extent, cathepsin B. In all cases, non-competitive inhibition kinetics were observed, as indicated by double reciprocal plots (data not shown). Ki values of 1.9x10–9 M for papain, 4.7x10–7 M for cathepsin H, and 3.3x10–6 M for cathepsin B, were determined from Dixon plots (1/V versus [I]) using BANA as substrate. The Ki value towards papain is one order of magnitude lower than those reported for other PhyCys, such as HvCPI from barley (Ki: 2.0x10–8 M) and OC-I from rice (Ki: 3.2x10–8 M). Other cystatins with similar molecular mass and the same gene structure with three introns have higher Ki against papain, such as those from sesame (Ki: 2.7x10–8 M) or soya (Ki: 1.9x10–7 M). However, FaCPI-1 is a weaker inhibitor towards cathepsin H (Ki: 4.7x10–7 M) than barley HvCPI (Ki: 3.7x10–8 M) and rice OC-I (Ki: 5.7x10–9 M) PhyCys. Its inhibitory activity against cathepsin B is similar to that of the barley cystatin HvCPI, in the order of 10–6 M (Kondo et al., 1990
; Misaka et al., 1996; Gaddour et al., 2001; Martinez et al., 2003; Shyu et al., 2004). The recombinant FaCPI-1 expressed in E. coli and purified as previously described (Fig. 4a) was tested in vitro against phytopathogenic fungi Botrytis cinerea and Fusarium oxysporum. Botrytis was chosen because, together with Colletotrichum, it is the most important fungal pathogen in strawberry crops, and attacks leaves, stolons, and fruits. Fusarium was chosen due to its economical importance (it is the fourth most important fungal pathogen worldwide) and because it is a soil-borne pathogen that is difficult to eradicate. The antifungal activity was confirmed by the inhibition of spore germination and of mycelial development (Fig. 4b, c). The effective concentration for 50% growth inhibition (EC50) was 1.90 µM for B. cinerea and 2.28 µM for F. oxysporum, respectively. The recombinant protein almost completely inhibited the growth of B. cinerea at 3 µM and a higher concentration (6 µM) was required for the same effect in F. oxysporum growth (Fig. 4b). The data were corroborated by microscopic observations (Fig. 4c). As expected, no effect was observed when protein purified from E. coli transformed with the expression vector without insert was used as the negative control (data not shown).
It has previously been demonstrated, after studying the antifungal and the inhibitory properties against papain of several site-directed mutants of the HvCPI from barley, that the inhibition of B. cinerea by the barley cystatin HvCPI is not associated with its cysteine proteinase inhibitory properties (Martinez et al., 2003). At the same time intra- or extra-cellular cysteine proteinases in Botrytis cinerea zymograms could not be detected (Martinez et al., 2003). The inability of the synthetic inhibitor E-64 and the chicken egg white cystatins (both specific inhibitors of cysteine proteinases) to inhibit the in vitro growth of Botrytis, Fusarium, Septoria, Colletotrichum, and Trichoderma (Pernas et al., 1999; Siqueira-Junior et al., 2002), also supports the suggestion that the antifungal properties of the plant cystatins are not mediated by cysteine proteinase inhibition.
Cystatins from barley, chestnut, and sugarcane also show antifungal activity (Pernas et al., 1999; Soares-Costa et al., 2002; Martinez et al., 2003). Probably all of them, plus the cystatin from strawberry described here, share the same mechanism of action against fungi. It has been speculated that alterations in the fungal membrane permeability could be the origin of the antifungal properties of PhyCys. The trypsin inhibitor SAP16 from Helianthus annuus modifies membrane permeability of Sclerotinia scleroticum and represses the germination of ascospores (Giudici et al., 2000). Alteration of membrane permeability was also detected when rice OC-I was expressed in the cytosolic compartment of tobacco leaf cells (van de Vyver et al., 2003). However, more work is needed to establish the mechanism of action of PhyCys against fungi.
In conclusion, the FaCPI-1, the first characterized strawberry cystatin, encoded by the gene Cyf1 has three introns interrupting its ORF. Its mRNA is abundantly expressed in the seeds of the achenes, but not in the pseudocarp, and in fully expanded leaves and in roots.
The fact that the Ki value of this strawberry cystatin against papain is very low (Ki: 1.9x10–9 M) suggests a defence role for this inhibitor and this is corroborated by its antifungal properties against two important phytopathogenic fungi, Botrytis cinerea and Fusarium oxysporum. These inhibitory properties make Cyf1 an important potential transgene for disease control.
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Acknowledgements |
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We acknowledged Dr A Aharoni and the Plant Research International B.V., Wageningen (The Netherlands) for kindly providing the D8 EST. We thank Mar Gonzalez for technical assistance. M Martinez and Z Abraham are recipients of a Ramon y Cajal contract from the Ministerio de Ciencia y Tecnologia (Spain) and a contract from Comunidad de Madrid (Spain), respectively. M Gambardella was financed form the Alßan Programme (European Union Programme of High Level Scholarship for Latin America, ID: E03E25105CL). The financial support from the Comunidad Autonoma de Madrid, Spain (07M/0050/2002) and Minsiterio de Ciencia y Tecnologia of Spain (AGL03-0335) are gratefully acknowledged.
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Footnotes |
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* These authors have equally contributed to this work.
Present address: Departamento de Producción Agrícola, Facultad de Ciencias Agronómicas, Universidad de Chile, Casilla 1004, Santiago, Chile.
Abbreviations: BANA, N-Benzoyl-DL-Arginine-ß-NaphthylAmide; EST, Expressed Sequence Tag; nt, nucleotide; ORF, Open Reading Frame; PhyCys, phytocystatins.
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References |
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