Planta (2002) 216: 193–202 DOI 10.1007/s00425-002-0902-6
R EV IE W
Bart P.H.J. Thomma Æ Bruno P.A. Cammue Karin Thevissen
Plant defensins
Received: 5 June 2002 / Accepted: 22 August 2002 / Published online: 8 October 2002 Ó Springer-Verlag 2002
Abstract Plant defensins are small, basic peptides that have a characteristic three-dimensional folding pattern that is stabilized by eight disulfide-linked cysteines. They are termed plant defensins because they are structurally related to defensins found in other types of organism, including humans. To date, sequences of more than 80 different plant defensin genes from different plant species are available. In Arabidopsis thaliana, at least 13 putative plant defensin genes (PDF) are present, encoding 11 different plant defensins. Two additional genes appear to encode plant defensin fusions. Plant defensins inhibit the growth of a broad range of fungi but seem nontoxic to either mammalian or plant cells. Antifungal activity of defensins appears to require specific binding to membrane targets. This review focuses on the classification of plant defensins in general and in Arabidopsis specifically, and on the mode of action of plant defensins against fungal pathogens. Keywords Arabidopsis Æ Cysteine-rich peptide Æ Defensin Æ Innate immunity Æ Permeabilization Æ Thionin
Introduction All living organisms, ranging from microorganisms to plants and mammals, have evolved mechanisms to actively defend themselves against pathogen attack. The most sophisticated of those mechanisms deploy antibodies and killer cells to recognize and eliminate specific invaders, respectively. These adaptive immune responses act on the recognition of foreign molecules and are only elaborated in a small subset of living species, namely
B.P.H.J. Thomma (&) Æ B.P.A. Cammue Æ K. Thevissen Centre of Microbial and Plant Genetics (CMPG), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, 3001 Heverlee-Leuven, Belgium E-mail:
[email protected] Fax: +32-16-321966
higher vertebrates stemming from the jawed vertebrate lineage (Matsunaga and Rahman 1998). Innate immunity, on the other hand, is a much more widespread, ancient defense strategy involving, among other responses, the production of antimicrobial peptides (Boman 1995). These antibiotics lack the antigen-recognition specificity of antibodies. However, because these peptides are simply produced by transcription and translation of a single gene, they can be delivered relatively rapidly after infection with a limited input of energy and biomass. Although the innate immune response of plants has been regarded as a primordial defense system, it is able to display differential activity against different types of microorganism (Thomma et al. 2001).
Structure of defensins In the innate immune response, only one class of peptide seems to be conserved between plants, invertebrates and vertebrates, namely defensins (Fig. 1A). At the beginning of the 1990s, the first plant defensins were isolated from wheat and barley grains (Colilla et al. 1990; Mendez et al. 1990). At that time these proteins were called c-thionins since their size and cysteine content were found to be similar to formerly described thionins (Carrasco et al. 1981). Subsequent structure analysis has demonstrated, however, that c-thionins are not related to thionins (Bruix et al. 1993). Because of their structural similarity to mammalian and insect defensins, c-thionins were renamed plant defensins (Terras et al. 1995). Plant defensins are small (45–54 amino acids) highly basic cysteine-rich peptides that are apparently ubiquitous throughout the plant kingdom. All plant defensins identified so far have eight cysteines that form four structure-stabilizing disulfide bridges. Study of the threedimensional structure of a number of plant defensins has shown that the structure comprises a triple-stranded b-sheet with an a-helix in parallel (e.g. Rs-AFP1, Fig. 1A, B; Bruix et al. 1993, 1995; Bloch et al. 1998; Fant et al. 1998, 1999; Almeida et al. 2002).
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Fig. 1 A Three-dimensional structure of defensins of plant, invertebrate (insect and mollusc) and vertebrate (mammalian) origin. Structures were downloaded from the protein data bank (http://www.rcsb.org/pdb; PDB accession ID numbers: MGD-1: 1FJN, defensin A: 1ICA, drosomycin: 1MYN, Rs-AFP1: 1AYJ, HNP-3: 1DFN, HBD-2: 1FD3, RTD-1: 1HVZ). Pictures were generated using Rasmol software. Alpha-helices and b-sheets are shown in yellow and red, respectively. B Amino acid sequence of mature Rs-AFP1 and 2. Dashes indicate identical amino acid residues. Connecting lines between cysteine residues represent disulfide bonds and the spiral and arrows indicate the location of the a-helix and b-strands, respectively
In insect defensins, an a-helix is combined with a double-stranded b-sheet, stabilized by three disulfide bridges between six cysteine residues (e.g. insect defensin A, Fig. 1A; Bonmatin et al. 1992; Cornet et al. 1995). Interestingly, the two pathogen-inducible peptides drosomycin (Fig. 1A) and heliomicin (not shown) isolated from the fruit fly Drosophila melanogaster and from the lepidopteran Heliothis virescens, respectively, combine a triple-stranded b-sheet with the a-helix (Landon et al. 1997, 2000; Lamberty et al. 2001). Heliomicin, like insect defensins, is stabilized by three disulfide bridges, while the structure of drosomycin, like plant defensins, is stabilized by four disulfide bridges (Landon et al. 1997, 2000; Lamberty et al. 2001). Defensins have also been identified in a different phylogenetic group of invertebrates, namely mollusks (Mitta et al. 2000). From the blue mussel Mytilus edulis, defensins containing six cysteines have been characterized, while from the Mediterranean mussel, M. galloprovincialis, a defensin (MGD-1) containing eight cysteines was isolated (Charlet et al. 1996; Hubert et al. 1996). The structure of MGD-1 consists of an a-helix and two antiparallel b-strands stabilized by four disulfide bridges (Fig. 1A; Yang et al. 2000). The core of the global fold of plant defensins as well as invertebrate defensins includes a cysteine-stabilized a-helix b-sheet (CSab) motif (Cornet et al. 1995). In this motif, two cysteine residues, located one turn apart in the a-helix, form two disulfide bridges with two cysteine
residues separated by a single amino acid in the last strand of the b-sheet. The protein super-family containing such a CSab motif also includes some scorpion neurotoxins (Bontems et al. 1991; Kobayashi et al. 1991; Polikarpov et al. 1999; Jablonsky et al. 2001) and the plant protein brazzein, known for its intrinsic sweet taste (Caldwell et al. 1998). Before they were identified in invertebrates and plants, defensins were first identified as a family of peptides in rabbits (Selsted et al. 1984) and subsequently in other vertebrate species including humans. Up to now, three types of defensin have been identified in mammals (Fig. 1A). Apart from the 18-amino-acid cyclic peptide only discovered in macaques and termed h-defensin (Tang et al. 1999; Trabi et al. 2001), a- and b-defensins occur generally. These latter two subgroups differ in size and the arrangement of cysteines within their sequences (Lehrer et al. 1993; Selsted et al. 1993). Mammalian defensins of the a-class do not comprise an a-helix (Hill et al. 1991; Pardi et al. 1992) and thus do not share the CSab motif found in invertebrate defensins and plant defensins. Structure analysis of two mammalian defensins of the b-class, namely human b-defensin (HBD)-1 and HBD-2, revealed that these defensins combine an a-helix with a triple-stranded anti-parallel b-sheet (Hoover et al. 2000, 2001; Sawai et al. 2001). Nevertheless, the global fold of these defensins does not comprise a CSab motif because the a-helix is located at the N-terminus of the peptide (abbb-fold in mammalian defensins versus babb-fold in plant defensins). However, the size and spatial orientation of the triple-stranded b-sheet in mammalian defensins is comparable to that found in plant defensins (Broekaert et al. 1995). Moreover, the a-helix appears to be approximately in the same position relative to the b-sheet (Hwang and Vogel 1998). Because three genes encoding the human b-defensins HBD1, HBD2 and HBD3 have been localized close to a region carrying a-defensins, it was suggested that at least these types of defensin descend from a shared ancestral gene (Harder et al. 1997; Liu et al. 1997; Jia et al. 2001).
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But based on the overall three-dimensional structure there is a closer relationship between plant defensins, insect defensins and mammalian b-defensins than between mammalian a- and b-defensins. This suggests that defensins are ancient peptides conserved across the eukaroytic kingdom, originating before the evolutionary divergence of plants and animals. Possibly, defensins have evolved from a single precursor, being a molecule with an overall structure resembling that of plant defensins.
Classification of plant defensins In the second half of the 1990s it was already recognized that the plant defensin family was quite diverse regarding amino acid composition as well as biological activity (Osborn et al. 1995; Broekaert et al. 1995, 1997; Segura et al. 1998). Some defensins were not found to display any antimicrobial activity, while others were found to have antifungal or occasionally antibacterial activity in vitro. Among defensins that have antifungal effects, some defensins cause hyperbranching associated with antifungal activity, at least on particular fungi and under certain conditions, while others do not produce morphological changes to filamentous fungi (Osborn et al. 1995; Broekaert et al. 1995, 1997; Segura et al. 1998). Based on the amino acid sequence alignment of 17 defensins a division into 2 main groups sharing only 25% similarity was proposed. Both groups were divided again into up to four subgroups, within each group displaying at least 45% similarity (Harrison et al. 1997). The sequence-based groupings appeared to correlate with differences in their biological activities. Since then, the number of reports on plant defensins and their activities has increased and even more defensins have been de-
duced from cDNA sequences. Taken together, plant defensins show a clear, but limited, sequence conservation restricted to the eight structurally important cysteines. In the past, glycines at positions 13 and 34, a glutamate at position 29 and an aromatic residue at position 11 were also considered to be conserved (numbering relative to Rs-AFP1, see Fig. 1B), but it now appears this is not true for all plant defensins. Using the ClustalW-algorithm a multiple alignment of over 80 defensin sequences from different plant species was performed and a molecular phylogeny tree was constructed. From the topology of the tree it is clear that the earlier division into two main groups (Harrison et al. 1997) is not apparent anymore (Fig. 2). Moreover, although some of the earlier proposed subgroups seem to cluster, the bootstrap values at the nodes demonstrate that only a few small subgroups can be discriminated. Fig. 2 Unrooted phylogenetic tree for known and putative plant defensins. Protein sequences were aligned with the ClustalWalgorithm. Topologies for a 50% majority rule consensus tree were attributed by maximum parsimonial analysis with 1,000 bootstrap replicates using the PHYLIP software package. Bootstrap values above 70% on the central line are indicated at the appropriate nodes. Protein accession codes are preceded by italic two-letter codes which refer to the plant species the peptide has been identified in: Ah, Aesculus hippocastanum; At, Arabidopsis thaliana; Bv, Beta vulgaris; Bn, Brassica napus; Bo, Brassica oleracea; Br, Brassica rapa; Ca, Capsicum annuum; Cc, Capsicum chinense; Cf, Cassia fistula; Cp, Citrus paradisi; Ct, Clitoria ternatea; Dm, Dahlia merckii; Gm, Glycine max; Ha, Helianthus annuus; Hs, Heuchera sanguinea; Hv, Hordeum vulgare; Le, Lycopersicon esculentum; Ms, Medicago sativa; Ne, Nicotiana excelsior; Np, Nicotiana paniculata; Nt, Nicotiana tabacum; Os, Oryza sativa; Pi, Petunia integrifolia; Pc, Phaseolus coccineus; Pa, Picea abies; Ps, Pisum sativum; Pp, Pyrus pyrifolia; Rs, Raphanus sativus; Sa, Sinapis alba; St, Solanum tuberosum; Sb, Sorghum bicolor; So, Spinacia oleracea; Ta, Triticum aestivum; Vf, Vicia faba; Vr, Vigna radiata; Vu, Vigna unguiculata; Wj, Wasabia japonica; Zm, Zea mays
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The resolving power of molecular phylogeny is so poor because sequences are small, share only a few conserved residues, and have accumulated multiple substitutions at the same site. Interestingly, there is no general pattern of clustering versus dispersal of the various defensins identified in a single species. The radish defensins, for example, are tightly grouped, suggesting recent diversification, whereas the Arabidopsis defensins, for example, are spread all over the tree. This phenomenon has previously also been observed for insect defensins (Charlet et al. 1996).
Arabidopsis defensins In previous studies, five different plant defensin genes have been identified in Arabidopsis thaliana (PDF1.1, 1.2, 2.1, 2.2 and 2.3; Penninckx et al. 1996; Epple et al. 1997; Thomma and Broekaert 1998). By performing a TBLASTN search on the fully sequenced genome of A. thaliana using the amino acid sequence of the previously identified Arabidopsis plant defensin PDF1.2 as a template, a total of 13 putative plant defensin genes was identified. Based on a phylogenetic analysis, the deduced Fig. 3 A Alignment of putative Arabidopsis defensins. Deduced amino acid sequences from putative defensin genes are aligned together with the carboxy-terminus of the putative fusion proteins encoded by Arabidopsis genes At5g38330 and At4g30070. Predicted signal sequences are in lower case. Shading indicates the degree of conservation: 100%, 75%, 40%. The consensus motif for Arabidopsis plant defensins is shown in the bottom line. B Alignment of the predicted proteins encoded by the genes At5g38330 and At4g30070. Both proteins share a carboxy-terminal plant defensin domain (double underlined). Predicted signal sequences are in lower case. Identical amino acids are shaded in black
putative protein sequences can be classified into two families (Fig. 3A). The first family contains seven peptides (PDF1.1 to 1.5), five of which are highly similar (PDF1.1 to 1.3). Moreover, the predicted mature peptides of three of them are identical (PDF1.2a, b and c). Remarkably, the genes encoding PDF1.2a and PDF1.2c are present in a tandem repeat on chromosome 5, while the gene encoding PDF1.2b is present on chromosome 2 in tandem array with the gene encoding PDF1.3. Possibly, segmental duplication is the cause of this spatial organization. Based on the chromosomal organization of the genome sequence, it was found that 58% of the Arabidopsis genome shows large segmental duplications. Moreover, it was observed that 17% of all Arabidopsis genes are arranged in tandem arrays (The Arabidopsis Genome Initiative 2000). The high homology between members within this gene family suggests that duplication and subsequent diversification occurred evolutionarily quite recently. The putative protein sequences within the second family (PDF2.1 to 2.6) show more variation at the amino acid level (Fig. 3A). This suggests that duplication and subsequent segregation of these members occurred before the diversification of the members of the first family. Also for the second family a number of members are found in close proximity. PDF2.1, 2.3 and 2.6 occur in a tandem array and PDF2.2 is separated from this array by one gene. Possibly this separation is caused by shuffling, a phenomenon that frequently happened in the Arabidopsis genome (The Arabidopsis Genome Initiative 2000). Previous studies on five different plant defensins genes (PDF1.1, 1.2, 2.1, 2.2 and 2.3) have shown that these genes display distinct organ-specific expression patterns. While one gene (PDF1.2) is induced in leaves upon pathogen
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challenge, the other four genes are expressed constitutively. As a result, most plant tissues constitutively express two or more defensin genes. This suggests that individual peptides are expressed under specific circumstances or at specific sites. Interestingly, while most peptides carry a predicted signal peptide, one protein from each family (PDF1.4 and 2.4) appears not to contain such a sequence. This suggests that these two peptides stay in the cytoplasm while the others are secreted. As an alternative, different Arabidopsis defensins can cover gaps in each-others’ activity spectrum or act synergistically. In addition to the 13 putative plant defensin genes, 2 genes were identified that encode proteins containing a plant defensin domain. Gene At4g30070 encodes a protein of 129 amino acids of which the C-terminal domain displays the conserved cysteine pattern shared by all plant defensins in addition to a number of residues that are conserved among the Arabidopsis plant defensins (Fig. 3A). Gene At5g38330 encodes a similar protein consisting of 122 amino acids, sharing 56% identical residues with the protein encoded by gene At4g30070. Both proteins have a putative 26-amino-acid signal sequence. The amino-terminal domains of both proteins are rich in cysteine residues but do not share significant homology to any previously characterized protein (Fig. 3B). Possibly, these proteins are fusion proteins. Alternatively, these proteins could be precursors, as has been found for a tobacco defensin and for thionins (Bohlmann and Apel 1991; Gu et al. 1992). The precursor of these proteins is characterized by the presence of a C-terminal acidic domain that has been proposed to neutralize the toxicity of the basic effector domain and thus mitigate toxicity during synthesis (Bohlmann and Apel 1991; Gu et al. 1992). However, in the case of the two Arabidopsis proteins, the basic defensin domain is at the C-terminus and the N-terminal domain has an excess of one basic residue. Further research will have to elucidate the function of the N-terminal domain.
Antimicrobial activity of plant defensins Most plant defensins isolated to date exhibit antifungal activity against a broad range of fungi, including various plant pathogens. Some plant defensins, however, do not inhibit fungal growth but rather inhibit a-amylase activity and protein synthesis (Colilla et al. 1990; Mendez et al. 1990; Bloch and Richardson 1991; Osborn et al. 1995). Since a-amylase is an insect gut enzyme, it is proposed that inhibition of a-amylase activity results in indigestibility of plant material and thus in defense against feeding insects (Shade et al. 1994). Remarkably, plant defensins that inhibit a-amylase activity do not appear to display antifungal activity and vice versa (Osborn et al. 1995). A common observation for most antifungal plant defensins, as well as for other types of defensin, is the reduction of antifungal activity when the cationic strength of the medium is increased (Lehrer et al. 1988;
Terras et al. 1992, 1993; Cociancich et al. 1993; Osborn et al. 1995). This antagonistic effect of cations is strongly dependent on the test fungus, indicating that electrostatic interactions probably change the conformation of the target site on the fungal membrane rather than the conformation of the defensin itself (Broekaert et al. 1995). With some exceptions (Moreno et al. 1994; Osborn et al. 1995; Segura et al. 1998), plant defensins do not display antibacterial activity. In contrast, the 6-cysteine type defensins isolated from insects as well as defensins isolated from mussels are mainly active against Grampositive bacteria and only occasionally against Gramnegative strains or fungi (Dimarcq et al. 1998; Mitta et al. 2000). Possibly, this reflects the relative importance of infection pressure from fungal as opposed to bacterial pathogens on plants.
Structure–activity relationship The plant defensins Rs-AFP1 and Rs-AFP2 are constitutively expressed in radish (Raphanus sativus) seeds. These defensins only differ in two amino acids, resulting in an increase in positive charge for Rs-AFP2 (Fig. 1B). Interestingly, the antifungal potency of Rs-AFP2 is, depending on the test fungus, 2–30 times higher than that of Rs-AFP1 (Terras et al. 1992). Via analysis of RsAFP2 variants, the contribution of basic amino acids to its antifungal activity was demonstrated (De Samblanx et al. 1997). At two positions (glycine-9 and valine-39), the substitution of neutral residues by arginines increased the activity of Rs-AFP2 against the filamentous fungus Fusarium culmorum. Analogously, replacement of a basic lysine residue at position 44 by a neutral glutamine residue decreased antifungal activity (De Samblanx et al. 1997). The difference in antifungal activity of these Rs-AFP2 variants, compared to the antifungal activity of native Rs-AFP2, was found to be dependent on the test fungus, which suggests a very specific recognition mechanism between the plant defensin and its fungal target site. Based on a mutational analysis, it was shown that RsAFP2 possesses two adjacent sites important for antifungal activity against Fusarium culmorum. The first one is the loop connecting b-strands 2 and 3 forming a highly hydrophobic patch (De Samblanx et al. 1996, 1997; Schaaper et al. 2001). The second one is composed by the loop connecting b-strand 1 and the a-helix and residues on the a-helix and b-strand 3, together forming a path of contiguous residues despite their scattered positions along the Rs-AFP2 sequence (De Samblanx et al. 1997). These two regions important for antifungal activity might constitute two sites contacting a single putative receptor. Alternatively, the presence of two sites could be indicative of two distinct features necessary for the antifungal activity of Rs-AFP2, such as binding of Rs-AFP2 to its receptor on one hand, and subsequent permeabilization of the membrane on the other hand.
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Mode of antimicrobial activity Multiple antimicrobial molecules throughout the animal and plant kingdom, belonging to different families, display similar modes of action against a wide range of bacteria and fungi. These molecules, including defensins, share common properties including broad-spectrum antimicrobial activity and cationic charge at physiological pH. There are currently two models describing the mode of antimicrobial activity of such cationic peptides. One model postulates the formation of multimeric pores within microbial membranes. After initial electrostatic binding of these positively charged peptides to negatively charged (phospho)lipids on the target cell surface, they insert into the energized cell membrane and most likely form multimeric ion-permeable channels in a voltage-dependent manner (Kagan et al. 1990; Cociancich et al. 1993; Lehrer et al. 1993; Wimley et al. 1994; Hristova et al. 1996; Maget-Dana and Ptak 1997). The second model postulates an electrostatic chargebased mechanism of membrane permeabilization, as in the case of magainins, cecropins and HBD2, antimicrobial peptides isolated from frog skin, insect hemolymph and human skin, respectively (Ludtke et al. 1996; Bechinger 1997; Harder et al. 1997). They are thought to bind as monomers onto anionic lipid headgroups of the membrane, thereby covering the membrane in a ‘carpetlike’ manner. The subsequent neutralization of the anionic lipid headgroups disrupts the integrity of the lipid bilayer, causing transient gaps and allowing ions and larger molecules to cross the membrane (Oren and Shai 1998; Shai 1999; Hoover et al. 2000). Recently, the theory that many cationic peptides exert their antimicrobial activity not only through permeabilization of the membrane but also through cytoplasmic targets has gained support (Wu et al. 1999; Xiong et al. 1999). It was shown that apidaecins, short proline–arginine-rich insect peptides, enter bacterial cells through stereospecific interactions with the outer membrane and, once into the interior of the cell, affect protein synthesis (Castle et al. 1999). This and other evidence has led to the suggestion that membrane disruption by itself is not the primal cause of antimicrobial activity of possibly many cationic peptides, but rather inhibition of DNA, RNA or protein synthesis (Friedrich et al. 2000). Thus, the ability of cationic peptides in general to permeabilize cytoplasmic membranes might be a means to reach an intracellular target. In contrast to mammalian and insect defensins, plant defensins that display antifungal effects do not interact directly with plasma membrane phospholipids (Thevissen et al. 1996; Caaveiro et al. 1997). The plant defensins Dm-AMP1, isolated from Dahlia merckii, and Rs-AFP2 induce an array of relatively rapid responses in fungal cells, including increased K+ efflux and Ca2+ uptake, membrane potential changes and membrane-permeabilization (Thevissen et al. 1996, 1999). Remarkably, a variant of Rs-AFP2 that displays enhanced antifungal
activity induced even more Ca2+ uptake, while a variant that displays almost no antifungal activity caused no Ca2+ uptake (De Samblanx et al. 1997). This demonstrates a direct link between ion fluxes and antifungal activity. Moreover, using radiolabeled defensins from Heuchera sanguinea (Hs-AFP1) and dahlia (Dm-AMP1), the existence of high-affinity binding sites on fungal plasma membranes was shown (Thevissen et al. 1997, 2000a). The precise mode of action of plant defensins is still unclear, and for most plant defensins molecular components involved in signaling and putative intracellular targets remain unknown. Only for Dm-AMP1 has a putative binding target been identified in the yeast Saccharomyces cerevisiae. The gene that determines Dm-AMP1 sensitivity appeared to be IPT1, a gene encoding inositol phosphotransferase that is involved in the last step of the biosynthesis of the sphingolipid mannose-(inositol-phosphate)2-ceramide (M(IP)2C; Dickson et al. 1997; Thevissen et al. 2000b). IPT1 determines not only binding capacity, but also Dm-AMP1mediated permeabilization and growth inhibition, thus linking all three phenomena. Elimination of IPT1 not only leads to Dm-AMP1 resistance but also to an increased resistance to the antibiotic cycloheximide and syringomycin E, an antifungal lipopeptide from the bacterium Pseudomonas syringae (Leppert et al. 1990; Stock et al. 2000). Sphingolipids are, together with sterols and phosphoglycerolipids, the major types of lipid found in eukaryotic membranes. They associate with sterols in the plasma membrane to form patches or rafts that are highly enriched in glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins (Bagnat et al. 2000). Based on mutational analysis it was concluded that resistance to Dm-AMP1 has mainly to do with modifications in sphingolipid composition rather than with alterations in IPT1 itself (Thevissen et al. 2000b). Therefore, it is not likely that IPT1 is the binding site for Dm-AMP1 on the plasma membrane. IPT could, however, be involved in constituting the Dm-AMP1 binding site. Specific sphingolipids might interact directly with Dm-AMP1 or, alternatively, attach particular GPI-anchored membrane proteins that act as Dm-AMP1 docking sites (Thevissen et al. 2000b). This interaction facilitates the insertion of these defensins in the plasma membrane, leading to alterations in membrane permeability and finally fungal growth arrest. Likewise, mammalian GPI-anchored proteins have been shown to act as receptors for a bacterial toxin, aerolysin, that causes pore formation (Diep et al. 1998; Abrami et al. 2002). It was shown before that binding of Dm-AMP1 could be competed for by the highly homologous defensins Ah-AMP1 and Ct-AMP1, isolated from Aesculus hippocastanum and Clitoria ternatea, respectively (Thevissen et al. 2000a). This suggests that these defensins share the same binding site. Indeed, elimination of IPT1 not only leads to Dm-AMP1 resistance but also to AhAMP1 and Ct-AMP1 resistance (our unpublished data).
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More-distantly related plant defensins, like Heuchera sanguinea Hs-AFP1 and Raphanus sativus Rs-AFP2, are not able to compete for the Dm-AMP1 binding site and elimination of IPT1 does not lead to enhanced resistance to Hs-AFP1. This demonstrates that these defensins probably have distinct binding sites on the plasma membrane (Thevissen et al. 2000a; and unpublished data). Moreover, for plant defensins, like for cationic peptides in general, it is still unclear whether they have specific intracellular targets. Both c- and x-hordothionin, plant defensins isolated from barley endosperm, were found to inhibit protein synthesis in eukaryotic as well as prokaryotic cell-free systems (Mendez et al. 1990, 1996). This mode of action could support the theory of intracellular targets for plant defensins, but it is not known whether these defensins actually exhibit antifungal effects. Apart from a working mechanism involving membrane permeabilization, some plant defensins have been reported to exert a different mode of action. It was demonstrated that c1- and c2-zeathionins, isolated from maize, inhibit sodium currents in a rat tumor cell line without changing the kinetics or voltage dependence of activation or inactivation of these currents (Kushmerick et al. 1998). Similarly, some structurally related scorpion neurotoxins are known for blocking potassium channels (Garcia et al. 2001). Since the overall structure of ion channels is conserved among eukaryotes, the native target of zeathionins may be insect or fungal ion channels (Hille 1992; Kushmerick et al. 1998). However, it is unclear whether these zeathionins are active against fungi or insects.
A role in plant defense: possible applications Most plant defensins isolated in the first half of the 1990s are seed-derived. For radish seeds it was shown that although plant defensins represent only 0.5% of the total seed proteins, the defensins accounted for about 30% of the proteins released upon mechanical wounding (Terras et al. 1995). The amount of defensin released from a single seed was estimated to be at least 1 lg, enough to cause fungal growth inhibition. Therefore, it has been proposed that plant defensins contribute to the protection of seeds or seedlings against attack by soilborne pathogens and thus enhance seedling survival rates (Terras et al. 1995). In addition, it was recognized that plant defensins are also expressed in vegetative tissues (Penninckx et al. 1996; Thomma and Broekaert 1998). Plant defensins appear to be mainly expressed in peripheral cell layers, which is consistent with a role in a first line of defense against pathogens. Defensins are found in surface walls of radish germlings (Terras et al. 1995), in peripheral cell layers of tobacco flower organs (Gu et al. 1992) and in the epidermal cell layer and leaf primordia of potato tubers (Moreno et al. 1994). Defensins are also found in stomatal cells and in the cell
walls lining substomatal cavities of beet leaves (Kragh et al. 1995), which is interesting since stomata are wellknown entry points for fungal pathogens. A role in plant defense is also supported by the pathogen-inducibility of some plant defensin genes (Chiang and Hadwiger 1991; Gu et al. 1992; Terras et al. 1995; Penninckx et al. 1996). Arabidopsis wild-type plants express the leaf-specific defensin PDF1.2 upon pathogen challenge (Penninckx et al. 1996; Thomma et al. 1998, 1999). Mutants that are impaired in either jasmonate- or ethylene-signaling do not express this pathogen-inducible defensin and display a hyper-susceptibility towards the fungal pathogen Botrytis cinerea. This susceptibility cannot be attributed to PDF1.2 alone as other proteins with known antifungal properties are induced together with this defensin (Thomma et al. 1998, 1999). In other studies it has been demonstrated that transgenic expression of plant defensins leads to protection of vegetative tissues against pathogen attack. Constitutive expression of a radish defensin clearly enhanced resistance of tobacco plants to the fungal leaf pathogen Alternaria longipes (Terras et al. 1995) and similarly in tomato to Alternaria solani (Parashina et al. 2000). Canola (Brassica napus) constitutively expressing a pea defensin showed slightly enhanced resistance against blackleg (Leptosphaeria maculans) disease (Wang et al. 1999). Finally, it was shown that constitutive expression of an alfalfa defensin in potato provided a robust resistance against the agronomically important fungus Verticillium dahliae under field conditions (Gao et al. 2000). Possibly, the antimicrobial activity of defensins in vivo can even be enhanced due to the synergistic interaction with other defense components. In addition, variants of these naturally occurring antimicrobial peptides may provide peptides with improved characteristics (De Samblanx et al. 1997; Lamberty et al. 2001). For the antifungal insect peptide heliomicin it was shown that by substituting two basic amino acids located at the end of the a-helix with two leucines, this peptide gained antibacterial activity while retaining its antifungal activity. These particular changes give rise to an amphiphilic character in the helix, a common feature of helices of several antibacterial peptides (Lamberty et al. 2001). All these results demonstrate that plant defensins are important components of host defense. Moreover, they can be used to generate transgenic crops with improved pathogen resistance. Acknowledgements The authors thank Drs. M. Lambrecht and A. Parret for assistance with data analysis and Drs. M. De Bolle, I. Franc¸ois and I. Penninckx for critical comments. B.P.H.J.T. is a postdoctoral fellow of the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’.
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