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Critical Reviews in Plant Sciences

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Antimicrobial Peptides from Plants

a a a a
Willem F. Broekaert , Bruno P. A. Cammue , Miguel F. C. De Bolle , Karin Thevissen ,
a b c
Genoveva W. De Samblanx , Rupert W. Osborn & Dr. K. Nielson
a
F. A. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, Willem de Croylaan
42, B-3001 Heverlee, Belgium
b
ZENECA Agrochemicals, Jealott's Hill Research Station, Bracknell Berkshire, RG42 6ET, U.K.
c
Danisco Biotechnology, Copenhagen, Denmark
Version of record first published: 22 Sep 2010.

To cite this article: Willem F. Broekaert , Bruno P. A. Cammue , Miguel F. C. De Bolle , Karin Thevissen , Genoveva W. De
Samblanx , Rupert W. Osborn & Dr. K. Nielson (1997): Antimicrobial Peptides from Plants, Critical Reviews in Plant Sciences,
16:3, 297-323

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 Critical Reviews in Plant Sciences, 16(3):297-323 (1997)

Antimicrobial Peptides from Plants

Willem F: Broekaert,’ Bruno I? A. Cammue,’ Miguel F: C. De Bolle,’
Karin Thevissen,’ Genoveva W. De Samblanx,’ and Rupert W. Osborn2
F. A. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, Willem de Croylaan
42, B-3001 Heverlee, Belgium; 2ZENECA Agrochemicals, Jealott’s Hill Research Station,
Bracknell Berkshire RG42 6ET, U.K.

* Address for correspondence: Willern F. Broekaert, F. A. Janssens Laboratory of Genetics, Katholieke Universiteit
Leuven, Willern de Croylaan 42, B-3001 Heverlee, Belgium.

Referee: Dr. K. Nielson, Danisco Biotechnology, Copenhagen, Denmark

ABSTRACT: Peptides with antimicrobial properties are present in most if not all plant species.
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All plant antimicrobial peptides isolated so far contain even numbers of cysteines (4, 6, or S),
which are all painvise connected by disulfide bridges, thus providing high stability to the
peptides. Based on homologies at the primary structure level, plant antimicrobial peptides can be
classified into distinct families including thionins, plant defensins, lipid transfer proteins, and
hevein- and knottin-type antimicrobial peptides. Detailed three-dimensional structure informa-
tion has been obtained for one or more members of these peptide families. All antimicrobial
peptides studied thus far appear to exert their antimicrobial effect at the level of the plasma
membrane of the target microorganism, but the different peptide types are likely to act via
different mechanisms. Antimicrobial peptides can occur in all plant organs. In unstressed organs,
antimicrobial peptides are usually most abundant in the outer cell layer lining the organ, which
is consistent with a role for the antimicrobial peptides in constitutive host defense against
microbial invaders attacking from the outside. Thionins are predominantly located intracellularly
but are also found in the extracellular space, whereas most plant defensins and lipid transfer
proteins are deposited exclusively in the extracellular space. In a number of plant species, a strong
induction of genes expressing either thionins, plant defensins, or lipid transfer proteins has been
observed on infection of the leaves by microbial pathogens. Hence, antimicrobial peptides can
also take part in the inducible defense response of plants. Constitutive expression in transgenic
plants of heterologous antimicrobial peptide genes has been achieved, which in some cases has
led to enhanced resistance to particular microbial plant pathogens.

KEY WORDS: antifungal, defense, defensin, hevein, knottin, lipid transfer protein, molecular
breeding, pathogen, resistance, thionin, transgenic plant.

1. INTRODUCTION effective mechanisms to restrict the growth

Despite the fact that animals and plants
are continuously exposed to microbes dur-
ing their lifespans, microbial infections which
threaten their survival as individuals do not
occur very frequently. This implies that
higher organisms must have evolved highly
0735-2689/97/$.50
0 1997 by CRC Press LLC
of microorganisms inside their tissues. The
complexity of these defense mechanisms
generally correlates with that of the organ-
ism, with higher vertebrates possessing the
most multifarious system. An important com-
ponent of the higher vertebrate defense sys-
tem is the adaptive immune response, which

297
 is unique in the sense that it displays speci-
ficity toward the infectious agent and fea-
tures a memory function enabling it to re-
pulse more efficiently subsequent challenges
by the same agent. Such an immunoglobu-
lin-based system does not operate in plants.
However, plants do share with animals sev-
eral components of the so-called innate im-
munity. These include the capacity to pro-
duce, upon perception of microbial signals,
various antimicrobial substances such as
hydrogen peroxide, digestive enzymes and
antimicrobial proteins and peptides, or alter-
natively, to produce some of these defense
molecules constitutively in certain special-
ized cell types or tissues. On the other hand,
some defense strategies used by plants are
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not or are less well developed in animals, for
example, the ability to produce in a constitu-
tive or inducible way antimicrobial second-
ary metabolites or to alter and reinforce the
extracellular matrix in response to microbial
attack.
As this review focuses on antimicrobial
peptides as defense components, we will
briefly highlight some of the well-studied
antimicrobial peptides implicated in the de-
fense system of animals. A first group of
antimicrobial peptides are the short linear
peptides (20 to 40 amino acids) that form
amphipathic a-helices in solution, for ex-
ample, the cecropins and the magainins.
These peptides are known to associate with
lipid membranes of target cells and to tran-
siently form ion channels (Duclohier, 1994;
Matsuzaki et al., 1995). Cecropins accumu-
late in the hemolymph of many invertebrates
in response to injury or infection (reviewed
by Boman and Hultmark, 1987), but ho-
mologs of cecropins have also been found in
the intestines of mammals (Lee et al., 1989).
Magainins and related compounds occur in
the skin and in the gastrointestinal system of
amphibians and are thought to take part in
the shielding of these organs against micro-
organisms (reviewed by Bevins and Zasloff,
298
1990; Nicolas and Mor, 1995). A second
type of antimicrobial peptide is formed by
the cysteine-rich peptides, which in contrast
to cecropins and magainins, have a complex
disulfide-bond stabilized folding pattern of-
ten involving antiparallel P-sheets. Insect
defensins, for instance, are short peptides
(34 to 43 residues) with three disulfide bonds.
They are folded into a compact structure
with a double-stranded antiparallel P-sheet
and a short a-helix (reviewed by Hoffmann
and HCtru, 1992. Like cecropins, insect
defensins are produced in a pathogen-induc-
ible manner by the insect fat body and se-
creted in the hemolymph (Cociancich et al.,
1994). Mammalian defensins are 29 to 34
amino acids long and have three disulfide
bridges that consolidate a triple-stranded
P-sheet structure (reviewed by Lehrer et al.,
1991; Ganz and Lehrer, 1995). Mammalian
defensins are stored at high concentrations
in granules of phagocytic blood cells (Rice
et al., 1987). When the phagocytes ingest
microorganisms, the granules release their
content in the phagocytosis vesicles, where
killing of the engulfed microorganisms is
accomplished by the combined action of
antimicrobial proteins and inorganic chemi-
cals (e.g., hydrogen peroxide, hypochlorous
acid, nitric oxide). Defensins are also se-
creted by specialized cells in the epithelium
of intestines and airways, where they may
help maintain the normal microbial flora in
a steady state (Selsted et al., 1992; Diamond
et al., 1993). The importance of defensins in
innate immunity of humans is underscored
by the observation that certain disorders that
are characterized by recurrent infections are
associated with a drastic reduction of defensin
content of blood phagocytes (Ganz et al.,
1988). Moreover, transposon mutants of a
pathogenic Salmonella strain known to in-
fect and proliferate inside phagocytes were
shown to have simultaneously lost their re-
sistance to defensins (as well as to other
antimicrobial proteins) and their virulence,
 8 - c y s t e i n e type t h i o n i n s K . C C b . . . ........ . C . .
. . R . C Y . . C ................. .C.C.. .hG. . C P . .aP.

6 - c y s t e i n e type t h i o n i n s . . C C P . . . .R. .a. . C...............

. ....... . . C f . . .... . IC . . ....
...........
..C..

Plant def e n s i n s ... C . . ....a . G . C . . .. . C.............................

............. . . . C . . .L.. . . G . C . . ......C . C . . .C

Lipid t r a n s f e r p r o t e i n s ..C.
1
... .......
.............
.V.. .Cf . a . ..CC. . f . .L.. .f.h.. .b.. .c.c

r.....................
L-f....... .......
..f fP. ......f .
.c. . . f . a . f . . ..C..f ...
a
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. . C ........C ....C C S . a G a C G ....

Hevein-type p e p t i d e s .............

Knottin-type p e p t i d e s ....c . . ... . C . . ..... . C C . . .C.. ....... .C..

Ib-AMPS ..cc.. ...... c . . .c

Imp-1 ......c . . . c .............c . . . c ....

FIGURE 1. Conserved residues in primary structures of different antimicrobial peptides. Cys-
teine connectivities are indicated by full lines. Segments that vary in length between different
members of an antimicrobial peptide family are indicated by dashed underlining. Dots represent
positions that are not fully conserved; a represents aromatic amino acids (F, W, Y); b represents
basic amino acids (H, K, R); h represents hydroxy amino acids (S, T); f represents hydrophobic
amino acids (A, I, L, M, V). For the knottin-type peptides, Ib-AMPS and MBP-1, only the cysteines
are shown as the limited number of available sequences does not yet allow consensus motives
to be forwarded. Eight- and six-cysteine type thionins: the conserved residues are derived
from a comparison of sequences in Bohlmann (1994) (excluding the truncated thionin encoded
by the wheat thionin gene TthV), Schrader-Fischer and Ape1 (1993), Schrader-Fischerand Ape1
(1994), and Epple et al. (1995). Numbering is relative to the Pyrdaria pubera thionin (Vernon et
al., 1985). Plant defensins: the conserved residues are derived from a comparison of sequences
in Broekaert et al. (1995) and Kragh et al. (1995). Numbering is relative to Rs-AFP1 from radish
seed (Terras et al., 1995). Lipid transfer proteins: the conserved residues are based on a
comparison of the sequences in Cammue et al. (1995). Numbering is relative to LTP from wheat
seeds (Simorre et al., 1991). Hevein-type peptides: the conserved residues are derived from a
comparison of the sequences shown in Broekaert et al. (1992, 1994). Some hevein-type pep-
tides, such as Ac-AMP2 from amaranth seed, lack the C-terminal segment encompassing the
seventhth and eighth cysteine. Numbering is relative to hevein (Walujono et al., 1975). Knottin-
type peptides: numbering relative to Mj-AMP1 from Mirabilisjalapa seed (Cammue et al., 1992).
It is assumed that Mj-AMP1 has the same cysteine connectivities as AAI, the a-amylase inhibitor
from amaranth seeds (Chagolla-Lopez et al., 1994). Ib-AMPs: numbering relative to Ib-AMP1
(Tailor et al., 1997). MBP-1: sequence as described in Duvick et al. (1992).

299
 Thionins

Plant defensins

Lipid transfer proteins

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Hevein-type peptides

Knottin-type peptides

FIGURE 2. Three-dimensional structures of different antimicrobial peptides. Peptide backbones are shown
in molscript presentation with helices representing a-helices and arrows representing p-strands. Disulfide
bridges are shown as black tubes with balls at the C or S atom positions. Structures assumed to be
representative for the peptide families are shown for the thionin p-purothionin from wheat seed (Stec et al.,
1995), the plant defensin Rs-AFP1 (Fant et al., 1997), the LTP from wheat seed (Gincel et al., 1994), the
hevein-type peptide hevein from the rubber tree (Andersen et al., 1993), and the knottin-type peptide AAI
(amaranth a-amylase inhibitor; Chagolla-Lopez et al., 1994).

suggesting that the virulence of this strain
relies in part on its ability to overcome the
toxic action of the antimicrobial defensins
(Groisman et al., 1992). In Drosophila flies,
a mutant has been identified which is im-
paired in the induced production of several
antimicrobial peptides, including insect
defensins. The mutant flies showed a lower
survival rate relative to wild-type flies when
challenged with bacteria (Lemaitre et al.,
1995).
In accordance with the terminology used
in the literature on animal antimicrobial pep-
tides (Boman, 1995; Ganz and Lehrer, 1999,
we will refer to proteins as “peptides” if
their length is below the arbitrary limit of
100 amino acids. Emphasis in this review
will be put on those classes of plant anti-

300
 microbial peptides for which members have
been shown to be inducible on microbial
infection of plant tissues. The rationale be-
hind this option is that the sole demonstra-
tion of antimicrobial properties in an in vitro
assay is insufficient as an argument to claim
the possible involvement of a protein in host
defense. Full proof for a functional role in
defense must rely on multiple lines of evi-
dence, and ultimately include the demon-
stration that a mutant or engineered plant
with a reduced content of the antimicrobial
peptide shows enhanced susceptibility to-
ward microbial attack in comparison with a
wild-type plant. Up to now, however, such
evidence has not yet been provided for any
of the antimicrobial peptides.
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II. THlONlNS
A. Protein Structure
Thionins represent a family of peptides
that mostly range from 45 to 47 amino acids
in length. There are at least two well-dis-
criminated subgroups of thionins, namely
those containing eight cysteines intercon-
nected by four disulfide bridges and those
possessing six cysteines involved in three
disulfide bonds. A third subgroup, which
will not be further considered in this section,
consists of thionins that are truncated rela-
tive to the above subgroups and lack a C-
terminal nonapeptide portion (Castagnero et
al., 1992, 1995). A comparison of all pub-
lished thionin amino acid sequences reveals
that the thionin sequences are highly diver-
gent. Residues conserved among all thionins
are restricted to the six cysteines at positions
3,4, 16,27,33, and 41, the aromatic residue
at position 13, and the arginine at position 10
(numbering as in Figure 1). However, if the
six- and eight-cysteine type thionins are
looked on as separate subgroups, a couple of
additional consensus residues become ap-
parent (Figure 1).
The three-dimensional structure has been
elucidated for several thionins including both
six- or eight-cysteine type thionins (Hen-
drickson and Teeter, 1981; Clore et al., 1987;
Rao et al., 1995; Stec et al., 1995). All these
studies have revealed a very similar global
fold which most probably applies to all
thionins. This common structure is charac-
terized by an L shape, the long arm of which
is formed by two antiparallel a-helices and
the short arm by a P-sheet consisting of two
short antiparallel P-strands (Figure 2).
Thionins have also been shown to have an
amphipathic structure. The hydrophobic resi-
dues are clustered at the outer surface of the
long arm of the L, whereas hydrophilic resi-
dues mainly occur at the inner surface of the
L and at the outer surface of the corner of the
L (Clore et al., 1986; Hendrickson and Tee-
ter, 1981). The eight-cysteine type thionins
al-purothionin and P-purothionin from
wheat grains appear to have a binding site
for phospholipids, which may be implicated
in their toxic activity (Rao et al., 1995; Stec
et al., 1995).
B. Antimicrobial Activities
It has been known for over 50 years that
thionins inhibit the growth of bacteria and
fungi in vitro (Stuart and Harris, 1942).
Fernandez de Caleya et al. (1972) first dem-
onstrated that thionins are inhibitory to a
number of plant pathogenic bacteria, thus
suggesting that thionins may fulfill a protec-
tive role inplanta. In subsequent studies, the
antimicrobial activity spectrum of thionins
has been analyzed more extensively and
shown to include several Gram-positive and
Gram-negative plant pathogenic bacteria
(Cammue et al., 1992; Florack et al., 1993;
Molina et al., 1993a), as well as about 20
different phytopathogenic fungi with IC,,
values (concentrations required for 50%
growth inhibition) generally ranging from 1
to 15 yg/ml (Cammue et al., 1992; Molina et
301
 al., 1993a). Some Gram-negative bacteria
such as a number of Pseudomonus and
Erwinia species are, however, apparently
insensitive to thionins (Fernandez de Caleya
et al., 1972; Cammue et al., 1992; Florack et
al., 1993; Pineiro et al., 1995). The antifun-
gal activity of thionins is inhibited by Ca+at
concentrations above 5 mM, but not by Mg2+
or Ba2+at concentrations up to 10 mM, nor
by monovalent cations at concentrations up
to 50 mM (Okada et al., 1970; Terras et al.,
1992b; Cammue et al., 1995). The antifun-
gal potency of thionins has been found to be
strongly potentiated by the addition of
subinhibitory concentrations of other cys-
teine-rich proteins or peptides, namely the
seed storage 2 s albumins and related pro-
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tease inhibitors (Terras et al., 1993a), lipid
transfer proteins (Molina et al., 1993b), and
the wheat puroindolinesdescribed by Blochet
et al. (1993) (Broekaert W. F., unpublished
results). On the fungus Alternuria brussi-
cicola, for instance, a thionin from wheat
seeds was inhibitory down to 0.32 pg/ml in
the presence of 50 pg/ml of rapeseed 2 s
albumin, whereas the same thionin was only
active down to 11 pg/ml in the absence of
the 2s albumin, which corresponds to a po-
tentiation by a factor of 34 (Terras et al.,
1993a). Interestingly, all proteins known to
potentiate the activity of thionins, namely,
2 s albumins, lipid transfer proteins, and puro-
indolines, share a similar cysteine skeleton
and possibly also a similar folding pattern
(Gautier et al., 1994). All antimicrobial ac-
tivities described above have been observed
for eight-cysteine type thionins from cere-
als. However, we have recently isolated a
six-cysteinetype thionin from the radish stor-
age organ and shown that it inhibits the
growth of the fungus Fusurium culmorum in
vitro (Terras et al., 1996). However, when
tested in parallel with an eight-cysteine type
thionin from barley seed, the radish thionin
was much less potent, and, in addition, did
not act synergistically with 2 s albumins. It
is not known, however, whether the rela-
302
tively low antifungal potency of the radish
thionin applies to other fungi as well, nor
whether it can be generalized toward other
six-cysteine type thionins.
In addition to their effects on microor-
ganisms, thionins have also been shown to
exert adverse effects, often involving
permeabilization, on various cultured mam-
malian and insect cells and plant protoplasts
(reviewed in Florack and Stiekema, 1994).
Moreover, thionins are toxic to insects
(Kramer et al., 1979) and mammals (Coulson
et al., 1942; Rose11 and Samuelsson, 1966;
Evett et al., 1986;Mellstrand and Samuelsson,
1973) when injected in their body fluids, but
not when administered orally (Coulson et al.,
1942; Samuelsson, 1974).
The mechanism underlying the antimi-
crobial activity of thionins was first investi-
gated in the yeast Succharomyces cerevisiae
(Okada and Yoshizumi, 1973). It was found
that a wheat seed thionin causes per-
meabilization of the yeast cells as evidenced
by leakage into the culture medium of K+,
PO:-, and cellular components absorbing at
265 and 280 nm. A drop in cytoplasmic
PO,3- content is known to result in adeno-
sine triphosphatase (ATP) hydrolysis, which
in turn entails a block of energy-requiring
processes (Guihard et al., 1993). Also for the
filamentous fungus Neurosporu crassa it was
found that a barley seed thionin can cause a
substantial release of 14C-isoaminobutyric
acid from hyphae preloaded with this
nonmetabolizable compound (Thevissen et
al., 1996).Moreover, the dose-response curve
of the isoaminobutyric acid release corre-
lated well with that of fungal growth inhibi-
tion, suggestingthat growth arrest may directly
or indirectly result from the permeabilization
process. Thionin was also shown to mediate
a transient influx of Ca2+into Neurosporu
crussu hyphae, a steady efflux of K+, and a
steady alkalinization of the culture medium,
the latter possibly due to increased H+influx.
All these ion fluxes could be observed within
1 min after addition of the protein (Thevissen
 et al., 1996). It was also shown that a com-
bination of thionins and 2 s albumins that
exerted synergistic antifungal effects also
caused a synergistic increase of K+ efflux
from hyphae of Fusarium culmorum (Terras
et al., 1993a). The changes in the permeabil-
ity of the fungal membrane toward ions and
other small solutes may be the result of a
direct interaction between membrane phos-
pholipids and thionins. Indeed, using artifi-
cial planar lipid bilayers it was shown that
thionins can alter electrical and physico-
chemical properties of the membrane at rela-
tively low concentrations (1 pg/ml). This
process does not seem to involve the forma-
tion of bona fide ion channels by the thionins
since current-time plots showed highly ir-
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regular current spikes, but not square-like
fluctuations as usually observed for bona
fide channel-forming proteins (Thevissen et
al., 1996). Such interaction with the mem-
branes may require specific phospholipid
domains, most likely consisting of patches
with acidic phospholipids such as phos-
phatidylserine (Vernon, 1992). A radiola-
beled thionin from Pyrularia pubera was
shown to bind efficiently to artificial lipo-
somes composed of phosphatidylserine
(Vernon, 1992). In addition, physicochemi-
cal analysis of the incubation of this thionin
with phospholipid bilayers at varying lipid
compositions has shown that inclusion of
phosphatidylserine in the bilayers makes
them more susceptible to thionin-induced
structural disturbance (Gasanov et al., 1993).
The binding of Pyrularia thionin to
phosphatidylserine containing lipid bilayers
does not appear to involve insertion of the
thionin into the membrane (Wallet al., 1995).
Interestingly, 2s albumins, which are able to
potentiate the antimicrobial activity of
thionins, also interact with phospholipid
vesicles composed of acidic phospholipids,
thereby causing lipid mixing and membrane
permeabilization (Onaderra et al., 1994). It
is possible that thionins and 2s albumins
somehow cooperate during the process of
membrane insertion or membrane binding.
Cooperativity during the process of inser-
tion in artificial liposomes has previously
been observed between different types of
magainins (Vaz Gomes et al., 1993).
C. Expression Pattern and
Localization
Thionins have been isolated from a wide
range of plant species, including both mono-
cots and dicots, and are therefore expected
to be present in most if not all plant species.
In barley, for instance, thionins have been
shown to be expressed in the endosperm of
seeds (Ponz et al., 1983, 1986), in leaves
(Gaming, 1987; Bohlmann and Apel, 1987),
and in roots (Steinmuller et al., 1986). The
thionins expressed in these different organs
are encoded by different genes displaying
organ-specific expression (Garcia-Olmedoet
al., 1992). A similar situation holds true for
dicotyledonous species such as Crambe
abyssinica, which contains seed-, cotyledon-,
and leaf-expressed thionin genes (Schrader-
Fischer and Apel, 1994), and mistletoe
(Viscum album), which contains both seed-
and leaf-expressed thionin genes (Schrader-
Fischer and Apel, 1993). In Ara-bidopsis
thaliana, at least two genes are present, one
of which is expressed predominantly in flow-
ers, siliques, and rosette leaves, whereas the
second is expressed at low levels in seed-
lings and rosette leaves. Expression of the
former gene is strongly induced in seedlings
infected with Fusarium oxy-sporum f s p .
matthiolae (Epple et al., 1995). Also in bar-
ley, expression of leaf thionin genes was
shown to be strongly upregulated upon chal-
lenge of the leaves with Erysiphe graminis
f.sp. hordei, both in compatible (successful
infection) and incompatible (unsuccessful
infection) interactions (Bohlmann et al.,
1988).
303
 Induction of the expression of thionin
genes after pathogen stress might involve
methyl jasmonate as an endogenous signal
transducer. Indeed, both in barley and
arabidopsis leaves thionin transcripts accu-
mulate upon external application of methyl
jasmonate (Andresen et al., 1992; Molina
and Garcia-Olmedo, 1993; Epple et al.,
1995). Methyl jasmonate is a hormone-like
compound which is synthesized upon re-
lease of a-linolenic acid from phospholipid
membranes (Farmer and Ryan, 1992) and
has been shown to accumulate after wound-
ing (Creelman et al., 1992) or exposure of
plant cells to microbial elicitors (Mueller et
al., 1993) or invading microorganisms
(Penninckx et al., 1996). On the other hand,
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application of salicylic acid, another endog-
enous hormone-like compound which medi-
ates expression of a subset of defense-re-
lated genes upon microbial infection (Klessig
and Malamy, 1994), did not induce thionin
expression in barley leaves (Garcia-Olmedo
et al., 1995) or in arabidopsis leaves (Epple
et al., 1995).
Accumulation of thionin transcripts in
barley leaves has been shown to be strongly
repressed by light (Reimann-Philipp et al.,
1989a). However, as the meristem of barley
leaves is protected from light by the sheath
of the leaf underneath, thionins can be syn-
thesized in the meristematic cells even when
plants are exposed to the light. Their pres-
ence in differentiated cells of healthy barley
leaves exposed to the light might be ex-
plained by their exceptional stability. In
healthy leaves, thionins have been found by
immunocytochemistry to be most abundant
in epidermal cells, which is consistent with
a role in first line defense (Reimann-Philipp
et al., 1989a).
Studies on the subcellular localization of
thionins in barley and wheat seed (Carmona
et al., 1993a) and in Viscum album seed and
leaves (Schrader-Fischer and Apel, 1993)
have revealed that thionins are present in
304
vacuolar compartments. Also in etiolated
barley leaves, over 95% of thionins are
present intracellularly in vacuoles, whereas
a minor fraction is present in the cell walls,
to which they appear to be tightly bound via
ionic interactions (Rei-mann-Philipp et al.,
1989b; Bohlmann et al., 1988). Sequencing
of thionins isolated from cell walls and vacu-
oles indicated that similar isoform mixtures
occur in both compartments (Reimann-
Philipp et al., 1989b). It is therefore possible
that a fraction of the thionins is deposited in
the cell wall as a result of overflow from the
main trafficking route that leads them to the
vacuoles.
Most plant pathogens grow extracellu-
larly during at least some stage of the infec-
tion process. During that stage, they are un-
likely to become affected by antimicrobial
peptides which are stored in vacuoles. How-
ever, in many plant pathogen interactions,
plant cells and their vacuoles rupture due to
either the hypersensitive response or to the
action of necrosis-inducing toxins from the
pathogen. As a result of this rupture, vacu-
olar antimicrobial compounds can reach the
pathogen and exert their antibiotic effect.
D. Biosynthesis
In all cases studied thus far, thionins
have been found to be synthesized as
preproproteins (Ponz et al., 1983), with the
mature thionin domain located centrally be-
tween the signal sequence domain at the
N-terminus and the prodomain at the C-ter-
minus (Ponz et al., 1986; Bohlmann et al.,
1988; Castagnero et al., 1992; Schrader-
Fischer and Apel, 1993, 1994; Epple et al.,
1995). One remarkable feature of the
prodomain of thionins is that, upon compari-
son of different thionin genes or cDNAs
from the same species, it is always better
conserved than the thionin domain itself,
indicating that it might play an important
 role (Bunge et al., 1992, Castagnero et al.,
1992; Schrader-Fischer and Apel, 1993,
1994; Epple et al., 1995). Strikingly, the
physicochemical properties of the thionin
domain appear to be correlated with those of
the prodomain. Indeed, thionin precursors
with a positively charged mature domain
consistently have acidic prodomains, whereas
neutral and hydrophobic mature domains are
linked to neutral and hydrophobic pro-
domains (Schrader-Fischer and Apel, 1994),
suggesting that both domains interact during
the biosynthetic process (Reimann-Philipp
et al., 1989a). The prodomain may act as a
transient intramolecular chaperone and as-
sist the mature domain during the folding
process or, alternatively, it may help to pre-
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vent harmful interactions between the ma-
ture domain and components of the intracel-
lular trafficking machinery. Florack et al.
(1994) have analyzed transgenic tobacco
plants transformed with barley thionin gene
constructs either with or without the
prodomain. No difference was found in the
subcellular targeting of thionins in tobacco
plants transformed with either type of con-
struct, arguing against a role of the prodomain
in targeting. However, a tenfold lower ex-
pression level was reached in the plants trans-
formed with the construct lacking the
prodomain vs. plants transformed with the
full-length construct. This observation is
consistent with a role of the prodomain in
protein maturation. An alternative interpre-
tation may be that transgenic individuals
expressing the prodomain-less thionin above
a certain threshold are not viable and there-
fore underrepresented in the transgenic popu-
lation.
E. Molecular Breeding
Transgenic tobacco plants expressing a
barley thionin gene (with the prodomain re-
gion) under control of the constitutive cauli-
flower mosaic virus 35s RNA promoter
(CaMV35S) were analyzed for their resis-
tance against bacterial pathogens. One
transgenic line expressing the thionin at about
20 pg/g fresh leaves showed reduced lesion
symptoms when challenged with either
Pseudomonas syringae pv. tabaci or Pseudo-
monas syringae pv. syringae. The bacterial
titer of the transgenic plants measured 2 d
after infection with Pseudomonas syringae
pv. tabaci was two orders of magnitude lower
relative to similarly treated untransformed
plants (Carmona et al., 1993b). Transgenic
tobacco plants expressing a barley thionin
were also independently obtained by Florack
et al. (1994), but infection tests performed
on these plants with Pseudomonas syringae
pv. tabaci failed to indicate a significant
difference in resistance between transgenic
and control plants (Florack, 1994). The in-
consistency between both studies is pres-
ently unclear, but is likely to be due to the
use of different bacterial strains.
111. PLANT DEFENSINS
A. Protein Structure
Plant defensins resemble thionins in that
they are about the same size (45 to 54 amino
acids) and, like some but not all thionins,
possess eight disulfide-linked cysteines. The
first plant defensins to be isolated were ini-
tially considered as a novel thionin subgroup
and were therefore called y-thionins (Colilla
et al., 1990; Mendez et al., 1990). However,
subsequent work has established that thionins
and plant defensins are structurally unre-
lated. Whereas thionins contain two antipar-
allel a-helices and an antiparallel double-
stranded P-sheet, plant defensins are typified
by a triple-stranded antiparallel P-sheet struc-
ture with only one a-helix (Figure 2, Bruix
et al., 1993, 1995; Fant et al., 1997). A char-
acteristic feature of plant defensins is that
305
 the two cysteines in the CXXXC segment of
the a-helix are connected by two disulfide
bridges to the two cysteines in the CXC
segment of the C-terminal P-strand, a struc-
tural motif known as the cysteine-stabilized
a-helix motif (Kobayashi et al., 1991). Re-
markably, the cysteine-stabilized a-helix
motif also occurs in insect defensins, which
overall have a very similar fold as plant
defensins except that they lack the domain
correspondingto the amino-terminal P-strand
of plant defensins (Bruix et al., 1993;
Broekaert et al., 1995).
When the amino acid sequences of a
series of plant defensins originating from 13
different plant species are compared, it be-
comes clear that relatively few residues are
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conserved in all plant defensins (Broekaert
et al., 1995). Indeed, the conserved residues
are restricted to the eight cysteines, two gly-
cines at positions 13 and 34, an aromatic
residue at position 11, and a glutamic acid at
position 29 (numbering as in Figure 1).
Hence, as in the case of the thionins, the
majority of the nonstructural residues is vari-
able, thus sustaining the divergence in bio-
logical activities.
B. Antimicrobial Activities
The first plant defensins that were dem-
onstrated to possess antifungal activity were
the two plant defensin isoforms Rs-AFPl
and Rs-AFP2 isolated from the radish seed
(Terras et al., 1992b). To date, a whole range
of plant defensins have been thoroughly ana-
lyzed to determine their antimicrobial activ-
ity spectrum. Based on the antimicrobial
effects observed on fungi, at least two groups
of plant defensins can be distinguished. The
“morphogenic” plant defensins cause reduced
hyphal elongation with a concomitant in-
crease in hyphal branching, whereas the
“nonmorphogenic”plant defensins only slow
down hyphal elongation but do not induce
306
marked morphological distortions. The anti-
fungal activity of plant defensins, whether
morphogenic or not, is reduced by increas-
ing the ionic strength of the fungal growth
assay medium. This antagonism was found
to be due to the cations, with divalent cations
being at least one order of magnitude more
potent than monovalent cations (Terras et
al., 1992b, 1993b). Ca2+,Mg2+,and Ba2+are
equally effective at inhibiting the antifungal
activity of plant defensins (Terras et al.,
1993b; Broekaert, W. F., unpublished re-
sults). The antagonistic effect of cations is
strongly dependent on the fungus and on the
plant defensin type (Osborn et al., 1995).
When comparing different plant defensins
for their relative activity on various fungi,
marked differences in activity spectrum can
be observed, and such differences are even
accentuated after increasing the ionic strength
of the medium. For instance, in a potato
dextrose broth medium supplemented with
1 mM CaC1, and 50 mM KC1, the plant
defensin from horse chestnut (Ah-AMP1) is
active against Leptosphaeria maculans with
an IC,, value of 6 pg/ml, but is basically
inactive against Fusarium culmorum. How-
ever, a plant defensin from Heuchera
sanguinea (Hs-AFP 1) is inactive against the
former fungus, but inhibits the latter with an
IC,, value of 3 pg/ml (Osborn et al., 1995).
In general, plant defensins are less active
against bacteria, with some exceptions such
as a Clitoria ternatea plant defensin (Ct-
AMPl), which is active against Bacillus
subtilis (Osborn et al., 1995), and a potato
tuber plant defensin, which inhibits Pseudo-
monas solanacearum and Clavibacter
michiganensis (Moreno et al., 1994). So far,
none of the plant defensins has been found
to cause detrimental effects on cultured hu-
man or plant cells (Terras et al., 1992;
Broekaert, W. F. and Cammue, B. P. A., un-
published results).
It is not known at present how exactly
plant defensins inhibit fungal growth. It was
 shown that the plant defensins from radish
seed (Rs-AFP2) and dahlia seed (Dm-AMP1)
do not cause substantial permeabilization of
Neurospora crussu hyphae to isoaminobu-
tyric acid, unless the plant defensin concen-
tration was raised tenfold above the minimal
concentration required for growth inhibition
(Thevissen et al., 1996). In addition, these
plant defensins had no effect on electrical
currents measured in artificial membrane
systems (Thevissen et al., 1996). Hence,
permeabilization through direct protein-lipid
interactions does not seem to be the primary
cause of fungal growth inhibition by plant
defensins, unlike in the case of thionins. On
the other hand, plant defensins did cause a
marked and sustained increased Ca2+influx
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when added to Neurospora crassa hyphae,
reaching up to tenfold the level of Ca2+in-
flux in untreated hyphae (Thevissen et al.,
1996). Concomitant with the Ca2+influx, the
plant defensins caused efflux of K+and alka-
linization of the external medium (Thevissen
et al., 1996). Such ion fluxes were not ob-
served with a recombinant Rs-AFP2 variant
in which the tyrosine at position 38 was
replaced by a glycine and which lacked sub-
stantial antifungal activity (De Samblanx, et
al., 1997). It is noteworthy that similar ion
fluxes, including Ca2+influx, K+ efflux, and
extracellular alkanization, are also observed
upon treatment of plant cells with peptides
of fungal origin known to elicit defense re-
sponses in the plant (Nurnbergeret al., 1994).
C. Expression Pattern and
Localization
Plant defensins have now been isolated
from over a dozen plant species (Broekaert
et al., 1995) and most probably occur in
most if not all plant species. The tissues in
which they occur range from leaves (Kragh
et al., 1995; Terras et al., 1995), tubers
(Moreno et al., 1994), flower organs (Gu et
al., 1992; Moreno et al., 1994; Karunanandaa
et al., 1994; Kragh et al., 1995), and pods
(Chiang and Hadwiger, 1991) to seeds
(Mendez et al., 1990; Colilla et al., 1990;
Bloch and Richardson, 1991; Terras et al.,
1992b, 1993b; Osborn et al., 1995). In
arabidopsis, at least four different plant
defensin genes exist, all of which have a
distinct organ-specific expression pattern
(Thomma, B., Manners, J. M., and Broekaert,
W. F., unpublished results). Localization of
plant defensins in their tissue of origin by
immunological or in situ hybridization tech-
niques has, in many of the cases reported so
far, revealed that they accumulate preferen-
tially in the peripheral cell layer. In radish
seed, the highest concentrations of plant
defensins occur in the outer cell wall lining
the epidermis of cotyledons, hypocotyls, and
endosperms (Terras et al., 1995). Unopened
tobacco flower buds transcribe a plant
defensin gene in the epidermis of the adaxial
surface of petals and in the peripheral cell
layers of the style, the ovary, the stamen
filaments, and anthers (Gu et al., 1992).Like-
wise, in potato tubers, the highest transcrip-
tion of a plant defensin gene is in the epider-
mis and in the leaf primordia (Moreno et al.,
1994). In healthy sugar beet leaves, on the
other hand, plant defensins were located pre-
dominantly in the xylem, in stomatal cells,
and in cell walls lining the substomatal cavi-
ties (Kragh et al., 1995). The preferential
location of plant defensins in peripheral cell
layers is consistent with a role in protection
of the organ against microbial challenge.
Furthermore, their occurrence in stomatal
cells and cells surrounding the substomatal
cavity also fits in with a role in defense,
because stomata are well-known entry points
for fungi.
In at least four different plant species,
namely pea (Chiang and Hadwiger, 1991),
tobacco (Gu et al., 1992), radish (Terras et
al., 1995), and arabidopsis (Penninckx et al.,
1996), plant defensins have been shown to
307
 be induced after fungal infection of a vegeta-
tive tissue. In contrast, no overall increase in
plant defensin content was observed in sugar
beet leaves infected with the fungal patho-
gen Cercospora beticola. However, in this
case the infection triggered the appearance
of extracellular globular bodies containing
plant defensins in the leaf mesophyl cells
surrounding the infection zone, whereas such
bodies were apparently absent from the cor-
responding tissue in healthy leaves (Kragh
et al., 1995). At least in radish and in
arabidopsis, the induction by fungal patho-
gens is systemic in the sense that it can be
detected not only in infected leaves, but also
in uninfected leaves (Terras et al., 1995;
Penninckx et al., 1996). Accumulation of
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leaves can be induced after external applica-
tion of methyl jasmonate, but not by sali-
cylic acid (Penninckx et al., 1996).
It has been proposed that plant defensins
also play an important role in the protection
of seed or seedlings against invasion by soil-
borne fungi (Terras et al., 1995). This hy-
pothesis is based on the observation that
radish seed germinating on a medium sup-
porting the growth of a fungal colony cause
a growth inhibition halo which can be mim-
icked by application on the medium of drops
containing as little as 1 pg of the purified
radish plant defensins Rs-AFPl and Rs-
AFP2. Seed that were kept dormant by ex-
ternal application of abscisic acid did not
produce the inhibition halo unless their seed

plant defensins in radish and arabidopsis coats were mechanically perforated. Analy-

FIGURE 3. Disease symptoms caused by Alfernaria longipes on leaves of untransformed

tobacco plants (upper two rows) and on the leaves of a transgenic line expressing Rs-AFP2
at a level of approximately 0.2% of total protein (lower two rows). Leaves of in vitro
propagated tobacco plants were inoculated while still attached to the plants with 5 pl drops
containing 1000 spores per milliliter in 50 m M glucose (see Terras et al., 1995). Five days
after inoculation, the leaves were detached and photographed.

308
 sis of the imbibition solution of seed with a
mechanically incised seed coat revealed that
plant defensins accounted for 30% of re-
leased proteins, although Rs-AFPs are mi-
nor proteins in the seed (0.5% of total seed
proteins). The amount of plant defensins
released from a single seed was estimated to
be at least 1 pg, which is the amount of
peptide required to mimic the inhibition halo
formed around a germinating seed. All of
these experiments indicate that plant
defensins are released from radish seeds when
the seed coat is perforated (either by the
radicle of the germinating embryo under
natural conditions or artificially with the aid
of a scalpel). Moreover, these experiments
also indicate that the released amounts are
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sufficient to create a zone around the seed in
which fungal growth is suppressed. Hence,
these findings strongly suggest that plant
defensins play a role in the protection of
seedling tissues during the early stage of
emergence, and thus may contribute to the
enhancement of seedling survival rates. The
simple fact that chemical fungicides are com-
monly used for the coating of crop seed to
increase seedling stand illustrates that soil-
or seed-borne fungi form a considerable
threat to germinating seed.
D. Biosynthesis
Most plant defensins studied thus far are
synthesized with a signal peptide, but with-
out a propeptide (Stiekema et al., 1988;
Ishibashi et al., 1990; Chiang and Hadwiger,
1991; Karunanandaa et al., 1994; Terras et
al., 1995). However, some plant defensins,
such as the flower-specific plant defensins
from tobacco (Gu et al., 1992) and tomato
(Milligan and Gasser, 1995), are synthesized
as preproproteins with the propeptide being
located C-terminally and consisting of about
30 residues.
E. Molecular Breeding
To investigate the ability of plant
defensins to confer increased disease resis-
tance in transgenic plants, tobacco plants
were transformed with a chimeric gene con-
struct consisting of the coding region of the
radish (Rs-AFP2) plant defensin cDNA
driven by a constitutive promoter. T,-gen-
eration tobacco plants expressing Rs-AFP2
at a level of 0.2% of total leaf protein showed
a sevenfold reduction in lesion size upon
infection with the foliar fungal pathogen
Alternuria Zongipes relative to untransformed
plants (Figure 3, Terras et al., 1995).
IV. LIPID TRANSFER PROTEINS
A. Protein Structure
Lipid transfer proteins (LTPs) in plants
form a family of homologous peptides con-
taining 90 to 93 amino acid residues, among
which are eight disulfide-linked cysteines.
The first LTPs were isolated based on an in
vitro bioassay that measures transfer of phos-
pholipids from artificial liposomes to mito-
chondria (Kader et al., 1984), which explains
the name they have been given. LTPs or
proteins homologous to LTPs have since been
detected in at least a dozen different plant
species. One of these LTP-like proteins,
namely, an antimicrobial protein from onion
seed, does not transfer phospholipids in the
classical LTP bioassay, indicating that this
activity may not be shared by all members of
the LTP family of peptides (Cammue et al.,
1995). It was originally postulated that LTPs
play a role in the intracellular trafficking of
lipids from the endoplasmic reticulum, the
site of phospholipid biosynthesis, to other
cellular organelles (Arondel and Kader,
1991). However, this hypothesis has now
been largely abandoned because it is not
309
 consistent with a number of features of this
group of peptides, e.g., the fact that they are
not located in the cytosol (see below).
When LTPs from different plant spe-
cies are compared at the amino acid se-
quence level, about 30% of the residues
appear to be conserved. Conserved posi-
tions include those of the eight cysteines as
well as 12 positions which are invariably
occupied by hydrophobic or aromatic resi-
dues (Figure 1). Tertiary structure determi-
nation of an LTP from maize by X-ray crys-
tallography (Shin et al., 1995) and of another
LTP from wheat seed by proton-nuclear
magnetic resonance (NMR) (Gincel et al.,
1994) has revealed a similar folding pattern
consisting of a bundle of four a-helices
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linked by flexible loops (Figure 2). The
structure displays a hydrophobic cavity
which can accommodate a fatty acyl chain,
as shown by crystallography of an LTP-
palmitate complex (Shin et al., 1995).
B. Antimicrobial Properties
Antimicrobial properties of LTPs were
almost simultaneously discovered by our
group (Terras et al., 1992a) and Garcia-
Olmedo and co-workers (Molina et al.,
1993b; Segura et al., 1993). Meanwhile, it
has become clear that different LTPs from
different plant species can exert different
antimicrobial activities. For instance, an
onion seed LTP is highly active against a
broad range of fungi (with IC,, values rang-
ing from 1 to 6 pg/ml), whereas a radish
seed LTP is only moderately active (1C5,
values ranging from 7 to 100 pg/ml) and
maize and wheat seed LTPs are inactive
against most fungi (Cammue et al., 1995).
Also, the sensitivity of their antifungal ac-
tivity to the presence of cations varies with
the LTP type. The onion seed LTP was
almost as potent in a low ionic strength
medium as in the same medium supple-
310
mented with 1 mM Ca2+and 50 mM K+,
whereas the radish LTP showed drastically
reduced antifungal activity in the presence
of cations (Cammue et al., 1995). LTPs
have also been shown to possess antibacte-
rial activity. Those from barley and spinach
leaves were about tenfold more active than
wheat thionins against the Gram-positive
bacterium Clavi-bacter michiganensis
subsp. sepedonicus and three to fivefold
more active relative to thionins against the
Gram-negative bacterium Pseudomonas
solanacearum (Molina et a1.,-1993b; Segura
et al., 1993). The most active LTP from
barley leaves had an IC,, value of 0.6 pg/
ml on Clavibacter michiganensis subsp.
sepe-donicus. The onion seed LTP, which
exhibits strong antifungal activity, was also
shown to be inhibitory to Gram-positive
bacteria but not to a range of Gram-nega-
tive bacteria (Cammue et al., 1995). In con-
trast, none of the LTPs from the seed of
onions, radishes, maize, or wheat caused
lysis of human erythrocytes, nor did they
affect the viability of cultured human fibro-
blasts (Cammue et al., 1995), indicating
that the cytotoxicity of LTPs is more re-
stricted than that of thionins.
C. Expression Pattern and
Localization
The spatial expression pattern of LTP
genes has been studied in a wide range of
plant species including arabidopsis (Thoma
et al., 1994), barley (Molina and Garcia-
Olmedo, 1993), broccoli (Pyee and Kolat-
tukudy, 1995), carrot (Sterk et al., 1991),
tobacco (Koltunow et al., 1990; Fleming et
al., 1992), and maize (Sossountzov et al.,
1991). LTPs have been found to be expressed
in a variety of plant organs including em-
bryos, cotyledons, leaves, stems, siliques,
and various flower organs. In the majority of
the cases, highest expression levels were
 found in the epidermal or peripheral cell
layer surrounding the organ (Sossountzov et
al., 1991; Sterk et al., 1991; Fleming et al.,
1992; Clark and Bohnert, 1993; Molina and
Garcia-Olmedo, 1993; Thoma et al., 1993,
1994; Gaming, 1994); and in organ abscis-
sion zones (Thoma et al., 1994). Localiza-
tion studies by immunocytochemical elec-
tron microscopy have revealed that LTPs are
located in the cell walls, at least in various
arabidopsis organs and in broccoli leaves
(Thoma et al., 1994; Pyee et al., 1994). In
broccoli leaves, an LTP was shown to be the
major protein in the outer wax layer (Pyee et
al., 1994). In addition, a substantial amount
of LTP could be washed from barley leaves
by simple imbibition of leaves in aqueous
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buffer (Molina and Garcia-Olmedo, 1993),
indicating that at least in these species leaf
LTPs are present at an external position which
is directly accessible.
The fact that LTPs are most often pre-
dominant in epidermal layers of cutinized
organs has led to the hypothesis that they
may be involved in the deposition of cutin
monomers or other highly lipophilic sub-
stances (Sterk et al., 1991). However, this
hypothesis is not consistent with the obser-
vation that at least in some cases, for ex-
ample, in tobacco stems (Fleming et al.,
1992), LTPs are uniformly distributed
throughout the organ, notwithstanding the
fact that not all cells of this organ actively
secrete lipophilic substances in the apoplast.
The preferred occurrence of LTPs in outer
cell layers can also, as in the case of plant
defensins, be interpreted as an argument in
favor of a role of LTPs in repulsion or sup-
pression of microorganisms invading from
the outside. Moreover, as we pointed out
previously (Terras et al., 1992a), both pro-
posed functions are not necessarily mutually
exclusive. Indeed, LTPs may well fulfill a
protective function after being deposited in
the cell wall with cotransported lipophilic
substances.
In barley leaves, LTP genes are induced
after challenge with both a virulent and an
avirulent strain of Erysiphe graminis f.sp.
hordei and with avirulent, but not with viru-
lent strains of Rhynchosporium secalis
(Molina and Garcia-Olmedo, 1993; Garcia-
Olmedo et al., 1995). However, in contrast
to barley thionin genes, which are also in-
duced by these fungi, the LTP genes are
downregulated rather than upregulated af-
ter external application of methyl jasmonate
(Molina and Garcia-Olmedo, 1993). This
suggests that pathogen induction of LTP
and thionin genes most probably proceeds
via different pathways that may be acti-
vated in concert during infection. On the
other hand, both LTP and thionin genes are
induced by abscisic acid (Molina and
Garcia-Olmedo, 1993). Abscisic acid is a
pleiotropic endogenous stress hormone that
is implicated in the induction of defense
genes after wounding (Pena-Cortes et al.,
1991), but is also implicated in the response
of plants to drought stress and osmotic stress
(Zeevaart, 1988). It has also been shown
that an LTP gene from tomato can be in-
duced by external application of abscisic
acid, and by drought stress (Torres-
Schumann et al., 1992). If LTPs are indeed
involved in cutin or wax deposition, then
the enhanced accumulation of LTPs after
drought stress may be seen as part of an
adaptation process leading to reduced wa-
ter evaporation via the surfaces of aerial
organs.
D. Biosynthesis
LTP genes or cDNAs have been cloned
from over ten different plant species and in
all cases the encoded proteins featured an
N-terminal signal sequence followed by the
mature domain. The sole exception up to
now is the cDNA encoding the onion seed
LTP-like protein that, in addition to a signal
311
 sequence domain, also has a short 12-amino
acid C-terminal propeptide domain (Cammue
et al., 1995). The role of this C-terminal
extension is currently unknown.
E. Molecular Breeding
The potential of LTPs to enhance dis-
ease resistance is just beginning to be ex-
plored. Garcia-Olmedo and co-workers have
succeeded in obtaining transgenic tobacco
plants which constitutively express a barley
LTP under control of the CaMV35S pro-
moter. Several transgenic lines showed a
drastic reduction of disease symptoms after
infection of the leaves with the bacterial
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pathogen Pseudomonus syringae (Garcia-
Olmedo, personal communication).
V. HEVEIN- AND KNOTTIN-TYPE
ANTIMICROBIAL PEPTIDES
A. Protein Structure
Most, if not all, plants contain proteins
that are able to bind to chitin (poly-(J-1,4-N-
acetylglucosamine). These chitin-binding
proteins exist in different types, varying con-
siderably in length and structure (see Raikhel
et al., 1993 for review). Many of them are
multidomain proteins sharing in common a
cysteine-rich domain of about 40 residues
that has been shown to harbor the site re-
sponsible for chitin binding (Lee et al., 1991;
Iseli et al., 1993).
Some chitin-binding proteins do not ex-
hibit multidomain structures and consist
solely of the chitin-binding domain. The best
known example of such small chitin-binding
peptides is hevein, a 43-residue peptide that
is one of the most abundant proteins in rub-
ber tree latex (Archer et al., 1969; Walujono
et al., 1975). Hevein has been shown to pos-
sess rather weak antifungal properties (Van
312
Parijs et al., 1991). More recently, however,
peptides homologous to hevein, but that are
much more potent than hevein itself in in-
hibiting microbial growth in vitro, have been
found in the seed of amaranth (Broekaert et
al., 1992) and sweet pepper (Broekaert et al.,
1994). Hevein, as well as the hevein-type
antimicrobial peptide from sweet pepper,
contains eight cysteines that are all linked by
disulfide bonds. In contrast, the hevein-
type peptides from amaranth seed contain
only six cysteines as they lack a portion
corresponding to the C-terminal part of
hevein encompassing the seventh and
eighth cysteine residue. The three-dimen-
sional structure of hevein has been deter-
mined by NMR and found to be dominated
by a triple-stranded (J-sheet and a short single
turn a-helix connecting the second to the
third p-strand (Figure 2, Andersen et al.,
1993).
From the seed of Mirubilis julupu, two
antimicrobial peptides were isolated of 36
and 37 residues, respectively, which both
contain six disulfide-linked cysteines
(Cammue et al., 1992). The cysteines of
these peptides can be relatively well aligned
with those of a group of peptides collec-
tively termed “knottins” (Le-Nguyen et al.,
1990; Chagolla-Lopez et al., 1994). The
knottins include an a-amylase inhibitor from
amaranth seeds, calcium channel-binding
toxins from Conus snails, and a sweet-
taste modifying peptide from Gymnema
sylvestre. Knottins have a knot-like fold char-
acterized by a compact triple-stranded
(J-sheet and a long loop connecting the first
to the second (J-strand (Figure 2, Chagolla-
Lopez et al., 1994). The knottin fold is also
adopted by the cellulose-binding domain of
fungal cellobiohydrolases (Kraulis et al.,
1989). From a structural point of view,
knottin-type peptides are distantly related to
hevein-type peptides in that their cysteine
motif (C-C-CC-C-C) and cysteine con-
 nectivities are identical to those found in the
hevein portion encompassing the first six
cysteines (Figure 1, Chagolla-Lopez et al.,
1994).
B. Antimicrobial Properties
The hevein- and knottin-type peptides
whose antimicrobial properties have been
studied most extensively are those from
amaranth and Mirabilis jalapa seed, respec-
tively. Both types of peptides have a similar
antimicrobial spectrum and inhibit a whole
range of fungi and Gram-positive bacteria
(Broekaert et al., 1992; Cammue et al., 1992).
Typical for these peptides is the finding that
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their antimicrobial effects are reversed when
the concentration of divalent cations in the
medium is raised above 1 mM (Broekaert et
al., 1992; Cammue et al., 1995). In media
containing 1 mM CaC1, and 50 mM KC1,
these peptides do not affect fungal growth at
concentrations below 100 pg/ml, whereas
they generally inhibit fungi at concentra-
tions below 10 pg/ml in low ionic strength
media. The antifungal activity of the hevein-
and knottin-type peptides is clearly more
susceptible to inhibition by cations compared
to the activity of eight-cysteine type thionins,
most plant defensins, and the LTP from on-
ion seed.
The knowledge of the expression pat-
terns of hevein- and knottin-type antimicro-
bial peptide genes in plants is at present still
fragmentary. RNA blot analysis of different
tissues of amaranth (De Bolle et al., 1993) or
Mirabilis jalapa (De Bolle et al. 1995) probed
with the respective hevein- or knottin-type
peptide cDNAs revealed that in both cases
the expression was restricted to the seeds.
The hevein gene is expressed in leaves and
stems, but not in roots, of rubber tree seed-
lings, and hevein transcript levels accumu-
late in leaves and stems after wounding or
application of abscisic acid (Broekaert et al.,
1990).
C. Biosynthesis
The cDNA encoding hevein consists of
an N-terminal signal sequence domain, fol-
lowed by the hevein domain and a 144-amino
acid C-terminal domain (Broekaert et al.,
1990). The C-terminal domain, which is
partially cleaved off during posttranslational
processing and can be detected as such in
rubber tree latex (Lee et al., 1991), is ho-
mologous to the pathogenesis-related pro-
tein PR-4 from tobacco. The cDNA of the
amaranth hevein-type peptide also encodes
a preproprotein, but in comparison to the
hevein cDNA, the C-terminal propeptide is
much shorter (3 1 amino acids) and therefore
unlikely to possess catalytic activity (De
Bolle et al., 1993). The Mirabilis jalapa
knottin-type peptide, on the other hand, is
encoded as a preprotein without a C-termi-
nal propeptide (De Bolle et al., 1995). The
possible role of the short C-terminal
propeptide of the amaranth hevein-type pep-
tide in cellular trafficking was investigated
using tobacco plants transformed with con-
structs containing the amaranth hevein-type
peptide coding sequence with or without the
C-terminal propeptide domain. Surprisingly,
it was found that the antimicrobial peptide
was expressed equally well in both sets of
transgenic plants, and in both cases the
hevein-type peptide was delivered in the
apoplast, indicating that the propeptide prob-
ably does not play a role in cellular targeting
(De Bolle et al., 1996). Likewise, the knottin-
type peptide from Mirubilis jalapa was also
located extracellularly in leaves of transgenic
tobacco plants transformed with the corre-
sponding cDNA construct linked to a consti-
tutive promoter (De Bolle et al., 1996).
D. Molecular Breeding
It was also demonstrated that the ama-
ranth hevein-type peptide and Mirabilis
jalapa knottin-type peptide were produced
313
 in a biologically active form in the trans-
genic tobacco plants carrying the constructs
described above. However, none of the trans-
genic lines constitutively expressing either
the amaranth hevein-type peptide or the
Mirabilis jalapa knottin-type peptide proved
more resistant than control lines when chal-
lenged with the fungal tobacco pathogen
Alternaria longipes (De Bolle et al., 1996).
The high susceptibility of the antifungal ac-
tivity of hevein- and knottin-type peptides to
the presence of inorganic cations might ex-
plain their apparent lack of activity when
expressed in planta (De Bolle et al., 1996).
On the other hand, Lee and Raikhel (1995)
have reported that the fruit from transgenic
tomato plants, transformed with a hevein
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cDNA hooked to a constitutive promoter,
were less susceptible than control plants to
infection by the opportunistic fungus
Trichoderma hamatum. Because the hevein
precursor was poorly processed in tomato, it
is not known whether the resistance is due to
the action of the hevein portion or the PR4-
like C-terminal domain.
VI. FOUR CYSTEINE-TYPE
ANTIMICROBIAL PEPTIDES
A 33-residue antimicrobial peptide with
four disulfide-linked cysteines has been iso-
lated from maize seed (Duvick et al., 1992).
This peptide, termed MBP-1, contains four
cysteine residues which are organized in two
CXXXC segments (Figure 1). MBP-1 is in-
hibitory to several fungi and at least one
Gram-positive (Clavibacter michiganense)
and one Gram-negative (Escherichia coli)
bacterium.
A novel class of antimicrobial peptides,
unrelated to any other protein or peptide
sequence reported to date, has been discov-
ered recently in Impatiens balsamina seed
and was called Ib-AMPS(Tailor et al., 1997).
Four homologous but nonidentical peptides
were purified, all of which are 20 amino
314
acids in length, making them the smallest
antimicrobial peptides found so far in plants.
The peptides contain four cysteines that are
clustered pairwise in a CC and a CXXXC
motif (with X representing any amino acid
residue), respectively (Figure 1). The Ib-
AMPSare inhibitory to a wide range of fungi
and Gram-positive bacteria, but they do not
affect most Gram-negative bacteria or cul-
tured human cells. The isoform with the high-
est isoelectric point is least sensitive to inhi-
bition of its antifungal activity by inorganic
cations. This particular isoform is still in-
hibitory to several fungi in a medium con-
taining 1 mM CaC1, and 50 mM KC1 (Tailor
et al., 1997).
Interestingly, all four Ib-AMP peptides
appear to be processed from a single
multipeptide precursor as deduced from the
cloned cDNA. This multipeptide precursor
features, next to an N-terminal prepeptide,
six repeated domains each corresponding to
one of the four mature Ib-AMP peptides.
The mature peptide domains are separated
from each other by moderately conserved
propeptides (Tailor et al., 1997). It is note-
worthy that apidaecins, a group of pathogen-
induced antimicrobial peptides from the
honey bee, are also processed by multiple
cleavages of a multipeptide precursor
(Casteels-Josson et al., 1993). This gener-
ates a set of homologous peptides with
slightly varying antimicrobial properties
(Casteels et al., 1994). Multipeptide precur-
sors can hence be regarded as a means to
diversify and broaden the activity spectrum
of the defensive peptides.
VII. ENZYME-INHIBITING PEPTIDES
HOMOLOGOUS TO ANTIMICROBIAL
PEPTlDES
As pointed out above, the families of
antimicrobial peptides show strong conser-
vation among their members in terms of the
three-dimensional structure of their peptide
 backbones, but are highly divergent at the
primary structure level. It is therefore not
surprising that not all members of these pep-
tide families have antimicrobial properties
and that some exhibit indeed quite different
biological activities. A biological activity
common among cysteine-rich peptides is
inhibition of enzymes via direct peptide-pro-
tein interactions. In particular, a-amylases
have been frequently used to assess the in-
hibitory activity of plant peptides. Peptides
capable of inhibiting a-amylases of animal
origin have been found among members of
the plant defensin family (Bloch and
Richardson, 1991),LTP family (Campos and
Richardson,-l984), and the knottin-type pep-
tide family (Chagolla-Lopez et al., 1994). It
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is noteworthy that plant defensins that in-
hibit a-amylases, such as those found in
sorghum seed, are not or only very weakly
active against fungi, whereas those that dis-
play antifungal activity are without effect on
a-amylases (Moreno et al., 1994; Osborn et
al., 1995). a-Amylases are important
digestive enzymes in herbivores, and plant
a-amylase inhibitors might serve as anti-
feedant compounds to limit herbivore dam-
age. This notion is supported by the observa-
tion that transgenic pea plants expressing an
a-amylase inhibitor from common bean are
protected from feeding damage by a bruchid
beetle (Shade et al., 1994). Hence, plant
defensins exhibiting a-amylase inhibitor
activity may possibly have evolved from a
role in protection against microorganisms
toward protection against herbivores.
Another striking but yet unexplained
observation is that many antimicrobial pep-
tide gene families count peculiar members
that encode hybrid proteins consisting of a
cysteine-rich domain linked to a proline-rich
domain. The best known example of such
chimera is the potato lectin that contains
both a hevein-like domain and a domain
with proline-rich repeats (Kieliszewski et al.,
1994). The potato lectin proline-rich domain
is posttranslationally modified by proline
hydroxylation and glycosylation (Allen et
al., 1978) and therefore strongly resembles
extensins, a class of ubiquitous cell wall
proteins that is supposed to play a structural
role (Showalter, 1993). Other examples of
hybrid proteins are those encoded by two
anther-specific cDNAs from the sunflower,
featuring a plant defensin domain and a pro-
line-rich domain (Domon et al., 1990) and
an embryo-specific maize gene harboring a
lipid transfer protein-like domain and a pro-
line-rich domain (Jose-Estanyol et d., 1992).
The significance of these chimeric proteins
is unclear, but it is tempting to speculate that
they are somehow anchored in the extracel-
lular matrix via their proline-rich domain,
whereas their cysteine-rich domain may in-
teract with other endogenous or alien pro-
teins.
VIII. CONCLUSION
In the last decade it has become clear
that plants produce different types of antimi-
crobial peptides. All plant antimicrobial pep-
tides characterized so far contain multiple
disulfide bridges and can therefore be con-
sidered as the counterparts of cysteine-rich
antimicrobial peptides from animals. Strik-
ingly, no homologs of the linear a-helical
antimicrobial peptides from animals have
yet been discovered in plants. For at least
some types of antimicrobialpeptides, namely
thionins, plant defensins, and LTPs, there is
compelling but as yet indirect evidence that
they play a role in some defense mecha-
nisms mounted by plants against microbial
invaders. Some antimicrobial peptides ap-
pear to take part in preformed defense mecha-
nisms. This is supported by the observation
that they are mostly predominant in periph-
eral cell layers surrounding plant organs thus
contributing to the formation of an antimi-
crobial shield. Other antimicrobial peptides
315
 are produced by plant tissues in response to
microbial invaders and hence are compo-
nents of the induced defense system. The
potential of antimicrobial peptides for engi-
neering disease resistance in crops is begin-
ning to be explored. Successful examples of
this approach have been obtained with chi-
meric transgenes based on a thionin, a plant
defensin. and a LTP.
ACKNOWLEDGMENTS
The authors acknowledge the financial
support by the Commission of the European
Community (AGRE-0005; AIR2-CT94-
1356) and by the Belgian National Fund for
Downloaded by [Ryerson University] at 10:47 10 March 2013
Scientific Research (G.2103.94). W.F.B. is
a Research Associate and G.W.D.S. and K.T.
are Research Assistants of the latter fund.
The authors thank Anita Vermassen for
preparation of the manuscript and Jos Desair
and Franky Fant for their help in the prepa-
ration of the figures.
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