Letters in Applied Microbiology 2000, 31, 163ÿ168

Design of self-processing antimicrobial peptides for plant

protection
W.A. Powell, C.M. Catranis and C.A. Maynard
SUNY, College of Environmental Science and Forestry, Syracuse, NY, USA

84/00: received 7 January 2000, revised 15 April 2000 and accepted 12 May 2000

Small antimicrobial peptides are

W . A . P O W E L L , C . M . C A T R A N I S A N D C . A . M A Y N A R D . 2000.
excellent candidates for inclusion in self-processing proteins that could be used to confer
pathogen resistance in transgenic plants. Antimicrobial peptides as small as 22 amino acids
in length have been designed to incorporate the residual amino acids left from protein
processing by the tobacco etch virus'(TEVs') NIa protease. Also, by minimizing the length
of these peptides and the number of highly hydrophobic residues, haemolytic activity was
reduced without affecting the peptide's antimicrobial activity.

INTRODUCTION technical problems with designing and building large con-

There has been an extensive amount of research on antimi-
crobial peptides in the medical area to develop peptides as
topical antimicrobial agents, antitumour agents, and for
enhanced wound healing (Maloy and Kari 1995). Also, sig-
ni®cant progress is being made to develop these types of
peptides for use in the area of plant protection (Rao 1995).
Previously, we designed magainin-like, synthetic peptides
that demonstrated growth inhibitory effects on both fungal
and bacterial plant pathogens (Powell et al. 1995). Our cur-
rent interests lie in designing genes that can deliver multi-
ple resistance products that would enhance sustainable
plant pathogen resistance in long-lived, woody plant spe-
cies (Powell and Maynard 1997). In this report, antimicro-
bial peptide designs are described that could be used in
self-processing protein precursors. Transgenic plants con-
taining genes encoding these precursor proteins could be
made to release a `cocktail' of antimicrobial peptides, possi-
bly with antimicrobial enzymes, that could act synergisti-
cally against the pathogen. Using a single self-processing
gene has an advantage over standard multiple-gene pyra-
miding because the gene products would be produced
under the control of a single gene promoter, therefore their
expression controlled as a unit. Also, when a single gene is
used to deliver multiple antimicrobial products, there is no
possibility of segregation during plant breeding as there is
when using multiple genes for pathogen resistance. A sin-
gle self-processing polyprotein product also simpli®es the
Correspondence to: W.A. Powell, SUNY College of Environmental Science
and Forestry, 1 Forestry Drive, Syracuse, NY 13210-2788, USA (e-mail:
wapowell@mailbox.syr.edu).
= 2000 The Society for Applied Microbiology
structs with multiple promoters and terminators ¯anking
the coding regions. One way to design this type of gene
system is to utilize the self-processing protein mechanisms
found in potyviruses. The NIa proteases from two of these
viruses have been shown to process cloned gene products
in vitro (Marcos and Beachy 1994) and in vivo (von
Bodman et al. 1995; Ceriani et al. 1998). One potential pro-
blem with using this system is that the proteases need a
recognition site for cleavage that leave amino acid
sequences on the resulting protein or peptide products. On
a large protein, these small amino acid `tails' are usually tri-
vial and often have little or no effect on the protein's activ-
ity. However, for small antimicrobial peptides, like our
ESF designs (Powell et al. 1995), these residual sequences
would account for about a third of the peptide's total
amino acid sequence and therefore could signi®cantly effect
the peptide's activity. Our hypothesis is that residual
amino acid residues retained on a self-processed peptide
can be incorporated into the overall peptide design so that
it retains its high antimicrobial activity and low haemolytic
activity. To test this hypothesis, we initially determined
the lower size limit of active ESF peptides in order to keep
gene design as compact as possible. Then selected amino
acid changes assessed for their effect the haemolytic and
antimicrobial activities of the peptides. Then, the self-pro-
cessing NIa protease system from tobacco etch virus (TEV)
was chosen as a model for modifying the ESF peptides.
Using the known protease recognition sequence from this
system (Carrington and Dougherty 1988; Dougherty et al.
1988), it was determined if residual amino acid sequences
from the protease left on the ®nal peptide products would
change their antimicrobial and haemolytic activities.
164 W . A . P O W E L L E T A L .
MATERIALS AND METHODS
Peptides
As an antimicrobial peptide control (Ala8,13,15) Magainin 2
amide (Chen et al. 1988) was purchased from Sigma. The
ESF peptide designs were aided by using the Garnier-
Robson and Chou-Fasman structural analysis, Eisenberg
hydrophobicity (H), alpha moment (a), and beta moment
(b) (Eisenberg et al. 1984), pI, and charge functions of
Protean computer software (DNASTAR, Madison, WI,
USA). ESF peptides were synthesized (> 70% purity) by
Genosys Biotechnologies (The Woodlands, TX, USA).
Fungal and bacterial culture
Two plant-pathogenic fungi were assayed. Cryphonectria
parasitica (Murrill) Barr, strain EP42, was obtained from
American Type Culture Collection (ATCC 38751) and
maintained on potato dextrose agar supplemented with
methionine and biotin (PDAmb) (Anagnostakis 1982).
Fusarium oxysporum Schlechtend: Fr. f.sp. lycopersici
(Sacc.), strain 73 was obtained from Felice Cervone, Plant
Biology Department, University of Rome, Italy and main-
tained on potato dextrose agar (PDA).
Two plant-pathogenic bacteria were assayed.
Agrobacterium tumefaciens (wild-type strain Bo542) was pro-
vided by Eugene Nester (Sciaky et al. 1978). Pseudomonas
syringae pv syringae bacteria (Collection PSS34, streptomy-
cin resistant) were provided by Thomas Burr (Department
of Horticultural Sciences, Cornell University, New York
State Agricultural Experiment Station, Geneva, New
York). Stock cultures of Ps. syringae were grown on a spe-
ci®c, de®ned nutrient medium (Atlas and Parks 1993). Ag.
tumefaciens was maintained on PDA.
Determination of the minimal inhibitory concentration
(MIC)
Conidial suspensions were aseptically prepared from agar
plate cultures of Cryphonectria parasitica grown at 25  C,
16-h photoperiod, or shaking liquid cultures for Fus. oxy-
sporum grown at 25  C, in the dark. Conidia were sus-
pended from agar plates with 10 ml sterile 1% Tween 20
(Sigma). All culture suspensions were ®ltered through four
layers of sterile cheesecloth, collected in sterile 15 ml
Falcon tubes, centrifuged for 3 min at 1900 g and the
supernatant was then poured off. The pellets were sus-
pended in 10 ml sterile deionized water, centrifuged for 3
min at 1900 g in a IEC Centra-4B centrifuge at room tem-
perature and then the supernatant was poured off. These
`washed' pellets were suspended in 5 ml sterile deionized
water and stored on wet ice while the conidial concentra-
tion was determined using a haemacytometer (Fisher ultra
= 2000 The Society for Applied Microbiology, Letters in Applied Microbiology, 31, 163ÿ168
plane, Neubauer ruling). Conidial suspensions were diluted
to 1
0  104 conidia mlÿ1. Bacterial suspensions of Ag.
tumefaciens and Ps. syringae were obtained from liquid cul-
tures grown in potato dextrose broth (pH 5
2) (Difco).
Cultures were grown at 25  C on a Lab-Line orbital shaker
(110 rev minÿ1) for 14 h. Bacterial cell suspensions with an
O.D.600 between 0
7 and 0
85 were used for assay inocula-
tions.
Dilutions (0, 0
625, 1
25, 2
5, 5, 10, 15, 20, 25, 50, 100,
150, 200 and 250 mmol lÿ1, except where noted) of peptides
tested against fungi and bacteria were aseptically prepared
in sterile double-distilled water. Media for fungal growth
(PDAmb) was prepared with deionized distilled water,
Difco potato dextrose broth (12 g lÿ1), Sigma D,L-methio-
nine (0
05 g lÿ1), biotin (2 mg lÿ1), low melting agarose (20
g lÿ1) and adjusted to pH 5
2 (before the addition of the
agarose). Aliquots (20 ml) of peptide solution were asepti-
cally placed in Corning disposable sterile polystyrene
ELISA plates (96-well, high binding) which had been pre-
treated with bovine serum albumin (BSA, Sigma) by rin-
sing each well with 200 ml of a 1
0-mg mlÿ1 BSA solution.
Sterilized media (80 ml) was added to each well to make a
100-ml volume and allowed to solidify prior to inoculation
with 10 ml of conidial or bacterial cell suspension.
Inoculated microtitre plates were incubated in ambient
light at room temperature in a moist chamber (plastic box
containing wet paper towels and covered with clear plastic
wrap). Plates were scored for growth by looking for myce-
lium for the fungi or colonies for the bacteria at 6 d. The
plates were then photographed.
The MIC of the fungi and bacteria was the lowest pep-
tide concentration that totally prevented growth. All tests
were repeated four or more times. Controls without pep-
tides were included with all tests. The highest concentra-
tion tested was 250 mmol lÿ1 except where noted. If
germination or bacterial growth occurred at this concentra-
tion, the MIC is yet undetermined, but it is known to be
greater than 250 mmol lÿ1.
Peptide haemolytic activity
Three millilitres of human blood cells (Carolina Biological
Supply, Burlington, NC, USA) were gently mixed, poured
into a sterile 15 ml polystyrene screw-cap tube and centri-
fuged 5 min, 850 g. The supernatant was poured off and
the viscous pellet washed three additional times with 5 ml
of chilled (4  C) sterile isotonic phosphate-buffered saline
(PBS) solution (amounts g lÿ1: NaCl, 8; KH2PO4, 0
2;
Na2HPO4, 1
2; and KCl, 0
2, adjusted to pH 7
4, mixed
for 60 min to stabilize pH). The washed cells were sus-
pended in a ®nal volume of 20 ml chilled, sterile PBS and
the cells counted on a haemacytometer (Fisher ultra plane,
Neubauer ruling). The blood cell suspension was main-
 ANTIMICROBIAL PEPTIDES 165

tained on wet ice and diluted with sterile PBS to 7
068 
108 cells mlÿ1 for each assay. Aliquots of 20 ml of peptide
were aseptically placed into 2
0 ml microfuge tubes. For
each assay, 0
1% Triton X-100 was the positive, 100%
lytic control and PBS was the negative, background (0%
lysis) control. Aliquots of 180 ml diluted blood cell suspen-
sion were aseptically placed into each 2-ml tube and gently
mixed three times with a wide mouth pipette tip. The pep-
tide concentration tested was 250 mmol lÿ1. Tubes were
incubated for 35 min at 37  C with agitation (80 rev minÿ1).
Immediately following incubation, the tubes were placed
on ice for 5 min then centrifuged for 5 min at 1310 g.
Aliquots of 100 ml of supernatant were carefully collected,
placed into a sterile 1
5 ml microfuge tube, and diluted
with 900 ml chilled, sterile PBS. All tubes were maintained
on wet ice after dilution. Absorbance at 576 nm was mea-
sured on a Bausch and Lomb Spectronic 1001 spectrophot-
ometer using a quartz cuvette. Three replicates were run
for each peptide per assay. Two to ®ve assays were per-
formed for each peptide.
Statistical analyses
Data were analysed with SAS1 software (SAS, Cary, NC,
USA; 1994) using the general linear models (GLM) proce-
dure. A multivariate analysis of variance (MANOVA) model
with main effects was used. Tukey's Studentized Range
Test (alpha ˆ 0
01) was performed on all main effect
means generated from minimum inhibitory concentration
data and haemolysis data. Means were compared among
replicates within each peptide treatment (within treatment
variance) and among means between peptide treatments
(between treatment variance). Tukey's Studentized Range
Test controls the maximum experiment-wise error rate and
is applicable to unequal sample sizes.
RESULTS
To determine if size effects the ESF peptide designs, pep-
tides were synthesized containing 22 (ESF22A and B), 20
(ESF1B), 15 (ESF15), 13 (ESF13A) and nine (ESF9A)
amino acids (Table 1). These were compared with ESF12
(18 amino acids), ESF17 (17 amino acids), and a control
(Ala8,13,18) magainin 2 which have previously been studied
(Powell et al. 1995). MICs were determined as described in
the methods section and reported as an average of four or
more repeats (Table 2). Average MICs within a column
containing the same superscript letter were not signi®cantly
different as determined by the Tukey's Studentized Range
Test. Haemolytic bioassays (Table 3) were performed with
human red blood cells and report the average lysis with
respect to the triton X-100 control.
The results of the MIC bioassays indicate a signi®cant
loss of antimicrobial activity when reducing the ESF pep-
tide size from 18 to 17 amino acids for all four of the plant
pathogens tested. Interestingly, antimicrobial activity of the
15 amino acid derivative was signi®cantly higher than that
of the 17 amino acid form, but still signi®cantly lower than
the 18 amino acid form. The one exception to these results
was with the bacterial assay using Ps. syringae, in which the
15 amino acid form's MIC was not statistically different
than the 18 amino acid form. In this assay it was still more
active than the 17 amino acid peptide. The only difference
between ESF17 and ESF15 is the loss of an alanine from

Table 1 Antimicrobial peptides

Peptide AA sequence a* charge H{

ESF peptides
ESF22A SRAAGLAARLARLALRELRYAQ 0
56 ‡ 3
91 ÿ 0
12
ESF22B SRAAGLARRLARLARRELRYAQ 0
68 ‡ 5
91 ÿ 0
42
ESF1B MVSRAAGLAARLARLALRAL 0
56 ‡ 3
91 ‡ 0
07
ESF6 MAARAAGLAARLAALALRAL 0
41 ‡ 2
91 ‡ 0
24
ESF5 MASRAAGLARRLARLARRAL 0
67 ‡ 5
91 ÿ 0
25
ESF12 MASRAAGLAARLARLALR 0
53 ‡ 3
91 ‡ 0
01
ESF17 ASRAAGLAARLARLALA 0
45 ‡ 2
91 ‡ 0
10
ESF15 SRAAGLAARLARLAL 0
47 ‡ 2
91 ‡ 0
06
ESF13A SRAAGLAARLARL 0
46 ‡ 2
91 ÿ 0
01
ESF9A SRLARLAAR 0
62 ‡ 2
91 ÿ 0
23
Control peptide
(Ala8,13,18) magainin 2 GIGKFLHAAKKFAKAFVAEIMNS 0
46 ‡ 3
07 ‡ 0
20

*Alpha moment, {hydrophobicity.

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166 W . A . P O W E L L E T A L .

Table 2 Average minimum inhibitory concentration (MIC), in micromoles per litre (mmol lÿ1), of ESF peptides and control for four plant
pathogens, Fusarium oxysporum, Cryphonectria parasitica, Agrobacterium tumefaciens and Pseudomonas syringae. The same superscript letter
within a column indicates no signi®cant difference in the average MIC as determined by Tukey's Studentized Range Test

Fusarium Cryphonectria Agrobacterium Pseudomonas

Peptide No. AA oxysporum parasitica tumefaciens syringae

ESF22A 22 2
8d 11b 6
3c,d 88a,b

ESF22B 22 5
0c,d 13b 3
8d 50a,b
ESF1B 20 1
9d 6
3b 3
1d 100a,b
ESF12 18 7
9c,d 8
8b 25c 150a,b
ESF17* 17 > 100 > 100 > 100 > 100
ESF15* 15 35a 36a 50b 23b
ESF13A* 13 > 100 > 100 > 100 > 100
ESF9A* 9 > 100 > 100 > 100 > 100
(Ala8,13,18) magainin 2 23 13b,c 13b 100a 250a
r2 0
977 0
949 0
983 0
847

*Highest concentration tested for these peptides was 100 mmol lÿ1 instead of 250 mmol lÿ1 used for the rest of the peptides.
a,b,c,d
Average MICs within a column containing the same superscript letter were not signi®cantly different as determined by the Tukey's
Studentized Range Test.

the amino and carboxyl ends of the ESF17 peptide to form that the smaller peptide's activity could be due to a differ-
ESF15 (Table 1). The decrease in activity of ESF17 and ent mechanism as has been reported for other synthetic
regaining of some activity in ESF15 as the size suggests peptides (Bessalle et al. 1993). There was little difference
in the MICs of the peptides varying in size between 18 and
22 amino acids, with the exception of ESF12 in the Ag.
tumefaciens bioassays where ESF12 was signi®cantly less
Table 3 Haemolytic activity, as a percentage of haemolysis caused active (Table 2). The relative consistency of antimicrobial
by 0
1% triton X-100, for 250 mmol lÿ1 of ESF peptides and activity among the different peptide sequences indicate
controls. The same superscript letter within the haemolysis
some ¯exibility in design within this size range without
column indicates no signi®cant difference in the average MIC as
determined by Tukey's Studentized Range Test loss of antimicrobial activity.
All the ESF peptides which were 18 amino acids in
Peptide No. AA #Leu or Val Haemolysis* length or larger had similar MICs as the (Ala8,13,18) magai-
nin 2 (Chen et al. 1988) control in the fungal and Ps. syrin-
ESF22A 22 5 70b gae bioassays. In the Ag. tumefaciens bioassays, the ESF
ESF22B 22 4 20c peptides were signi®cantly more inhibitory than (Ala8,13,18)
ESF1B 20 6 73b magainin 2. ESF15 was less active than (Ala8,13,18) magai-
ESF6 20 5 16c,d
nin 2 in the fungal bioassays but more active than
ESF5 20 4 4
7e,f
ESF12 18 4 3
1f
(Ala8,13,18) magainin 2 in the bacterial bioassays.
ESF17 17 4 1
3f Differences in peptide size did cause a statistically signif-
ESF15 15 4 0
93f icant change in haemolytic activity (Table 3). ESF peptides
ESF13A 13 3 1
2f of 18 amino acids or less were not signi®cantly different in
ESF9A 9 2 0
75f haemolytic activity than the PBS buffer control. ESF pep-
(Ala8,13,18) magainin 2 23 N/A 10d,e tides of 20 amino acids or larger, including the (Ala8,13,18)
PBS buffer 0
32f magainin 2 control, were signi®cantly more haemolytic
0
1% Triton-X 100 100a than the PBS buffer control. Therefore, size effects haemo-
lytic activity in general. However, by examining other dif-
* Tukey's Studentized Range Test grouping (A±F) of lysis by
peptide, r2 ˆ 0
997. ferences among ESF1B, ESF6 and ESF5, which were all
a,b,c,d
Average MICs within a column containing the same 20 amino acids in length, other in¯uencing factors could be
superscript letter were not signi®cantly different as determined by discerned. The most consistent factor among these three
the Tukey's Studentized Range Test. peptides was the number of highly hydrophobic amino
= 2000 The Society for Applied Microbiology, Letters in Applied Microbiology, 31, 163ÿ168

acids in the design. By manipulating these amino acid
sequences larger peptides can be designed with lower hae-
molytic activities than smaller peptides, as demonstrated by
comparing ESF22B and ESF1B (Table 3).
ESF22A and ESF22B were designed to contain amino
acid residues of the TEV NIa protease's recognition
sequence that would be retained by the resulting peptide if
digested with this protease. The antimicrobial activity of
both these peptides grouped with the most active peptides
tested (Table 2). ESF22A had a signi®cantly higher haemo-
lytic activity than ESF22B.
DISCUSSION
The primary hypothesis tested was that the residual amino
acids retained on a self-processed peptide could be incorpo-
rated into a peptide design so that it retains a high antimi-
crobial activity yet poses a low haemolytic activity.
Consideration of both these activities is important to target
the peptides to microbial pathogens while having a minimal
effect on higher organisms that might consume plant pro-
ducts containing these peptides. In testing this hypothesis,
we ®rst determined the lower size limit of active ESF pep-
tide before adding the residual amino acid sequences which
would be left after cleavage by the self-processing NIa pro-
tease system from TEV (Carrington and Dougherty 1988;
Dougherty et al. 1988). The lower size limit for antimicro-
bial activity of the ESF peptide design was determined to
be 18 amino acids. The exception was ESF15, which had
measurable antimicrobial activity but was only 15 amino
acids in length. The antimicrobial activity of ESF15 is
likely due to a different mechanism than the larger ESF
peptides. Changes in activity due to size reductions have
been reported with other synthetic peptide designs
(Bessalle et al. 1993). However, there were no signi®cant
structural differences in ESF15 predicted using the
Garnier-Robson and Chou-Fasman structural analysis
functions of Protean computer software (DNASTAR).
After determining a minimal size that would retain anti-
microbial activity, a peptide sequence was designed that
would prevent amino terminal methionine cleavage during
peptide expression in transgenic plants. ESF1B was
designed by replacing the alanine at position two in ESF1
(Powell et al. 1995), with a valine (Table 1). This change
did not signi®cantly change the MIC as compared with the
other most active ESF peptides in this study (Table 2).
However, ESF1B did have one of the highest haemolytic
activities of the peptides tested (Table 3). The haemolytic
activity of ESF1B was compared with two other 20-amino
acid peptides, ESF5 and ESF6. ESF1B had the highest
haemolytic activity, followed by ESF6 and ESF5. The
hydrophobicity, alpha moment or overall charge (Table 1)
of the three peptides does not appear to be associated with
= 2000 The Society for Applied Microbiology, Letters in Applied Microbiology, 31, 163ÿ168
ANTIMICROBIAL PEPTIDES 167
these haemolytic differences. The only discernible differ-
ence in these three peptide designs that follow the change
in haemolytic activity was the total number of highly
hydrophobic residues. ESF1B has six (1 Val, 5 Leu) as
compared with ESF6 that has ®ve (5 Leu) and ESF5
which has four (4 Leu). In other studies of synthetic anti-
microbial peptides, increases in hydrophobicity have been
associated with increases in haemolytic activity (Dathe et
al. 1997; Weiprecht et al. 1997). However, our data show
that in some cases a more hydrophobic peptide of equal
size can be less haemolytic. For example, ESF6 is more
hydrophobic than ESF1B, yet it is less haemolytic. ESF6 is
more hydrophobic because it contains more alanines (H ˆ
0
62) in place of some hydrophilic amino acids present in
ESF1B. But ESF1B has a greater number of highly hydro-
phobic amino acids such as valine (H ˆ 1
10) even though
its overall hydrophobicity is less then ESF6. Therefore,
from these results we ®nd it is more important to limit the
number of highly hydrophobic amino acids than the overall
hydrophobicity when trying to reduce haemolytic activity
in antimicrobial peptide designs.
ESF1B had a relatively high haemolytic activity (73% as
lytic as the 0
1% triton X-100 control) when tested at a
concentration of 250 mmol lÿ1. However, when the concen-
tration of ESF1B was reduced to 25 mmol lÿ1, the relative
haemolytic activity was not signi®cantly different from
PBS buffer control. This concentration (25 mmol lÿ1) is
approximately a magnitude higher than ESF1B's average
MIC (1
9±6
3 mmol lÿ1 for the fungal pathogens) and
therefore indicates this might still be a useful antimicrobial
peptide in some applications.
To employ antimicrobial peptides in self-processing
polypeptide systems in plants, they must be designed to be
active despite containing residual amino acids left by the
processing reaction. As a model system, we chose to design
peptides containing residues from NIa protease recognition
site Glu-X-X-Tyr-X-Gln/(Gly or Ser)(Carrington and
Dougherty 1988; Dougherty et al. 1988). This recognition
sequence has the advantage of placing a glutamic acid on
the hydrophobic side of the amphipathic alpha helix, simi-
lar to the natural magainins. This con®guration has been
suggested to in¯uence the magainin's differential lytic
activity between animal and bacterial cells, possibly due to
interaction with cholesterol (Tytler et al. 1995). Our ®rst
peptide design to incorporate these residues was ESF22A
(Table 1). The design incorporated all the amino acids of
ESF12 except the amino terminal methionine and alanine.
In addition, amino acids were added to the carboxyl end
that ®t the recognition site pattern while still maintaining
the amphipathic alpha helical structure of the overall pep-
tide. ESF22A retained a high antimicrobial activity (Table
2), indicating that a NIa protease recognition site could be
incorporated with no signi®cant effect on this activity.
168 W . A . P O W E L L E T A L .
However, the haemolytic activity of ESF22 was among the
highest activities of the peptides tested in this study
(Table 3). To reduce this activity, ESF22B (Table 1) was
designed by changing the eighth and ®fteenth amino acids
from an alanine and leucine, respectively, to arginines.
This increased the positive charge, decreased the overall
hydrophobicity, and lowered the number of highly hydro-
phobic amino acids. With these changes, ESF22B retained
its antimicrobial activity to all of the plant pathogens tested
and its haemolytic activity decreased signi®cantly (Tables 2
and 3). This demonstrated that if a new peptide design has
a high haemolytic activity, small changes in its sequence
could be made to reduce the haemolytic activity without
compromising its antimicrobial activity.
From these experiments, we conclude that small antimi-
crobial peptides can be designed that incorporate self-pro-
cessing recognition site residues. When designing these
peptides, there is ¯exibility in the size and amino acid
sequence used with respect to antimicrobial activity.
However, to minimize haemolytic activity, the size and
number of highly hydrophobic amino acids should be kept
to the minimum needed to maintain antimicrobial activity.
Other factors, as yet unidenti®ed, must also contribute to
the haemolytic activity because the control peptide
(Ala8,13,18) magainin 2, had a longer amino acid sequence
and also had more highly hydrophobic amino acids than
the ESF peptides and yet had only a moderate haemolytic
activity. These factors could not be identi®ed in this study
due to many differences in the amino acid sequence. Our
next step is to design a gene encoding an antimicrobial
peptide precursor using the ESF22 peptide design and test
its ability to be processed in vitro and in vivo by the TEV
NIa protease.
ACKNOWLEDGEMENTS
This research was funded in part by the USDA
Cooperative State Research Service, McIntire-Stennis
Program, and the New York Chapter of the American
Chestnut Foundation.
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