www.fems-microbiology.org

FEMS Microbiology Letters 243 (2005) 205–210

The mode of action of the plant antimicrobial peptideMiAMP1 differs from that of its structural homologue,

the yeast killer toxin WmKT

Camilla Stephens a,*, Kemal Kazan b, Ken C. Goulter a,Donald J. Maclean a, John M. Manners b

a Cooperative Research Centre for Tropical Plant Protection, University of Queensland, Brisbane, Qld 4072, Australiab Commonwealth Scientific Industrial Research Organisation (CSIRO) Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road,

St. Lucia, Brisbane, Qld 4067, Australia

Received 11 August 2004; received in revised form 10 November 2004; accepted 8 December 2004

First published online 16 December 2004

Edited by Dr. L.F. Bisson

Abstract

The plant antimicrobial peptide MiAMP1 from Macadamia integrifolia and the yeast killer toxin peptide WmKT from Williopsis

mrakii are structural homologues. Comparative studies of yeast mutants were performed to test their sensitivity to these two anti-

microbial peptides. No differences in susceptibility to MiAMP1 were detected between wild-type and several WmKT-resistant

mutant yeast strains. A yeast mutant MT1, resistant to MiAMP1 but unaffected in its susceptibility to plant defensins and hydrogen

peroxide, also did not show enhanced tolerance towards WmKT. It is therefore probable that the Greek key b-barrel structureshared by MiAMP1 and WmKT provides a robust structural framework ensuring stability for the two proteins but that the specific

action of the peptides depends on other motifs.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Macadamia integrifolia; Williopsis mrakii; Antimicrobial peptide; Mode of action

1. Introduction

MiAMP1 is a novel antimicrobial peptide (AMP)

found in the nut kernels of Macadamia integrifolia [1].

It consists of 76 amino acids, including six cysteine resi-

dues engaged in three intramolecular disulphide bridges,

and has an estimated isoelectric point of 10.1 [1]. The cys-teine residue spacing in MiAMP1 does not match that of

any described antimicrobial peptide, andMiAMP1 is the

0378-1097/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2004.12.007

* Corresponding author. Mailing address: Plant Genetic Engineer-

ing Laboratory, Level 4 John Hines Bldg., University of Queensland,

St Lucia, Qld 4072, Australia. Tel.: +61 7 33659172; fax: +61 7

33651699.

E-mail address: camilla.stephens@uq.edu.au (C. Stephens).

first functionally characterized member of a new class of

plant defence proteins. Other plant sequence homo-

logues in this class include a protein found in blister

rust-resistant genotypes of western white pine, Pinus

monticola [2] and deduced proteins from pathogen-in-

duced genes in Scots pine, Pinus sylvestris [3]. MiAMP1

has been shown to be highly inhibitory to a wide range ofphytopathogens, but has no affect on the growth of plant

and mammalian cells [1]. In addition, transgenic expres-

sion of MiAMP1 in canola, Brassica napus L. provided

enhanced resistance against blackleg disease caused by

the fungus Leptosphaeria maculans [4]. Therefore,

MiAMP1 is potentially a useful tool for genetic engineer-

ing of disease resistance in crop plants.

. Published by Elsevier B.V. All rights reserved.

206 C. Stephens et al. / FEMS Microbiology Letters 243 (2005) 205–210

The three-dimensional structure of MiAMP1 was

determined to consist of eight b-strands arranged in

two Greek key motifs, each containing four antiparallel

b-sheets, that form a Greek key b-barrel [5]. This struc-ture is unique amongst plant antimicrobial peptides, but

corresponds to the single domain bc-crystallin precursorfold found in a number of diverse proteins including

some with antimicrobial activity [6–10]. The structure

of MiAMP1 shows particularly strong resemblance to

that of theWilliopsis mrakii (formerly known asHansen-

ula mrakii) yeast killer toxin, WmKT (formerly known

as HM-1 toxin) [5,6]. McManus et al. [5] noted that

not only do MiAMP1 and WmKT share a common

structural framework (see Fig. 9 in [5]), they also bothhave two similarly located large protein loops that are

tethered to the b-sheets via disulphide bonds. These dis-

tinct structural features, common to antimicrobial pep-

tides in different phyla, led these authors [5] to

speculate that the three-dimensional structural conser-

vation may indicate a direct role in the mode of action

of the peptides. The three-dimensional structural homol-

ogy and similar antimicrobial function is remarkable inthat MiAMP1 (NCBI Accession # P80915) and WmKT

(NCBI Accession # P10410) share no significant protein

sequence homology and even differ both in the number

of cysteine residues they contain and their respective

spacing. Cysteine composition and spacing are often

conserved in structurally related proteins.

The WmKT toxin is believed to interfere with cell

wall biosynthesis in susceptible yeast cells by inhibitingb-(1,3)-glucan synthesis [11–16] while the mode of action

of MiAMP1 is unknown. In this study we have adopted

an approach using yeast mutants with respective resis-

tance to WmKT and MiAMP1 to investigate whether

these two structurally related peptides may share the

same antifungal mode of action.

2. Materials and methods

2.1. Yeast Strains

The Saccharomyces cerevisiae strains used in this

study are described in Table 1. Initially, twelve yeast

Table 1

Yeast strains used in this study

Strain Description

BJ1824 MAT a ura3 trp1 leu2 pep4

TK201 rhk1D:::URA3 in BJ1824

rhk2 WmKT-resistant mutant derived from BJ1824

rhk3 WmKT-resistant mutant derived from BJ1824

COP161 MAT a ade1 lys1 ura3

MT1 MiAMP1-resistant mutant derived from COP1

BY4741 MAT a his3 D1 leu2 D0 met15 D0 ura3 D04007 ipt1D:::kanMX4 in BY4741

strains commonly used in laboratory research were

screened for their sensitivity to MiAMP1 and sensitivi-

ties expressed as IC50 values (peptide concentration re-

quired to inhibit 50% cell growth) ranged from 5 to

>100 lg/mL (0.6 to >12.3 lM) [17]. The strain

COP161 [18] was the most sensitive and was used to de-rive a mutant resistant to MiAMP1. Subsequently,

WmKT-resistant yeast mutants TK201, rhk2, rhk3 and

wild-type strain BJ1824 were obtained from Dr. T.

Komiyama (Niigata College of Pharmacy, Niigata, Ja-

pan) while yeast deletion mutant 4007 (Invitrogen Inc.)

was derived from the wild-type strain BY4741 [19].

Coincidently, the BJ1824 and BY4741 wild-type strains

had IC50 values of approximately 5 lg/mL (0.6 lM) forMiAMP1 and were therefore comparable to COP161 in

their sensitivity to this peptide.

2.2. Protein samples and in vitro bioassays

The WmKT peptide from W. mrakii was kindly pro-

vided by Dr. T. Komiyama (Niigata College of Phar-

macy, Niigata, Japan). The DmAMP1 peptide fromDahlia merckii [20] was kindly provided by Dr. B. Cam-

mue (Catholic University of Leuven, Belgium). The

plant defensin, CtAMP1 [20] was obtained by microbial

expression, using the vector pPIC9K (Invitrogen Inc.) in

Pichia pastoris and purified by standard protein purifica-

tion procedures. MiAMP1 was purified from M. integri-

folia nut kernels as previously described [1]. Peptide

purity was verified using gel electrophoresis and massspectrometry. Antimicrobial activity of protein samples

against S. cerevisiae was quantitatively assayed by spec-

trophotometry of liquid cultures grown in microtiter

plates as described previously [1]. Cell suspensions were

grown at 30 �C in SD (Synthetic Defined) growth med-

ium [0.8 g/l CSM (Complete Supplement Mix; Bio101/

26.7 g/l DOB (Drop Out Base; Bio101) with glucose]

to logarithmic growth phase and then diluted to a con-centration of 105 cells/mL determined by cell counts

using a haemocytometer and light microscopy. Cell

growth in the presence and absence of specified antimi-

crobial peptides or hydrogen peroxide was measured

spectrophotometrically at 24 h intervals over a 96-h time

period. All experiments were repeated independently at

Mutated ORF Source

– [16]

Ybl082c [16]

Ygl022w [15,16]

Unknown [15,16]

– [18]

61 Unknown This study

– [19]

Ydr072c [19]

C. Stephens et al. / FEMS Microbiology Letters 243 (2005) 205–210 207

least twice and means and standard errors from 2–3 rep-

licates are shown.

0

20

40

60

80

100

0 20 40 60 80 10

Gro

wth

in

hib

itio

n (

%)

0

COP161

MT1

MiAMP1 concentration (µg/ml)

Fig. 2. Growth inhibition of S. cerevisiae strains COP161 and MT1 in

the presence of MiAMP1. Yeast cells were grown in SD growth

medium supplemented with MiAMP1 for 48 h. Each value ± standard

error is shown.

3. Results and discussion

3.1. Effect of MiAMP1 on WmKT-resistant S. cerevisiae

mutants

The effects of purified MiAMP1 and WmKT on

wild-type S. cerevisiae strain BJ1824 and three

WmKT-resistant yeast mutant strains were tested.

The yeast mutants, termed TK201, rhk2 and rhk3,

were all derived from the parental strain BJ1824[15,16]. No differences in growth rate were detected be-

tween the TK201, rhk2 and rhk3 mutants and the wild-

type strain in plain SD growth medium (data not

shown).

Growth of the BJ1824 (wild-type) strain was strongly

inhibited by WmKT, with an IC50 value of less than 1

lg/mL (0.1 lM, Fig. 1(a)). The three mutant strains

showed varying degrees of increased resistance towardsWmKT, confirming previous findings [15]. The TK201

mutant appeared to be almost unaffected by the pres-

ence of up to 5 lg/mL (0.6 lM) of WmKT (Fig. 1(a)).

MiAMP1 displayed weaker inhibitory activity than

WmKT against the BJ1824 in terms of the peptide con-

centration required to inhibit cellular growth. As the

molecular masses of the mature MiAMP1 and WmKT

peptides are comparable (8138 Da and 9528 Da, respec-tively) these results demonstrate that MiAMP1 is less

potent than WmKT. Nevertheless, strong growth inhibi-

tion of the wild-type strain was detected in the presence

of MiAMP1, with an IC50 value of approximately 5 lg/mL (0.6 lM, Fig. 1(b)). No significant difference in

MiAMP1 susceptibility was detected between any of

the mutant strains and wild-type BJ1824 cells (Fig.

1(b)). The similar growth response of wild-type andWmKT-resistant mutant cells in the presence of

0

20

40

60

80

100

0 1 2 3 4 5

Gro

wth

in

hib

itio

n (

%)

BJ1824

TK201

rhk2

rhk3

WmKT concentration (µg/ml) (a) (

Fig. 1. Growth inhibition in S. cerevisiae strains by MiAMP1 and WmKT. Y

or (b) MiAMP1 for 48 h. Bars indicate ± standard error. A concentration

respectively.

MiAMP1 implies that the mode of action of MiAMP1

differs from that of WmKT and involves other targets

in yeast.

3.2. Isolation and characterisation of a MiAMP1-

resistant S. cerevisiae mutant

The S. cerevisiae strain COP161 [18] is highly sensi-

tive to MiAMP1 and was used for studies of its mode

of action. The IC50 value of MiAMP1 for COP161

was determined by in vitro bioassays to be approxi-

mately 5 lg/mL (0.6 lM) after 48 h incubation (Fig.

2). A spontaneous yeast mutant, termed MT1, was iso-

lated from COP161 cells grown on solid growth mediumsupplemented with 100 lg/mL (12.3 lM) MiAMP1.

DNA fingerprinting showed that this strain was derived

from COP161 (data not shown). The mutant showed a

highly significant increase in MiAMP1 tolerance com-

pared to the wild-type strain (Fig. 2). A 10-fold increase

in IC50 value was detected for the MT1 mutant after 48

h incubation with MiAMP1 compared to that of the

wild-type COP161 strain. No difference in growth rate

0

20

40

60

80

100

0 5 10 15 20 25

Gro

wth

in

hib

itio

n (

%)

BJ1824

TK201

rhk2

rhk3

MiAMP1 concentration (µg/ml) b)

east strains were grown in SD medium supplemented with (a) WmKT

of 1 lg/mL is equal to 105 and 123 nm of WmKT and MiAMP1,

208 C. Stephens et al. / FEMS Microbiology Letters 243 (2005) 205–210

was detected between the MT1 mutant and the COP161

wild-type strain in the absence of MiAMP1 (data not

shown).

To test whether the MT1 mutant had generally in-

creased tolerance to abiotic stresses we tested its re-

sponse to oxidative stress using hydrogen peroxide. Nodifference in the sensitivity to hydrogen peroxide was

observed between MT1 and the wild-type COP161

strain (Fig. 3(a)). To examine whether MT1 showed en-

hanced resistance to cytocidal peptides structurally

unrelated to MiAMP1, the MT1 mutant was also as-

sayed against the plant defensins, DmAMP1 and

CtAMP1 [20]. In contrast to its resistance to MiAMP1

(Fig. 2), the MT1 mutant did not show any enhancedresistance to the plant defensins (Fig. 3(b) and (c)).

Interestingly, MT1 was found to be more susceptible

to DmAMP1 than the wild-type strain whereas there

was no difference in susceptibility to CtAMP1 between

the wild-type and the mutant strain (Fig. 3(b) and (c)).

To further test whether there was any relationship be-

0

20

40

60

80

100

0 10 20

Gro

wth

in

hib

itio

n(%

)

COP161

MT1

H2O2 concentration (µM)

0

20

40

60

80

100

0 5 10 15 20 25

Gro

wth

inh

ibit

ion

(%

)

COP161

MT1

CtAMP1 concentration (µg/ml)

(a)

(c)

Fig. 3. Growth inhibition in S. cerevisiae wild-type and mutant strains b

(COP161) and mutant (MT1) strains were grown in SD growth medium suppl

WmKT for 48 h. Each value ± standard error is shown. 1 lg/mL of DmAMP

105 nm.

tween the mode of action of plant defensins that inhibit

S. cerevisiae and the action of MiAMP1 we also exam-

ined the effect of MiAMP1 on a yeast deletion mutant

(Table 1) that has enhanced resistance to DmAMP1

[21,22]. Sensitivity to DmAMP1 is determined by the

IPT1 gene that encodes an inositol transferase involvedin the final step of synthesis of the sphingolipid manno-

syldiinositolphosphorylceramide [21,22]. A yeast dele-

tion mutant for the IPT1 gene and the wild-type strain

had IC50 values of 4.2 ± 0.1 lg/mL (0.5 lM) and

3.8 ± 0.3 lg/mL (0.5 lM), respectively, for MiAMP1

and therefore the IPT1 gene and this sphingolipid class

are not involved in MiAMP1 action.

These results suggest that MT1 does not have en-hanced resistance towards oxidative stress and other

cytocidal peptides in general, but that the mutation in

MT1 probably affects MiAMP1 tolerance specifically.

Therefore, an experiment was conducted to examine

whether the enhanced resistance of MT1 for MiAMP1

also corresponded to resistance to WmKT. Importantly,

0

20

40

60

80

100

0 5 10 15 20

Gro

wth

in

hib

itio

n(%

)

COP161

MT1

DmAMP1 concentration (µg/ml)

0

20

40

60

80

100

0 2 4 6

Gro

wth

in

hib

itio

n (

%)

COP161

MT1

WmKT concentration (µg/ml)

(b)

(d)

y hydrogen peroxide, DmAMP1, CtAMP1 and WmKT. Wild-type

emented with (a) hydrogen peroxide, (b) DmAMP1, (c) CtAMP1 or (d)

1 and CtAMP1 is equivalent to 200 nM and 1 lg/mL of WmKT equals

C. Stephens et al. / FEMS Microbiology Letters 243 (2005) 205–210 209

the MT1 mutant showed no significant difference in sus-

ceptibility to WmKT compared to the wild-type strain,

COP161 (Fig. 3(d)), further substantiating the notion

that MiAMP1 and WmKT differ in their mode of

action.

4. Conclusion

Extensive studies on the cytocidal effects of WmKT

have demonstrated that WmKT perturbs cell wall bio-

synthesis in sensitive yeast, by inhibiting b-glucan syn-

thesis at budding sites or conjugation tubes, which

leads to cell lysis [13,14,16]. Based on the resemblancein globular folding between MiAMP1 and WmKT [5]

it was hypothesized that MiAMP1 and WmKT may

have functional similarities. However, no increase in

resistance towards MiAMP1 was detected in any of

three WmKT-resistant yeast mutant strains. Two of

the mutant yeast strains, TK201 and rhk2, are defective

in different parts of protein N-glycosylation, resulting in

underglycosylation. This is presumed to cause altera-tions in cell wall or mannoprotein properties that may

act as receptors. Modification of such putative receptors

is thought to prevent or impede WmKT from binding to

the cell and interfering with b-(1,3)-glucan synthesis [16].

The mutation in the third yeast mutant, rhk3, is cur-

rently unknown. The MT1 mutant with increased resis-

tance to MiAMP1, isolated in the present study, also did

not show increased resistance to WmKT. The observa-tion that mutations for resistance to WmKT have no ef-

fect on sensitivity to MiAMP1 suggests that there is little

functional similarity in the mode of action of WmKT

and MiAMP1. It is most probable that the structural

resemblance between MiAMP1 and WmKT is unrelated

to protein function and probably reflects a robust struc-

tural framework that provides stability for the two pro-

teins with the specific mode of action determined byspecific protein motifs. It is possible that the different re-

sponses of the yeast mutants to MiAMP1 and WmKT

may reflect altered receptor specificities for the two pep-

tides. Fungal receptors for AMPs are as yet unknown,

while receptors for some bacteriocin AMPs have been

identified in bacteria via mutational studies [23,24].

The screening of yeast deletion mutant libraries, now

established for studying the mode of action of chemicalinhibitors of yeast [25,26] represents a new and powerful

approach to identify key genes determining fungal sus-

ceptibility to antimicrobial peptides such as MiAMP1

and WmKT.

Acknowledgements

We thank Dr. T. Komiyama for provision of WmKT

and WmKT-resistant yeast strains and Dr. B. Cammue

for providing DmAMP1. This study was supported by

an Australian Postgraduate Award scholarship.

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