FEMS Microbiology Letters 228 (2003) 27^31

www.fems-microbiology.org

Isolation of a novel antimicrobial peptide gene (Sp-AMP)

homologue from Pinus sylvestris (Scots pine) following infection with
the root rot fungus Heterobasidion annosum
a;b;

Frederick O. Asiegbu , Woobong Choi b , Guosheng Li a , Jarmila Nahalkova a ,
Ralph A. Dean b
a
Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, Uppsala, Sweden
b
Fungal Genomics Laboratory, North Carolina State University, Raleigh, NC, USA

Received 31 May 2003 ; received in revised form 1 September 2003; accepted 11 September 2003

First published online 2 October 2003

Abstract

A new family of antimicrobial peptide homologues termed Sp-Amp has been discovered in Pinus sylvestris (Scots pine). This is the first
report of such proteins to be characterized in a conifer species. Sp-AMP1 was identified in a substructured cDNA library of root tissue
infected with the root rot fungus Heterobasidion annosum and encodes a mature peptide of 79 amino acid residues. Three additional
members of the Sp-AMP family (Sp-AMPs 2^4) encode cysteine-rich proteins of 105 amino acids, each containing an N-terminal region
with a probable cleavage signal sequence. Northern analysis confirmed that Sp-AMP expression is elevated in Scots pine roots upon
infection with H. annosum. These peptides share 64% amino acid identity with a mature protein from Macadamia integrifolia (MiAMP1),
which allowed us to build a homology model for preliminary analysis. Southern analyses further confirmed that several copies of the gene
are present in the Scots pine genome. The potential significance of Sp-AMP in the H. annosum^conifer pathosystem is discussed.
: 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords : Subtraction hybridization ; Heterobasidion annosum; Expressed sequence tag; Pinus sylvestris

1. Introduction activity in vitro indicates that they may serve a general

Forest trees like other plant species are exposed to
pathogens at every stage in their life cycle and appear to
use a variety of defenses to resist attack. To minimize
damage by pathogens, conifers have evolved a large array
of defense mechanisms that include preformed structural
barriers and antimicrobial chemicals (resins/phenolics/pep-
tides), activation of a battery of defenses (often called the
hypersensitive reaction) and intra-organismic responses re-
sulting in systemic induction of defense compounds [1^3].
Antimicrobial peptides (AMPs) have been detected in a
wide variety of agricultural plant species and have been
implicated in resistance of such plants to microbial infec-
tions [2,4]. The localization of antimicrobial peptides in a
wide array of plant tissues and their potent antimicrobial
* Corresponding author. Tel. : +46 (18) 67 15 98;
Fax : +46 (18) 67 35 99.
E-mail address : fred.asiegbu@mykopat.slu.se (F.O. Asiegbu).
0378-1097 / 03 / $22.00 : 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00697-9
protective role against plant pathogens. These peptides
are highly expressed both locally and systemically during
pathogen attack supporting their role in plant protection
[5].
The designation of all of these proteins as AMPs is
actually an oversimpli¢cation. While all do have antimi-
crobial activity, they fall into a number of di¡erent struc-
tural and functional categories [2]. Thionins and plant
defensins are two well-known subclasses found in many
di¡erent plants [6,7]. Other AMPs that have been isolated
include protease inhibitors [8], chitin-binding proteins [2]
and knottin-type peptides [9] among others. In recent
years, much attention has been focused on the potential
use of AMPs in the design of novel environmentally
friendly fungicides. They are also a possible source of
genes for engineering disease resistance in plants, which
could reduce the need for using additional chemical fungi-
cides [10].
One of the most damaging plant pathogens of conifers
is Heterobasidion annosum, the causative agent of root and

FEMSLE 11232 30-10-03

28 F.O. Asiegbu et al. / FEMS Microbiology Letters 228 (2003) 27^31
butt rot. Several conifer species (Norway spruce, Scots
pine, Douglas ¢r) serve as hosts to the three forms of
H. annosum, the S, P and F types, respectively. The fact
that these organisms are white rot fungi (i.e. they degrade
both lignin and cellulose components of wood) contributes
to their action as serious parasites and saprophytes able to
infect and destroy living conifer roots and stems of all
ages, as well as dead trees [3]. It has been estimated [11]
that root rot causes a direct annual loss of nearly E800
million in EU countries, of which Sweden alone accounted
for at least E35 million. When the indirect impacts of
economic losses are included, the loss to Swedish forestry
is even greater, estimated to lie in the range of E50^100
million per year. Pretreatment of stumps with chemicals or
with the biocontrol agent Phlebiopsis gigantea is presently
considered the only feasible means of controlling the dis-
ease in forest plantations. In recent years, the interest in
environmentally friendly alternatives to chemical pretreat-
ment has increased. For example, phenotypic selection for
resistance and breeding among recognized resistant trees is
feasible, but identi¢cation of useful resistance markers re-
mains a major challenge in breeding programs.
Recent analysis of gene expression in pine trees after
challenge with H. annosum led to the isolation and char-
acterization of a chitin-binding protein (Pinus nigra lectin,
PNL [12]). Here, we report the isolation and character-
ization of members of a gene family that are highly tran-
scribed in infected pine roots with very high sequence ho-
mology to a known AMP from Macadamia integrifolia [4].
This is the ¢rst report of AMPs of this type from a conifer
species, here designated Sp-AMP.
2. Materials and methods
2.1. Host material and pathogen isolate
Scots pine (Pinus sylvestris) seeds were purchased from
Assidomain AB, Sweden. H. annosum (FP5) was obtained
from K. Korhonen (Finland) and maintained on Hagem
agar at 20 N 1‡C. Mycelial of H. annosum used for inocu-
lation was obtained from cultures cultivated in liquid Ha-
gem medium (glucose 5 g l31 , NH4 NO3 0.5 g l31 , KH2 PO4
0.5 g l31 , MgSO4 0.5 g l31 , malt extract 5 g l31 ) for 10
days at static conditions.
2.2. Root rot inoculation
Seedlings of P. sylvestris (16 days old) were transferred
to sterile ¢lter paper pre-laid on 1% water agar in Petri
dishes. The root regions of the seedlings were inoculated
with clumps of mycelia without homogenization (about
0.1 g). For every three seedlings, one clump of mycelium
was added together with 0.5 ml of distilled water with a
total of 10 seedlings per 90 mm diameter plate. Control
seedlings were mock inoculated with sterile distilled water.
FEMSLE 11232 30-10-03
After inoculation, seedlings were exposed to a 16-h photo-
period and used for RNA extraction at time intervals in-
dicated in Section 3.
2.3. Subtractive hybridization and cDNA library
construction
Seedling root materials were used directly after harvest.
Total RNA was extracted from roots infected with H. an-
nosum at 6 days post inoculation (d.p.i.) and from un-
infected Scots pine seedling roots following the procedures
of Chang et al. [13]. cDNA was synthesized using a
SMART cDNA synthesis kit (Clontech, USA). Excess
cDNA from the uninfected seedling roots was used for
the subtraction in two rounds of hybridization, adapters
were then ligated followed by polymerase chain reaction
(PCR) ampli¢cation to enrich cDNA from infected roots
as described in the PCR select cDNA subtraction kit
(Clontech).
2.4. Storage of cDNA clones in microtiter plates and on
nylon membrane ¢lters
Recombinant cDNA clones (12 288) were stored in 384-
well microtiter plates and were also replicated onto Hy-
bond Nþ membranes (Amersham Pharmacia Biotech,
USA) using a Q-bot automated workstation (Genetix,
USA) as described by Zhu et al. [14].
2.5. Sequencing of cDNA clones encoding Sp-Amp gene
from subtractive library and sequence analyses
Plasmid DNA template puri¢cation was performed us-
ing 96-well format ¢lter plates from 2 ml Terri¢c Broth
cultures [14]. For DNA sequencing, each reaction was
performed with 2 Wl of Bigdye terminator chemistry (Per-
kin-Elmer, USA) in a 10 Wl reaction with T7 primer using
ABI Prism 3700 (96-well) capillary automated DNA se-
quencers. Nucleotide sequence and data analysis was per-
formed using a scheme developed at the Fungal Genomics
Laboratory, North Carolina State University, Raleigh,
NC, USA. Raw sequence ¢les were uploaded to a Sun
Ultrasparc UNIX server and read into Phrep and Phrap.
After deleting vector and contaminating sequence, cDNA
sequences were compared with GenBank database sequen-
ces using BlastX. Phylogenetic analysis was done with the
Jotun Hein method (DNASTAR [www.dnastar.com]
mega-align [version 4.03] expert sequence analysis soft-
ware) using data obtained from this study and from Gen-
Bank (Table 1).
2.6. Southern analyses
Genomic DNA (10 Wg) from Scots pine roots was di-
gested with HindIII, EcoRI or BamHI separated by elec-
trophoresis on an agarose gel and blotted onto a nylon
 F.O. Asiegbu et al. / FEMS Microbiology Letters 228 (2003) 27^31 29

Table 1
GenBank accession numbers of sequences used in this study
Gene Source Accession number
Sp-Amp1 P. sylvestris (Scots pine) AF410952 (this study)
Sp-Amp2 P. sylvestris (Scots pine) AF410953 (this study)
Sp-Amp3 P. sylvestris (Scots pine) AF410954 (this study)
Sp-Amp4 P. sylvestris (Scots pine) AF410955 (this study)
Amp1 M. integrifolia Y10903
Mi-Amp M. integrifolia ICO1_A
Defensin Drosophila XM 081000
Defensin Arabidopsis NM_101817
Defensin Mus musculus NM_010031

membrane (Hybond-Nþ ). The membrane was hybridized

with Sp-AMP1 as a probe.

2.7. Northern analysis Fig. 1. The Sp-Amp1 cDNA sequence and predicted amino acid se-
quence. The nucleotide sequence data have been deposited in GenBank.
For Northern hybridizations, 30 Wg of RNA samples
was electrophoresed on a 1.2% formaldehyde agarose
gel. The gel was transferred to Hybond-Nþ nylon mem- agreement with observations for the Macadamia AMP
branes according to the manufacturer’s instructions. Sp- (MiAMP).
AMP1 cDNA was labeled and used for hybridization ac-
cording to the AlkPhos direct labelling kit (Amersham 3.2. Sp-Amp1 is a member of the gene family
Pharmacia Biotech). Blots were hybridized at 55‡C and
post-hybridization stringency washes were done according Screening a macro-array nylon membrane ¢lter contain-
to the manufacturer’s instructions. Hybridization signals ing 12 288 clones from the subtraction library with the Sp-
were detected using CDP-Star chemiluminescence (Amer- AMP1 cDNA clone as a probe resulted in 81 strong pos-
sham Pharmacia Biotech). Nylon membrane ¢lters con- itive AMP clones representing 0.7% of genes in the sub-
taining 12 288 clones were treated similarly. traction cDNA library. Phrap clustering of sequences from
these clones resulted in four contigs with 93^97% nucleo-
tide sequence identity to each other suggesting the exis-
3. Results and discussion tence of a family of antimicrobial peptide genes in Scots
pine (Table 2). The sequences (Fig. 1) have been deposited
3.1. Identi¢cation of the AMP gene in GenBank (accession numbers AF410952, AF410953,
AF410954, AF410955). Alignment of full-length amino
Expression of AMPs is elevated when plants are ex- acid sequence of all four Sp-Amps revealed few amino
posed to biotic stresses such as microbial infections [4] acid di¡erences (Fig. 2). At the amino acid level Sp-
suggesting their importance in host defense reactions dur- Amp2 and Sp-Amp4 are identical (Fig. 2), the only di¡er-
ing pathogenic attack. However, despite the importance of ence being in the untranslated region of the sequence.
tree species to timber production, there has been no re-
search directed towards identifying and characterization of
AMP genes in conifer trees. Evaluation of host genes ob-
tained following subtractive hybridization [15^17] of Scots
pine root infected with H. annosum revealed a cDNA
clone with homology to AMP [4] (Fig. 1). Further BlastX
analysis of 200 randomly sequenced expressed sequence
tags from the subtraction library revealed additional
clones with homology to AMPs. Such redundancy sug-
gests a possible involvement in the host^parasite interac-
tion in the conifer pathosystem. This putative Scots pine
AMP designated Sp-AMP1 showed high sequence homol-
ogy (64% amino acid identity) to AMP in M. integrifolia
[4]. The Sp-AMP1 sequence contained an open reading Fig. 2. Polypeptide sequence comparison between Sp-Amp1^4 genes of
frame encoding a 105-amino acid polypeptide. The ¢rst Scots pine and Mi-Amp gene of M. integrifolia. Conserved regions are
26 amino acids revealed a predicted signal sequence, in highlighted in dark gray.

FEMSLE 11232 30-10-03

30 F.O. Asiegbu et al. / FEMS Microbiology Letters 228 (2003) 27^31
Table 2
Nucleotide sequence identities of Sp-Amp genes
A dendrogram was produced on the aligned amino acid
sequences of the Sp-AMPs used in this study and the
defensin genes from other sources (Fig. 3). The dendro-
gram con¢rms the close relationship to the two MiAMPs
and that other subclasses of AMPs such as plant, insect
and animal defensins are more distantly related.
3.3. Genomic Southern analysis
To determine the copy number of the Sp-AMP in the
Scots pine genome, the Sp-AMP1 cDNA probe was hy-
bridized to Scots pine genomic DNA digested with various
restriction endonucleases by Southern analysis (Fig. 4).
The presence of several intense and faint bands suggests
that Sp-AMP exists as several copies in the Scots pine
genome. A large number of copies likely indicates this
gene family confers a signi¢cant biological function(s).
Other authors have also reported high copy numbers of
functionally important genes in eukaryotic genomes [18].
3.4. Accumulation of the Sp-AMP mRNA in roots during
host^pathogen interaction
The expression pro¢le of Sp-AMP mRNA accumulation
in Scots pine seedlings inoculated with H. annosum was
examined in a time course experiment (Fig. 5). An increase
in accumulation of the Sp-AMP1 mRNA transcript was
apparent at 1 d.p.i. The transcript increased progressively
and accumulated to considerable levels by 7 d.p.i., there-
after levels remained high through the ¢nal time point (15
d.p.i.). Symptomatically, no visible evidence of infection
was apparent until 3^7 d.p.i., at which time necrosis of
roots was observed. Late stages of infection (15 d.p.i.)
coincided with the period of vascular colonization and
Fig. 3. A dendrogram representation of the sequence similarities be-
tween the di¡erent antimicrobial peptide genes and defensin genes from
plant and animal sources. The dendrogram was produced with the Jo-
tun Hein method using data obtained from this study and from Gen-
Bank (see Table 1).
FEMSLE 11232 30-10-03
Fig. 4. Southern blot analysis of the AMP gene in the Scots pine ge-
nome. A 10-Wg aliquot of genomic DNA was completely digested with
BamHI, EcoRI and HindIII, and electrophoresed through a 1% agarose
gel. The DNA was transferred onto a Hybond nylon membrane. AMP
gene PCR products used as probe were labeled and hybridized with the
AlkPhos direct labelling kit (Amersham Pharmacia Biotech).
disintegration of host cellular tissues by the pathogen as
documented by histochemical observation [19]. The en-
hanced accumulation of the gene transcript during the
necrosis formation stage strongly suggests a role for this
gene during disease development. Low, but detectable
transcript levels were also recorded in uninoculated con-
trol seedlings suggesting a low level of constitutive expres-
sion of the gene within healthy root tissues.
3.5. Potential role of Sp-AMP in the H. annosum^conifer
pathosystem
Based on results from Northern analyses, we reasoned
that the higher transcript levels of Sp-AMP in the infected
roots as compared to control could be an asset to the tree
when combating root pathogens such as H. annosum. Oth-
er authors have also demonstrated that overexpression of
this class of AMP (MiAMP) in canola plants was e¡ective
in conferring resistance against the fungal pathogen Lep-
tosphaeria maculans [20]. MiAMP is capable of inhibiting
the growth of a variety of fungi and Gram-positive bacte-
rial phytopathogens [4]. By analogy to other AMPs, the
structures of Sp-AMPs are expected to be based on the
extremely stable L-barrel fold found in MiAMP1 [21]. In
MiAMP, the residues important in the core structure and
disul¢de bonds are highly conserved. However, surface
Fig. 5. Northern blot analysis of AMP gene expression. Total RNAs
were extracted from various developmental stages of roots infected with
H. annosum and controls. A 30-Wg aliquot of total RNA was electro-
phoresed on 1.2% formaldehyde agarose gel. The RNA was transferred
onto a Hybond nylon membrane. PCR-ampli¢ed AMP cDNA gene
products were used as probe and hybridized with the AlkPhos direct la-
belling kit (Amersham Pharmacia Biotech). C1, C3, C7, C10, C15: con-
trols (days after inoculation with sterile water). S1, S3, S7, S10, S15: in-
fected samples (days after inoculation with H. annosum).
 F.O. Asiegbu et al. / FEMS Microbiology Letters 228 (2003) 27^31
loops that have been proposed to be involved in function
di¡er greatly between the proteins [21]. More recently, the
nuclear magnetic resonance structure of a much more dis-
tantly related protein has been solved, that of the Strepto-
myces killer toxin-like protein, SKLP [22]. The basic struc-
tural unit is the same L-barrel fold, but again, the surface
structure and charge properties are very di¡erent from
those of MiAMP1. Like MiAMP1, the cysteine residues
in all four Sp-AMPs are highly conserved. Cysteine-rich,
low molecular mass antimicrobials isolated from several
plant, animal and microbial sources have been shown to
possess inhibitory activity toward phytopathogens and ex-
pression of these peptides has the potential to confer dis-
ease resistance in transgenic plants [10,20]. Functionally,
MiAMP1 has been proposed to interfere with cell wall
synthesis, by analogy with the action of the yeast toxin,
WmKT, on the membrane-bound L-1,3-glucan synthase
[21]. However, the target of Sp-AMP is currently un-
known. Our future work with Sp-AMPs will address the
question of the biological target. In addition, characteriz-
ing the structure and the surface characteristics of Sp-
AMPs will also be a primary focus.
Acknowledgements
This work was supported by grants from the Swedish
Research Council for the Environment, Agricultural Sci-
ences and Spatial Planning (FORMAS), by the Swedish
Organization for International Cooperation in Research
and Higher Education (STINT) and by Akademiens Sa-
mordnade Fonder (KSLA).
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