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Department of Forest SciencesFaculty of Agriculture and Forestry
and
Doctoral Programme in Plant Sciences (DPPS)
University of Helsinki
PATHOBIOLOGY OF HETEROBASIDION-CONIFER TREE
INTERACTION: MOLECULAR ANALYSIS OF ANTIMICROBIAL
PEPTIDE GENES (Sp-AMPs)
By
Emad Jaber
ACADEMIC DISSERTATION
To be publicly discussed, with the permission of the Faculty of Agriculture and Forestry of theUniversity of Helsinki, in the lecture room LS3, B-Building (Latokartanonkaari 7), on October 31st
2014, at 12 o'clock
Helsinki 2014
Supervisor Prof. Fred O. AsiegbuFaculty of Agriculture and ForestryDepartment of Forest SciencesUniversity of Helsinki, Finland
Pre-examiners Prof. Johanna WitzellUniversity of Eastern Finland
School of Forest SciencesP.O. Box 111, 80101 JoensuuFinland
Dr. Jun-Jun LiuPacific Forestry Centre506 Burnside Road WestVictoria, British Columbia
V8Z 1M5, Canada
Opponent Prof. Joerg BohlmannUniversity of British Columbia2185 East Mall Vancouver
British Columbia V6T 1Z4, Canada
Custos Prof. Fred O. AsiegbuFaculty of Agriculture and ForestryDepartment of Forest SciencesUniversity of Helsinki, Finland
ISSN 2342-5423 (Print)ISSN 2342-5431 (Online)ISBN 978-951-51-0228-7 (Paperback)ISBN 978-951-51-0229-4 (PDF)
Hansaprint Printing HouseHelsinki 2014
To my parents: for their limitless love and support
4
TABLE OF CONTENTS
ABBREVIATIONS ............................................................................................................................6
LIST OF ORIGINAL PUBLICATIONS AND SUBMITTED MANUSCRIPTS .................................8
ABSTRACT ..................................................................................................................................... 10
1. INTRODUCTION ...................................................................................................................... 12
1.1. Plant innate immunity ................................................................................................................. 12
1.2. Forest tree defense responses ...................................................................................................... 15
1.3. The conifer root and butt rot pathogen Heterobasidion annosum ................................................. 17
1.3.1. Infection biology of Heterobasidion annosum ...................................................................... 17
1.3.2. Control strategies of Heterobasidion annosum ...................................................................... 18
1.4. Plant antimicrobial peptides (AMPs) ........................................................................................... 20
1.4.1. Plant antimicrobial peptide (AMP) evolution ........................................................................ 21
1.4.2. Role of plant antimicrobial peptides (AMPs) in innate immunity .......................................... 22
1.4.3. Plant antimicrobial peptide (AMP) mode of action ............................................................... 24
1.5. Scots pine antimicrobial peptides (Sp-AMPs) ............................................................................. 25
2. AIMS OF THE PRESENT STUDY ............................................................................................ 26
3. HYPOTHESES .......................................................................................................................... 27
4. MATERIALS AND METHODS ................................................................................................ 28
5. RESULTS AND DISCUSSION ................................................................................................. 30
5.1. Comparative pathobiology of Heterobasidion annosum s.s during challenge on Scots pine and
Arabidopsis roots (II) .................................................................................................................. 30
5.2. Analysis of defensin gene expression in H. annosum-Scots pine/Arabidopis pathosystems (II) .... 33
5.2.1. Defensin gene expression in Scots pine versus Arabidopsis during challenge with pathogens or
non-pathogens ...................................................................................................................... 34
5.2.2. Defensin gene expression in Scots pine versus Arabidopsis in the presence of fungal cell wall
elicitors and hormones .......................................................................................................... 36
5.3. Molecular regulation of Scots pine antimicrobial peptide (Sp-AMP) (III) .................................... 37
5.3.1. Sp-AMP regulation during fungal interactions ....................................................................... 37
5.3.2. Sp-AMP regulation in response to exogenous application of hormones.................................. 38
5.3.3. Sp-AMP is induced by glucan and other elicitors................................................................... 39
5.3.4. Sp-AMP antifungal activity against H. annosum ................................................................... 39
5
5.3.5. Sp-AMP binds to β-glucan sugars in both soluble and insoluble forms .................................. 40
5.3.6. Sp-AMP homology model and a proposed binding site for β-1,3 glucans .............................. 41
5.4. Scots pine pathogenesis-related protein 19 (PR-19) confers increased tolerance against Botrytis
cineria in transgenic tobacco (IV) ............................................................................................... 43
5.4.1. Generation of Sp-AMP2 transgenic tobacco plants ................................................................ 43
5.4.2. PR-19 tobacco plants exhibit increased tolerance to B. cinerea ............................................. 44
6. SUMMARY AND FUTURE DIRECTIONS .............................................................................. 47
ACKNOWLEDGEMENTS .............................................................................................................. 49
REFERENCES ................................................................................................................................. 51
6
ABBREVIATIONS
ACC 1-Aminocyclopropane-1-carboxylic-acid
AMP Antimicrobial protein
BLAST Basic local alignment search tool
cDNA Complementary DNA
CRP Cysteine-rich peptides
DAMPs Damage-associated molecular patterns
DEFLs Defensin-like sequences
DNA Deoxyribonucleic acid
ET Ethylene
ETI Effector-triggered immunity
GM Genetically modified
HR Hypersensitive response
ISGs Intersterility groups
JA Jasmonic acid
MAMPs Microbe-associated molecular patterns
MAS Marker-assisted selection
MiAMP1 Macadamia integrifolia antimicrobial protein family
MeJA Methyl jasmonate
mRNA Messenger RNA
NGS Next-generation sequencing
PAMPs Pathogen-associated molecular patterns
PCD Programmed cell death
PCR Polymerase chain reaction
PDB Protein Data Bank
PR Pathogenesis-related
PRRs Pattern recognition receptors
7
PsDef1 Pinus sylvestris defensin 1
PTI Pattern-triggered immunity
qRT-PCR Real-time quantitative reverse transcription PCR
QTL Quantitative trait loci
LRR-RLKs Leucine-rich repeat receptor-like kinases
LRR-RLPs Leucine-rich repeat receptor-like proteins
SA Salicylic acid
SAR Systemic acquired resistance
SDS–PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SE Somatic embryogenesis
Sp-AMP Scots pine antimicrobial peptide/protein
8
LIST OF ORIGINAL PUBLICATIONS AND SUBMITTED
MANUSCRIPTS
This dissertation is based on the following original publications. The publications in the text
will be referred to by their Roman numerals, I-IV:
I. Andriy Kovalchuk, Susanna Keriö, Abbot Oghenekaro, Emad Jaber, Tommaso
Raffaello, Fred O. Asiegbu (2013). Antimicrobial defenses and resistance of forest
trees: challenges and perspectives in a genomic era. Annual Review of
Phytopathology 51: 221-244
II. Emad Jaber, Chaowen Xiao, Fred O. Asiegbu (2014). Comparative pathobiology of
Heterobasidion annosum during challenge on Pinus sylvestris and Arabidopsis roots:
an analysis of defensin gene expression in two pathosystems. Planta 239:717-733
III. Sanjeewani Sooriyaarachchi*, Emad Jaber*, Adrian Suárez Covarrubias, Wimal
Ubhayasekera, Frederick O. Asiegbu, Sherry L. Mowbray (2011). Expression and β-
glucan binding properties of Scots pine (Pinus sylvestris L.) antimicrobial protein (Sp-
AMP). Plant Molecular Biology 77 (1-2): 33 - 45 * Joint first authors
IV. Emad Jaber, Teemu Teeri, Frederick O. Asiegbu (2014) Scots pine pathogenesis-
related protein 19 (PR-19) confers increased tolerance against Botrytis cineria in
transgenic tobacco. Submitted
The original publications and all figures in this dissertation are reprinted with the kind permission
of the copyright owners.
9
Author’s contribution:
I. The author contributed in drafting the following sub-sections: breeding for tree resistance,
genetic engineering of tree resistance, transcriptomics of tree-pathogen interaction and
emerging model system for forest pathology in the genomic era. The author also
contributed to data collection, generated tables for the review and provided images for the
“Impact of Heterobasidion annosum infection on wood quality” figure.
II. The author planned the experiment and performed the laboratory work. The author
analyzed the data, interpreted the results and wrote the article. CX contributed to drafting
the manuscript. FOA contributed to the experimental design and to drafting the article.
III. The author planned and conducted the laboratory work concerning Sp-AMP expression in
response to pathogen, non-pathogen or fungal protoplast exposure and the effects of
exogenous application of hormones, carbohydrate and yeast mutant treatments on Sp-AMP
expression; analyzed the data and interpreted the results. The author also contributed to
drafting the paper.
IV. The author planned the experiment and performed the laboratory work. The author
analyzed the data, interpreted the results and wrote the article. TT contributed to the
experimental design and manuscript drafting. FOA conceived the study and contributed to
the experimental design and article drafting.
10
ABSTRACT
Little information is available concerning the interaction of Heterobasidion annosum
with the roots of herbaceous angiosperm plants. We investigated the infection biology of H.
annosum during challenge with the angiosperm model Arabidopsis and monitored the host
response after exposure to various hormone elicitors, chemicals (chitin, glucan and chitosan)
and fungal species. This necrotrophic pathogen of conifer trees was able to infect the Col-8
(Columbia) ecotype of Arabidopsis in laboratory inoculation experiments. The germinated H.
annosum spores had appressorium-like penetration structures that attached to the surface of
the Arabidopsis roots. The subsequent invasive fungal growth led to the disintegration of the
vascular region of the root tissues. The progression of root rot symptoms in Arabidopsis was
similar to the infection development that was previously documented in Scots pine seedlings.
To better understand the regulation of the defensin gene in Scots pine, we analyzed
host defensin gene expression in response to various biotic and chemical treatments.
Furthermore, to gain better insight into the regulatory pattern of defensin in gymnosperms
compared with angiosperms, we repeated these analyses using the Arabidopsis thaliana
ecotype Columbia (Col-8) as a non-host model and as a potential alternative new pathosystem
model. Scots pine PsDef1 and Arabidopsis DEFLs (AT5G44973.1) and PDF1.2 were induced
at the initial stage of the infection. However, differences in the expression patterns of the
defensin gene homologs in the two plant groups were observed under various conditions,
suggesting functional differences in their regulation.
In parallel to the above study, the expression patterns of other closely related
proteins, Scots pine antimicrobial proteins (Sp-AMPs) and the structure and function of the
encoded proteins were investigated. The Sp-AMPs exhibited increased levels of expression
specifically when challenged with H. annosum but did not show increased levels when
challenged with non-pathogens, consistent with a function in conifer tree defenses. The Sp-
AMPs were up-regulated after treatment with salicylic acid (SA) and with ethylene (ET). The
Sp-AMPs possessed antifungal activity against H. annosum and caused morphological
changes in its hyphae and spores. The Sp-AMPs directly bind soluble and insoluble β-(1,3)-
glucans specifically and with high affinity. Furthermore, the addition of exogenous glucan is
associated with increased levels of Sp-AMP expression in the conifer tree. Homology
modeling and sequence comparisons suggest that a conserved patch on the surface of the
globular Sp-AMP protein is a carbohydrate-binding site that can accommodate approximately
four sugar units. It was concluded that Sp-AMPs belong to a new family of antimicrobial
11
proteins (PR-19) that are likely to act by binding the glucans, which are a major component of
the fungal cell walls.
To evaluate the potential of Sp-AMP as a molecular marker for resistance tree
breeding, we developed transgenic tobacco plants expressing the Sp-AMP gene. A bioassay of
transgenic tobacco (Nicotiana tabacum L. cv. SR1) plants over-expressing Sp-AMP2
challenged with the necrotrophic tobacco pathogen Botrytis cinerea was further investigated.
The necrotic lesions caused by B. cinerea on the non-transgenic tobacco leaves were severe
and larger than those lesions formed on the transgenic line. The results suggest that Scots pine
pathogenesis-related protein 19 (PR-19) confers increased tolerance against Botrytis cineria in
transgenic tobacco. This study provided insight concerning the initial molecular
characterization of the expression and regulation of this protein family. The potential utility of
the Sp-AMP genes as resistance markers in the conifer tree H. annosum pathosystem merits
further investigation.
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1. INTRODUCTION
1.1 Plant innate immunity
Microbial life consists of beneficial mutualists and saprotrophs as well as a countless
number of potential pathogens. Plants are constantly exposed to numerous pathogens, such as
fungi, oomycetes, bacteria, insects, nematodes, viruses and viroids. Plant pathogens utilize
various strategies to attack and colonize the plant host tissues. Unlike mammals, which
possess both acquired immunity and innate immunity, plants rely solely on the innate
immunity of each cell and on systemic signals emanating from infection sites to impede their
attackers by employing several layers of defense to minimize damage by pathogens (Dangl
and Jones, 2001, Spoel and Dong, 2012). The strategy of innate immunity is based on the
recognition of constitutive and conserved molecules from pathogens by specific receptors,
triggering defense responses. Punctual and specific recognition is crucial for efficient and
active defense mechanisms (Janeway and Medzhitov, 2002, Monaghan and Zipfel, 2012). The
first profile of innate immunity occurs at the plant cell surface through receptors called pattern
recognition receptors (PRRs), which recognize slowly evolving microbial- or pathogen-
associated molecular patterns (MAMPs or PAMPs). Activation of these PRRs leads to active
defense responses (MAMP/PAMP-triggered immunity (PTI) or basal immunity) (Ausubel,
2005, Jones and Dangl, 2006).
Many PAMPs that have been identified are essential for microbial metabolism or for
penetration and invasion of a host cell and are therefore broadly conserved and required for
microorganism fitness and dispersal. These PAMPs include lipopolysaccharides from Gram-
negative bacteria, peptidoglycans from Gram-positive bacteria, bacterial elongation factor Tu
(EF-Tu), bacterial flagellin, glucans, chitins and proteins derived from fungal cell walls
(Nurnberger and Brunner, 2002, Parker, 2003, Boller and Felix, 2009). Other signals are plant
endogenous elicitors, which are currently described as damage-associated molecular patterns
(DAMPs) (Lotze et al., 2007). Some of these compounds and their hydrolysis products are
able to elicit plant defense responses. For example, chitin and its hydrolysis products are
considered as PAMPs that induce plant defenses via chitin receptor-like kinases
(Schwessinger and Ronald, 2012).
Recognition is often initiated upon ligand binding by pattern recognition receptor
complexes, which are typically cell surface-localized receptor kinases, leucine-rich repeat
receptor-like proteins (LRR-RLPs) and receptor-like kinases (LRR-RLKs) (Altenbach and
13
Robatzek, 2007). Some of the well-investigated PRRs in plants include the flagellin receptor
FLS2 and the EF-Tu receptor EFR from Arabidopsis, the rice chitin binding protein CEBiP,
the Arabidopsis chitin receptor CERK1 and the rice receptor-like kinase XA21 (Zipfel, 2009).
Recognition of the pathogen triggers multiple signaling pathways through a network
employing altered cytoplasmic Ca2+ levels, reactive oxygen species (ROS) and nitric oxide
(NO) as well as post-translationally regulated mitogen-activated protein kinase (MAPK) and
calcium-dependent protein kinases (Nicaise et al., 2009). In addition, the signal-specific
activation of plant PRRs by various MAMPs leads to seemingly generic responses, including
transcriptional changes and the production of antimicrobial compounds, such as pathogenesis-
related (PR) proteins and phytoalexins. ROS production is required for hypersensitive cell
death (HR), a type of programmed cell death thought to restrict the access of the pathogen to
water and nutrients (Neill et al., 2002, Asai and Yoshioka, 2008, Spoel and Dong, 2012). Ca2+
elevation in the cytosol controls SA production and stomatal closure (Nomura et al., 2008, Du
et al., 2009). In Arabidopsis, MAPK activation leads to the activation of the WRKY family of
transcription factors (Pandey and Somssich, 2009). The DNA binding domain WRKY
subsequently interacts with the W-box (TTGACC/T) motif present in promoters of defense-
associated genes and activates the expression of early defense-related genes (Ishihama and
Yoshioka, 2012). The accumulation of callose and the biosynthesis of SA, jasmonic acid (JA)
and ET are other indicators of PTI (Tsuda and Katagiri, 2010, Luna et al., 2012).
The second profile of plant innate immunity occurs inside the cell. This form of
immunity is triggered by the recognition of pathogen effectors and is called effector-triggered
immunity (ETI). Pathogen effectors from diverse kingdoms are recognized by intracellular
and extracellular nucleotide-binding leucine-rich repeat (NB-LRR) proteins in a highly
specific fashion and activate similar defense responses. Some plant cultivars have evolved
resistance proteins (R proteins) to recognize particular effectors directly or indirectly leading
to ETI, typically involving an accelerated and amplified PTI response and a hypersensitive
response (HR)-related programmed cell death (PCD) at the infection site (Jones and Dangl,
2006).
PTI and ETI extensively share downstream signaling machinery mediated by an
integrated signaling network (Tsuda and Katagiri, 2010). This network includes the activation
of a downstream MAPK cascade; activation of WRKY transcription factors; biosynthesis of
SA, JA and ET; activation of a string of PR genes; cell wall strengthening; lignifications; and
the production of various antimicrobial compounds (Boller and Felix, 2009, Eichmann and
Schafer, 2012). The key signal molecules mediating both basal and specific defense responses
14
are SA, JA and ET. SA is required for local and systemic acquired resistance (SAR) and
together with NO and ROS, acts synergistically in activating defense responses (Klessig et al.,
2000, Wang et al., 2005, Tsuda et al., 2008).
PTI appears to cause basal disease resistance, which is in contrast to the strong and
more prolonged disease resistance conferred by ETI. Generally, ETI is more associated with
HR and SAR than PTI. However, examples of PTI, inducing HR and activating SAR, were
observed in Arabidopsis; both ETI and PTI can be robust or weak, depending on the
specificity of the host and pathogen interaction (Thomma et al., 2011). Resistance resulting
from ETI is effective against pathogens that can grow only on living host tissue (obligate
biotrophs) or against hemi-biotrophic pathogens due to programmed cell death in the host and
the associated activation of defense responses regulated by the SA–dependent pathway.
However, resistance resulting from ETI is not effective against pathogens that kill host tissue
during colonization (necrotrophs) and indirectly benefit from the host cell death (Glazebrook,
2005, Jones and Dangl, 2006). As a countermeasure to plant defense mechanisms, numerous
pathogens have evolved a method to avoid recognition by masking PAMPs and/or interfering
with signaling and defense induction. Likewise, pathogens have evolved to overcome the
latest protective strategy of host defenses. All pathogens carry MAMPs that may be
recognized by plants; however, plants remain susceptible to virulent pathogens, such that the
activation and suppression of PTI is a fundamental principle central to plant-microbe
interactions. In fact, disease may result from either the failure of the pathogen recognition
event or the ability of the pathogen to avoid or overcome the resistance response (Ferreira et
al., 2006, Boller and He, 2009).
15
1.2 Forest tree defense responses
Most of the current knowledge concerning molecular interactions between plants and
pathogens was gained through studies on herbaceous angiosperm models, which have
advanced our understanding of the genes implicated in disease resistance (Boyd et al., 2013).
However, molecular and genomic studies in tree pathosystems remain in their infancy
(Asiegbu et al., 2005a).
When exposed to pathogens, forest trees employ several layers of defense to
minimize damage by pathogens. Conifers have developed both constitutive and inducible
defenses, including preformed structural barriers (physical defense), antimicrobial chemicals
(resins/phenolics/peptides), the activation of a battery of defenses (often called the
hypersensitive reaction) and intra-organismic responses resulting in the systemic induction of
defense compounds to ward off attacks from pathogens (Pearce, 1996, Asiegbu et al., 2005b,
Bonello et al., 2006, Kolosova and Bohlmann, 2012).
Constitutive defenses are present before colonization and are composed of several
physical and chemical barriers. The first and typically most effective layer of defense in
conifer trees is the bark, which consists of periderm, cortex, phloem and cambial tissues. The
combination of the mechanical properties of suberized and tough lignified cell layers, which
provides a hydrophobic barrier, and the chemical properties of phenolics forms a
multifunctional barrier to the external environment (Franceschi et al., 2005).
Along with constitutive defenses, which can repel or inhibit the invasion of tissues,
other defense responses are induced to compartmentalize the invading pathogen or to seal and
to repair the resulting damage. Inducible defenses are generated upon the perception of
foreign invaders once an attack has begun. The induced defense system is composed of both
structural and biochemical elements, including cell wall alterations (lignification and
suberization), lytic enzyme production (chitinases and glucanases) and de novo synthesis or
activation of a wide range of antimicrobial compounds (phenols, stilbenes, lignans, flavonoids
and terpenoids), phytoalexins, PR proteins and other enzymes (Keeling and Bohlmann, 2006,
Eyles et al., 2010).
There has been growing emphasis on transcriptional and chemical studies of
phenolics and terpenoids, which are abundant in conifer tissues and are derivatives of the
phenylpropanoid and terpenoids pathways, and their implications in tree defenses (Danielsson
et al., 2011, Hall et al., 2011, Keeling et al., 2011). Signaling molecules constitute another
group of plant metabolites with an important role in tree defense systems. Regulatory
16
pathways that coordinate host responses to diverse biotic threats are mediated by JA, SA and
ET (Zulak and Bohlmann, 2010, Robert-Seilaniantz et al., 2011).
The synthesis of low molecular weight proteins and peptides that have antifungal
activities is one of the most important inherent inducible defense mechanisms of the tree
system. Many of these proteins are also classified as PR proteins based on their induction by
pathogen attack and are categorized into several different structural and functional classes
(Broekaert et al., 1997, Van Loon and Van Strien, 1999). The PR protein family consists of
multifunctional proteins, such as glycoside hydrolases (chitinases and β-1,3-glucanases),
endoproteinases, putative ribonucleases, peroxidases, proteinase inhibitors, oxalate oxidases,
lipid-transfer proteins, and small cationic antimicrobial peptides (thionins and defensins)
(Veluthakkal and Dasgupta, 2010).
Tree defense responses are extremely diverse, and their complex dynamics are
effective against a broad range of organisms. Recognition mechanisms help to identify the
invader and activate specific defenses against the pathogenic organism. Actually, resistance
and susceptibility do not depend exclusively on the ‘quality’ of the activated defense genes or
on differences in the timing and magnitude of their expression but also on the contemporary
expression of different sets of genes (Tao et al., 2003) (for a more extensive review of tree
defense responses, see Paper I: (Kovalchuk et al., 2013).
17
1.3 The conifer root and butt rot pathogen Heterobasidion annosum
The Heterobasidion annosum species complex, which is referred to as H. annosum
sensu lato (s.l.), is regarded as the most destructive pathogen causing root rot and stem decay
in the coniferous forests of the Northern Hemisphere and huge economic and ecological losses
in the forestry industry (Asiegbu et al., 2005a). H. annosum sensu lato (s.l.) is one of the most
intensively studied forest fungi. The complete genome sequence of the fungus is now
available, making H. annosum s.l. the first sequenced plant pathogenic homobasidiomycete
(Olson et al., 2012). H. annosum s.l. is a basidiomycetous fungus classified in the family
Bondarzewiacae in the order Russulales, under the class Agaricomycetes in the subphylum
Agaricomycotina, phylum Basidiomycota (Woodward et al., 1998, Matheny et al., 2007).
The H. annosum species complex is composed of five species that are necrotrophic
white rot fungi pathogens with an ability to saprotrophically colonize dead wood. Each
species complex, previously known as intersterility groups (ISGs) that are now formally
described as species, is characterized by a distinct host preference. Three Eurasian groups
have been described: H. annosum sensu stricto (s.s.), H. abietinum and H. parviporum,
whereas North American groups have been named H. irregulare and H. occidentale
(Garbelotto and Gonthier, 2013).
H. annosum has a broad host spectrum of over 200 wood species (Schmidt, 2006).
The host preference for H. annosum s.s. (known as P type) is primarily pine (Pinus sylvestris).
However, H. annosum s.s. can also attack other conifers, some broad-leaf tree species and
more rarely other angiosperm trees, such as alder, maple, birch, pear and many others in
addition to species of shrubs, including the cranberry, blueberry, and bilberry (Ryvarden and
Gilbertson, 1993, Hüttermann and Woodward, 1998, Niemelä and Korhonen, 1998). The host
preference for H. parviporum is spruce (Picea abies), whereas fir (Abies) species are the
target host for H. abietinum (Asiegbu et al., 2005a). In North America, H. irregulare
generally attacks pines, junipers and incense cedar, whereas H. occidentale exhibits a broader
host range and attacks the genera Abies, Picea, Tsuga, Pseudotsuga and Sequoiadendron
(Otrosina and Garbelotto, 2010).
1.3.1 Infection biology of Heterobasidion annosum
The primary infection of H. annosum s.l. occurs through airborne basidiospores that
land on stumps or wounds on the roots or the stem. . Following spore germination, the fungal
mycelium proceeds to undergo wood colonization. The growth of H. annosum s.l. within the
18
tree stem and roots depends, to a varying extent, on the tree species. The secondary infection
often spreads through root contacts to the adjacent healthy trees (Redfern and Stenlid, 1998,
Stenlid and Redfern, 1998). However, basidiospore deposition could travel hundreds of
kilometers to infect freshly cut stump surfaces (Gonthier et al., 2001). The effective spore
dispersal gradient of H. annosum s.l. ranges from 0.1 to 1.25 kilometers, indicating that the
presence of basidiospore-producing fruit bodies during thinning and cutting increases the risk
of stump infection within the forest. In fact, temporal patterns of the availability and
abundance of viable airborne inoculum and the risk of primary infections vary greatly among
forests in different climatic zones (Garbelotto and Gonthier, 2013). The role of asexual
conidiospores produced by the fungus in transmission is unknown; however, asexual
conidiospores are most likely important for short distance transmission in substrates or
vectored by root-feeding insects. H. annosum can remain infectious in stumps for up to
several decades after felling. The fungus can also persists in the root system of diseased trees
for decades and efficiently can spread from one forest generation to the next (Asiegbu et al.,
2005a).
1.3.2 Control strategies of Heterobasidion annosum
As a necrotroph, H. annosum s.l. is capable of infecting and destroying living conifer
roots and stems of all ages as well as dead trees. Current control methods of root and butt rot
do not provide absolute protection against the pathogen. However, spread in the attacked root
system and transfers between trees can be reduced to minimize economic losses. Because
transmission is the major driver of the infection, several chemicals, biocontrol agents and
silvicultural measures are currently employed to control the disease in forest plantations
(Asiegbu et al., 2005a).
Stump removal, including careful removal of all roots, is an effective silvicultural
control strategy against Heterobasidion root and butt rots. Other measures aimed to prevent
the disease or limit airborne infections include replacing susceptible tree species with broad-
leaved trees, which are relatively less susceptible, as well as decreasing the number of
thinnings/stand rotation and performing thinning and logging in periods of low risk of spore
infection (Garbelotto and Gonthier, 2013). Chemical fungicides, such as urea and borates, are
also efficient at reducing the severity and dispersal of the disease if applied immediately on
stump surfaces when logging is practiced in periods of sporulation (Oliva et al., 2008, Pratt,
2000). Both chemicals affect fungal metabolism, resulting in the inhibition of spore
germination (Lloyd et al., 1997). However, increasing environmental concern regarding the
19
effect of chemical agents, such as borates, on surrounding vegetation has been noted
(Westlund and Nohrstedt, 2000). A biological control approach using the fungus Phlebiopsis
gigantea is equally effective when applied on the stumps, leading to hyphal interference and
competition for the substrate (Mgbeahuruike et al., 2011). Investigating and finding new,
more effective and environmentally friendly alternative methods are major pre-requisites for
long-term strategies to control and manage Heterobasidion root and butt rots if existing
methods fail or if the pathogen develops tolerance to these methods.
20
1.4 Plant antimicrobial peptides (AMPs)
Antimicrobial peptides (AMPs) are gene-encoded natural antibiotics that form an
ancient and evolutionary conserved defense strategy in all living organisms (Shai and Oren,
2001). AMPs are considered important components of the innate defense response of plants
and animals that exert a broad spectrum of microbicidal activities against pathogenic microbes
(Ajesh and Sreejith, 2009, Pasupuleti et al., 2012).
Although AMPs differ in their amino acid composition and structure (Padovan et al.,
2010), AMPs share fundamental structural properties, such as small size, positive net charge
and clustering of cationic and hydrophobic amino acids within distinct domains of the
molecule (Hancock and Sahl, 2006). Approximately 1228 AMPs have been reported from
living organisms, as documented in the antimicrobial peptide database APD2 (Wang et al.,
2009). AMPs can be categorized into linear peptides often adopting helical structures,
cysteine-rich open-ended peptides containing disulfide bridges and cyclopeptides forming a
peptide ring. Linear and cyclic peptides may link fatty acid chains (lipopeptides) or other
chemical substitutions, resulting in complex molecules (Montesinos, 2007).
The actions of plant AMPs are initially directed against fungi, oomycetes and
bacteria (Benko-Iseppon et al., 2010). However, certain members of the AMP class can be
directed against other targets, including herbivorous insects (Howe and Jander, 2008).
Antimicrobial compounds may be synthesized in plant cells either constitutively in specialized
tissues or organs or induced by pathogen challenge (Osbourn, 1996). Certain criteria,
including in vitro antimicrobial activity, gene induction and peptide accumulation in planta
and gene up-regulation are crucial for classifying any peptide as an AMP. AMPs are
categorized into distinct families primarily from their sequence identity, number of cysteine
residues and their spacing (Garcia-Olmedo et al., 1998). The AMP definition does not include
enzymes that are induced upon pathogen infection. This definition excludes enzymes with
hydrolytic activities (e.g., lysozymes, chitinases, glucanases, etc.), although many are
classified as PR proteins (van Loon et al., 2006).
Plant AMPs are assigned to different classes. The most common classes are thionins
and defensins, and the less common classes include cyclotides, 2S albumins, lipid transfer
proteins, hevein-like proteins knotins, snakins and glycine-rich proteins (Benko-Iseppon et al.,
2010, Egorov and Odintsova, 2012, Sarika et al., 2012). AMPs that have primarily been
isolated from various plant species include thionins and plant defensins (Ponz et al., 1983,
Terras et al., 1992, Osborn et al., 1995, Games et al., 2008, Finkina et al., 2008), proteinase
21
inhibitors (Joshi et al., 1998), lipid-transfer proteins (Cammue et al., 1992, Regente and de la
Canal, 2003), chitin-binding proteins (Broekaert et al., 1992, Nielsen et al., 1997) and knottin-
type peptides (Chagolla-Lopez et al., 1994). Several 4-cysteine-type peptides (Broekaert et al.,
1997) and snakin proteins (Segura et al., 1999) have been detected in other tissues with
different classes (Asiegbu et al., 2003, Fujimura et al., 2005, de Beer and Vivier, 2008) based
on sequence similarities. Other plant AMPs that do not fit into these categories are
documented in PhytAMP, which is a curated online database of plant AMPs that focuses on
AMPs with experimentally verified expression profiles (Hammami et al., 2009).
Thionins and plant defensins are two well-known subclasses found in many different
plants; both are 45–54 amino acids in length with low molecular mass (~ 5 kDa), cysteine-rich
peptides and minor sequence similarity. Defensins, which are ubiquitous within the plant
kingdom, are integrated in the plant innate immune system and regarded as the PR-12 family.
In diverse plant species, representatives of plant defensins have been previously described as
complex and sophisticated peptides with functions that extend beyond their role in the defense
of plants against microbial infection (Carvalho Ade and Gomes, 2009). By contrast, thionins,
which are referred to as the PR-13 family, have broad in vitro antifungal and antibacterial
activities that promote the cell membranes permeabilization of phytopathogenic bacteria and
fungi (Sels et al., 2008).
1.4.1 Plant antimicrobial peptide (AMP) evolution
AMPs are highly divergent among different species. Given their diverse structure,
AMPs demonstrate potential for therapeutic and resistance applications. The rapid
development of transcriptomics and next-generation sequencing (NGS) has revealed
surprising secrets of plant genomes that has led to the identification of several dozens to
several hundreds of AMP-like genes, underscoring the importance of AMPs in the eukaryote
immune system, particularly in plants that are sedentary and that do not have acquired
immunity (Schutte et al., 2002, Higashiyama, 2010).
Franco (2011) relates plant defensive peptides to promiscuity, in which multiple
functions are associated with a single peptide structure. These findings suggest that this
phenomenon is extremely common with regard to plant antimicrobial peptides, defensins,
cyclotides, and 2S albumin, which exhibit an enormous multiplicity of biological activities.
Family promiscuity is commonly observed in plant defenses, indicating its importance to
plant survival and evolution. Family promiscuity represents also a starting point for the
22
divergence of novel functions so that the broad specificity of the protein served as the ancestor
for multiple specialized polypeptides (Franco, 2011, Khersonsky et al., 2012).
Cysteine residues in AMPs often form disulfide bonds important for their molecular
structure; thus, cysteine codons are expected to be more conserved than other sequence
regions. Other modifications, such as amidation, also occur in some peptides (Andreu and
Rivas, 1998, Padovan et al., 2010). The arrangement of cysteine-rich peptide sequences in
plant genomes suggests that plant genomes have high adaptive potential and are evolutionarily
dynamic. Some cysteine-rich peptide (CRP) sequences may have multiplied in some plant
genomes, whereas other CRP sequences have been lost. This possibility was demonstrated in
a study conducted by Silverstein et al. (2007), wherein CRP sequences in the rice and
Arabidopsis genomes were compared. For example, with 323 members, defensin-like
sequences are the most abundant CRP sequences in the Arabidopsis thaliana genome,
whereas only 93 defensin-like genes are present in rice. By contrast, the rice genome has 13
CRP sequences for Bowman-Birk protease inhibitor CRPs, whereas these sequences are
missing from the A. thaliana genome. Therefore, with regard to plant-microbe interactions,
plants possess considerable adaptive potential for the development and selection of altered
repertoires of CRP molecules with roles in plant defenses against more evolutionary flexible
pathogen populations. Such redundancy may represent a multi-pronged defense system
required to counter the strong evolutionary potential of microbial pathogens, to ensure
functional diversity and to provide adaptation to the plant immune system.
1.4.2 Role of plant antimicrobial peptides (AMPs) in innate immunity
In both animals and plants, innate immunity is triggered after recognition of
conserved MAMPs by pattern recognition receptors (Ausubel, 2005). Innate immunity
triggered by initial recognition events is multifaceted, involving local (at the site of infection)
and systemic responses (throughout the host), and is specific for different taxa. However, the
primordial importance of the induced production of AMPs after infection with microbes in
innate immunity is conserved among all host organisms and reflects the ancient origin of this
type of defense response (Zasloff, 2002). In plants, AMPs are most likely either constitutively
expressed in specific sensitive organs or are systemically induced by microbes at the site of
infection (Sels et al., 2008). As products of single genes, antimicrobial peptides can be
synthesized in a swift and flexible manner. Given their small size, AMPs can be produced by
the host with a minimal input of energy and biomass (Broekaert et al., 1997).
23
To date, the first and the only application of AMPs originating from plants to be
utilized in plant protection was developed and introduced by the Monsanto Company. This
method was achieved by generating a transgenic potato carrying a defensin Alfalfa antifungal
peptide (alfAFP) isolated from seeds of Medicago sativa, which displays strong activity
against Verticillium dahliae (Gao et al., 2000). Applications of AMPs from various sources
other than plants have been demonstrated to confer resistance against fungal and bacterial
pathogens in an array of genetically engineered plant species, including Arabidopsis, tobacco,
rice, potato, tomato, cotton, pear, banana, ornamental crops, geranium (Pelargonium sp.),
American elm and hybrid poplar (Keymanesh et al., 2009, Zhou et al., 2011).
The defensin RsAFP peptides from Raphanus sativus are secreted into the middle
lamella region of plant cell walls. Studies to understand their role were performed in
Neurospora crassa. In vitro assays demonstrated a pathogen response, and ionic changes in
the fungal membrane resulted in increased K+ efflux and Ca2+ uptake, thereby altering the
membrane potential. These defensins may interact with membrane receptors, acting as signal
molecules to ion channels. In addition, RsAFP expression is induced after pathogen challenge,
and the constitutive expression of RsAFP in transgenic tobacco resulted in increased
resistance against the foliar pathogen Alternaria longipes (Terras et al., 1995).
The Arabidopsis defensin gene PDF1.2 represents an important marker gene to study
the activation of the JA/ET signaling pathway (Manners et al., 1998, Zander et al., 2010).
PDF1.2 is regulated by an amplification loop that involves the recognition of the endogenous
peptide elicitors AtPEP1-6 by the receptors AtPEPR1 and AtPEPR2 (Yamaguchi et al., 2010).
Another AMP with two knottin motifs was isolated from the cycad (Cycas revolute). The
recombinant peptide is capable of binding to chitin, which is a component of the fungal cell
wall and has antifungal and antibacterial activities, implying a recognition function in the
plant defense response along with its antimicrobial actions (Yokoyama et al., 2009).
Certain plant AMPs, such as thionins and cyclotides, are inherently toxic, whereas
defensin and LTPs fulfill important functions in plant signaling as intricate parts of the plant
immune system in addition to their activity in killing pathogens. Interestingly, wheat LTP1
binds to a plasma membrane-located receptor for elicitins, which trigger plant defense
responses reminiscent of SAR (Keller et al., 1996). Some LTPs mediate pathogen recognition
and play essential roles in SAR (Stotz et al., 2013).
24
1.4.3 Plant antimicrobial peptide (AMP) mode of action
Limited information exists concerning regulation of the expression of plant AMPs as
well as AMP processing and posttranslational modification. However, an understanding of the
mechanism of action of antimicrobial peptides has evolved over time. As amphipathic,
cationic peptides, AMPs clearly target the membranes of the microbes, which are then killed
by these peptides, and this proposed mode of action is consistent with studies utilizing model
membranes (Amiche and Galanth, 2011).
The mechanisms of action for AMPs are as varied as their sources and include fungal
cell wall polymer degradation, membrane channel and pore formation, damage to cellular
ribosomes, inhibition of DNA synthesis, inhibition of fungal protein synthesis, blocking of
fungal ion channels and cell cycle inhibition (Hernández et al., 2005, Wong et al., 2007). The
plant defensins Dm-AMP1 from Dahlia merckii and Rs-AFP2 from Raphanus sativus
increase K+ efflux and the uptake of H+ and Ca2+ ions and evoke membrane potential changes
and membrane permeabilization (Thevissen et al., 1999). Another example is alfalfa
(Medicago sativa) defensin (MsDefl), which strongly inhibits the growth of Fusarium
graminearum in vitro. MsDefl blocks L-type Ca2+ channels. MsDefl and the Ca2+ channel
blocker 1,2-bis [(2 aminophenoxy) ethane-N,N,N,N-tetraacetate] EGTA inhibit hyphal growth
and induce hyperbranching of fungal hyphae (Spelbrink et al., 2004). Other AMPs are non-
membrane disruptive: the peptides cross the cell membrane to interact with intracellular
targets and inhibit nucleic acid or protein synthesis and enzymatic activity (Brogden, 2005).
Different mechanisms have been suggested for AMP actions. In some instances,
these mechanisms involve the translocation of these peptides across the plasma membranes of
target cells to attack intracellular targets, such as bacterial DNA, thereby inhibiting
intracellular functions via interference with nucleic acid synthesis (Cho et al., 2009, Auvynet
et al., 2009). However, AMP actions include direct attacks on the membrane of target cells
and generally involve membrane disruption and permeabilization (Al-Benna et al., 2011).
Numerous models have been proposed to describe the mode of action of AMPs, including the
barrel stave pore model; the toroidal, disordered toroidal pore model; the carpet and tilted
peptide mechanism; and the Shai, Huang and Matsazuki model (Wimley, 2010, Wimley and
Hristova, 2011). Resistance to AMPs is unlikely to be acquired by microbes due to
redundancy and to the non-specific nature of the actions (Conlon and Sonnevend, 2011).
25
1.5 Scots pine antimicrobial peptides (Sp-AMPs)
Recent analysis of gene expression in pine trees led to the identification of a novel
family of antimicrobial proteins, the so-called Sp-AMPs, in Pinus sylvestris (Scots pine). Sp-
AMPs were identified in a subtractive cDNA library of Scots pine roots infected with the root
rot fungus H. annosum. At least five genes were identified by Southern blotting of Hind III-
digested pine genomic DNA, of which four (Sp-AMP1-4) genes with 93–100% nucleotide
sequence identity have been described (Asiegbu et al., 2003). Sp-AMPs encode cysteine-rich
proteins, and each contains an N-terminal region with a probable cleavage signal sequence.
The cellular localization of Sp-AMP1 revealed substantial accumulation of the
peptide in the cell wall region at 15 d.p.i. of H. annosum (Adomas et al., 2007). The
abundance of Sp-AMP on the cell surface and its high expression during pathogen attack
indicates a redundancy that suggests possible direct involvement in the conifer-H. annosum
interaction. In addition, the Sp-AMP1 gene is also up-regulated in Scots pine by non-
pathogens at early stages of infection, suggesting that Sp-AMP is employed as a response
against a wide range of organisms (Adomas et al., 2008a). The up-regulation continued in the
roots infected with the pathogen but did not continue with non-pathogenic fungi. To date,
little or no research has been performed regarding the identification and characterization of
AMP genes in conifer trees.
The novel Sp-AMP1 gene exhibits a relatively high sequence similarity to the
antimicrobial protein MiAMP1, which was originally isolated from the seeds of Macadamia
integrifolia. MiAMP1 is a functional, well-characterized member of the AMP class. MiAMP1
is a prototypic plant member of a structural superfamily of AMPs also found in other
eukaryotes and prokaryotes conserved across the plant kingdom from lycophytes and
gymnosperms to early angiosperms (e.g., Amborella and Papaver) and various monocots (e.g.,
Zantedeschia, Zea, and Sorghum). This superfamily is implicated in the defense against fungal
pathogens in gymnosperms (Manners, 2009). The MiAMP1 family is highly inhibitory to a
wide range of phytopathogens. In addition, the transgenic expression of MiAMP1 in canola
Brassica napus L. provides enhanced resistance against blackleg disease caused by the fungus
Leptosphaeria maculans (Kazan et al., 2002). A comparison of MiAMP1 with its structural
homolog in the yeast model, yeast killer toxin (WmKT), indicated that the two proteins did
not have the same mode of action, suggesting that the actual mechanism by which MiAMP1
inhibits fungal growth is unknown (Stephens et al., 2005).
26
2. AIMS OF THE PRESENT STUDY
Heterobasidion annosum is one of the most harmful and economically important
forest pathogens in the Northern Hemisphere. Molecular and genomic studies in the H.
annosum–tree pathosystem remain in the early stages, and many aspects of the H. annosum–
tree interaction remain unclear. Several studies have explored the possibility of using
Arabidopsis as the principal model host to exploit the wealth of genetic and molecular tools
available for this model plant to allow comparative analyses of pathogenicity mechanisms and
defense responses between tree and plant models. Investigating the pathobiology of H.
annosum during challenge in the Arabidopsis model would be an extremely promising
strategy to facilitate mechanistic studies of the conifer pathosystem. Furthermore, an
additional challenge in this conifer pathosystem is to determine a resilient control and
management strategy in the continuous co-evolutionary battle between the tree host and the H.
annosum pathogen. It is important to investigate and identify new, more effective and
environmentally friendly alternative methods to manage Heterobasidion root and butt rots.
The first objective of this study was to conduct a thorough literature review on the
antimicrobial defences of forest trees to pests and diseases in order to have a broader overview
of mechanisms of tree resistance. The review (paper I: kovalchuk et al., 2013) provided novel
insights on the developments, achievements and potential limitations in this research area. The
acquisition of such knowledge contributed enormously in shaping the plan of my research
study.
The second objective is to conduct a detailed molecular characterization of the
antimicrobial proteins in P. sylvestris (Scots pine), to investigate their potential utility as new
methods of fighting fungal diseases and to explore their potential use as resistance markers in
conifer trees.
The specific objectives of this study are as follows:
A. To conduct comparative pathobiology of H. annosum during challenge on P. sylvestris
and A. thaliana.
B. To study biochemical and molecular factors regulating Sp-AMP expression in the
conifer host.
C. To study the role of Sp-AMP in plant resistance by heterologous expression of Sp-
AMP in tobacco.
27
3. HYPOTHESES
Our primary hypothesis is that the conifer pathogen H. annosum is capable of
infecting the angiosperm model plant A. thaliana, thereby making it a suitable host model for
use in molecular studies in conifer pathosystems. Our additional hypothesis is that the Scots
pine antimicrobial peptide (Sp-AMP) possesses inhibitory effects against phytopathogenic
fungi.
28
4. MATERIALS AND METHODS
The methods, fungal strains and plant material used in this study are summarized in
Tables 1, 2 and 3:
Table 1: Methods used in this study.
Methods Publications
Scots pine growth conditionsFungal strain growth conditionsFungi inoculationProtoplast generationHormone treatmentDNA isolationDNA sequencing and data analysisqPCR conditions and data analysisPrimer designsPCR conditionsGene cloningRNA isolationcDNA synthesisSequence alignmentNorthern analysisQuantification of fungal rate of infectionDetermination of antifungal activityHomology modeling of Sp-AMP3Protein expression and purificationCarbohydrate binding assaysElectron microscopyTobacco transformationSelection of transgenic plantsPathogen bioassays on transgenic plants
II, IIIII, III, IVII, III, IV
II, IIIII, IIIII, IVII, IVII, III
II, III, IVII, III
II, III, IVII, III, IVII, III, IV
II, IIIIIIIIIIIIIIIIIIIIIIVIVIV
29
Table 2: Fungal strains used in this study.
Fungal strains Strain/Genotype Publications
Heterobasidion annosums.s.
Stereum sanguinolentum
Stereum rugosum
Lentinellus vulpinus
Lactarius rufus
Saccharomyces cerevisiae
Saccharomyces cerevisiae(∆chs5 mutant)
Saccharomyces cerevisiae(Δexg mutant)
Botrytis cinerea
Isolate Dragstjard 05044, heterokaryotic
Isolate FBCC1148, (FBCC)
Isolate FBCC1190, (FBCC)
Isolate FBCC605, (FBCC)
Isolate from METLA
Wild type BY4742 (MATα; his3Δ1; leu2Δ0;lys2Δ0; ura3Δ0)
Strain BY4741 (MATα; his3Δ1; leu2Δ0;lys2Δ0; met15∆0; ura3∆0;YLR330w::kanMX4)
Strain BY4741 (MATα; his3Δ1; leu2Δ0;met15Δ0; ura3Δ0; YLR300w::kanMX4)
Isolate B05.10
II, III
II, III
II
II
II, III
II, III
II, III
II, III
IV
Table 3: Plant material used in this study.
Plant materials Strain/Genotype Publications
Pinus sylvestris
Arabidopsis thaliana
Nicotiana tabacum
Svenska Skogsplantor (Saleby FP-45, Sweden)
Ecotype Columbia Col-8 (N60000, NASC)
Cv. Petit Havana SR1
II, III
II, III
IV
30
5. RESULTS AND DISCUSSION
5.1. Comparative pathobiology of H. annosum s.s. during challenge on
Scots pine and Arabidopsis roots (II)
The screening of several fungal isolates belonging to the Russulales group with
diverse trophic levels following a challenge on Scots pine roots led to the selection of a subset
representing parasite (Heterobasidion annosum), mutualist (Lactarius rufus) and saprotroph
(Stereum sanguinolentum) habits. Although both the mycorrhizal and saprotrophic fungi
induced slight necrosis on Scots pine roots, neither fungus hindered lateral root formation or
led to mortality. By contrast, H. annosum induced a strong necrotic reaction on the roots,
which led to mortality after prolonged incubation of some of the seedlings. Both the tree
pathogen (H. annosum) and the saprotroph (S. sanguinolentum) infected Arabidopsis Col-8 in
the laboratory inoculation experiments, whereas L. rufus did not cause visible symptoms of
infection or restricted growth over time and remained viable for 15 d.p.i. Evidence of
appressorium-like penetration structures, which were attached to the surface of Arabidopsis
roots inoculated with H. annosum within 24 h, was documented by scanning electron
microscopy (Fig. 1).
Figure 1: Scanning electron micrograph of Arabidopsis roots inoculated with an H. annosum spore suspension at
1 d.p.i. revealing spore adhesion followed by hyphal branching and penetration between epidermal cells. (II,
Figure 3).
31
Heterobasidion annosum hyphal penetration was visible within cortical cells at early
(1 d.p.i.) and late (15 d.p.i.) inoculation. H. annosum provoked rapid cell wall degradation
within the vascular tissues. The invasive growth led to the disintegration of tissues and
cellular structures, thereby promoting extensive colonization (Fig. 2).
Figure 2: Transmission electron micrograph of transverse sections of Arabidopsis roots representing various
stages of cellular colonization of roots infected with the mycelia homogenate of H. annosum and showing hyphal
penetration of H. annosum at 1 d.p.i. in the cortical cell (a, b). The advanced stage of H. annosum infection (15
d.p.i.) (c, d, e, f) shows fungal hyphae proliferation within the intercellular spaces of cortical cells and cell wall
disruptions that result in the complete collapse of the root architecture (II, Figure 4).
Previous studies on the infection process of H. annosum in Norway spruce (Asiegbu
et al., 1994) and Scots pine (Li and Asiegbu, 2004) also documented the formation of a germ
tube and appressoria accompanied by the formation of papillae and lignifications in the host
(Asiegbu et al., 1999, Asiegbu et al., 1993). H. annosum spores easily adhere to fine roots
32
prior to germination. Direct contact with the host cell tissues beneath the slime and
mucilaginous covering on the root material was established. In some roots, appressoria were
formed within ridges on the root surfaces (Asiegbu, 2000). In Scots pine, the first signs of
epidermal and cortical penetration were observed at 48 h and 72 h p.i., respectively, followed
by extensive disintegration of cortical cells as the fungi reached the endodermal and vascular
regions at 6–9 d.p.i. At 10–15 days post inoculation, disintegration of the meristematic tissues
and vascular system of some of the root tissues was visible (Li and Asiegbu, 2004).
The electron microscopy results demonstrated the susceptibility of the angiosperm
model A. thaliana to the necrotrophic conifer parasite H. annosum (Jaber et al., 2014). The
electron microscopy observations and the rate of infection quantitatively studied in H.
annosum in Scots pine and Arabidopsis at two time points (1 and 5 d.p.i.) (II, Supplementary
material S4) indicated that the advancement of root rot infection stages in Arabidopsis was
similar to the key sequence of events during infection, as was previously documented by Li
and Asiegbu (2004). H. annosum s.s. fungal biomass was detectable in infected Scots pine at 5
d.p.i., whereas H. annosum s.s. could be detected in the Arabidopsis seedlings at 1 and 5 d.p.i.
However, the progression rate of H. annosum s.s. colonization in Scots pine was faster than
the progression of the pathogen colonization in Arabidopsis based on the slope values of
1.1058 and 0.3425 of the linear regression for infected Scots pine and infected Arabidopsis,
respectively. The interaction between the tested fungi, which belong to the fungal group
Russulales, and the Scots pine seedlings generated similar general reactions and recognition
patterns as reported in earlier studies (Asiegbu et al., 1999, Adomas et al., 2008).
Pathogens can specifically colonize particular host organs or cell types (Schulze-
Lefert and Robatzek, 2006). However, H. annosum s.s. exhibits an extremely wide host range,
as discussed earlier. H. annosum s.s. also infects and causes necrosis not only on the roots but
also on the needles (Adomas and Asiegbu, 2006). H. annosum s.s. and Magnaporthe grisea
(Sesma and Osbourn, 2004) are two examples of pathogens that infect other tissues apart from
the organ the pathogen has typically been reported to attack.
The requirement for a functional model system for genomic studies is critical for
understanding the biochemical and molecular studies in Heterobasidion-conifer pathosystems
(Li and Asiegbu, 2004, Asiegbu et al., 2005a). Arabidopsis has been a model host for many
necrotrophic pathogens of diverse plant species (Glazebrook, 2005). Several researchers have
tested non-adopted pathogens of specific hosts not related to Brassicaceae utilizing
Arabidopsis as a host (II, Table 1).
33
The successful infection of H. annosum s.s. reported here, based on the colonization
of Col-8 Arabidopsis root, makes it a promising pathosystem that would facilitate the
mechanistic study of conifer pathosystems, allowing the elucidation of the signaling networks
and the identification of genes with roles in the regulation of disease resistance responses. A
successful example of adopting the Arabidopsis model in the identification of genes with roles
in the regulation of the disease resistance responses in tree pathology was reported in
Eucalyptus. A Eucalyptus bacterial wilt isolate from South Africa, Ralstonia solanacearum,
was shown to be pathogenic on Arabidopsis (Deslandes et al., 1998). The expression data of
the Arabidopsis transcriptome revealed a suppressed subset of basal defense genes, which
were targeted by specific R. solanacearum effectors (Naidoo et al., 2011). The availability of
the genome sequence of Eucalyptus grandis will further boost basic research on the molecular
interaction between E. grandis and R. solanacearum.
For the Heterobasidio pathosystem, the availability of mutant Arabidopsis lines
further underscores the huge potential for such a new pathosystem to facilitate resistance
research in conifer tree pathologies. In our study, investigating inducible defense systems,
including JA/ET- or SA-dependent pathways, in the tested H. annosum-Arabidopsis
pathosystem was a first step to reveal the key players of the signaling cascade of inducible
defense (SA in regulating DEFL genes). Future experiments should screen for mutants in this
type of signaling pathway. The findings from the tested Heterobasidion-Arabidopsis/ conifer
pathosystem models may not strictly apply to all forest trees due to possible differences in the
physical structure and longevity of the host in addition to the type of pathogens. Additional
inoculation experiments with other ecotypes and mutants may help to further demonstrate
non-host resistance, which will be of great interest for elucidating the cellular and genetic
basis of the H. annosum-conifer pathosystem.
5.2. Analysis of defensin gene expression in H. annosum s.s.-Scots
pine/Arabidopsis pathosystems (II)
To resist pathogen invasion, forest trees utilize defense strategies and mechanisms
similar to short-lived herbaceous crops; however, variations are likely to occur in gene
regulation and signaling pathways (Adomas, 2007). Defensin, which has been identified in
both angiosperms and gymnosperms, represents the largest and the most characterized family
of antimicrobial proteins. Defensin exhibits multiple biological activities (Jenssen et al.,
2006). The number of defensin genes in the Arabidopsis thaliana genome was originally
34
estimated at 15 defensins and more than 300 defensin-like genes (DEFLs) (Thomma et al.,
2002, Silverstein et al., 2007). In gymnosperms, defensins have been identified in Ginkgo
biloba (Shen et al., 2005), Picea abies (Fossdal et al., 2003), P. glauca (Pervieux et al., 2004)
and Pinus sylvestris (Kovaleva et al., 2009, Kovaleva et al., 2011). Scots pine defensin
PsDef1 BLAST searches against Arabidopsis genome sequences revealed many significant
alignments, one of which is the PDF1.2 gene (Blast score E value = 6e−06). The PDF1.2
gene is commonly used as a marker of the jasmonate-dependent defense response (Penninckx
et al., 1998). Arabidopsis DEFLs (Silverstein et al., 2005) (AT5G44973.1, DEFLs) were used
because their structure shares the closest homology to the Sp-AMPs. The alignment of PsDef1
with representatives of various defensin groups also revealed its high similarity to the SPI1-
putative gamma-thionin protein from Norway spruce (P. abies), which exhibited strong
antifungal activity and increased transcript accumulation after wounding and jasmonate
treatments (Pervieux et al., 2004) (II, Figure 5).
5.2.1. Defensin gene expression in Scots pine versus Arabidopsis during challenge with
pathogens or non-pathogens
In Scots pine, PsDef1 was slightly induced in response to inoculation with any of the
tested fungi at an early time point (1 d.p.i.) and strongly down-regulated at 5 d.p.i. in response
to pathogenic fungi. In Arabidopsis, the DEFLs (AT5G44973.1) were slightly induced upon
inoculation with the pathogen (H. annosum) and with the saprotroph (S. sanguinolentum) at 1
d.p.i. The expression was sustained over time by the pathogen. A strong induction of DEFLs
(AT5G44973.1) was also observed after a prolonged incubation at 5 d.p.i. with the mutualist
(L. rufus). By contrast, PDF1.2 was strongly induced by the pathogen (H. annosum) and
slightly induced by the saprotroph (S. sanguinolentum) at early and prolonged incubation
times (1 and 5 d.p.i., respectively). The pathogen provoked a stronger induction of defensin
genes in Arabidopsis compared with the Scots pine (Fig. 3-a).
Expression of the Scots pine defensin PsDef1 occurs during seed germination and in
response to pathogenic infection with H. annosum. A five-fold increase after 2 d.p.i. was
observed compared with healthy Scots pine seedlings (Kovaleva et al., 2011). In our study,
PsDef1 was only induced initially upon the first physical encounter (1 d.p.i.) with all fungi,
whereas the two Arabidopsis defensins (DEFLs, PDF1.2) were induced in response to H.
annosum infection (Fig. 3-a). PDF1.2 expression is induced both locally and systemically by
pathogen challenge (Penninckx et al., 1996). The strong induction of DEFLs (AT5G44973.1)
in Arabidopsis upon challenge with L. rufus suggests that DEFLs have other functions. It is
35
also possible that the DEFL (AT5G44973.1) genes share a motif with a set of largely nodule-
specific DEFLs from the model legume Medicago truncatula (Silverstein et al., 2005).
Mycorrhizal fungi have developed strategies to avoid the initiation of plant defense responses
and to suppress or evade host-induced responses by controlling the plant immune system and
nutrient transport (Veneault-Fourrey and Martin, 2013).
Figure 3: Transcript levels of Scots pine PsDef1 compared with the transcript levels of Arabidopsis DEFLs and
PDF1.2 genes during challenge with different fungi and in the presence of fungal cell wall elicitors and
hormones (II, Figure 6).
36
5.2.2. Defensin gene expression in Scots pine versus Arabidopsis in the presence of
fungal cell wall elicitors and hormones
To investigate the effect of fungal cell wall components on Scots pine and
Arabidopsis defensin gene expression and regulation, we monitored the plants’ responses after
treatment with chitin, chitosan or glucan. In Scots pine, elevated levels of the PsDef1 gene
transcript were observed after a prolonged incubation of 5 d.p.i. following treatments with
chitin, chitosan or glucans. By contrast, in Arabidopsis, glucan and chitin induced DEFL gene
expression at 1 d.p.i., and sustained induction was only observed with glucan (Fig. 3-b). In
addition, inoculation using yeast mutants with ~4-fold reduced levels of chitin (YCH) or with
increased levels of β-(1,6)-glucan (YG) led to a slight induction of PsDef1 in Scots pine after
a prolonged incubation of 5 d.p.i. However, in Arabidopsis, inoculation with each yeast
mutant provoked strong expression of the DEFL genes compared with the wild type (Fig. 3-
c).
As noted previously, fungal cell wall components, such as chitin, glucans and
mannoproteins as well as their hydrolysis products, are considered to be MAMPs that induce
plant defenses (Monaghan and Zipfel, 2012, Schwessinger and Ronald, 2012). The defensin
gene expression patterns from the two plant groups were somewhat different when exposed to
fungal cell wall components. This difference suggests that the fungal cell wall components
affect the induction of PsDef1 and DEFL (AT5G44973.1) transcripts in Scots pine and
Arabidopsis.
To study the role of the fungal cell wall and its importance in the regulation of
defensin, homogenized mycelia of various fungi were treated with cell wall-degrading
enzymes. Arabidopsis and Scots pine roots were exposed to the fungal protoplasts that were
devoid of cell walls. Protoplasts from all fungi induced PDF1.2 expression. Notably, the
protoplasts from the saprotroph induced a strong, significant level of PDF1.2 expression (II,
Figure 7), indicating that factors other than cell wall components, such as molecules secreted
by the fungal cells, may also trigger expression.
The roles of hormones (e.g., MeJA, SA and ET) and hydrogen peroxide (H2O2) in
defensin gene expression were further analyzed. In Scots pine, qRT-PCR results revealed a
slight up-regulation of PsDef1 1 day after treatment with the ET precursor ACC. In Arabidop-
sis, all hormone treatments induced DEFL gene transcription. No transcripts of PDF1.2 were
detected in response to the exogenous hormone treatments, except for an extremely slight
induction observed with the ET precursor ACC (Fig. 3-d). Interestingly, the DEFL genes
37
were significantly induced in Arabidopsis by H2O2, whereas PsDef1 remained unchanged in
Scots pine after a similar treatment.
The activation of the PDF1.2 gene occurs via the JA/ET-mediated signaling pathway
rather than via the SA-dependent pathway (Penninckx et al., 1996, Penninckx et al., 1998).
PDF1.2 expression induced specifically in response to H. annosum and S. sanguinolentum
correlated with the phenotypic symptoms of infection observed in Arabidopsis. However,
significant PDF1.2 transcription levels were not detected in response to exogenous hormone
treatment, except for a slight induction provoked by the ET precursor for all defensins. This
finding may be attributed to the in vitro nature of the exogenous application of the hormones.
Although PDF1.2 and DEFLs (AT5G44973.1) were induced specifically upon H.
annosum challenge in Arabidopsis, it is extremely difficult to draw any conclusion concerning
the variation documented in this new pathosystem. In Arabidopsis, there are over 300 defensin
gene homologs, suggesting that the defense process is complex (Silverstein et al., 2005). The
diverse functional roles of the defensins make it difficult to conduct transcript-level
comparisons among these genes under diverse treatments, as shown in the two plant hosts in
our study. The defensin genes examined in this study had a unique expression pattern that
further reflects their diverse biological function, regardless of the evolutionary separation
between Arabidopsis and Scots pine.
5.3. Molecular regulation of Scots pine antimicrobial peptide (Sp-AMP)
(III)
5.3.1. Sp-AMP regulation during fungal interactions
Sp-AMP gene expression was investigated in Scots pine challenged with pathogenic
(H. annosum), mutualistic/beneficial (L. rufus) or saprotrophic (S. sanguinolentum) fungi, all
of which belonged to the same basidiomycete group, Russulales. Northern-blot analysis at 1
day revealed no significant differences in Sp-AMP expression when the plants were
challenged with the three fungi. Sp-AMP expression over a longer period of infection (5 d.p.i.)
was considerably increased with pathogenic fungi compared with either mutualistic or
saprotrophic fungi, both of which were only modestly increased over the control. Sp-AMP
expression was further investigated using quantitative reverse-transcriptase (qRT)-PCR (III,
Figure 1), which showed an initial decrease at 1 d.p.i and then a strong increase during
infection with the pathogenic fungus at 5 d.p.i. Slight increases in Sp-AMP expression were
38
observed at 1 day after challenge with the mutualistic and saprotrophic fungi, but these levels
were not significantly changed at 5 d.p.i.
Scots pine roots responded differently when exposed to three Russulales fungi
belonging to distinct ecological functional groups. The differential expression indicates that
the host is able to distinguish among the diverse lifestyles of the inoculated fungi and suggests
a role for Sp-AMPs in defense. These results confirm and extend earlier work with more
distantly related fungi (Adomas et al., 2008); AMP induction occurred on a timescale similar
to that observed for the pathogen’s invasion into the plant tissues (Li and Asiegbu, 2004). The
delay in Sp-AMP expression during challenge with pathogenic fungi suggests the possibility
of some form of masking by the invading fungus as a means to evade host defenses (Jones and
Dangl, 2006).
The protoplasts from both pathogenic and mutualistic/beneficial but not saprotrophic
fungi induced strong Sp-AMP expression (III, Figure 1), also suggesting the possibility that
mutualistic and pathogenic fungi manipulate host cells through effectors and/or adopt
common infection strategies to evade recognition by the plant surveillance (Rafiqi et al.,
2012). Inoculation with yeast mutants having either ~4-fold reduced levels of chitin (∆chs5
mutant) or increased levels of β-(1,6)-glucan (∆exg mutant) caused increased transcription of
Sp-AMP genes in Scots pine roots relative to the wild type yeast control at 5 d.p.i. Notably,
inoculation with a yeast mutant that had increased levels of β-(1,6)-glucan induced higher
expression of Sp-AMP at the prolonged time of inoculation compared with inoculation with
yeast mutants with ~4-fold reduced levels of chitin at the same time point (5 d.p.i.) (III,
Figure 6).
5.3.2. Sp-AMP regulation in response to exogenous application of hormones
Exogenous applications of hormones can mimic the endogenous increases that occur
after pathogen challenge. A combination of pharmacological studies and genetic analyses
suggests that different pathogens elicit distinct host responses in model plants, such as
Arabidopsis and tobacco, with a tendency for necrotrophic pathogens to elicit JA-dependent
responses and for biotrophic pathogens to elicit SA-dependent responses (Davis et al., 2002).
In our study, to explore the role of MeJA-, SA-, ET- and H2O2-responsive pathways
in regulating Sp-AMP expression, Scots pine roots were treated with the respective
compounds. qRT–PCR results revealed up-regulation of Sp-AMP at 1 day after treatment with
SA or with ACC. Neither MeJA nor H2O2 induced Sp-AMP expression, indicating that Sp-
AMP gene expression is independent of the JA signaling pathways (III, Figure 2).
39
Furthermore, SA is known to induce specific sets of PR genes (Pieterse and van
Loon, 1999) before the establishment of systemic acquired resistance (Grant and Lamb,
2006). The induction of Sp-AMP by ACC suggests the involvement of ET in Sp-AMP
regulation during biotic and abiotic stress. ET signaling induces cellular and chemical
defenses in conifers and is vital in the activation of cells specialized for the formation of
defense-related terpenoids and phenolics in the outermost bark and phloem tissues (Hudgins
et al. 2006).
5.3.3. Sp-AMP is induced by glucan and other elicitors
The responses of Scots pine seedlings after treatment with chitin, chitosan or glucan
(laminarin) were monitored. All three compounds provoked strong discoloration in the roots
at 5 d.p.i., although glucan treatment provoked the greatest response at both 1 and 5 days.
Analyses of Sp-AMP expression by qRT-PCR showed a 2-fold induction of Sp-AMPs
expression 1 day after glucan treatment, which persisted for 5 days after glucan treatment (III,
Figure 5).
5.3.4. Sp-AMP antifungal activity against H. annosum
Acquiring Sp-AMP for direct biological and biochemical tests was an important step.
However, numerous strategies for E. coli expression produced only large quantities of
insoluble protein, which could not be refolded. The cDNA encoding the Sp-AMP3 protein
was cloned and expressed in Pichia pastoris. The yields of soluble Sp-AMP3 pure protein
were extremely variable and low at best (~0.4 mg of 99% pure protein per liter of induced
culture), although sufficient quantities were obtained to allow several key functional studies.
Sp-AMP3 strongly inhibited both the hyphal growth and spore germination of H.
annosum (Fig. 4). The addition of 10 µg ml-1 Sp-AMP3 caused almost complete inhibition
during a 3 day incubation period. Controls (samples taken before the transformed Pichia
strain induction in the same buffer as the protein sample and the buffer itself) did not inhibit
mycelial growth or spore germination. Sp-AMP3 samples with and without His-tags exhibited
marked inhibitory effects on fungal hyphae and spores (Sooriyaarachchi et al., 2011 and
unpublished microscopic studies).
AMPs are interesting compounds in plant health given the requirement for new
products in plant protection that fit into the new regulations (Montesinos, 2007). Indeed, the
inhibitory effects of Sp-AMP3 on spore and hyphal development of H. annosum suggests the
40
potential of Sp-AMPs for use as small molecular weight compounds with properties similar to
biocontrol agents.
Figure 4: Sp-AMP3 inhibition of H. annosum growth and spore germination. In panels (a), (b) and (c), the effects
of various samples on H. annosum growth are shown at 2 (A), 3 (B) and 5 (C) days, respectively. Spore
germination was investigated in panels (D) and (E); for spores treated with buffer (10 mM HEPES, pH 7.0) or
Sp-AMP3 without His-tag, respectively. Spores were incubated for week (III, Figure 3).
5.3.5. Sp-AMP binds to β-glucan sugars in both soluble and insoluble forms
To test the hypothesis that the biological function of the Sp-AMP involves some
component of the fungal cell wall, our binding activity assays included the primary
compounds in that structure as well as those components in plant cell walls. The results
indicated that β-glucan sugars in both soluble and insoluble forms bind to Sp-AMP3, whereas
sugars from the chitin, chitosan and cellulose classes did not bind (III, Figure 4).
The first test assessed whether Sp-AMP3 binds insoluble carbohydrates that are
common in the fungal cell walls of chitin, chitosan and β-(1-3)-D-glucan. Binding assay
revealed that Sp-AMP3 did not bind to chitin and chitosan but did bind to curdlan (an
insoluble β-(1,3)-D-glucan). Soluble sugar binding to Sp-AMP3 was assessed using the
fluorescence change of at least one tryptophan residue of the protein upon binding. The sugars
41
themselves did not fluoresce in the absence of Sp-AMP3. Significant binding was observed
for β-(1,3)-glucan containing sugars (including laminarioligosaccharides up to laminarihexose
and laminarin). In addition, the fluorescence results suggested that ligand binding promotes a
conformational change upon binding to the entire protein that is sensed by the tryptophan
residues (III, Figure 4).
5.3.6. Sp-AMP homology model and a proposed binding site for β-1,3-glucans
Sp-AMP3 was modeled using the Macadamia integrifolia antimicrobial protein
(MiAMP1) NMR structure (PDB entry 1C01, identity 64%) as the template. The homology
modeling studies indicated that Sp-AMPs are expected to have the Greek-key β-barrel fold
(Fig. 5). No obvious active site cavity or cleft similar to those observed in enzymes was noted,
and therefore no enzymatic reactions were expected. There are three conserved disulfide
bonds and several other conserved residues. Some of the conserved residues are clustered on
one face of the molecule, forming a surface patch as depicted in Figure 5. Sequences
identified in a BLAST search primarily represent plant proteins with amino-acid identities of
at least 39% over the entire Sp-AMP sequence. Several fungal proteins were also identified
with similar levels of sequence identity, although these sequences did not cover the first 16
residues (Fig. 5).
As noted above, the homology model of the Sp-AMP3 structure does not suggest its
involvement in an enzymatic activity, and thus a binding function was expected. The
combined results suggest an evolutionary relationship among the proteins mentioned above.
Therefore, we hypothesize that the residues in the hydrophobic patch on Sp-AMP3 shown in
Figure 5 are involved in β-glucan sugar recognition in this family and explain the action of
the various proteins on fungal cell walls. The results from homology modeling and sequence
comparisons suggest that a conserved patch on the surface of the globular Sp-AMP is a
carbohydrate-binding site that can accommodate approximately four sugar units.
The Sp-AMP is a novel pathogenesis-related protein 19 (PR-19). PR-19 is proposed
to be a non-catalytic β-glucan binding protein family that possesses antifungal activity against
the hyphal growth of H. annosum. PR proteins display antimicrobial activity in vitro and
contribute to plant pathogen resistance (van Loon et al., 2006). Antimicrobial properties and
increased expression of Sp-AMPs in response to pathogenic attacks suggest a major role of
these proteins in plant defense. Sp-AMPs are not similar to any other known PR protein
family but fulfill the requirements to be classified as a new family. In conifers, the PR
proteins identified in response to H. annosum infection include PR-2 (Glucanase (Asiegbu et
42
al., 1995)), PR-3 (Chitinase (Davis et al., 2002, Asiegbu et al., 1994), PR-5 (Thaumatin-like
protein (Asiegbu et al., 2005b), PR-9 (Peroxidase (Mensen et al., 1998, Adomas et al., 2007),
PR-12 (defensin: PsDef1 (Kovaleva et al., 2009), SPI1 (Fossdal et al., 2003) and PR-19. Other
PR proteins induced upon other pathogen infection and diverse abiotic stresses in forest tree
species are also implicated in systemic acquired resistance and tree resistance (as reviewed
elsewhere (Veluthakkal and Dasgupta, 2010)).
Figure 5: Sp-AMP3 sequence comparison and homology modeling. Sequence alignment of Sp-AMP1, Sp-
AMP2, MiAMP1 and a number of similar plant proteins (a). Ribbon cartoons of the Sp-AMP3 homology model
(b) and the molecular surface calculated based on the homology model (c) are shown. The proposed binding
surface with four sugar units (laminaritetraose) is modeled (d) (III, Figure 7).
Glucans are accessible in Heterobasidion cell walls (Asiegbu et al., 1995) and thus
are biologically reasonable targets for PR-19 proteins. The discovery that glucans are ligands
of interest also suggests that the problems in heterologous expression were due to
counterproductive interactions between Sp-AMPs and the expression hosts tested.
Streptomyces killer toxin protein also exhibits structural similarity to Sp-AMP3 based on the
relationship to MiAMP1 identified in our study. These killer toxins exhibit cytocidal effects
43
for both budding and fusion yeasts, which changes the yeast morphology despite the fact that
the actual target is obscure (Ohki et al., 2001). Another similar protein identified by the DALI
protein server (Holm, 1998), the yeast killer toxin secreted by Williopsis mrakii (WmKT),
inhibits β-glucan synthesis, which affects cell wall construction (Antuch et al., 1996).
Alternative expression systems merit exploration in future works. In our research study, the
Sp-AMP glucan binding property may cause, for example, interference with glucan assembly,
which could alter the fungal cell wall structure and result in the morphological distortion of
hyphae. Effects on the fungal cell wall glucan could also weaken the membrane and
compromise cell wall integrity. These effects could result in unusual spores, hyphal swellings
and subsequent burst.
5.4. Scots pine pathogenesis-related protein 19 (PR-19) confers increased
tolerance against Botrytis cineria in transgenic tobacco (IV)
For forest tree species, conventional seed orchard and phenotypic selection through
breeding practices are costly and time consuming; numerous prospects suggest that this
limitation could be overcome using molecular marker selection (OMalley et al., 1996). PR-19
family members are potential alternatives to current H. annosum control and management
strategies because these family members exploit built-in defense systems of the host trees.
Finding a gene that could serve as a molecular DNA marker would assist breeding programs
and facilitate the selection of naturally occurring genotypes with increased resistance against
root rot.
5.4.1. Generation of Sp-AMP2 transgenic tobacco plants
To evaluate the potential of the Sp-AMP genes as molecular markers for resistance
tree breeding, a correlation between resistance and Sp-AMP family members was initially
established. Under the control of a super promoter, Scots pine Sp-AMP2 cDNA was
transformed into tobacco (Nicotiana tabacum cv. Petit Havana SR1) using the Agrobacterium
tumefaciens-mediated transformation method (IV, Figure 1). The super promoter (Ni et al.,
1995) is a chimeric promoter derived from the octopine and mannopine synthase genes that is
approximately 156-fold stronger than the CaMV 35S promoter in tobacco leaf tissue, which
makes it useful for high level constitutive expression of genes.
44
The integration of the Sp-AMP2 gene into the genomic DNA of tobacco plants was
confirmed by PCR amplification of the super promoter and Sp-AMP gene sequences. A 358-
bp DNA fragment was detected in T0 transgenic tobacco lines, whereas no bands were
detected in the untransformed control tobacco plant (IV, Figure 2). Four tobacco lines tested
were selected for T1 seed collection via self-pollination and used for further analysis. T1
seeds were collected and verified for stable transgene integration into plants that were
regenerated on selective medium by PCR. Sp-AMP2 transcription was confirmed by
amplifying the cDNA of the wild type control, T0, non-transgenic and T1 transgenic plants
(IV, Figure 3). T0 and T1 transgenic lines revealed an abundance of the Sp-AMP transcript.
The present study demonstrates the successful transfer of a PR-19 encoding gene of
gymnosperms to angiosperms tobacco plants. No homolog of PR-19 was identified in
Nicotiana benthamiana or Nicotiana tabacum (Bombarely et al., 2011).
5.4.2. PR-19 tobacco plants exhibit increased tolerance to B. cinerea
The effects of infection caused by B. cinerea spores on the transgenic lines were
investigated and compared with non-transformant control and non-transgenic T1 tobacco
plants. The lesion sizes from the inoculation at two time points, 3 and 5 d.p.i., are indicated in
Figure 6. Necrotic lesions caused by B. cinerea on the control tobacco (non-transformants)
and non-transgenic T1 tobacco leaves were more severe and larger than those lesions formed
on the transgenic line after 3 d.p.i.. These lesions on the transgenic lines typically increased
slightly after 5 d.p.i.; however, none of the lesions achieved the same size as the non-
transformed control or the siblings of the transgenic lines with no Sp-AMP2 integration.
Together, transgenic tobacco plants exhibited a significant difference in lesion size in
response to infection by B. cinerea compared with the non-transgenic plants at two time
points (see Supplementary material S1).
The results suggest that Sp-AMP2 expression in transgenic tobacco conferred
enhanced resistance to the fungal pathogen B. cinerea. This finding is a further functional
demonstration of the potential role of PR-19 in plant defense. The potential of deploying PR-
19 as a breeding strategy for the development of pathogen resistant in conifers merits further
investigation. Increased expression of PR-19 in response to pathogen challenge, SA, ET and
other elicitors, as shown in our previous study, suggests that PR-19 is involved in the tree
defense against H. annosum.
The broad spectrum action of MiAMP1 protein family members on numerous
microorganisms (Marcus et al., 1997, Zamany et al., 2011) and the nature of the cysteine-rich
45
AMPs, which reduce the growth of major microbes (Marshall et al., 2011), makes PR-19
members potential candidates for the development of pathogen-resistant crops. Plants
expressing Sp-AMP2 showed a reduction in the spread and subsequent expansion of fungal
infection over the 5-day evaluation period, with the most significant difference being observed
at 5 d.p.i.. These results indicate that PR-19 is actively involved in the inhibition of B. cinerea
and suggest a broader spectrum of PR-19 action.
Figure 6: Disease evaluation in Sp-AMP2-transgenic tobacco plants (IV, Figure 4).
In herbaceous annuals, a fitness cost is associated with inducible defenses (Baldwin,
1998). Negative impacts for the constitutive expression of some defensins include reduced
46
cell growth, reduced efficiency of regeneration, reduced fertility and abnormal morphology of
regenerated transgenic plants (Stotz et al., 2009). However, it would be difficult to assess
whether such fitness costs of inducible defenses apply to long-lived conifer trees that may
have large nutrient reserves and a very different phenology. In addition, the low stability of
antimicrobial peptides is a main constraint associated with transgenic expression. The
problem arises from the small size and susceptibility to protease degradation. In addition, the
potential undesirable toxic effects of AMPs, if any, may limit their expression and activity
(Marcos et al., 2008). Nevertheless, the structure of PR-19 as a cysteine-rich AMP that
reduces the growth of major microbes without any toxic affects toward the host (Marshall et
al., 2011) makes PR-19 a promising candidate for the development of pathogen-resistant
plants with no risk of toxic effects.
In summary, Sp-AMP2 was inserted into the genome of the tobacco model plant. The
transformed tobacco plants exhibited increased tolerance against B. cineria. Future studies
will explore the possibility of transferring PR-19 into a related tree species and further assess
its role in conifer tree resistance. These studies will be facilitated by recent advances in spruce
tree genetic transformation and somatic embryogenesis (SE) to generate and propagate elite
recalcitrant genotypes of forest trees (Nehra et al., 2005).
47
6. SUMMARY AND FUTURE DIRECTIONS
Heterobasidion annosum is the most destructive pathogen for forest trees in the
Northern Hemisphere. Although the genome sequence of a close related species (H.
irregulare) was published in 2012, most of the studies focus on the pathogenicity aspects of
the fungus. Few studies have been conducted to provide insight concerning the molecular
regulation of pathogen defense and resistance in trees.
The nature of the pathogen, the limitations of available strategies for controlling the
disease, the economic and social importance of the forest trees and the paucity of molecular
and genomic studies necessitate the development of suitable model systems for basic
mechanistic understanding of the Heterobasidion-conifer pathosystem. This prompted the
study described in article II to determine whether the plant model Arabidopsis could be used
as a suitable host model for studies of H. annosum-host interaction.
The comparative study was the first report of the infection of Arabidopsis with a
necrotrophic pathogen that naturally occurs in conifer trees. However, findings from the tested
Heterobasidion- Arabidopsis/conifer pathosystem models may not strictly apply to all forest
trees due to possible differences in the physical structure of the host and the type of
pathogens. Additional inoculation experiments with other Arabidopsis ecotypes and mutants
may help to further exhibit non-host resistance, which will be of great interest for elucidating
the cellular and genetic basis of the H. annosum pathosystem. Advances in transcriptomics
and NGS would also be advantageous for conducting comparative genomics for identifying
differences in defense strategies between herbs and trees as well as between angiosperms and
gymnosperms. These advances would also aid in the development of alternative tree
pathosystem models for necrotrophic pathogens.
One of the investigated defense proteins is the Scots pine antimicrobial peptide. The
recombinant Sp-AMP has an inhibitory effect against the conifer pathogen. Based on
functional analysis, it was concluded that the studied Sp-AMP proteins belong to a new family
of antimicrobial proteins (PR-19) that are likely to act by binding glucans, which are a major
component of fungal cell walls (see Fig. 7).
48
Figure 7: Schematic view of Sp-AMP gene regulation in response to different organisms and triggers.
To explore the practical applications of the Sp-AMPs, further investigations of their
spectrum of antimicrobial action and development of a functional synthetic mimic are
important future research goals.
Finally, transgenic tobacco plants expressing Sp-AMP2 exhibited a significant
reduction in lesions due to B. cinerea infection, thereby further indicating that PR-19 is
actively involved in pathogen resistance in this non-host model. Future studies will explore
the possibility of transferring PR-19 into a related tree species and further assess its role in
conifer tree resistance.
49
ACKNOWLEDGMENTS
This work was conducted at the Department of Forest Sciences, Faculty of
Agriculture and Forestry at the University of Helsinki. I would like to thank the Finnish
Doctoral Program in Plant Science (FDPPS) that I joined in 2010 for the financial support that
I received during these years.
My sincere appreciation goes to my supervisor, Professor Fred Asiegbu, who is a
unique mentor and scientist in all respects. The knowledge, vision and creative thinking of
Prof. Fred have been a source of inspiration for me throughout this work, and he has greatly
enriched my knowledge with his exceptional insights. This thesis would never have been
possible without his kind guidance, invaluable assistance and support.
I would also like to thank the three members of my follow-up group, Professor
Teemu Teeri, Dr. Jing Li and Dr. Niina Stenval, for their valuable suggestions and critical
discussions to develop my PhD thesis. I am also grateful to the pre-examiners, Professor
Johanna Witzell and Dr. Jun-Jun Liu, for their revisions and suggestions to improve this
thesis.
It has been a pleasure to be a member of the forest pathology group. I am greatly
indebted to the teamwork spirit and enlightening discussions that have enriched my research
life. Therefore, I would like to thank Tommaso, Andriy, Susanna, Risto, Eeva, Hui, Chen,
Abbot, Chaowen, Sannakajsa and Anna-Maija. Moreover, I wish to acknowledge the
following colleagues for their help and support in the Department of Agricultural Sciences:
Shahid, Hany, Bahram, Iman, Mikko, Tian and all the others with whom I shared thoughts
and discussions.
A special acknowledgement goes to Jukka Lippu and all the administrative staff in
the Department of Forest Sciences for their very kind help. It is impossible to mention
everyone who has made a difference in my work; therefore, I am deeply thankful to everyone
who helped me to accomplish this thesis.
Thanks are also due to my wonderful friends who have made my stay in Helsinki
pleasant and enjoyable: Qasem, Nader, Rami, Zuhdi, Yehia, Murad, Mahmoud and Moukhlis.
I would further like to thank the following friends: Rana’a, Ghassan, Mohamad Arori and
Kareem along with all my friends who have been there for me here in Finland as well as back
home in Palestine and in Jordan. You keep my spirit alive, and I thank you for the constant
encouragement. Your continuing friendship means very much to me.
50
This journey could not have been possible without the support of my family. I cannot
express my full gratitude to my parents, my brothers and my sisters, who deserve all my
consideration for their love and moral support; I am deeply indebted to you forever.
Helsinki, August 2014
Emad
51
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