Pathobiology of Heterobasidion-conifer tree interaction: molecular … · 2017-03-11 · II. Emad Jaber, Chaowen Xiao, Fred O. Asiegbu (2014). Comparative pathobiology of Heterobasidion

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

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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.

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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).

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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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