Masaryk University
Faculty of Science
Department of Biochemistry
HABILITATION THESIS
Molecular Aspects of MAMP (Microbe-Associated
Molecular Pattern) Triggered Immunity in Plants
Jan Lochman
Brno 2016
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ACKNOWLEDGEMENTS
On the first place I would like to gratefully and sincerely thank deceased prof. Vladimír
Mikeš for his guidance, understanding, and most importantly, his friendship and support
during my graduate and post-graduate studies in the Department of Biochemistry. His
mentorship was essential for my further scientific work and carrier. I would also like to
thank all members of the Department of Biochemistry for their patience and all the
stimulating discussions we have had.
I would also like to thank Michel Ponchet from the INRA Sophia Antipolis for his
assistance and guidance in getting my post-graduate career started on the right foot and
for many inspirational discussions about the fascinating world of plant defence, science and
life. I also wish to thank Harald Keller from the INRA Sophia Antipolis for many valuable
advices in getting my graduate career. I am grateful to Francois Simon-Plas and Nathalie
Leborgne-Castel from the INRA Dijon, for introducing me into the field of membrane rafts.
My thanks go to all my colleagues and students for their inspiring thoughts, for
excellent work and cooperation and for creation of a stimulative working environment.
Finally, and most importantly, I would like to thank my wife Gabriela for her support,
encouragement, quiet patience and unwavering love, my daughters Dominika and Valerie
for being a source of motivation and pleasure.
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TABLE OF CONTENTS
PREFACE ............................................................................................................................................. 4
1. PLANT DEFENCE MECHANISMS.................................................................................................. 5
1.1 PLANT-PATHOGEN INTERACTIONS..................................................................................... 6
1.2 ZIG-ZAG MODLE OF PLANT IMMUNITY.............................................................................. 6
2. MICROBE-ASSOCIATED MOLECULAR PATTERNS TRIGGERED IMMUNITY (MTI)........................ 8
2.1 PERCPEPTION OF MAMPs .................................................................................................. 9
2.2 PATHOGENESIS RELATED PROTEINS AND SYSTEMIC PLANT IMMUNITY......................... 11
3. ERGOSTEROL AS AN EXAMPLE OF AN ORPHAN FUNGAL MAMP ............................................ 13
3.1 ERGOSTEROL INDUCED DEFENCE REACTION IN PLANTS ................................................. 14
3.2 SIGNALLING PATHWAY TRIGGERED BY ERGOSTEROL...................................................... 16
3.3 THE ROLE OF SA AND SPERMINE...................................................................................... 16
3.4 THE ROLE OF NO AND CALCIUM ...................................................................................... 17
3.5 CONTRIBUTION TO GIVEN PROBLEMATIC ................................................................... 20
4. ELICITINS AS AN EXAMPLE OF A TYPICAL OOMYCETE MAMP ................................................. 21
4.1 CHARACTERIZATION OF ELICITINS BINDING SITE............................................................. 24
4.2 THE ROLE OF STEROL TRAPING IN ELICITINS BIOLOGICAL ACTIVITY................................ 26
4.3 THE ROLE OF ELICITINS NET CHARGE IN THEIR BIOLOGICAL ACTIVITY............................ 32
4.4 THE ROLE OF ELICITINS CHARGE IN THEIR MOVEMENT ACROSS THE PLANT.................. 36
4.5 CONTRIBUTION TO GIVEN PROBLEMATIC ................................................................... 37
5. CONCLUSION............................................................................................................................ 38
6. REFRENCES ............................................................................................................................... 39
7. PUBLICATIONS PRESENTED IN HABILITATION THESIS.............................................................. 47
8. SUPPLEMENTS.......................................................................................................................... 49
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PREFACE
The present habilitation thesis is a comment summary of original scientific articles
published between the years 2005 and 2016 to which I have contributed as the first author,
corresponding author or co-author. All these publications are related to the
characterization of molecular mechanisms responsible for the defence reaction in plants
triggered by Microbial-Associated Molecular Patterns (MAMPs). The text part of the thesis
is divided into four main chapters. The first chapter presents a current concept of plant
defence mechanisms in plants, covering the generally accepted zig-zag model. The second
chapter is dedicated to the description of processes included in MAMPs-Triggered
Immunity (MTI) which affords a basic effective level of the resistance to plants. The third
chapter describes in detail molecular processes of ergosterol perception which represents
an orphan fungal MAMP. The last chapter presents a complexity of factors taking the role
in perception of elicitins, a typical oomycetes MAMP, by plants.
A comprehensive information on the studied plant-pathogen interactions can be
found in the supplement, together with the list of publications included in this habilitation
thesis. At the end of each third and fourth part a contribution to the given topic is presented
and in conclusion future prospects of the research are outlined.
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1. PLANT DEFENCE MECHANISMS
In their natural environment, plants serve as a rich source of nutrients and that is
why they host a great number of non-pathogenic and pathogenic microorganisms,
especially on their root system and shoots (leaves, reproductive structures, stem). In
contrast to vertebrates, plants are sessile organisms lacking neither specialized immune
cells nor are adaptive immune system against continuous attack of a vast array of
pathogenic microorganisms such as fungi, oomycetes, viruses, bacteria or nematodes
(Jones and Dangl, 2006). To deal with this challenge, plants are protected by mechanical
barriers such as a waxy cuticle or protective periderm and they have evolved a multilayered
immune system, in which each cell has a potential capacity to trigger immune
response against pathogen infection. This process called the innate immunity is
fundamental for survival, and it allows a plant to ward off pathogen both, in a rapid and
localized manner, and remember any previous infection (Reimer-Michalski and Conrath,
2016).
Plant pathogens use diverse strategies to colonise their hosts. Pathogenic bacteria
enter the plant through pores (stomata or hydathodes) or wounds and proliferate in the
intercellular space; nematodes and aphids directly insert a stylet into a plant cell and
pathogenic fungi or oomycetes are able to invaginate haustoria into the host cell plasma
membrane (Jones and Dangl, 2006). One of the common features of all these diverse
pathogen classes is the delivery of some type of effector molecules (virulence factors) into
the host cell to enhance microbial fitness (Cui et al., 2015; Reimer-Michalski and Conrath,
2016).
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1.1 PLANT-PATHOGEN INTERACTIONS
In general, the plant-pathogen interactions are either incompatible or compatible.
The compatible interaction (between a susceptible host and a virulent pathogen) results in
a successful pathogen infection and outbreak of the disease and the incompatible
interaction results in triggering of a defence response and elimination of the pathogen. In
the 1940s, Harold H. Flor proposed the gene-for-gene model to describe the interaction
between a pathogen and its host plant (Flor, 1971). This model predicted that a plant
resistant phenotype would occur only in that situation when the plant possessed a
dominant resistance gene (R) and the pathogen expressed a complementary dominant
avirulence gene (Avr). Even though the model holds true for most biotrophic plantpathogen
interactions, for many other pathogens an indirect activation of R proteins by
pathogen encoded-effectors was described. This fact implies that the R proteins are able
to recognise indirectly pathogen effectors by monitoring the integrity of host cellular
targets of effector action, which is similar to the recognition of “modified self” in “danger
signal” models of the mammalian immune system. Based on this knowledge, a new fourphased
“zig-zag” model was proposed (Jones and Dangl, 2006).
1.2 ZIG-ZAG MODLE OF PLANT IMMUNITY
This model describes, in essence, two branches of the plant immune system. One
uses surface pattern recognition receptors (PRRs) acting as initial radars that recognise
slowly evolving microbial-associated molecular patterns (MAMPs) to induce a basal
resistance response called PRR-triggered immunity (PTI) (Figure 1) (Böhm et al., 2014; Jones
and Dangl, 2006; Zipfel, 2008). PTI is considered as an ancient form of plant immunity and
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has the potential to fend off host non-adapted pathogens due to the conserved nature of
MAMPs (bacterial flagellin, chitin or oligogalacturonides) across species (Reimer-Michalski
and Conrath, 2016). The second takes a place largely inside the cells employing a family of
polymorphic intracellular nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins to
perceive secreted virulence effectors (previously called Avr protein) directly or indirectly as
a result of host-pathogen co-evolutionary cycle (Cui et al., 2015; Jones and Dangl, 2006).
The direct interaction of the pathogen effector with plant R proteins leads to gene-for-gene
immunity. The indirect recognition of pathogen effector by plant R proteins has been
described in the so-called guard hypothesis in which R proteins guard the integrity of
cellular proteins and when there is some modification/degradation of proteins by an
appropriate effector, they will trigger plant defence (Jones and Dangl, 2006; ReimerMichalski
and Conrath, 2016). Independent of whether pathogen effector is recognised
directly or indirectly, its perception results in the effector-triggered immunity (ETI)
amplifying PTI response and resulting in a resistance, and usually, a hypersensitive cell
death response at the infection site to avoid spreading of biotrophic pathogens to healthy
tissue of plants (Figure 1) (Reimer-Michalski and Conrath, 2016).
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Figure 1. The Zig-Zag model for evolution of innate immunity- and silencing-based plant defence
against viral and non-viral pathogens (adopted from Jones and Dangl, 2006). The ultimate
amplitude of disease resistance or susceptibility is proportional to [PTI – ETS + ETI]. During phase 1,
plants sense microbe-associated molecular patterns (MAMPs) and host danger-associated
molecular patterns (DAMPs) via pattern-recognition receptors (PRRs) to induce PRR-triggered
immunity (PTI). In phase 2, successful pathogens secret effectors/suppressors into the plant
interfering with PTI which results in effector-triggered susceptibility (ETS). In phase 3, plant
recognizes directly or indirectly the effector molecule (indicated in red) by an NB-LRR protein which
results in triggering effector-triggered immunity (ETI), a boost version of PTI passing a threshold for
induction of hypersensitive response (HR) and programmed cell death (PCD). In phase 4, a new
selection of pathogen isolates, that have lost the red effector and gained a new effector (indicated
in purple), is done. This process helps pathogens to overcome ETI, however selection pressure
favour new plants possessing new NB-LRR allele recognising one of the newly acquired effector,
resulting again in ETS.
2. MICROBE-ASSOCIATED MOLECULAR PATTERNS TRIGGERED IMMUNITY (MTI)
Evolutionary conserved microbial signatures (also termed as exogenous elicitors)
are counterparts of animal pathogen-associated molecular patterns (PAMPs). They occur
in both, pathogenic and non-pathogenic microbes, and because the basal line of plant
defence does not distinguish between mutualistic and parasitic symbiosis, the term
Microbe-Associated Molecular Patterns (MAMPs) has been introduced (Schwessinger and
Ronald, 2012). MAMPs are largely diverse in their chemical nature including
oligosaccharides, (poly)-peptides, (glyco)-proteins and lipids; such as chitin, a structural
component of the cell wall of fungi; flagellin, a protein subunit of bacterial flagella;
lipopolysaccharides from outer membrane of gram-negative bacteria; xylanase, Pep-13,
ergosterol, cold-shock proteins or oligogalacturonides (Boller and Felix, 2009; Henry et al.,
2012; Nürnberger et al., 2004). Some of the MAMPs are only perceived by a narrow range
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of plant species belonging to a single family (Felix and Boller, 2003; Ron and Avni, 2004).
Such an example is the EF-Tu (elongation factor thermo unstable) recognised only in the
Brassicaceae family (Kunze et al., 2004). On the other hand, other MAMPs such as chitin,
lipopolysaccharides and flagellin, trigger defence responses in various host species,
although there is difference to a certain extent in specificity and perception efficacy for a
plant family or species (Zipfel et al., 2006). For all these reasons, MTI typically wards off
multiple microbes, no matter whatever pathogenic or not, likely because of the conserve
structure of MAMPs across diverse microorganisms. Recent studies suggested that
endogenous danger-associated molecular patterns (DAMPs), released from the host plant
by enzymatic or mechanical processes controlled by the pathogen, help further amplifying
MTI to establish a robust systemic plant immune response (Reimer-Michalski and Conrath,
2016).
2.1 PERCPEPTION OF MAMPs
Today it seems that in plants the recognition of a wide range of proteinaceous,
carbohydrate or lipophilic MAMPs or DAMPs rely only on plasma membrane localized
receptor kinases (RKs) or receptor-like proteins (RLPs) (Böhm et al., 2014; Cui et al., 2015;
Zipfel, 2014). This is in contrast to mammals in which both, surface localized (e.g. TLRs) and
intracellular (e.g. NLRs) immune receptors, have been shown to recognise PAMPs
(Maekawa et al., 2011). A typical receptor like-kinase consists of a variable extracellular
leucine-rich repeat (LRR) ligand-binding domain, resembling that of Toll-like receptors in
animals, a single transmembrane domain and an intracellular serine/threonine kinasesignalling
domain and bear structural similarities to animal receptor-tyrosine kinases (RTKs)
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(Böhm et al., 2014; Shiu and Bleecker, 2001). Although numerous MAMPs and their
corresponding PRRs have been reported, only a few of them are well characterized. Some
of the best characterized MAMP-PRR recognitions in bacteria perception are Flg22:FLS2
and EF-Tu:EFR. Flg22 (a 22-amino-acid epitope from the N-terminus of flagellin) binding to
FLS2 (flagellin-sensitive 2) leads to the rapid forming of heterodimers with another LRR-RK
BAK1/SERK, what is required for the full activation of FLS2 and flg22-triggered immune
signalling (Figure 2) (Chinchilla et al., 2006; Zipfel et al., 2006). Interestingly, it is becoming
increasingly apparent that BAK1, and related SERK proteins, associate with several
additional LRR-RKs or LRR-RLPs like brassinolide (BL) receptor BR1 or elicitin receptor ELR
(Figure 2) (Du et al., 2015; Zipfel, 2014).
Figure 2. Various types of plasma membrane immune receptors for perception of MAMPs (adopted
from Böhm et al. 2014). Leucine-Rich Repeat Receptor Kinase (LRR-RK) and Leucine-Rich Repeat
Receptor-Like Proteins (LRR-RLP) type immune receptors sense proteinaceous patterns. The
conserved LRR-RKs FLS2 and EFR recognise flagellin (via the fls2 epitope) and Elongation Factor Tu
(EF-Tu), respectively and form heterodimeric complex with LRR-RK BAK1. The conserved LRR-RLP
ELR form heterodimer with LRR-RK BAK1/SERK3 and recognise elicitins domain. Lys-M type immune
receptors recognise GlcNAc-containing carbohydrate ligands by RLL-RK CERK1 or by RLL-RLP
forming heteromeric complexes with LysM-RK CERK1.
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In perception of fungi the recognition of chitin, a major constituent of fungal cell
walls and classical MAMP, has been studied for decades in plants. Lysine motif receptor
kinases, (LysM-RKs) with the extracellular LysM domain recognising N-acetylglucosamine
(fungal chitin, bacterial peptidoglycan or bacterial nodulation factor) and intracellular
Ser/Thr kinase domain, play a crucial role in perception of fungal chitin (Figure 2). However,
different plants use distinct mechanisms for chitin perception; for example, chitin elicitor
receptor kinase (CERK1) of Arabidopsis or chitin oligosaccharide elicitor-binding protein
(CEBiP) in rice exhibiting three and two LysM extracellular domains, respectively (Figure 2)
(Kaku et al., 2006; Miya et al., 2007).
In perception of oomycetes, recently a receptor-like protein ELR (elicitin response)
responsible for extracellular recognition of elicitins, a molecular pattern conserved in
Phytophthora species, was identified in the wild potato Solanum microdontum. Noticeably,
similarly to flg22, ELR associates with the immune co-receptors BAK1/SERK (ChaparroGarcia
et al., 2011; Du et al., 2015).
2.2 PATHOGENESIS RELATED PROTEINS AND SYSTEMIC PLANT IMMUNITY
After the perception of MAMPs, in many plant species, a massive induction of
inducible defence-related proteins has been described (Dadakova et al., 2015; Keller et al.,
1996; Klemptner et al., 2014; van Loon et al., 2006). These proteins are called
pathogenesis-related proteins (PRs) and due to the common features they have been
classified 17 families (Table 1).
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Table 1. Individual families of PR proteins (van Loon et al., 2006)
Family Type member Function
PR-1 Tobacco PR-1 unknown
PR-2 Tobacco PR-2 -1,3-glucanase
PR-3 Tobacco P, Q chitinase
PR-4 Tobacco `R' Chitinase
PR-5 Tobacco S thaumatin-like
PR-6 Tomato Inhibitor I proteinase-inhibitor
PR-7 Tomato P endoproteinase
PR-8 Cucumber chitinase Chitinase
PR-9 Tobacco `lignin-forming peroxidase' peroxidase
PR-10 Parsley `PR1` ribonuclease-like
PR-11 Tobacco class V chitinase
PR-12 Radish Rs-AFP3 Defensin
PR-13 Arabidopsis THI2.1 Thionin
PR-14 Barley LTP4 lipid-transfer protein
PR-15 Barley OxOa (germin) Oxalate oxidase
PR-16 Barley OxOLP Oxalate-oxidase-like
PR-17 Tobacco PRp27 Unknown
The common features of these proteins are usually antimicrobial activities in-vitro
through hydrolytic activities on cell walls, contact toxicity, and an involvement in defence
signalling. Three base signalling compounds included in the process of PRs induction are
salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The production of PR proteins is
related to the process of systemic plant immunity covering System Acquired Resistance
(SAR) and Induced Systemic Resistance (ISR) (van Loon et al., 2006). In SAR, which wards
off diverse biotrophic pathogens, the presence of SA-induced PR-type proteins is likely to
contribute to some extent to the enhanced defensive capacity. On the other hand, in ISR
that is activated by root-colonizing grow/yield promoting bacteria and fungi, no defencerelated
proteins are present in induced leaves before challenge, but upon infection, the
activation of JA- and/or ET-responsive genes in particular is accelerated and enhanced, in
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a phenomenon called defence priming (Conrath et al., 2015). Consequently, the activation
of both systemic immune responses, SAR and ISR, can result in an additive effect, however
a negative cross-talk of the SA- and JA/ET signalling pathways has beenreported as well
(van Loon et al., 2006; Reimer-Michalski and Conrath, 2016).
3. ERGOSTEROL AS AN EXAMPLE OF AN ORPHAN FUNGAL MAMP
Ergosterol (5,7 – diene oxysterol), a principal compound of the fungal plasma
membrane which has never been found in plants, is perceived by many plant species as an
elicitor (Amborabé et al., 2003; Granado et al., 1995; Klemptner et al., 2014; Laquitaine et
al., 2006; Rossard et al., 2010). Sterols are essential for the organisation and structure of
eukaryotic cells plasma membranes when ergosterol is the major component in lower
eukaryotes, whereas cholesterol and sitosterol are the most abundant sterols in higher
eukaryotes and plants, respectively (Roche et al., 2008; Xu et al., 2001). These steroids
differ structurally, with ergosterol having two additional double bonds (at the positions C7C8
and C22-C23) and a methyl group at the C24 position of the side chain (Figure 3).
Figure 3. Chemical structure of ergosterol (A), cholesterol (B) and -sitosterol (C) showing
similarities and differences. Compared to cholesterol, ergosterol has unsaturated positions C7-C8
and an additional methyl group at the position C24 and sitosterol has an additional ethyl group at
position C24.
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These structural differences presumably allow the plant cell to recognize ergosterol as a
typical MAMP, when it has been shown, that many aspects of the early response to
ergosterol treatment are similar to that of chitosan, including desensitization phenomenon
(Amborabé et al., 2003).
3.1 ERGOSTEROL INDUCED DEFENCE REACTION IN PLANTS
Previously, using various plant models, ergosterol treatment has been shown to
induce an early phase of defence reaction characterized by changes in membrane
potential, cytosolic calcium level and modification of H+ flux or production of reactive
oxygen species (ROS), even in nano-molar concentration (Amborabé et al., 2003;
Kasparovsky et al., 2004; Rossard et al., 2006; Vatsa et al., 2011). In the case of a plant
infiltration, the ergosterol insolubility in water was increased by dissolving it in 2hydroxypropyl-cyclodextrin,
which is generally used for solubilisation of different
lipophilic compounds (Lochman and Mikes, 2006; Uekama, 2004). Exogenous application
of ergosterol to tobacco plants induced an accumulation of salicylic acid (SA) together with
transcripts of phenylalanine-ammonia lyase (PAL), sesquiterpene cyclase (EAS) and acidic
pathogenesis-related proteins PR1a, PR1b, PR3Q and PR5 (Figure 4) (Lochman and Mikes,
2006).
The PAL represents an important regulatory enzyme of secondary metabolism that
has important roles in plants defence against pathogens (Dixon and Paiva, 1995; Howles et
al., 1996). As the synthesis of salicylic acid is closely related to PAL activation, the measured
15
regulation of enzyme activity after an ergosterol application is not surprising (Dadakova et
al., 2013).
EAS is an enzyme that plays an important role in the synthesis of sesquiterpene
phytoalexins in tobacco in a response to various stress stimuli, pathogen and fungal
elicitors. An increased concentration of capsidiol after an ergosterol treatment in cell
suspension has been demonstrated (Kasparovsky et al., 2004). However, compared to
previously used elicitors such as cryptogein or cellulase from Trichoderma viridae, which
show a maximum expression after 8 hours following the treatment, the induction time for
EAS in the case of ergosterol treatment shows a gradual increase within 24 hours (Lochman
and Mikes, 2006), similar to that previously observed for methyljasmonate (Keller et al.
1998; Mandujano-Chávez et al., 2000).
The increasing expression of the acidic PR1a protein is usually used as a marker of
systemic acquired resistance (SAR) although some recent studies demonstrated that its
expression may not be linked with neither development of SAR nor with an effective
resistance phenotype (Block et al., 2005; Conrath, 2006). For the group of PR3 proteins,
results of previous studies have indicated a chitinase activity leading to decreasing
susceptibility to infection by fungi with chitin cell walls (Sela-Buurlage et al., 1993). For the
group of PR5 proteins called thaumatin-like proteins, an antifungal activity has been
demonstrated (Vigers et al., 1992).
Noticeably, ergosterol treatment did not result in the accumulation of a transcript
for the basic PR1 protein and the expression of proteinase inhibitors I and II were
suppressed (Lochman and Mikes, 2006). Proteinase inhibitors (PIs) have been characterized
in various plant species. An induction of their expression after a wounding, TMV infections
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and application of jasmonic acid was demonstrated (Linthorst et al., 1993; Park et al., 2000).
A central signalling molecule in this pathway leading to the accumulation of PIs and basic
PR proteins is jasmonic acid produced in the octadecanoid pathway. Many studies assumed
the existence of a cross-talk between at least two main signalling pathways: SA dependent
and JA dependent pathways (Heil and Bostock, 2002). In both tobacco and tomato, SA
inhibited the octadecanoid pathway and JA synthesis (Baldwin et al.; Niki et al., 1998; PenaCortés
et al.). Moreover, the accumulation of PIs in tomato in a response to wounding was
strongly reduced by the application of salicylic acid (Doares et al., 1995). Similarly, SAmediated
inhibition of the octadecanoid pathway downstream of JA has been described
(Engelberth et al., 2000). However, an acceptable model for the cross-talk between these
two pathways has not been found up until now.
3.2 SIGNALLING PATHWAY TRIGGERED BY ERGOSTEROL
Unfortunately, the mechanisms of ergosterol recognition by plant cell have not
been described yet. It is hypothesised, that plants either possess an ergosterol receptor, or
that ergosterol uptake results in perturbations of a lipid raft structure because of the ability
of ergosterol to form very stable microdomains (Klemptner et al., 2014). Although the
signalling transduction pathway after perception of ergosterol has not been fully elucidated
to date, some general aspects were already described.
3.3 THE ROLE OF SA AND SPERMINE
The observed accumulation of the PAL transcript and an increase in lipoxygenase
(LOX) and phospholipase A2 (PLA2) activity after ergosterol treatment imply a possible
involvement of both, SA and JA signalling, in ergosterol perception (Dadakova et al., 2013;
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Lochman and Mikes, 2006). In the case of PAL, an up-regulation of the PAL gene caused an
increased PAL activity due to elevated protein levels. Furthermore, a very good correlation
between this increase and accumulation of SA was shown (Lochman and Mikes, 2006). On
the other hand, measured changes in the NaLOX3 transcript after ergosterol treatment
corresponded well to changes in LOX activity in Beta vulgaris leaves measured previously,
with a rapid and transient increase peaking in the first two hours and subsequent slight
decrease below basal level around 3 h (Dadakova et al., 2013; Rossard et al., 2010).
However, no JA accumulation after the ergosterol application was detected (Dadakova et
al., 2013). This observation corresponds to the results with a potato homolog (LOX-H3)
where its depletion does not result in a reduced accumulation of the wound-induced JA
level suggesting that LOX-H3 may play a regulatory role over other activities (Royo et al.,
1999) and the role of NaLOX3 in JA synthesis is contentious. Surprisingly, an elevated level
of free polyamine spermine, able to trigger acidic PR proteins accumulation in plants, was
measured after ergosterol application (Dadakova et al., 2013). These findings indicate that
SA and spermine signalling pathways are more likely involved in ergosterol elicitation than
JA signalling (Figure 4). This is further supported by the above mentioned observations that
ergosterol treatment triggers an accumulation of transcripts only for the acidic form of PR1
protein and suppresses accumulation of transcripts for PI-I and –II induced by wounding
(Lochman and Mikes, 2006).
3.4 THE ROLE OF NO AND CALCIUM
Among plants, NO plays a key role in diverse (patho) physiological processes, e.g.
hormonal, wounding and defence responses, stomatal closure, and cell death regulation.
In plants, NO is generated by NOS-like enzymes, nitrate reductase or non-enzymatic
18
processes (Wendehenne et al., 2004; Wilson et al., 2008). The response to NO may involve
a signalling through the cGMP, cADPR and Ca2+ pathways or activation of a MAP kinase
signalling pathway (Wendehenne et al., 2004; Wilson et al., 2008). The observed NO
accumulation in ergosterol treated cells by DAF-FM fluorescence probe and suppression of
ergosterol induced accumulation of PR1a, PR5 and tPOXC1 transcripts by the scavenger of
NO cPTIO and inhibitor of NOS-like activity L-NMMA implies that NO participates in
activating defence related transcripts after ergosterol treatment and its production is
controlled by a NOS-like enzyme (Dadakova et al., 2013). However, the inhibitor of NOSlike
enzymes L-NMMA did not modify ROS production after the application of ergosterol to
a tobacco cell suspension suggesting that NO does not directly participate in the activation
of NADPH oxidase activity which was measured previously in different plant species
(Dadakova et al., 2013; Kasparovsky et al., 2004; Rossard et al., 2006, 2010).
It has been shown that ergosterol induces a concentration-dependent elevation of
calcium in the cytosol. The ergosterol-triggered Ca2+ increase is biphasic, where the first
phase corresponds to the influx from the extracellular space and the second phase
corresponds to the release of calcium from internal stores via IP3 and cADPR sensitive
channels (Figure 4) (Vatsa et al., 2011). Moreover, it has been shown that the cADPR
channels are involved in both, NO- and SA-mediated pathways, leading to the activation of
the PR1a gene (Klessig et al., 2000). Ruthenium red, a known inhibitor of cyclic ADP-ribose
dependent Ca2+ channels, only inhibited the ergosterol-induced accumulation of PR1a
transcript (Dadakova et al., 2013). Durner et al. showed that addition of cADPR causes
increased the accumulation of mRNAs encoding PAL and PR1a protein in tobacco. The same
pathway is likely to be important in ergosterol-triggered PR1a transcript induction (Figure
4). On the other hand, low or no inhibition of ergosterol-induced accumulation of tPOXC1
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and PR5 transcripts indicates the involvement of other regulation mechanisms that are not
fully dependent on Ca2+ channels located on the membranes of internal stores (Dadakova
et al., 2013). Moreover, almost the same pattern of inhibition for the ergosterol-induced
accumulation of PR1a, PR5 and tPOXC1 transcripts was observed with the inhibitor of
calmodulin/Ca2+ dependent steps W7 (Dadakova et al., 2013).
Figure 4. Summary of known /putative mechanisms taking a role in ergosterol perception (adopted
from Klemptner et al. 2014). The scheme illustrates the perception of ergosterol by a hypothesised
receptor, based on the assumption that ergosterol may be perceived in the same manner like other
MAMPs. Ergosterol perception has been shown to trigger a Ca2+
influx as well as Ca2+
release from
intracellular stores. The already known pathways that are involved in the defence response
following ergosterol treatment are shown including putative WRKY transcription factors in the
nucleus activated through various upstream interactions by MAPK-based signalling. Sesquiterpene
cyclase (SQC), 5-epi-aristolochene synthase (EAS) and phenylalanine-ammonia lyase (PAL)
represent important metabolic enzymes contributing to the production of specific ergosterol-
20
induced metabolites. The genes, proteins and metabolites included in this scheme have resulted
from studies in tobacco and grapevine.
Recently, the role of both calcium-dependent and -independent pathways in
ergosterol elicitation has been shown (Vatsa et al., 2011). Whereas the activation of NAPDH
oxidase and inhibition of H+-ATPase were clearly calcium-dependent, the activation of two
mitogen-activated protein kinases (SIPK and WIPK) during the first minutes after ergosterol
treatment was shown to be calcium independent (Figure 4). With respect to the elevated
spermine content triggered by ergosterol (Dadakova et al., 2013) a good candidate for the
observed calcium independent pathway is, the spermine pathway, which role in the
induction of both transcripts, PR5 and tPOXC1 has previously been demonstrated (Hiraga
et al., 2000; Yamakawa et al., 1998).
3.5 CONTRIBUTION TO GIVEN PROBLEMATIC
Fungal pathogens are a significant threat to crop production. Ergosterol, a principal
compound of the fungal plasma membrane, which has never been found in plants, is one
of the classical MAMP molecule together with chitin, chitosan or -glucans. Therefore,
understanding the mechanism of its participating in the perception by plants is one of the
challenge in discovering of plant’s innate immunity.
Within our work, we firstly demonstrate the ability of ergosterol to trigger the
expression of some defence related genes in plants. We found a specific induction of acidic
forms of PR proteins families and enzymes participating in the defence response, such as
phenylalanine ammonia lyase and sesquiterpene cyclase. Further, a possible cross-talk
between the signalling pathways of salicylate and jasmonate was observed. Moreover, we
studied in detail the signalling pathway following the ergosterol perception in plants. To
21
understand the sequence of the signalling cascade, several representative steps involved
in the synthesis of crucial signalling molecules were targeted using specific inhibitors. The
results showed an important role of calmodulin-dependent protein kinases and nitric oxide
in SA-dependent pathway, resulting in the expression of defence-related genes. However,
at the same time the presence of SA-independent spermine pathway was demonstrated.
Our results from these studies significantly contributed to understanding the mechanism
following ergosterol perception in plants and have been cited several times cited in
prestigious journals.
4. ELICITINS AS AN EXAMPLE OF A TYPICAL OOMYCETE MAMP
Oomycetes are a distinct class of fungus-like eukaryotic microbes, many
economically significant diseases of crop species as the potato late blight, caused by the
pathogen Phythopthora infestans. On the other hand, oomycetes also include the
mycoparasite Pythium oligandrum, which is used as a biocontrol agent against pathogenic
fungi. Thus understanding the mechanisms taking place in the oomycal infection process
could lead to new methods providing a durable resistance. Oomycete plant pathogens
exhibit a biotrophic, necrotrophic or hemibiotrophic lifestyle. Among the biotrophic
oomycetes, completely reliant on the host tissue are Hyaloperonospora parasitica or
Plasmopara viticola causing the white rust. Typical representatives of hemibiotrophic
oomycetes are Phytophthora species, some of which have a potential to infect a wide range
of different plant species. Necrotrophs are represented e.g. Pythium ultimum (Fawke et al.,
2015). During the evolution, some parasitic Phytophthora and Pythium spp. have lost the
ability to synthetize their own sterols and must pick-up sterols from the host cell
22
membranes. From culture filtrates of non-pathogenic Phytophthora spp. on tobacco, P.
cryptogea and P. capsici, small proteinaceous elicitors named elicitins (namely cryptogein
and capsicein) were isolated (Ponchet et al., 1999). Elicitins are members of a family of
conserved lipid transfer proteins with sterol-binding and elicitor activity, catalysing an invitro
sterol transfer between liposomes and micelles (Mikes et al., 1997, 1998). However,
there is still missing a clear in-vivo evidence of their involvement in sterol uptake because
a P. infestans strain lacking elicitin INF1 remains pathogenic (Kamoun et al., 1997, 1998).
Today, elicitins are among the most well-known oomycetes MAMPs which have been
shown to induce the hypersensitive response (HR) in several plants, such as Nicotiana
species and some radish and rape cultivars (Kamoun et al., 1998; Panabieres et al., 1995;
Ponchet et al., 1999; Ricci et al., 1989). Elicitins represent a highly conserved family of
proteins characterised by the “elicitins signature” distinguished by their size (98
aminoacids), lack of amino acid tryptophan, histidine and arginine and abundance of a few
amino acids, such as serine, threonine (30 % of the protein), and leucine (about 10%), and
the presence of six cysteine residues located in conserved positions responsible for three
structurally determinant disulphide bridges. Based on the primary structure of elicitins,
three different classes have been identified. Elicitins from class I contain only the elicitin
domain of 98 amino acids and can be further separated according to their respective pI to
class I including acidic -elicitins (pI < 5) and class I including basic -elicitins (pI > 7.5).
Despite the presence of both within the genome of respective Phytophthora spp. subclasses
are usually present, compared to -elicitins, -elicitins were found to be secreted
by a restricted range of species and appear to be ancestors of other elicitins (Ponchet et
al., 1999). Class II contains hyperacidic elicitins with the sequence length of about 103
amino acids and net charges ranging from -6 to -10 which transcripts have not been
23
observed within the corresponding species up today. The last class III contains elicitinencoding
sequences from P. infestans having app. 70 amino acids long C-terminal sequence
rich in Ala, Ser and Thr residues, representing a possible O-glycosylation domain.
The three-dimensional structure of elicitins were firstly determined for a basic
elicitin cryptogein secreted by Phytophthora cryptogea. The three-dimensional structure
of cryptogein is composed of five α-helices, one β-sheet, and one ω-loop arranged in a
unique protein fold (Figure 5) (Boissy et al., 1996; Gooley et al., 1998). The ω-loop present
in the structure of cryptogein is very flexible and highly conserved, which suggests it has an
important function. The study of conformational changes, due to pH and cryptogein
concentration, demonstrated the ability of cryptogein to dimmerize (Gooley et al., 1998)
which was later proved by the published X-ray structure of the elicitin cinnamomin,
secreted by Phytophthora cinnamomi crystallizing as a homodimer (Figure 5) (Rodrigues et
al., 2006).
Figure 5. Structure of elicitin -cryptogein (A) and cinnamomin (B). Cryptogein structure composed
of five α-helices, one β-sheet, and one ω-loop is shown with a molecule of ergosterol located in the
hydrophobic cavity (Boissy et al., 1999). -Cinnamomin crystalized as a homodimer with opposite
orientation of monomer subunits (Rodrigues et al., 2006). Both structures were determined by Xray
crystallography.
24
Most of the further characterisation of interactions between the elicitins and plants
has been carried out on tobacco plants, by using cryptogein representing a very efficient elicitin
of class I secreted by Phytophthora cryptogea. In tobacco cells the perception step
is followed either activation of protein kinases (PK) or inhibition of protein phosphatases
(PP), triggering the Ca2+ influx, which, in turn, triggers ROS production, MAPK activation,
anion effluxes and plasma-membrane depolarization, and glucose (Glc) import inhibition,
or may lead to H+-ATPase inhibition (for a review, see Garcia-Brugger et al. 2006).
4.1 CHARACTERIZATION OF ELICITINS BINDING SITE
Consequent cryptogein binding studies and crosslinking experiments performed on
tobacco plasma membrane resulted in the characterization of a single, low-abundance
class of binding site with a Kd value of 2 nM, characterized as a glycosylated heterodimeric
protein (Bourque et al., 1999; Wendehenne et al., 1995). Further, Svozilova et al. studied
the interaction of cryptogein with high-affinity binding site in real-time using a piezoelectric
biosensor and found values of kinetic rate association ka = 5.74 · 106 M1 s-1 and kinetic rate
dissociation kd = 6.87 · 10-4 s-1 constants, respectively. The calculated kinetic equilibrium
dissociation constant (KD = 12.0 nM) was six times lower than that calculated in previous a
study but the obtained value of ka was substantially higher than the previous corresponding
value from the literature (ka = 1.35 · 105 M-1 s-1) (Bourque et al., 1999; Svozilová et al., 2009).
This discrepancy should be mainly because of using another approach compared to
previous studies (Bourque et al., 1999; Wendehenne et al., 1995) using radioactive labelling
and not real-time measurement. Moreover, in the study by Svozilova et al., the exact
concentration of binding sites was carefully determined, providing only the number of
25
binding sites accessible on the outer surface of membrane vesicles (Svozilová et al., 2009).
Recently, in the wild potato Solanum microdontum, a receptor-like protein ELR (elicitin
response) mediating the extracellular recognition of the elicitin domain was demonstrated,
although the binding to elicitins still needs to be proved (Du et al., 2015). ELR protein
associated with a well-known central regulator of cell surface-mediated immunity BAK1
(BRI1 associated kinase 1), also known as SERK3 in solanaceous plants, undergoes complex
formation with PRRs after ligand binding (Du et al., 2015). Previously, such a response to
elicitin INF1 from P. infestans was just dependent on BAK1/SERK3 in tobacco (ChaparroGarcia
et al., 2011).
Although - and -elicitins have been shown to bind with an equivalent affinity to
the same high affinity binding site on the plasma membrane (Bourque et al., 1998), one
important difference was determined; after an application on decapitated tobacco plants,
-elicitins were shown to be 50- to 100-fold more active in inducing a distal HR and systemic
resistance than  -isoforms. Moreover, Bourque et al. showed that -elicitins in a tobacco
cell suspension are at least 10-times more efficient in inducing extracellular pH changes,
reactive oxygen species (ROS) production or Ca2+ influx compared to elicitins (Bourque
et al., 1998). Even though the elicitin binding seems to be a prerequisite for the induction
of the plant defense response, e.g. the AVR9/Cf-9 interaction in tomato or NIP1/Rrs1 in
barley, an effective response is only observed in the presence of a third interacting
component (Bourque et al., 1999; van’t Slot et al., 2007; Wulff et al., 2009). Kanzaki et al.
showed that the elicitin INF1 could interact with the intracellular kinase domain of NbLRK1
kinase (Kanzaki et al., 2008). Although at first glance their results suggesting the
intracellular recognition of elicitins seem to be enigmatic, they fully correspond with the
measured stimulation of clathrin-mediated endocytosis by the elicitin cryptogein in
26
tobacco cells or localization of the elicitin quercinin inside cells of host oak plants by
immunocytology (Brummer et al., 2002; Leborgne-Castel et al., 2008). Finally, a ligandinduced
receptor endocytosis has been suggested to be involved in the activation of plant
defense mechanisms (Robatzek, 2007).
In the next chapters, three main factors with a suggested role in different biological
activity of - and -elicitins are discussed.
4.2 THE ROLE OF STEROL TRAPING IN ELICITINS BIOLOGICAL ACTIVITY
Naturally occurring elicitins show a similar binding characteristics represented by a
1:1 sterol:protein stoichiometry and the dissociation constants in the same range. Despite
these similarity, elicitins exhibit differences in the kinetics of sterol trapping and exchange
between liposomes or micelles (Mikes et al., 1998; Vauthrin et al., 1999). The most efficient
elicitin is the -elicitin cryptogein (from P. cryptogea), the less efficient being -elicitins
parasiticein and capsicein, secreted by P. parasitica and P. capsici, respectively. Together
with differences in triggering defence cell responses between - and -elicitins, the sterolbinding
hypothesis was proposed (Figure 6).
Osman et al. (2001) studied the link between elicitor and sterol-loading properties
with the use of site-directed mutagenesis of the 47 and 87 cryptogein tyrosine residues,
playing an important role in sterol binding based on crystal structure of an engineered
cryptogein-ergosterol complex (Boissy et al., 1999). The results demonstrated a link
between elicitor and sterol-carrier activity of elicitins when the authors suggested that the
sterol binding induced conformational changes in the -loop which “activate” elicitins and
allow them to bind effectively to high-affinity binding sites and trigger cell responses.
27
However, at the least, the affinities and activities of the Tyr47Gly mutant from this study
need to be interpreted with caution. According to the proposed working scheme of elicitin
action, an effect similar to that of sterol binding and hence similar protein activities should
be expected for Tyr47Phe and Tyr47Gly mutants. Nevertheless, high variances in individual
parameters between the Tyr47Phe and Tyr47Gly mutants were observed which could be
explained by a structural change in the helix C evoked by glycine, the amino acid with the
lowest tendency to form a helical structure (Pace and Scholtz, 1998) and unable to stack
with the side chains of Pro42 and Tyr33, in contrast to phenylalanine.
Figure 6. Sterol-binding hypothesis in activity of elicitins. Elicitin traps sterols from plant plasma
membrane (PM) and such an “activated elicitin” is able to bind to high affinity binding site (ELR
protein) on PM and activate plant defence response.
Thus, to confirm these results, Lochman et al. carried out a site-directed
mutagenesis on cryptogein to introduce mutations Met35Phe/Met59Trp,
Met35Phe/Met59Trp /Ile63Phe, Met35Trp/Met59Trp, Leu19Arg and Leu15Trp/Leu36Phe
located mainly in the hydrophobic cavity (Lochman et al. 2005). All mutants showed altered
abilities to bind sterols and fatty acids, nevertheless based on the results authors
suggested, that the conformational change of the ω-loop may be necessary to trigger early
28
defence responses, such as the synthesis of reactive oxygen species (ROS), but not for the
activation of later responses such as PR-protein expression and cell necrosis . However,
since the mutations considered in that study were targeted to the ω-loop region they not
only affected the sterol- and phospholipid-binding properties of cryptogein, but also
slightly modified both the ω-loop structure and the overall protein structure.
Figure 7. Superposition of the free wild type, dehydroergosterol bound wild type, and mutant
structures of cryptogein. (A) Binding of dehydroergosterol to the cavity of cryptogein wild type
induces a conformational change in the  loop (arrow). (B) Superposition of the structures of wild
type and the M35W/M59W mutant (arrow) of cryptogein. Changes induced by the mutation are
similar to those induced by sterol binding (adopted from Lochman et al. 2005).
Even though, conclusions from these studies indicated some role of sterol binding
in elicitins biological activity, the conclusions drawn they were not fully concordant and
apparent discrepancies remained to be explained. For this reason, the main goal of
Dokládal et al. 2013 was to finally support or contradict the role of the elicitin-sterol
complex in the induction of defence responses triggered by elicitins. Based on previous
results (Dobes et al., 2004), site-directed mutagenesis of the elicitin cryptogein was
performed in order to modify the residues that have been proposed to be responsible for
29
sterol- and/or phospholipid-binding without modifying the -loop or overall protein
structure. By this strategy three new cryptogein mutants were prepared; Leu41Phe mutant
with a reduced ability to transfer fatty acids, Val84Phe mutant having a reduced ability to
transfer sterols, and the double mutant Leu41Phe/Val84Phe having reduced ability to
transfer sterols and fatty acids. Contrary to previous studies (Lochman et al., 2005; Osman
et al., 2001), the Val84Phe mutant, with a substantially lowered ability to bind and transfer
sterols, was as efficient as wt cryptogein in stimulating ROS production and induction of
qualitative changes of intercellular proteome in proteins playing an important role in
resistance processes. Surprisingly, the Leu41Phe mutant, with only a slightly lowered ability
to bind and transfer sterols and Leu41Phe/Val84Phe double mutant showing almost no
ability to transfer and bind sterols, were far less efficient in inducing the synthesis of ROS
and proteome changes (Dokládal et al., 2012).
Figure 8. Extent of ROS production and structural alignment of the wild-type cryptogein (green) and
the L41F mutant (blue) (A) ROS synthesis in tobacco cells in suspension stimulated by 10 nM elicitins
- wt cryptogein (diamonds), V84F (filled triangles), L41F (squares), L41F/V84F (circles) and control
(X) (B) The side-chains of the mutated residues (red) adopt their conformation without a disruption
of the omega loop or entire protein structure (adopted from Dokládal et al. 2012).
30
This unexpected result was explained by the construction of a double mutant
Leu80Phe/Val84Phe and detailed characterization of mutants’ interaction with highaffinity
binding on the plasma membrane, when mutation of a small leucine residue 41 for
a large hydrophobic phenylalanine residue altered the interaction of the protein with this
high-affinity binding site (Dokládal et al., 2012).
All these findings contradict previous study of Osman et al. (2001) suggesting the
necessity of a change in the -loop for elicitins activation. At a first sight, the inconsistency
of the obtained data with those published by Lochman et al. (2005), which suggested the
lack of any relation between the ability of elicitins to induce the production of PR proteins
together with tissue necrosis and sterol-binding properties, is clear as well. But if we take
into consideration the fact that the residues Met35 and Leu36 in the -loop are highly
conserved among elicitins, as in the case of the Leu41Phe mutant, their mutation to large
bulky residues (tryptophan and phenylalanine) could have resulted in an altered interaction
with the high-affinity binding site, which was not determined. Consequently, this effect
could be responsible for a longer lag phase (especially at the lower concentration) before
the pH changes and ROS production, but it had a minimal effect in the late phase of cell
necrosis or expression of defence genes.
A final summary of all obtained results imply that the conformational change in
the -loop induced by sterol binding might not be crucial factor in determining an elicitins’
activity. So, it is obvious that the activity of elicitins is more dependent on the presence of
specific residues than on the loop structure; the most probable candidates being lysine
residues in helices A and D of basic elicitins (Dokládal et al., 2012, Ptáčková et al., 2015).
This assumption is further supported by the observed correlation between the necrotic
31
index and pI (Pernollet et al. 1993) and by a clear impact of the Lys13Val mutation in helix
A on induction of a defence response in tobacco plants (Plešková et al., 2011).
Figure 9. Enhancing of elicitin-induced ROS production in tobacco cells. Perception of MAMPs
represenred by cryptogein (Cry), flagellin (Flg22), and oligogalacturonides (OGs) triggers a kinase
activation leading to calcium influx and activation of the NADPH oxidase-driven ROS burst, which in
turn increases membrane order. In parallel, the sterol-trapping ability of cryptogein leads to an
increase in membrane fluidity which enhances the ROS burst intensity compare to Flg22 or OGs
(adopted from Sandor et al. 2016).
However, recently it was proved that the sterol-trapping activity of elicitins
influence PM fluidity and might act as an enhancing factor of elicitin-induced ROS
production in agreement with the synergistic effect obtained with a combination of
cyclodextrin and the potent elicitor methyl jasmonate (Lijavetzky et al., 2008). On the basis
of this results, a model assuming two roles for cryptogein was proposed. The first role is in
triggering of a signalling cascade including the modification of membrane order
independently on sterol-trapping activity and the second role is as a ROS production
32
enhancer through a sterol trapping from the PM, and thus increasing PM fluidity (Figure 9).
This model of co-operative effect could explain how cryptogein would exhibit a strong
ability to induce defence responses including cell death in tobacco cells (Hirasawa et al.,
2004; Kadota et al., 2004, Ptáčková et al., 2015), whereas other classical MAMPs as flg22
and OGs trigger a signalling cascade without inducing cell death (Figure 9).
4.3 THE ROLE OF ELICITINS NET CHARGE IN THEIR BIOLOGICAL ACTIVITY
A rapid comparison of - and -elicitins provides some evident characteristics for
each form, significantly differing in the induction of a distal hypersensitive response and
resistance induction in plants. The most pronounced difference appears in the net charge
which is the consequence of a different number of lysine residues. While the number of
negatively charged Asp and Glu residues is among the basic and acidic elicitins almost
similar, the presence of 6 Lys residues gives a positive charge to -elicitins and the presence
of only 2-4 Lys residues gives a negative charge to -elicitins (Figure 10). The importance
of elicitins net charge has been from the beginning by the observed correlation between
the necrotic index and pI (Pernollet et al., 1993).
Figure 10. A comparison of selected - and -elicitins. The multiple sequence alignment of selected
protein sequences of elicitins and cryptogein was performed using the CLUSTALW algorithm; the
corresponding lysine residues are highlighted in bold.
33
Other structural differences were found among the amino acid sequences in the A
helix of elicitins. The -elicitins residues 13 and 14 are always polar amino acids (Lys and
Thr) constituting for an interruption in the hydrophobic region, compared with -elicitins
(Figure 10). The importance of the residue Lys 13 in elicitins structure for the sterol trapping
activity and induction of a defence response in tobacco plants was repeatedly shown
(Hirasawa et al., 2004; O’Donohue et al., 1995; Plešková et al., 2011). Previously it was
demonstrated that a cryptogein mutant Lys13Val is able to bind both sterols and fatty acids
(Plešková et al., 2011) which correspond with the fact that the Kd values for sterol binding
by acidic elicitins (containing valine at the amino acid position 13) are similar to those of
the basic elicitins (Mikes et al., 1998). However, the sterol trapping capacity of Lys13Val
mutant and acidic elicitins (e.g. cactorein or parasiticein) was about 5-fold lower, compared
with the -elicitin cryptogein (Plešková et al., 2011; Vauthrin et al., 1999). Based on these
results it was proposed that the Lys13Val mutation eliminates the positive charge on the
surface of cryptogein, which could be important for a correct protein positioning in the
interaction between the protein and plasma membrane (Figure 11) (Plešková et al., 2011).
It is well known, that the electrostatic interactions between charged residues
located on a protein surface are known to be important for a long-range recognition in
biomolecular systems (Klein-Seetharaman, 2005). In an agreement with the previously
proposed sterol-binding hypothesis the Lys13Val induce only a slight resistance against P.
parasitica compared to cryptogein which was fully comparable with those induced by the
acidic elicitin capsicein having at the amino position 13 valine. Moreover, this result was
consistent with a significantly lower accumulation of chitinase (PR3Q) or thaumatin like
protein (PR5) transcripts, for which an important role was described in the defence reaction
against fungi (Plešková et al., 2011).
34
Figure 11. Electrostatic potential surface maps of cryptogein and the Lya13Val mutant (adopted
from Plešková et al. 2013). The Lys13Val mutation significantly alters the distribution of charges on
the surface of cryptogein (A) and the Lys13Val mutant (B). The position of the mutated residue is
indicated by a yellow circle.
Even though, this result could be interpreted as a supporting argument for the
sterol-binding hypothesis of elicitins biological activity, it must be interpreted with caution.
Recently, Uhlíková et al. 2016 investigated the role of individual Lys residues in the ability
to induce distal systemic resistance on a model of basic elicitin -cryptogein when using
site-directed mutagenesis of five Lys residues (Lys39, Lys48, Lys61, Lys62 and Lys94) were
systematically replaced by Thr residues. In this study, basic biochemical properties of the
proteins together with the induction of resistance to the pathogen P. parasitica were
studied on tobacco plants. Similarly to the study by Dokladal et al. 2012, the obtained
results did not support a crucial role of sterol binding for elicitins resistance induction when
no correlation of the sterol-trapping activity of the mutants with their resistance induction
activity was proved (Uhlíková et al., 2016). A mild decrease in the sterol transfer activity
measured in a majority of the mutants was probably the result of changes in the protein
surface charge, as discussed previously (Plešková et al., 2011).
35
In addition to the important role of lysine residues for elicitins sterol trapping
activity, their role in the previously shown homo-dimer formation was suggested (Ponchet
et al., 1999; Rodrigues et al., 2006). Particularly, the role of the residues 13 and 14, located
in the helix A responsible for dimer formation, seems to be appealing. Uhlíková et al. 2016
proved by quartz crystal microbalance (QCM) experiments cryptogein homodimer
formation in solution with a KD value of 2.21 M, which was much closer to physiological
conditions than calculated previously in NMR-based study (Gooley et al., 1998). However,
the detailed QCM measurement of the kinetic parameters of cryptogein dimer formation
resembled those of capsicein. Thus the homodimer formation does not seem to be a key
process explaining differences in the biological activity of - and -elicitins. On the other
hand, a chemical crosslinking of cryptogein dimer resulted in its largely reduced ability to
induce necrosis and resistance in plants. To explicate these incongruous results of the
homodimer role in biological activity of elicitins, a possible interaction of elicitins with
another partner in plant, non-specific Lipid Transfer Protein 1 (nsLTP1), was studied
(Uhlíková et al., 2016). The nsLTP1 protein was selected as a promising candidate because
it behaves as an elicitin antagonist with known superimposition of helix 3 with helix A of
cryptogein (Blein et al., 2002), its cell wall localization is assumed (Carvalho and Gomes,
2007) and an important role of LTPs in the key processes of plant physiology and plant
defence signalling was proved (Champigny et al., 2013). Noticeably, there a strong
correlation between the nsLTP1-elicitin complex formation kinetic and biological activity of
individual used proteins was measured, including cryptogein variants carrying mutation in
lysine residues (Lys39, Lys, 48 and Lys 94) and capsicein.
36
4.4 THE ROLE OF ELICITINS CHARGE IN THEIR MOVEMENT ACROSS THE PLANT
Most of the studies on elicitins have been carried out on tobacco plants with three
different methods of their application: on the stem of decapitated plants, on the petiole of
detached leaves or via a direct infiltration into the leaf mesophyll. The first two modes of
treatment are used for measurement of induced distal resistance because it leads to the
systemic movement of - and -elicitins across the plants or leaves (Devergne et al., 1992;
Dorey et al., 1997; Keller et al., 1996; Uhlíková et al., 2016). Based on this treatment, elicitins
were shown to be 50- to 100-fold more active in inducing of HR and systemic
resistance against pathogens, compared to - elicitins. In the latter mode, most of the and
-elicitins have approximately the same activity in inducing HR however they are
restricted to the infiltration site without any movement to surrounding areas, inducing only
local acquired resistance (LAR) against the pathogens (Dorey et al., 1997). Thus, it is clear
that not the movement of elicitins in the phloem but the consequent transport from the
phloem to companion and parenchyma cells plays a crucial role in their ability to induce
systemic resistance in plants. This fact could be supported by previous findings that
protein´s properties play an important role in phloem loading/unloading (Niu et al. 2011).
At the beginning, elicitins pI was considered as an important factor in this process because
the elicitin penetration through the negatively charged cell wall could contribute to delay
response of suspension cells to acidic elicitins despite their similar binding parameters to a
high affinity binding site on the plasma membrane (Bourque et al., 1998). Nevertheless, no
clear relationship between the charge of cryptogeins carrying mutation in Lys residues and
their activity to induce resistance and HR disprove this hypothesis (Uhlíková et al., 2016).
On the other hand, the noticeable role of Lys residues (Lys13, Lys39) and homodimer
covalent crosslinking in elicitins ability to induce systemic resistance suggest an important
37
role of some partner in plant, forming a heterodimer complex with elicitin, facilitating the
process of phloem unloading (Uhlíková et al., 2016). From this point of view, the described
interaction of the studied proteins with nsLTP1, with a clear involvement of specific lysine
residues, seems to be very promising for further studies.
4.5 CONTRIBUTION TO GIVEN PROBLEMATIC
Oomycetes, including Phytophthora infestans causing potato late blight, represent
one of the most emerging pathogens on food crop. Elicitins are structurally conserved
proteins secreted by Phytophthora and Pythium pathogenic species and they are
recognised as oomycetes MAMPs.
The results of our studies significantly improved our knowledge about the role of
the sterol-binding activity of elicitins in their ability to induce resistance in plants. By several
studies using mutants affected in sterol binding we disproved previous results suggesting
an essential role of sterol binding in elicitins biological activity, especially in their binding to
potential high affinity binding site on plasma membrane. However, we demonstrate that
the sterol-trapping activity could serve as an enhancer of ROS production through an
increase in the plasma membrane fluidity. On the other hand, by using elicitins mutants
affected in their surface charge, we demonstrated an important role of the lysine residues
13 and 39 in the ability of elicitins to induce systemic resistance in plants when we
suggested an important role of some partner in plants, forming a heterodimer complex
with elicitins and thus facilitating the process of phloem unloading. Within the framework
of this topic, several long-term stays of our students in the cooperating laboratories of INRA
in Dijon and Sophia-Antipolis have been made.
38
5. CONCLUSION
Today, in the period of an intensive agriculture practise, the cultivation of highly
fertilized crops in large monocultures is widely used. Unfortunately, these types of crops
are very sensitive to a wide spectrum of diseases and their cultivation requires the use of
extensive amounts of pesticides and fungicides. In the past decades, the via transfer of
resistance (R) genes was the main strategy to improve plant resistance. However, the
durability of this resistance has been shown to be very limited because of the process of a
rapid pathogen effectors evolution, and due to the species or strain-related specificity of
effectors which fails against a broad spectrum of the attacking pathogens.
On the other hand, PTI, providing a broad-spectrum immunity, seems to be a very
promising tool for engineering plants with an enhanced immunity. It is mainly due to the
fact that MAMPs, which are specific and conserved in microbes, are very often crucial for
their survival (e.g. flagellin for bacterial motility or ergosterol for fungal plasma membrane
structure). Noticeably, a generally enhanced resistance after the interfamily transfer of
genes encoding PRRs or other regulators of PTI was observed even though in some cases
undesirable side effects in growth and development were found.
Further investigations of MAMPs perception mechanisms by plant cells emerge using
novel large scale quantifiable tools (e.g. transcriptomics, proteomics or metabolomics).
This will certainly provide comprehensive insights into the understanding of MAMPs
interactions with plants. The long-time durable resistance should be achieved only through
a combination of multiple PTI- and ETI-related genes together with deploying of a proper
field management.
39
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Zipfel, C. (2014). Plant pattern-recognition receptors. Trends Immunol. 35, 345–351.
Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J.D.G., Boller, T., and Felix, G. (2006).
Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated
transformation. Cell 125, 749–760.
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7. PUBLICATIONS PRESENTED IN HABILITATION THESIS
ERGOSTEROL AS AN EXAMPLE OF AN ORPHAN FUNGAL MAMP
Lochman, J., Mikes, V. Ergosterol treatment leads to the expression of a specific set of
defence-related genes in tobacco (2006) Plant Molecular Biology, 62 (1-2), pp. 43-51
Contribution of author JL: Measurements of gene expression, WB analysis, Preparation of
manuscript
Dadakova, K., Klempova, J., Jendrisakova, T., Lochman, J., Kasparovsky, T. Elucidation of
signaling molecules involved in ergosterol perception in tobacco (2013) Plant Physiology
and Biochemistry, 73, pp. 121-127
Contribution of author JL: Measurements of gene expression, Design of experiments,
Preparation of relevant parts of manuscript
ELICITINS AS AN EXAMPLE OF A TYPICAL OOMYCETE MAMP
Lochman, J., Kasparovsky, T., Damborsky, J., Osman, H., Marais, A., Chaloupkova, R.,
Ponchet, M., Blein, J.-P., Mikes, V. Construction of cryptogein mutants, a proteinaceous
elicitor from Phytophthora, with altered abilities to induce a defense reaction in tobacco
cells (2005) Biochemistry, 44 (17), pp. 6565-6572
Contribution of author JL: Preparation of proteins, Northern blot analysis, Preparation of
relevant parts of manuscript
Svozilová, Z., Kašparovský, T., Skládal, P., Lochman, J. Interaction of cryptogein with its
binding sites in tobacco plasma membrane studied using the piezoelectric biosensor (2009)
Analytical Biochemistry, 390 (2), pp. 115-120
Contribution of author JL: Design of experiments, Measurements on QCM, Preparation of
manuscript
Literakova, P., Lochman, J., Zdrahal, Z., Prokop, Z., Mikes, V., Kasparovsky, T. Determination
of capsidiol in tobacco cells culture by HPLC (2010) Journal of Chromatographic Science, 48
(6), pp. 436-440
Contribution of author JL: Preparation of relevant parts of manuscript
Plešková, V., Kašparovský, T., Obořil, M., Ptáčková, N., Chaloupková, R., Ladislav, D.,
Damborský, J., Lochman, J. Elicitin-membrane interaction is driven by a positive charge on
the protein surface: Role of Lys13 residue in lipids loading and resistance induction (2011)
Plant Physiology and Biochemistry, 49 (3), pp. 321-328
Contribution of author JL: Design of experiments for protein expression and RT-qPCR,
Measurement of sterol trapping activity, Preparation of manuscript
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Dokládal, L., Obořil, M., Stejskal, K., Zdráhal, Z., Ptáčková, N., Chaloupková, R., Damborský,
J., Kašparovský, T., Jeandroz, S., Žd'Árská, M., Lochman, J. Physiological and proteomic
approaches to evaluate the role of sterol binding in elicitin-induced resistence (2012)
Journal of Experimental Botany, 63 (5), pp. 2203-2215
Contribution of author JL: Design of experiments, Evaluation of data from 2Delectrophoresis,
Preparation of manuscript
Ptáčková, N., Klempová, J., Obořil, M., Nedělová, S., Lochman, J., Kašparovský, T. The effect
of cryptogein with changed abilities to transfer sterols and altered charge distribution on
extracellular alkalinization, ROS and NO generation, lipid peroxidation and LOX gene
transcription in Nicotiana tabacum (2015) Plant Physiology and Biochemistry, 97, pp. 82-95
Contribution of author JL: Preparation of proteins, Preparation of relevant parts of
manuscript
Uhlíková, H., Obořil, M., Klempová, J., Šedo, O., Zdráhal, Z., Kašparovský, T., Skládal, P.,
Lochman, J. Elicitin- induced distal systemic resistance in plants is mediated through the
protein–protein interactions influenced by selected lysine residues (2016) Frontiers in Plant
Science, 7 (FEB2016), art. no. 59
Contribution of author JL: Design of experiments, Evaluation of data from QCM, Preparation
of manuscript
Sandor, R., Der, C., Grosjean, K., Anca, I., Noirot, E., Leborgne-Castel, N., Lochman, J.,
Simon-Plas, F. and Gerbeau-Pissot, P. Plasma membrane order and fluidity are diversely
triggered by elicitors of plant defence (2016) Journal of Experimental Botany,
doi:10.1093/jxb/erw284
Contribution of author JL: Preparation of proteins, Preparation of relevant parts of
manuscript
OTHERS
Racapé, J., Belbahri, L., Engelhardt, S., Lacombe, B., Lee, J., Lochman, J., Marais, A., Nicole,
M., Nürnberger, T., Parlange, F., Puverel, S., Keller, H. Ca2+-dependent lipid binding and
membrane integration of PopA, a harpin-like elicitor of the hypersensitive response in
tobacco (2005) Molecular Microbiology, 58 (5), pp. 1406-1420 Contribution of author JL:
Contribution of author JL: Northern Blot analysis, Preparation of relevant parts of
manuscript
Dadakova, K., Havelkova, M., Kurkova, B., Tlolkova, I., Kasparovsky, T., Zdrahal, Z.,
Lochman, J. Proteome and transcript analysis of Vitis vinifera cell cultures subjected to
Botrytis cinerea infection (2015) Journal of Proteomics, 119, pp. 143-153
Contribution of author JL: Design of experiments, Evaluation of data from 2Delectrophoresis
and RT-qPCR, Preparation of manuscript
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8. SUPPLEMENTS
ERGOSTEROL AS AN EXAMPLE OF AN ORPHAN FUNGAL MAMP