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BARLEY DEFENSE GENES AGAINST APHIDS

Aleksandra Losvik

Barley defense genes against aphids

Aleksandra Losvik ©Aleksandra Losvik, Stockholm University 2018

ISBN print 978-91-7797-145-0 ISBN PDF 978-91-7797-146-7

Cover photo by Aleksandra Losvik Printed in Sweden by Universitetsservice US-AB, Stockholm 2018 Distributor: Department of Ecology, Environment and Plant Sciences To my dearest Johan Thank you for the adventures of my lifetime

Sammanfattning

Bladlöss är skadeinsekter av stor betydelse över hela världen. Genom att livnära sig från växternas ledningsrör undandrar de direkt näring från växterna. Skador av angrepp syns ofta som bladinrullning, gula eller svarta bladfläckar eller andra växtdeformationer. Deras roll som skadeinsekter hänger också samman med att de överför virussjukdomar. Bladlusangrepp hanteras inom jordbruket med bekämpningsmedel, vilka kan utgöra ett hot mot miljön. Ett alternativt angreppssätt kan vara att identifiera och karakterisera genetiska faktorer som bidrar till bladlusresistens, samt medel som inducerar resistens, med avsikten att utnyttja denna kunskap i förädlingsprogram.

Syftet med denna avhandling var att identifiera sådana resistensgener och karakterisera deras roller i samspelet mellan växt och bladlus. Detta genomfördes med två angreppssätt. För det första klonades två förmodade bladlusresistensgener från korn och överfördes till Arabidopsis och korn och effekterna av transformationen undersöktes på två bladlusarter. Generna var

CI2c som kodar för en kymotrypsinhämmare och LOX2.2. som kodar för ett lipoxygenas. Bladlusarterna var havrebladlus (Rhopalosiphum padi L.) som är en skadegörare på viktiga sädesslag och generalisten persikbladlus (Myzus persicae Sulzer) som är skadegörare på växter tillhörande mer än 40 olika familjer. Effekterna av transformation studerades också med avseende på uttrycket av andra försvarsrelaterade gener i de transformerade plantorna.

Det andra angreppssättet var att behandla plantor med luftburna ämnen och studera i vad mån detta framkallade resistens, följt av tester med havrebladlus på de behandlade växterna.

Studierna med överuttryck av CI2c visade att genprodukten i Arabidopsis gav en övergående reducering av persikbladlusens fruktsamhet, men i korn indirekt minskade samma arts undvikande av plantan på grund av att plantans försvar trycktes ned. Transformationen hade ingen effekt på havrebladlusens beteende eller fruktsamhet. Överuttryck av LOX2.2. i korn påverkade uttrycket av andra gener som regleras av jasmonsyra och minskade kortvarigt fruktsamheten både av havrebladlus och persikbladlus.

Studierna med behandling av luftburna ämnen gav stöd för idén att bladlusresistens kan framkallas med denna metod. Flera försvarsgener visade högre uttryck efter behandling med metylsalicylat, metyljasmonat och (Z)-3- hexen-1-ol. Av dessa tre undersökta ämnen, visade metyljasmonat den största potentialen som försvarsframkallande ämne, då den orsakade kortvarig reducering av fruktsamhet hos bladlusen.

Sammanfattningsvis stöder denna avhandling idéerna att korngenerna

CI2c och LOX2.2. spelar en roll i bladlusresistens och att viss bladlusresistens kan framkallas genom externa faktorer. Bladlössen kan direkt påverkas av genprodukten eller det kan finnas en indirekt effekt, som orsakas av förändringar i genuttryck av andra gener som är inblandade i växtens försvar.

De observerade negativa effekterna på bladlössen var av måttlig omfattning och det är troligt att de var för sig inte har någon stark negativ effekt, men att de kan bidra till att förmedla bladlusresistens.

List of publications

This thesis is based on the work contained in the following papers and man- uscripts, referred to in Roman numerals in the text:

I: Losvik A, Beste L, Mehrabi S, Jonsson L (2017). The protease inhibitor

CI2c gene induced by bird cherry-oat aphid in barley inhibits green

peach aphid fecundity in transgenic Arabidopsis. International Journal of

Molecular Sciences 18: 1317.

II: Losvik A, Beste L, Stephens J, Jonsson L (2018). Overexpression of the

aphid-induced serine protease inhibitor CI2c gene in barley affects the

generalist green peach aphid, not the specialist bird cherry-oat aphid.

Accepted for publication in PLoS One.

III: Losvik A, Beste L, Glinwood R, Ivarson E, Stephens J, Zhu Li-Hua, Jons-

son L (2017). Overexpression and down-regulation of barley lipoxygen-

ase LOX2.2 affects jasmonate-regulated genes and aphid fecundity. In-

ternational Journal of Molecular Sciences 18: 2765.

IV: Losvik A, Glinwood R, Jonsson L. Effects of treatments of barley with

methyl salicylate, methyl jasmonate or (Z)-3-hexen-1-ol on barley gene

expression and fecundity of bird cherry-oat aphid (Manuscript).

Papers I, II and III are reproduced with the permission of their respective publisher.

My contributions to the papers

Paper I: Was main responsible for the experimental design, conducted

the experiments, except for the cloning and transformation and

carried out the analysis of data. Wrote the first draft of the man-

uscript.

Paper II: Was main responsible for the experimental design, conducted

the experiments, except for the cloning and transformation,

and analyzed the data. Contributed to writing the manuscript.

Paper III: Designed and conducted the experiments, except for the clon-

ing and transformation, and performed the analysis of data.

Contributed to writing the manuscript.

Paper IV: Participated in the experimental design, conducted the experi-

ments and the analysis of data. Wrote the first draft of the man-

uscript.

Contents

Introduction ...... 11

Aphids as plant pests ...... 13

Host specificity ...... 13

Bird cherry-oat aphid ...... 14

Green peach aphid ...... 16

Host recognition and feeding ...... 17

Reproduction ...... 18

Damage to barley ...... 19

Plant defenses against aphids ...... 19

Direct defenses ...... 20

Indirect plant defenses ...... 23

Triggering of plant defense response ...... 24

Pattern-triggered immunity (basal defense) ...... 25

Effector-triggered immunity (active defense) ...... 27

Aphid-associated effectors ...... 28

Defense signaling pathways ...... 29

The cross-talk of SA and JA pathways ...... 34

Aphid resistance genes...... 35

Aphid management in agriculture ...... 36 Aim of the thesis ...... 41

Main approaches ...... 43

Hypotheses for the thesis work...... 43

Putative resistance factors against aphids ...... 43

Induction of resistance ...... 45 Comments on methodology ...... 47

Plant transformation ...... 47

Selection and controls ...... 49

Confirmation of transformation ...... 50

Plant phenotype ...... 52

Aphid performance ...... 53

Antixenosis ...... 53

Antibiosis ...... 54 Main results ...... 57

Effects of transformation or plant treatments with volatiles ...... 57

Effects of transformation or treatments on aphid performance ...... 60

CI2c overexpression ...... 60

LOX2.2 overexpression and down-regulation ...... 63

Volatile treatments ...... 64

Other effects of transformation or treatments ...... 67

Differences in aphid performance ...... 70

Between aphid species: BCA and GPA on barley ...... 70

Between host plants: GPA on Arabidopsis and barley ...... 72 Conclusions ...... 73

Future perspectives ...... 75

Acknowledgements ...... 79

References ...... 81

Abbreviations

AOS Allene oxide synthase

BCA Bird cherry-oat aphid

BCI Barley chemically induced

BYDV Barley yellow dwarf virus

CI2c Barley chymotrypsin inhibitor

COI1 Coronatine-insensitive 1

CRISPR Clustered regularly interspaced short palindrome repeats

DAMPs Damage-associated molecular patterns dsRNA Double-stranded RNA

Eβf (E)-β-farnesene

EPG Electrical penetration graph

ETI Effector-triggered immunity

GLV Green leaf volatile

GMO Genetically modified organism

GPA Green peach aphid h Hour

HAMPs Herbivore-associated molecular patterns

HPL Hydroperoxide lyase

Hx Hydroxamic acid

JA Jasmonic acid

JAZ Jasmonate ZIM domain

LOX Lipoxygenase

MeJA Methyl jasmonate

MeSA Methyl salicylate

NPBT New plant breeding technique

NPR1 Non-expressor of PR genes 1

ODN Oligodeoxynucleotides

PAMPs Pathogen-associated molecular patterns

PI Proteinase inhibitor

PRR Pattern-recognition receptors ps Phloem specific

PTGS Post-transcriptional gene silencing

PTI Pattern-triggered immunity

R-gene Resistance gene

ROS Reactive oxygen species qPCR Quantitative polymerase chain reaction

RNAi RNA-mediated interference

SA Salicylic acid siRNA Small interfering RNA

TILLING Targeting induced local lesions in genomes

TPPE Thionin proprotein-processing enzyme

VIGS Virus-induced gene silencing

WT Wild type

Introduction

Barley (Hordeum vulgare L.) is a self-pollinating, diploid species, a member of the grass family (Poaceae) and a major cereal grain grown in temperate regions of the world. Domestication of barley began about 10,000 years ago from its wild progenitor H. vulgare ssp. spontaneum (C. Koch) (Zohary and Hopf,

2000). Barley has evolved to include several forms. The two–row and six–row varieties differ in the protein content; the two-row barley has lower protein content and more fermentable sugar and is used for brewing, while six-row barley is used to feed livestock (Ullrich, 2011). Traditionally, spring malting barley varieties were grown in regions with moderate temperatures and sufficient rainfall during the growth season, whereas winter barley was mostly grown in the warmer and dry parts of Europe (Pržulj et al., 1998). Barley was probably initially used as human food, but due to the rise in prominence of wheat and rice, eventually evolved into a feed and later into a source of fermentable material for the production of beverages (Ullrich, 2011). In 2014, barley ranked fourth in acreage among major cereal crops, after maize, rice and wheat, covering more than 49 million hectares worldwide, with a total average yield of 2,9 tons per hectare (FAOSTAT, 2014).

Barley is a host for numerous pathogens such as Puccinia species which are causal agents of steam, leaf and stripe rust, Xantomonas campestris causing bacterial blight, Blumeria graminis f.sp. hordei causing powdery mildew and

Fusarium fungi causing head blight infections (Paulitz and Steffenson, 2011).

Aside from bacterial and fungal pathogens, barley is also hosting a few virus species, among them barley yellow dwarf virus (BYDV). The most important insect pests on barley are aphids, which cause direct damage by feeding from

11 the plant’s phloem and indirect damage by transmitting plant viruses (e.g.

BYDV) (Miller and Rasochová, 1997; Plumb, 2002; Gray and Gildow, 2003).

Chemical control strategies are commonly used to constrain aphid populations, however in spite of strict regulations for pesticide transport and application, the many drawbacks of insecticide usage necessitate a search for alternative methods. Breeding for aphid resistance is a less harmful pest management strategy (Åhman et al., 2010). The pursuit to find aphid resistance factors is enhanced by the emergence of new molecular techniques in plant science.

The sequencing of the barley genome that was finished in 2012 facilitates molecular studies on this plant and simplifies the production of new resistant cultivars (Mayer et al., 2012). The genome size has been reported to be about

5.1 gigabases with around 40 thousand coding genes (Mayer et al., 2012).

A barley breeding program was initiated, attempting to introduce genes for aphid resistance from barley wild ancestor H. vulgare ssp. spontaneum into cultivated barley, and those attempts resulted in moderately aphid resistant double haploid lines (Åhman et al., 2000). The original resistance bioassays showed ca. 15% lower increase in nymphal weight on those lines, when compared to an aphid-susceptible barley cultivar (Åhman et al., 2000). The barley wild ancestor, an aphid resistant barley double haploid line (DH28:4) and two barley susceptible cultivars were used in a comparative transcript profiling study based on a microarray, which resulted in the identification of candidate genetic factors contributing to the observed aphid resistance. Among them were gene sequences coding for a protease inhibitor (PI), a lipoxygenase

(LOX) and thionins (Delp et al., 2009; Mehrabi et al., 2014). The work in this thesis aims to explain the contribution of two of the suggested resistance factors (PI and LOX) to aphid resistance by studying aphid responses to plants with overexpression or downregulation of the selected genes as well as responses of the transgenic plants to aphid feeding at the molecular level. In

12 addition, the effects of volatile treatments were studied on different cultivars of barley, with regard to activation of plant defense, followed by bioassays with bird cherry-oat aphid (BCA, Rhopalosiphum padi L.) on the treated plants.

Aphids as plant pests

Host specificity

Aphids are members of the superfamily Aphidoidea of the order Hemip- tera. They are small (1-10 mm), soft-bodied insect pests which feed on the phloem sap. There are more than 4000 species of aphids distributed worldwide (Dixon 1985a; Dixon et al., 1987). Most aphid species (95%) are monoph- agous or oligophagous and show high host specificity. Polyphagous species, such as the black bean aphid (Aphis fabae Scopoli) are associated with a wide range of primary and secondary hosts (Blackman and Eastop, 2007). Consid- ering aphids’ small body and limited energy reserves, the time they can survive without a suitable food source is narrow. Thus, aphids are associated mainly with abundant plants species and the greatest amount of aphid species occurs in temperate regions (Dixon, 1985b). Most aphid species are monoe- cious, completing their life cycle on one host and some of them, like the pea aphid (Acyrthosiphon pisum Harris) and grain aphid (Sitobion avenae Fabricius) are known to live only on herbaceous hosts or grasses, respectively. Only 10% of aphids are host-altering species (Figure 1) and spend the summer on different plant species than the primary (winter) host, where sexual reproduction occurs. The secondary host, where populations of parthenogenetic females develop, is often an herbaceous crop plant species, and those aphids are important from the agronomic point of view (Williams and Dixon, 2007).

Figure 1. Life cycle of host-altering BCA. A fundatrix (foundress) is a wingless parthenogenetic female that produces live offspring during spring; after one or two generations, winged offspring (alataes) migrate to the secondary host to establish a population of parthenogenetic females. With the approach of winter, a winged female (gynopara) migrates to the primary host and produces ovipara - wingless, sexual female that mates with a winged male to produce overwintering eggs (adapted from Finlay and Luck, 2011).

Bird cherry-oat aphid

The colour of the BCA (Figure 2) varies from yellow-green to green-olive and greenish-black with a rust-coloured pigmentation around the cornicles. It is a cosmopolitan, host-alternating insect species (Figure 1) occurring in temperate regions. Permanent parthenogenetic development can however result from warmer weather conditions or a decrease of the geographical range of the primary host plant (Simon et al., 1996; Williams and Dixon, 2007;

Strażyński and Ruszkowska, 2015). BCA with a holocyclic life cycle uses bird cherry (Prunus padus L.) as its primary host and all major cereals and pasture

14 grasses as secondary host. The overwintering eggs on the primary host hatch in the spring and after two to three wingless generations, winged emigrant morphs appear (Dixon, 1971; Glinwood and Pettersson, 2000a). Alataes fly to the summer host where they give rise to further parthenogenetic generations

(Wiktelius et al., 1990). Winged migrants are born and migrate to new host plants when the nutrient source becomes depleted. With the approach of cold weather and longer periods of darkness, the winged gynoparae and males return to bird cherry, whose senescing leaves provide a rich food source

(Dixon and Glen, 1971). Apterous oviparae are born and mate with the winged males to produce overwintering eggs (Figure 1) (Dixon, 1971).

Figure 2. Apterous adult BCA feeding on oat leaf. Photo by A. Losvik

15 Green peach aphid

The green peach aphid (GPA, Myzus persicae Sulzer) (Figure 3) is a cosmopolitan, polyphagous aphid species. The color varies from pale greenish-yellow and various shades of green, to pink, red, or even almost black (Blackman and Eastop, 2007). The life cycle includes primary and secondary hosts; however, it can reproduce asexually during the warmer months, and throughout the year in warmer climates. The primary host comprise stone fruits like peach

(Prunus persicae L.), Canadian plum (P. nigra Aiton), black cherry (P. serotine

Ehrhart), and dwarf Russian almond (P. tenella Batsch) (Blackman and Eastop,

2000). As secondary hosts GPA uses a large variety of plants, including crops belonging to Brassicaceae, Solanaceae and Cucurbitaceae families. Altogether, the host range comprises plant species belonging to more than 40 plant families (Blackman and Eastop, 2000; Louis and Shah, 2013). Furthermore, GPA is a vector for many viral plant diseases and its resistance to a large number of insecticides has been reported (Louis and Shah, 2013; Bass et al., 2014).

Figure 3. Apterous adult GPA feeding on kohlrabi leaf (Brassica oleracea L.). Photo by A. Losvik

16 Host recognition and feeding

Aphids are poor flyers and their migration depends strongly on air movement. Visual and olfactory cues can initiate the landing of a winged migrant, an alatae, chemical and nutritional cues available to the aphid may lead to host acceptance or rejection (Powell et al., 2006; Nam et al., 2013; Smith and

Chuang, 2014). To establish a feeding site, aphids scan the surface of a potential host to detect veins and penetrate the tissue. The stylet pathway is mostly intercellular and is facilitated by gelling saliva excreted by aphids at the time of probing (Will et al., 2012). Gel saliva is mainly composed of proteins (including phenoloxidases, peroxidases, pectinases, β-glucosidases), phospho- lipids and conjugated carbohydrates (Urbanska et al., 1998; Miles, 1999;

Cherqui and Tjallingii, 2000). While progressing toward the phloem, stylets briefly puncture many cells and after withdrawal the punctured site is quickly sealed by gelling saliva (Tjallingii and Esch, 1993; Powell et al., 2006; Tjallingii,

2006). There are indications that aphids discriminate between host and non- host plants before reaching the phloem, by uptake of small amounts of intracellular substance. Those studies were based on electrical penetration graph

(EPG) recordings, combined with observations of aphid behavior (Powell et al., 2006; Goggin, 2007). In the EPG technique, an aphid and plant are made parts of an electric circuit, which is completed when the aphid inserts its stylet into the plant. Voltage fluctuations in the circuit create distinct EPG wave- forms, patterns of amplitude, frequency, and voltage level, which correlate with the insect´s stylet activities (Tjallingii and Esch, 1993). During the phase of superficial piercing, as well as during initial establishment in the phloem, aphids secrete watery saliva, believed to aid in gustatory exploration and prevent plant defense responses (Miles, 1999; Powell et al., 2006; Will et al., 2009;

Moreno et al., 2011). Watery saliva contains a complex mixture of proteins e.g. hydrolases, oxidases and calcium-binding proteins, the latest involved in the

17 inhibition of calcium-mediated occlusion of phloem sieve elements (Urbanska et al., 1998; Miles, 1999; Carolan et al., 2009). Saliva composition differs between aphid species and is dependent on aphid’s diet (Cherqui and Tjallingii,

2000; Carolan et al., 2009; Will et al., 2009). The successful recognition and host acceptance may result in a compatible or incompatible reaction, where respectively aphids reach reproductive success or where they are not able to exploit a resistant plant (Powell et al., 2006; Nam et al., 2013). The herbivore-specific compounds of aphid salivary secretions can have elicitor-like properties and serve as herbivore-associated molecular patterns (HAMPs) (Mithöfer and

Boland, 2008). Additionally, saliva components and stylet progress through plant tissue may potentially lead to a release of damage-associated molecular patterns (DAMPs) from plant cells. The induction of plant innate immune response by HAMPs and DAMPs is discussed in more detail in section “Trig- gering of plant defense responses”.

Aphids feed passively on phloem sap, which is rich in sugars and poor in amino acids, especially the essential ones (Dinant et al., 2010). Because of this unbalanced diet, aphids need to ingest large amounts of food and the result is the excreted honey dew (Douglas, 2006). The diet is further supplemented with essential amino acids produced by the primary obligate prokaryotic symbiont from the genus Buchnera, present in bacteriocytes – specialized fat body cells localized near the aphid’s gut (Baumann et al., 1995; Douglas, 1998).

Reproduction

With few exceptions, aphids live in colonies, with both advantages and drawbacks. Aggregation provides increased protection from natural enemies, however colonies can grow to a size at which the colony loses sustainability on the host plant (Pettersson et al., 2007). This extremely rapid growth of a colony is secured by parthenogenesis, where offspring is produced from

18 unfertilized eggs, and telescopic generations, where as a result, fully developed nymphs are born. Aphid nymphs molt during growth and pass through

3-4 instars before becoming adults (Awmack and Leather, 2007). For most aphid species, parthenogenesis is cyclical, with periods of asexual reproduction alternating with sexual reproduction (holocyclic life cycle, Figure 1).

Some species, such as GPA are examples where all generations can comprise of asexual (parthenogenetic) females (Dixon, 1985c; Williams and Dixon,

2007).

Damage to barley

There are a few aphid species associated with barley and they cause different injury symptoms. The Russian wheat aphid (Diuraphis noxia Kurdjumov) causes leaf rolling and discoloration, whereas chlorosis and necrosis of plant leaves are characteristic for greenbug (Schizapis graminum Rondani) attack

(Quisenberry and Ni, 2007). BCA is a pest on grasses, causing no visible inju- ries to plants, but can reduce yield directly by withdrawing nutrients from the plant phloem and by transmitting plant viruses, for example the BYDV. This virus is the causal agent of one of the most important viral disease of barley and other small grain cereal crops worldwide (Paulitz and Steffenson, 2011).

It causes leaf discolouration and dwarfing and the infection may lead to losses approaching 100% if appearing early in the season (Irwin and Thresh, 1990;

Mathre, 1997; Paulitz and Steffenson, 2011; Ordon and Perovic, 2013).

Plant defenses against aphids

Non-host resistance includes traits preventing a plant from being a host and is the basic defense used by plants. It can be defined as the immunity, which is shared by all genotypes of a plant species including susceptible plants and is able to limit non-adapted herbivore populations. This type of

19 resistance has considerable value for crop improvement, since it is typically both broad-spectrum and durable (Lee et al., 2016). Traits that deter herbivore performance on host plants contribute to host plant resistance (Goggin, 2007).

Insect responses to host resistance characteristics can be of behavioral

(antixenosis) or physiological (antibiosis) nature (Smith, 2005a; 2005b). Plants can employ direct and indirect defense mechanisms for protection against pathogens and herbivores, both can be present constitutively before insect attack or can be induced.

Direct defenses

Surface barriers

The main determinants of plant resistance to herbivores are direct mechanisms, which include physical and chemical traits. The physical barriers can be created by the presence of leaf surface wax, cutin layers and trichomes

(Hanley at al., 2007; War et al., 2012). This line of defense was shown to have variable effects on aphids. A high density of trichomes was shown to have negative effects on GPA feeding on wild species of tomato (Lycopersicon)

(Wójcicka, 2015), and the presence of waxes impaired probing behavior of S. avenae on triticale (× Triticosecale Wittmack) plants (Wójcicka, 2015), however there exist also reports showing no effects of trichomes on aphids (Åhman et al., 2000; Sato and Kudoh, 2015; Karley et al., 2016).

Lignin is a phenolic heteropolymer deposited predominantly in the walls of cells showing secondary thickening, especially abundant in vascular (xy- lem tracheids, vessel elements) and support tissues (sclereid cells). Lignin depositions provide rigidity and imperviousness to the cells (Vanholme et al.,

2010). In addition, lignin biosynthesis can be induced upon various biotic and abiotic stress conditions and plays an important role in plant defense against

20 pathogens and insects, by hindering pathogen entry or increasing leaf tough- ness to reduce feeding by herbivores (War et al., 2012). It was shown that the chrysanthemum aphid (Macrosiphoniella sanborni Gillette) induced CmMYB19, a transcription factor important in the regulation of lignin synthesis. Overex- pression of CmMYB19 restricted the multiplication of aphids by enhancing accumulation of lignin (Wang et al., 2017). Callose and lignin depositions were also induced by melon/cotton aphid (Aphis gossypii Glover) in the cell walls of aphid-resistant melon genotypes, carrying the Vat gene and hindering aphids from reaching the vascular tissue, but not in aphid susceptible lines

(Villada et al., 2009).

Biochemical defense

Chemical barriers comprise a big group of plant secondary metabolites which are present constitutively or are induced by pathogen/herbivore attack.

These compounds belong to various chemical classes such as phenolics, terpenoids and nitrogen-containing organic compounds (Mithöfer and Boland,

2012). Some attention has been directed toward understanding the involvement of cereal secondary metabolites in resistance to aphids (Leszczynski and

Dixon, 1990; Forslund et al., 1998; Åhman et al., 2000; Larsson et al., 2006;

Goławska et al., 2008; Larsson et al., 2011).

Gramine is an indole alkaloid which occurs in leaves of certain barley cultivars (Forslund et al., 1998; Larsson et al., 2006). It’s involvement in defense against aphids has been suggested, however results from different studies do not provide clear evidence to support this idea. It has been shown that gramine is present in epidermis and mesophyll parenchyma cells of barley seedlings and can modify S. graminum and BCA feeding behavior (Zúñiga et al.,

1988). It has been correlated with barley seedling resistance to the two above- mentioned aphid species (Zúñiga et al., 1985; Zúñiga and Corcuera, 1986).

21 More recent studies however show no negative effect of high gramine content on BCA settling (Forslund et al., 1998) or growth (Åhman et al., 2000).

Another group of secondary metabolites present in barley is the cyanogenic glycosides, which release hydrogen cyanide upon tissue damage. Simi- larly to gramine, no conclusive evidence has been provided with regard to their involvement in defense against aphids. In one of the studies, the cyanogenic glycoside dhurrin occurring in sorghum (Sorghum bicolor Moench) was shown to be deterrent to S. graminum (Dreyer et al., 1981) in experiments where it was supplemented to aphids’ artificial diet. This effect was however not observed for the corn leaf aphid (Rhopalosiphum maidis Fitch) feeding on different sorghum varieties with varying content of dhurrin in the field

(Woodhead et al., 1980). It was further demonstrated that BCA settling did not differ on cultivars with different levels of cyanogenic glucosides (Forslund et al., 1998).

Cyclic hydroxamic acids (Hx) are a group of secondary metabolites characteristic for the family Poaceae and occur in wild grasses, as well as in cultivated crops such as maize (Zea mais L.), wheat (Triticum aestivum L.), rye (Se- cale cereale L.), and triticale, but are absent from barley, oats (Avena sativa L.) and rice (Oryza sativa L.) (Zúñiga et al., 1983; Niemeyer, 1988; Slesak et al.,

2001). Hx seem to be defensive factors against insects (Ahmad et al., 2011;

Meihls et al., 2013). It was shown by using genetically-defined maize lines, that the growth and survival of BCA, as well as reproduction of R. maidis were negatively affected on plants accumulating 2,4-dihydroxy-7-methoxy-2H-1,4- benzoxazin-3(4H)-one and its glucoside form (Ahmad et al., 2011; Meihls et al., 2013).

Another example of plant defense compounds with suggested protective functions are the thionins. They are small (~5kDa), cysteine-rich peptides and can be divided into the families of α/β-thionins and γ-thionins (plant

22 defensins) (Westermann and Craik, 2010). The barley leaf thionins are encoded by a large gene family, with more than 50 members per haploid genome

(Bohlmann et al., 1988; Bunge et al., 1992). Thionins are involved in protection against pathogenic bacteria and fungi by targeting the pathogen’s membrane

(Stec, 2006). Their involvement in protection against insects has also been suggested (Jones et al., 1985; Bohlmann et al., 1988; Charity et al., 2005; Pelegrini and Franco, 2005). Four barley thionin sequences were found to be constitutively expressed in moderately aphid resistant barley genotypes and all were upregulated by aphid feeding (Delp et al., 2009). It was shown that GPA progeny production on Nicotiana benthamiana (Domin) leaves transiently overexpressing barley thionin was reduced nearly 50% when compared to the vector control, the mechanism of this reduction remains however unknown

(Escudero-Martinez et al., 2017). Barley leaf thionins have been localized both in cell walls and intracellularly (Charity et al., 2005), which creates the possibility for aphids to encounter thionins during penetration or probing.

It has long been suggested that secondary metabolites might protect against generalists, but not against specialists (van Emden, 1972). Glucosin- olates constitute a much-studied example of this idea. Upon tissue damage, these compounds are broken down by the enzyme myrosinase to toxic isothi- ocyanates, nitriles and other compounds with documented anti-herbivore effects (Kelly et al., 1998; Renwick, 2002). The specialized cabbage aphid (Brevi- coryne brassicae L.) can however use the glucosinolates for its own protection against predators (Bridges et al., 2002; Kazana et al., 2007).

Indirect plant defenses

Attracting natural enemies, predators and parasitoids that prey upon her- bivorous insects, is an indirect defensive strategy employed by plants. Two particularly widespread indirect defense traits are extrafloral nectar and

23 volatile organic compounds (Thaler, 1999; Heil, 2008; Radhika et al., 2008).

Host plant-specific and herbivore-specific release of herbivore induced plant volatiles comprises the cue for predators and parasitoids used to locate prey

(de Vos and Jander, 2010). Volatile organic compounds released by plants can guide foraging behavior, especially significant for specialist insects, which have to invest more time in searching for host and prey than generalists. It has been shown that plant volatiles such as terpenoids and green leaf volatiles

(GLVs) induce electroantennographic responses of hoverfly (Episyrphus balteatus De Geer). Larvae of E. balteatus are insectivores and prey on aphids. The oviposition site is selected by the female and has important impact on offspring performance. It has been demonstrated that (Z)-3-hexenol increases the motility of females, plant acceptance and oviposition activity even in the ab- sence of prey. The same effect on E. balteatus behavior has been observed for the aphid alarm pheromone, (E)-β-farnesene (Eβf) (Verheggen et al., 2008).

Seven-spot ladybird (Coccinella septempunctata L.) is considered an important beneficial insect, an aphid predator. The ladybird is known to make use of volatile chemical cues in locating prey (Pettersson et al., 2008). It responds positively to volatiles from aphid-infested and previously attacked barley plants, but not to uninfested plants and undisturbed nonfeeding aphids (Ninkovic et al., 2001). Moreover, the ladybird C. septempunctata has been shown to be able to learn to associate the odor of aphid-infested plants with the presence of prey (Glinwood et al., 2011a).

Triggering of plant defense response

Plant perception of pathogens and hemipteran insects share some common features. There are two types of receptors involved in triggering plant defense response, which have distinct cellular localization: plasma membrane pattern recognition receptors (PRRs) which induce pattern-triggered immunity (PTI)

24 and intracellular receptors, recognizing effectors delivered inside the plant cell, which results in effector triggered immunity (ETI) (Figure 4) (Smith and

Clement, 2012; Kaloshian and Walling, 2016). PTI usually activates basal resistance mechanisms, whereas ETI is the main mechanism for host resistance gene (R-gene) mediated resistance (Lee et al., 2016).

Figure 4. Schematic representation of PTI and ETI in plant–aphid interactions. (a) HAMPs are recognized by membrane-bound PRRs to trigger PTI. (b) Delivery of effectors that interfere with PTI leading to increased susceptibility.(c) Effectors may be recognized by intracellular R-proteins (encoded for example by Mi or Vat genes) resulting in activation of ETI (figure from Hogenhout and Bos, 2011; reproduced with the permission from the publisher).

Pattern-triggered immunity (basal defense)

The plant-attacker interaction begins after recognition of molecular patterns associated with the microbes (microbe associated molecular patterns,

MAMPs) or upon perception of HAMPs, by plant PRRs (Kaloshian and

Walling, 2016). HAMPs may function in concert with herbivory-induced molecules originated from plants themselves, DAMPs. PTI is an important

25 component of plant innate immunity to insects. No aphid-associated molecular pattern has yet been identified, however it was shown that extract of the

GPA as well as treatments with GPA saliva triggered responses characteristic of PTI in Arabidopsis (de Vos and Jander, 2009; Prince et al., 2014). PTI was also shown to be activated by patterns associated with the bacterial endosym- bionts. Buchnera’s most abundant protein GroEL has been identified in aphid saliva and honeydew (Baumann et al., 1996; Sabri et al., 2013; Chaudhary et al., 2014; Vandermoten et al., 2014). The perception of aphid effectors involves

BAK1 (BRASSINOSTERIOD INSENSITIVE 1-ASSOCIATED RECEPTOR KI-

NASE 1), which is known to interact with PRRs to induce defense responses.

HAMPs induce influx of Ca2+ ions as part of the signal transduction chain (Wu and Baldwin, 2010). The subsequent accumulation of superoxide anion (O2−), hydrogen peroxide (H2O2), singlet oxygen (1O2) and hydroxyl radical (·OH), collectively called ROS, marks the early post-invasive plant defense to biotic stress (Wojtaszek, 1997; Jones and Dangl, 2006; Wu and Baldwin, 2010;

Jaouannet et al., 2014; Prince et al., 2014). ROS are produced in mitochondria, chloroplasts, and peroxisomes, as well as on the plasma membrane’s external surface. The herbivore-induced production of ROS is dependent on plasma membrane-bound NADPH oxidases (Wu and Baldwin, 2010). ROS production was shown to differ in Arabidopsis depending on the attacking aphid species. ROS amounts were lower in compatible Arabidopsis-GPA interactions, than in the case of poor- or non-host interactions with black cherry aphid (Myzus cerasi Fabricius) or BCA, respectively (Jaouannet et al., 2015). In addition, Arabidopsis NADPH oxidase knock-out lines with hindered production of ROS showed increased populations of GPA and M. cerasi, as well as increased survival of BCA (Jaouannet et al., 2015).

With regard to plant damage, aphids which display characteristic feeding behavior with active suppression of plant defense responses do not cause

26 plant tissue damage comparable to damage caused by chewing insects. Com- ponents of aphid saliva and the process of probing may potentially lead to formation of DAMPs (Miles, 1999). The largest and best characterized class of plant DAMPs are polypeptides/peptides produced from larger precursor proteins, such as the systemin of Solanaceous species (Choi and Klessig, 2016).

Different DAMPs are most likely recognized by yet unknown PRRs, which triggers the synthesis of jasmonic acid (JA) in the chloroplast and activates JA- signaling (discussed in detail in the section “JA and the LOX pathway in plant response to aphids”). Alternatively, Ca2+-influxes which can, among others, be triggered by the damage-associated perception of extracellular ATP, initiate the formation of ROS by NADPH oxidase and downstream activation of defense genes (Wu and Baldwin, 2010; Heil and Land, 2014).

Effector-triggered immunity (active defense)

Some pathogens or insect pests have evolved to suppress the basal defence by deploying effectors into plant cells. In turn, some plant hosts have evolved a second line of defence, ETI, in response to such effectors. This type of plant response requires resistance (R) genes in plants which can interact with insect effector molecules (avirulence factors, Avr). Most plant R genes providing resistance to pathogens and pests encode nucleotide-binding leucine rich repeat proteins (NB-LRR) (Jones and Dangl, 2006; Hogenhout and Bos, 2011). Three

R genes coding for proteins with NB-LRR structure and conferring resistance to aphids have been cloned and characterized: the Mi-1.2 gene conferring resistance to potato aphid in tomato (Rossi et al., 1998), the melon Vat gene providing resistance to melon/cotton aphid (Dogimont et al., 2014; Smith and

Chuang, 2014), and Ra gene from lettuce providing resistance to lettuce root aphid (Pemphigus bursarius L.) (Wroblewski et al., 2007). Those mentioned R-

27 gene encoded proteins lack organellar localization signals and thus are pre- sumed to be confined to the cytosol (Kaloshian and Walling, 2016).

Aphid-associated effectors

A number of potential effectors in aphid saliva and salivary glands have been identified by proteomics (Harmel et al., 2008; Carolan et al., 2009; Cooper et al., 2010; Carolan et al., 2011; Cooper et al., 2011; Nicholson et al., 2012; Rao et al., 2013; Vandermoten et al., 2014) and functional genomics studies (Mutti et al., 2006; Mutti et al., 2008; Bos et al., 2010; Pitino et al., 2011; Pitino and

Hogenhout, 2013; Elzinga et al., 2014; Wang et al., 2015). These aphid-specific proteins can inhibit plant defences, prevent phloem sieve-element occlusion, and promote phloem feeding by aphids (Elzinga and Jander, 2013). For example, a salivary protein Mp55 from GPA increased aphid reproduction when overexpressed in cultivated tobacco (Nicotiana tabacum L.) (Elzinga et al.,

2014). A C002 protein from salivary gland of A. pisum was shown to be crucial in the feeding establishment of this aphid on fava bean (Vicia faba L.) (Mutti et al., 2008). RNAi-based knockdown of A. pisum and GPA C002 transcripts significantly reduced the life span of pea aphids on fava bean leaves and fecundity of GPA (Mutti et al., 2006; Pitino et al., 2011).

It is known that chitin is a fungal PAMP; it is also an important component of the aphid stylets and exoskeleton structures (Uzest et al., 2010) and thus aphid saliva may contain chitin fragments detached during stylet movement

(van Bel and Will, 2016). It has been suggested that plants detect aphid chitin as non-self molecules to strengthen the plant-defense response, however this aspect remains to be further investigated (Jaouannet et al., 2014; van Bel and

Will, 2016).

28 Defense signaling pathways

The defense response in plants involves phytohormone signaling. Phloem feeders have been shown to modulate plant responses regulated by three key compounds of the plant defense system: salicylic acid (SA), JA and ethylene

(ET) (Thompson and Goggin, 2006; Kuśnierczyk et al., 2011; Morkunas et al.,

2011).

SA in plant response to aphids

SA is a product of the shikimic acid biosynthesis pathway important for the hypersensitive response (Waling 2000) and known to promote a broad- range resistance to pathogens (Reymond and Farmer, 1998; Morkunas et al.,

2011). SA-dependent responses use SA and MeSA to activate a number of defense-related genes, among them pathogenesis related (PR) genes, whose products localize in the apoplast (Moran and Thompson, 2001). Signaling downstream of SA is largely dependent on the regulatory protein NPR1 (non- expressor of PR genes 1) (Figure 5). SA presence in induced cells (e.g. after pathogen attack) leads to monomerization of NPR1 and its translocation to the nucleus. In the nucleus, NPR1 monomers interact with TGA transcription factors, which bind to the promoters of SA-responsive genes, such as PR-1, resulting in their activation. In addition, many WRKY transcription factor genes are induced, which can activate or repress SA responses. During this process, NPR1 becomes phosphorylated, ubiquitinylated and degraded in the proteasome (Figure 5) (Kesarwani et al., 2007; Pieterse et al., 2012).

29 related genes in barley (Beβer et al., 2000; Forslund et al., 2000; Li et al., 2005;

Glinwood et al., 2007).

MeSA is also known as an aphid repellent. It was shown to inhibit BCA attraction to aphid-infested oat (Pettersson et al., 1994), reduce BCA settling on barley, both in greenhouse and field conditions (Ninkovic et al., 2003;

Glinwood et al., 2007), as well as decrease infestation on wheat by S. avenae and rose-grain aphid (Metopolophium dirhodum Walker) by 40 to 50%

(Pettersson et al., 1994).

JA and the LOX pathway in plant response to aphids

JA is a low molecular mass regulator of inducible defense gene expression and plays an important role in a range of developmental processes (seed germination, tuber formation, senescence, flower development) (Wasternack,

2007). JA and its derivatives are products of the allene oxide synthase (AOS) branch of the LOX pathway (Wasternack, 2007; Mosblech et al., 2009; Lyons et al., 2013; Wasternack and Hause, 2013; Borrego and Kolomiets, 2016). Lev- els of JA rise after wounding and herbivore attack, which triggers the plant defense responses. A similar effect can be obtained by exogenous application of JA or MeJA to plants (Glinwood et al., 2007; Morkunas et al., 2011). JA can be readily metabolized to MeJA or conjugated to amino acids such as isoleucine via the JA conjugate synthase JAR1. Binding of JA-Ile to COI1 (coronatine-insensitive 1), a component of the ubiquitin ligase in induced cells (e.g. after herbivore attack) (Figure 6) leads to ubiquitylation and subsequent deg- radation of JAZ (jasmonate ZIM domain) transcriptional repressor proteins via the proteasome. This disables the physical interaction between JAZ proteins and transcriptional activators (Figure 6) (Thines et al., 2007). In Ara- bidopsis, two major branches of the JA signaling pathway are recognized: the

MYC branch associated with the wound response and defense against insect

31 herbivores, and the ERF branch associated with enhanced resistance to necrotrophic pathogens (Howe and Jander, 2008; Pieterse et al., 2012). In Arabidop- sis, VEGETATIVE STORAGE PROTEIN2 (VSP2) and PLANT DEFENSIN1.2

(PDF1.2) are the two marker genes for the two respective branches, MYC and

ERF (Figure 6) (Pieterse et al., 2012).

Figure 6. Schematic overview of the JA signaling pathways in induced cells, as summarized in the text. Abbreviations: SCF COI1, multi-protein E3- ubiquitin ligase named for the SKP/cullin/F-box proteins of the complex where COI1 is the F-box component; Ub, ubiquitinylated protein; (figure adapted from Pieterse et al., 2012).

JA and its derivatives were shown to induce expression of defense-related genes (Hause et al., 1999; Beβer et al., 2000; Bachmann et al., 2002; Glinwood et al., 2007; Walia et al., 2007). It has been suggested that JA regulates effective defenses against phloem feeders in dicot plants (Ellis et al., 2002; Gao et al.,

2007; Brunissen et al., 2010). Studies on monocot plants suggest that JA-

32 mediated pathway might affect aphid acceptance or feeding, without strong antibiosis effects (Slesak et al., 2001; Glinwood et al., 2007; Cao et al., 2014).

The hydroperoxide lyase (HPL) branch of the LOX pathway directs the formation of C6-volatiles (Bate and Rothstein, 1998; Matsui, 2006), which play a role in plant defense against pathogens and herbivores (Scala et al., 2013;

Naeem ul Hassan et al., 2015; Matsui and Koeduka, 2016). The involvement of C6 volatiles in defense against aphids has been shown in the case of tobacco aphid (Myzus nicotianae Blackman) and GPA (Hildebrand et al., 1993;

Vancanneyt et al., 2001).

ET in plant responses to aphids

ET and JA can act synergistically and activate defense responses in plants, which are different and often antagonized by responses induced by SA (Smith and Boyko, 2007). Both ET and JA are involved in induced systemic resistance activated in plants in response to non-pathogenic bacteria (van Loon et al.,

1998). ET signaling is in many cases dependent on JA induction (Von Dahl and Baldwin, 2007). It was shown that numbers of GPA were greatly reduced on Arabidopsis mutant cev1 with constitutive expression of JA and ET responses (Ellis et al., 2002). Aphid feeding was also shown to significantly increase ET production in an aphid-resistant barley cultivar, as compared to susceptible plants. This has been shown for S. graminum and BCA

(Aragandona et al., 2001) as well as for D. noxia (Miller et al., 1994). In addition, it was shown that feeding by potato aphid (Macrosiphum euphorbiae

Thomas) induces expression of ET biosynthetic genes both in susceptible

(mi/mi) and resistant (Mi-1/Mi-1) tomato cultivars, which correlated with a burst of ET after aphid feeding (Mantelin et al., 2009).

33 The cross-talk of SA and JA pathways

In summary, the above-mentioned signaling pathways do not act inde- pendently of each other, but rather are part of a complex network with positive and negative regulatory interactions, to fine tune the defense responses.

The SA response pathway is typically recognized as effective against microbial biotrophic pathogens, whereas JA and its derivatives, as well as ET, act against necrotrophic pathogens and herbivores (Glazebrook, 2005). It has been suggested that SA inhibits the biosynthesis of JA and subsequent induction of gene expression; similarly, that JA inhibits the effects of SA (Kunkel and Brooks, 2002; Morkunas et al., 2011; Thaler et al., 2012; Vidhyasekaran,

2015). Down-regulation of the JA-pathway was shown for Arabidopsis mutants that accumulate high levels of SA (Kachroo et al., 2003a; 2003b).

The NPR1 has been shown to be involved in the suppressive action of SA against JA signaling. In the cytosol, the SA-activated NPR1 pool may be involved in facilitating the delivery of negative regulators of JA-responsive genes to the nucleus or by inhibiting positive regulators of JA-responsive gene expression (Spoel, 2003). In Arabidopsis, the transcription factor WRKY70 is known to be involved in determining the balance between SA and JA signaling pathways (Li et al., 2004; Li et al., 2006). It was shown to inhibit expression of JA-responsive genes (Li et al., 2004). JA is known to regulate WRKY70 transcript levels negatively, whereas SA acts as positive regulator (Li et al., 2004).

Gene expression profiling analysis has shown that SA and JA act coordi- nately to induce defense in response to aphid feeding (Moran and Thompson,

2001). Up-regulation of both SA and JA/ET-responsive genes was shown in

Arabidopsis after feeding by B. brassicae and A. gossypii (Moran and

Thompson, 2001). It was proposed, however, that while the activation of the two signaling pathways is harmonized and temporarily controlled

(Kuśnierczyk et al., 2008), aphids can manipulate plant-induced resistance, to

34 suppress JA-regulated defenses by the induction of the less efficient SA-regulated responses (Kuśnierczyk et al., 2008; Giordanengo et al., 2010). Genes coding for JAZ proteins which act as negative feedback regulators of JA signaling were activated at 6 h after B. brassicae attack, with subsequent regres- sion of JA-connected responses at 12 h (Kuśnierczyk et al., 2008). This “decoy” hypothesis was supported also by results from studies on Arabidopsis mutants, where GPA resistance was shown independent of high levels of SA

(Pegadaraju et al., 2005). Similarly, no differences were observed in GPA reproduction between wild-type plants and mutants compromised in SA signaling (Moran and Thompson, 2001).

Aphid resistance genes

Previous studies have shown that gene up-regulation caused by aphid feeding provides resistance against aphids. Examples are the phytoalexin deficient 4 (PAD4) gene, the trehalose phosphate synthase 11 (TPS11) gene and

Myzus persicae-induced lipase 1 (MPL1) gene, all shown to control antibiosis and antixenosis against the GPA (Louis et al., 2010; Singh et al., 2011; Louis and Shah, 2015). PAD4 knockout and TPS11 or MPL1 overexpression were shown to support respectively higher and lower aphid population growth on

Arabidopsis (Louis et al., 2010; Singh et al., 2011; Louis and Shah, 2015). Sim- ilarly, a tomato α-dioxygenase, induced by potato aphids was shown to contribute to plant defense against this aphid and silencing of this gene resulted in higher aphid numbers (Avila et al., 2013).

In barley, BCA-induced gene expression was compared between two moderately aphid-resistant and two susceptible barley genotypes in a microarray-based study (Delp et al., 2009). One of the resistant genotypes used in that study was an accession of wild barley H. vulgare L. ssp. spontaneum

(Hsp5). The other, double haploid breeding line DH28:4 (full number 5172-

35 28:4), was an offspring from a cross of Hsp5 with the susceptible cultivar Lina and backcrossed to Lina (Åhman et al., 2000). The resistance in Hsp5 and

DH28:4 was demonstrated as decreased (up to 40% and 15%, respectively) nymphal growth (Åhman et al., 2000). Results from this comparative transcriptome profiling revealed that four genes were up-regulated by BCA only in the resistant genotypes and not in the susceptible barley. Those genes were encoding for: a protease inhibitor (CI2c, known also as BCI-7), a lipoxygenase

(LOX2.2), a calcium-binding EF-hand protein (BCI-4) and a Ser/Thr kinase

(Delp et al., 2009). Those four genes were considered candidate aphid-resistance factors and two of them, the CI2c and LOX2.2, were in the main focus of this thesis.

Aphid management in agriculture

Aphid management is commonly based on pesticide applications, however they have adverse environmental effects (Dewar, 2007; Hillocks, 2012).

The active compounds of insecticides, the organophosphates and carbamates, inhibitors of acetylcholine esterase, tend to have high toxicity to beneficial insects and mammals. As a consequence, many have been withdrawn from use

(Dewar, 2007). Control of viruses has also proven to be difficult using chemical applications. Insecticides are usually applied after aphid infestation has reached a certain threshold and at this point virus transmission has already occurred. In addition, a number of aphid species have been reported to have developed insecticide resistance, some of them belonging to the most problematic pest species (Foster et al., 2007; Bass et al., 2014; Bass et al., 2015;

Bettaibi et al., 2016).

Breeding for constitutive and inducible aphid resistance have been suggested as an alternative for more sustainable aphid control (Åhman, 2009).

With regard to inducible resistance, a variety of biological and chemical

36 agents have been successfully used, in the form of foliar sprays or seed treatments, under controlled and field conditions, to handle pathogen infections and insect pests populations, including aphids (Pushpalatha et al., 2011;

Worrall et al., 2011; Walters et al., 2013; Hamada et al., 2017). Volatile treatments show similar potential (Shulaev et al., 1997; Kishimoto et al., 2005;

Ninkovic and Åhman, 2009). Plants release volatile organic compounds from reproductive and vegetative parts and the amounts released increase upon mechanical damage or herbivore infestation (Bernasconi et al., 1998; Turlings et al., 1998; Paré and Tumlinson, 1999). However, there exist chemical interactions also between undamaged plants (Glinwood et al., 2011b). The term

“allelobiosis” was introduced to describe chemical interaction between undamaged plants, which is beneficial for the receiving plant and which involves organisms from other trophic levels (Ninkovic et al., 2006). It has been shown that mixing different plant species or different genotypes of the same species can reduce incidences of herbivory (Andow, 1991; Ninkovic et al.,

2002; Ninkovic and Åhman, 2009). Volatile-induced resistance was shown to be a heritable trait and presents the option to breed for allelobiotic effects

(Ninkovic and Åhman, 2009). Mixed cultivation is however problematic in agriculture and there exists a risk that allelobiosis will not be easily trans- ferred into practice (Ninkovic and Åhman, 2009).

The potential of Eβf, an aphid alarm pheromone, in the control of pest aphids has also been recognized. It was shown that Arabidopsis plants expressing Eβf synthase gene cloned from Mentha × piperita and emitting Eβf, caused alarm and repellent responses of GPA; additionally, transgenic plants became attractive to aphid parasitoid Diaeretiella rapae (M'Intosh). Similarly,

Eβf was shown to be an effective kairomone for the two-spot ladybird (Adalia bipunctata L) (Francis et al., 2004).

37 Breeding for aphid resistance has resulted in cultivars resistant to D. noxia,

S. graminum (Berzonsky et al., 2002), lettuce aphid (Nasonovia ribisnigri Mos- ley) (Liu and McCreight, 2006) and soybean aphid (Aphis glycines Matsumura)

(Li et al., 2007). Moreover, wheat cultivars which reduce BCA populations or tolerate the effects of aphid feeding have also been identified (Girvin et al.,

2017). The selection of a resistant cultivar was based on observed plant symptoms after aphid infestation. This can be difficult in a situation when the aphid, such as BCA, does not cause visible symptoms. In barley and wheat, quantitative trait loci for BCA resistance have been reported (Cheung et al.,

2010; Crespo-Herrera et al., 2014) and a number of gene sequences proposed as candidate resistance factors (Delp et al., 2009; Mehrabi et al., 2014).

Identification of genes related to aphid resistance would be of high value in breeding programs. Conventional breeding develops new plant varieties expressing desirable traits, by crossing between parental lines, followed by selection of progeny. This process is time and labor consuming, since it might need several rounds of backcrossing, inbreeding and selection. Introducing resistance genes into cultivars could be facilitated with the use of transgenic approaches (Gepts, 2002). Additionally, identification of resistance genes would enable marker-assisted selection, instead of time consuming and labor- intensive insect bioassays.

More recent approaches in biotechnology aiming to produce aphid-resistant plants include the plant-mediated RNAi strategy, where transgenic plants expressing double stranded RNA (dsRNA) designed to knock-down insect target genes show increased protection against insect herbivory (Yu et al., 2016). A decline in GPA fecundity was reported due to plant-mediated

RNAi of salivary effectors MpC002, MpPIntO1 and MpPIntO2 (Pitino et al.,

2011; Pitino and Hogenhout, 2013). GPA showed also impaired reproduction on Arabidopsis plants expressing dsRNA of the GPA serine protease gene

38 MySP (Bhatia et al., 2012) and on tobacco plants expressing dsRNA against the gap gene hunchback (hb), which regulates insect axial patterning (Mao and

Zeng, 2014). Promising results are observed when approaches based on pesticide usage are combined with RNAi knock-down of aphid genes involved in insecticide detoxification. An example of this concept is the plant-mediated

RNAi silencing of the carboxylesterase CbEE4, which reduces the tolerance of

S. avenae to Phoxim insecticides (Xu et al., 2014). The new methods of plant- mediated RNAi insect control need however to face a few challenges such as the persistence of the RNAi effect, delivery and production of dsRNA by transgenic plants, potential off-target RNAi effect and potential development of aphid resistance towards RNAi plants (Yu et al., 2016)

The advantage of the transgenic breeding is that a particular desirable trait may be introduced, or the level of expression of such traits can be modified.

Application of such methods is however hindered in the European Union, by the Directive 2001/18/EC of the European Parliament and European Council on non-contained production and propagation of genetically modified organisms (GMOs). The new plant breeding techniques (NPBTs) however, such as the clustered regularly interspaced short palindrome repeats (CRISPR)/Cas9 nuclease technique or RNA-based DNA methylation, which differ from classical breeding and conventional transgenic approaches, give opportunities for crop genetic improvement. Those methods escape one of the main arguments concerning hazard connected with GMOs, which is the uncontrolled integra- tion of recombinant DNA into the genome (Hartung and Schiemann, 2014).

40 Aim of the thesis

The aim of this thesis was to evaluate the candidate aphid-resistance genes,

CI2c and LOX2.2 in barley. To achieve this goal, the following investigations in the four papers were made:

I. A barley cDNA encoding the protease inhibitor CI2c was expressed in

Arabidopsis and the performance of the generalist GPA was studied in

control and transgenic plants, with constitutive or phloem-specific ex-

pression of the transgene.

II. Barley CI2c was overexpressed in barley and bioassays were carried

out with the specialist BCA and the generalist GPA. Aphid tests were

followed by analysis of aphid-induced plant responses related to de-

fense in plants.

III. The effects of the JA-responsive barley LOX2.2 gene overexpression

and silencing were studied with regard to consequences on expression

of other JA-responsive genes, changes in volatile emissions from trans-

genic lines, as well as BCA and GPA performance.

IV. The effects of volatile treatments on different cultivars of barley were

studied with regard to activation of plant defense responses on molec-

ular level, followed by bioassays with BCA on the treated plants.

BCA and GPA represent aphids with different host ranges and were exam- ined in detail in Papers II and III, to provide information regarding involvement of the studied resistance factors in host and non-host resistance.

42 Main approaches

Hypotheses for the thesis work

The work in Papers I, II and III is based on the hypothesis, that genes induced by aphids confer increased aphid resistance. It has been previously suggested that genes specifically induced in moderately aphid-resistant plants, and not in susceptible cultivars, may function as resistance factors in barley

(Delp et al., 2009).

For paper IV we hypothesized that treatments with volatiles would affect gene expression in barley in a way that aphid population growth would be reduced. The volatiles of special interest were those with known functions in plant defense: MeSA, in plants synthetized in the shikimic acid biosynthesis pathway, MeJA and (Z)-3-hexen-1-ol which both are produced via the LOX pathway. The genes of interest were those with suggested function in plant defense against aphids, of special interest were barley LOX genes, LOX2.1 and

LOX2.2, induced by BCA (Delp et al., 2009) and involved in production of JA or GLVs.

Putative resistance factors against aphids

With regard to the first hypothesis, the supporting evidence comes from the previously mentioned microarray study, where two moderately aphid- resistant and two susceptible barley genotypes were compared regarding

BCA-induced gene expression (Delp et al., 2009). One of the four genes induced by BCA in resistant genotypes and studied in Papers I and II was CI2c, which encodes a protease inhibitor. It belongs to a family of six chymotrypsin

43 inhibitor 2 (CI2) genes localized in the Mla (powdery mildew) resistance locus

(Wei et al., 2002). PIs are small molecular weight proteins involved in plant defense responses (Solomon et al., 1999; Hartl et al., 2011; Fluhr et al., 2012;

Jamal et al., 2012). They are mainly located in seeds or tubers and can be induced in leaves and roots (Habib and Fazili, 2007). They can be involved in regulation of endogenous plant protease activities or inhibition of exogenous pathogen- or insect-derived proteases during plant defense responses (Ryan,

1990; Carrillo et al., 2011a).

The second sequence suggested as a putative aphid resistance factor and studied in Paper III was a LOX2.2 gene. Plant LOXs are non-heme iron-containing enzymes which catalyse the addition of molecular oxygen to polyun- saturated fatty acids thus generating hydroperoxy fatty acids. This step initi- ates the synthesis of group of active compounds collectively called oxylipins.

Molecular oxygen can be incorporated to either the 9 or the 13 position of the

C18 chain of linoleic and linolenic acid by 9-LOXs and 13-LOXs respectively.

This results in synthesis of 9- or 13-hydroperoxy fatty acids, which are further channelled to different branches of the LOX pathway (Hildebrand, 1989;

Howe and Schilmiller, 2002; Porta and Rocha-Sosa, 2002). Another base of classification of LOXs is the localization. Type 1-LOXs do not have the chloroplast transit peptide and show high (>75%) sequence similarity and type 2-

LOXs have a chloroplast transit peptide and are characterized by lower

(>35%) sequence similarity to other LOXs (Shibata et al., 1994; Liavonchanka and Feussner, 2006; Andreou and Feussner, 2009). In general type 2-LOXs have been shown to exhibit 13-LOX activity and type 1-LOXs show 9-LOX activity, however there exist enzymes with no or dual position specificity

(Wang et al., 2008; Andreou and Feussner, 2009). Downstream of plant 13-

LOXs are two biosynthetic pathways: the AOS branch leading to the synthesis of JA and the HPL branch resulting in formation of GLVs (Matsui, 2006;

44 Mwenda and Matsui, 2014). End products from both pathways are important in plant defense against insects (Matsui, 2006; Scala et al., 2013; Wang and Wu,

2013; Naeem ul Hassan et al., 2015). Barley leaves contain at least three distinct forms of JA-responsive 13-LOX localized in chloroplasts: LOX2.1, LOX2.2 and

LOX2.3 (Vörös et al., 1998; Hause et al., 1999; Bachmann et al., 2002). LOX2.1 and LOX2.2 were shown to be induced by aphid infestation in barley (Delp et al., 2009). It has been shown that treatment of floating barley leaves with JA or JA-isoleucine conjugate caused accumulation of barley LOX transcripts, with LOX2.2 being induced prior to up-regulation of the LOX2.1 and LOX2.3 genes (Bachmann et al., 2002).

Induction of resistance

Regarding the second hypothesis, it has been previously shown for SA, JA, their methyl esters and GLVs in various grass species, that plant volatiles affect expression of defense genes in exposed plants (Vörös et al., 1998; Hause et al., 1999; Bachmann et al., 2002; Farag et al., 2005; Li et al., 2005; Xu et al.,

2005; Glinwood et al., 2007; Kakei et al., 2015; Yang et al., 2015). MeSA was of special interest in our study since it has been shown previously to negatively affect BCA preference and reduce BCA settling on barley, as well as infestation on wheat by S. avenae and M. dirhodum (Pettersson et al., 1994). Results from studies on plants after treatment with MeJA or JA suggest that JA regulates effective defenses against phloem feeders in dicot plants (Gao et al., 2007;

Brunissen et al., 2010). For monocots, MeJA treatment was shown to reduce settling and negatively affect feeding behavior of BCA in wheat (Slesak et al.,

2001) and acceptance in no-choice tests in barley after exposures during daylight (Glinwood et al., 2007). With regard to (Z)-3-hexen-1-ol, the treatment has been shown to up-regulate genes related to the JA pathway in maize

(Farag et al., 2005; Xu et al., 2005; Engelberth et al., 2013). Fecundity of tobacco

45 aphid M. nicotianae was reduced on tobacco leaves exposed to C6 volatiles

(Hildebrand et al., 1993). More evidence for the role of GLVs in plant defense comes from a study where potato plants were depleted in HPL. Silenced lines showed lower emission levels of hexanal and 3-hexenal, which caused an increase in GPA fecundity when compared to wild type plants (Vancanneyt et al., 2001).

46 Comments on methodology

Plant transformation

The work in Papers I, II and III is based on the use of transformed Ara- bidopsis or barley plants. The protocol for Arabidopsis transformation using the floral dip method is well established and the transformation process sim- ple, with no need for tissue culture (Zhang et al., 2006). Barley transformation was carried out using plant immature embryos, which produces plants with low transgene copy numbers and supports stable expression (Bartlett et al.,

2008). In the above cases, the transformation was mediated by Agrobacterium tumefaciens, a gram-negative soil bacterium, which can incorporate a fragment of its Ti plasmid (T-DNA) into the genome of the plant host (Klee et al., 1987;

Ziemienowicz, 2014). The possibility to engineer plants to overexpress or down-regulate expression of selected genes is a tool of great value to study gene functions and gives possibilities for applications, i.e. to improve crop resistance to abiotic and biotic stresses.

A barley CI2c gene sequence was cloned and transformed into Arabidopsis ecotype Columbia (Col-0) under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter (Paper I) and to barley under the control of the maize Ubi-1 promoter (Paper II). For monocots the CaMV 35S promoter is usable, although its activity has been reported as relatively low (Battraw and Hall, 1990). The maize Ubi-1 was shown to be comparatively stronger in monocots (Christensen et al., 1992). Additionally to constitutive transgene expression, CI2c was transformed to Arabidopsis and to barley under the control of rolC promoter from A. rhizogenes, with phloem-restricted expression reported in both dicot and monocot plants (Paper I and II) (Schmülling et al.,

47 1989; Saha et al., 2007; Srivastava et al., 2009). The use of a phloem-specific promoter was considered highly relevant for our studies. Expression of resistance factors in phloem tissues would provide valuable knowledge about the effects on aphids which successfully established feeding, narrowing the putative negative effects to antibiosis-based resistance.

For LOX2.2 overexpression and silencing, the Ubi-1 promoter was used

(Paper III). The down-regulation of LOX2.2 was accomplished by antisense transformation. Expression of transgenic antisense RNA was the first method of initiating post-transcriptional gene silencing (PTGS) in plants (Bourque,

1995). Antisense constructs consist of an inverted gene coding sequence. The homologous pairing of sense and antisense RNAs produces dsRNA, which was shown to be the initiator of silencing via production of small interfering

RNA (siRNA) (Di Serio et al., 2001). Antisense approach was used in Paper III for the reason that the sequence of the target gene to be silenced was known.

Other methods commonly used for PTGS are the heritable RNAi and

CRISPR/Cas9 techniques. Transient methods of gene silencing include virus- induced gene silencing (VIGS) or the use of synthetic antisense oligodeoxynucleotides (ODN). The above-mentioned methods differ regarding simplicity of application and level and specificity of silencing. Antisense RNA, RNAi,

VIGS and ODN are known to result only in partial silencing of the target gene.

The ODN method is not designed for intact plants and thus not suitable to study plant-aphid interactions (Gilchrist and Haughn, 2010; Dinc et al., 2011).

Moreover, siRNA-based approach can affect RNAs that are not perfectly com- plementary and considered off-targets. To secure a small number of targets with high sequence similarity, approaches based on micro RNA, short non- coding RNA, constitute alternatives (Schwab et al., 2006). In the CRISPR/Cas9 method the inactivation of genes is done by genome editing, which results in complete knock-out of the target gene. This method however needs

48 development for each species and is thereby more labor and time consuming than the above-mentioned techniques (Lawrenson et al., 2015; Lowder et al.,

2015). Another possible approach for gene silencing and used to investigate gene function is the targeting induced local lesions in genomes (TILLING) procedure. An advantage of TILLING is that it is a non-transgenic reverse genetic approach. It requires the production of mutants, amplification of a target gene and the recognition of the plants carrying a mutant allele (Talamè et al.,

2008).

PTGS has become a powerful method for studying gene function in plant interactions with phloem-feeding insects. For example, VIGS was used in wheat to silence resistance and susceptibility genes and study their function against D. noxia (van Eck et al., 2010; Anderson et al., 2014). Antisense-mediated transformation was used to silence the HPL gene in potato plants

(Vancanneyt et al., 2001) or OsHI-LOX gene in rice (Zhou et al., 2009), to study their involvement in resistance against aphids and the phloem-feeding brown plant hopper, respectively.

Selection and controls

Choosing a suitable control for studying the effects of transformation or treatment is important in order to avoid producing non-conclusive evidence.

For transgenic plants, few alternatives are possible. Wild type (WT) plants are used often (Vancanneyt et al., 2001; Aharoni et al., 2003; Ribeiro et al., 2006;

Wang et al., 2008; Zhou et al., 2009; Carrillo et al., 2011b), however those plants are not the most suitable controls to transgenic plants, since they do not expe- rience the same treatments (transformation process) and growth conditions.

Alternatives to WT plants are plants transformed with empty vector or azygous lines. Empty vector controls will show any possible effects of the vector itself on gene expression in the target organism. Azygous lines are those

49 which segregated as non-transformants and thus experienced the exact same conditions of transformation and selections as transformants, securing the same “seed history” effects, i.e., the same induced defenses carried from previous generations (Pastor et al., 2013).

With the selection process, we aimed to identify homozygous transgenic plants with a single insertion of the transgene and a corresponding azygous control, coming from the same transformation event. This was accomplished by repeated selections on medium containing kanamycin (Paper I) or hygro- mycin (Papers II and III). For experiments with aphids we aimed to select homozygous transgenic plants with varying level of transgene expression. Due to this process, the number of available lines for aphid bioassays was limited to five CI2c-overexpressing Arabidopsis lines in Paper I (two with constitutive and three with phloem-specific transgene expression), four CI2- transgenic lines in Paper II (two with constitutive and two with phloem-specific transgene expression), and two LOX2.2-transgenic lines in Paper III (one with overexpression and one with down-regulation of the gene). More transgenic lines were identified, but experimental restrictions led us to focus on a limited number.

Confirmation of transformation

To confirm transformation in Arabidopsis (Paper I) and barley (Paper II and III) we analyzed accumulation of the transgene using RT- qPCR. CI2c and

LOX2.2 gene expression was analyzed also in azygous lines, to detect levels of native expression of those sequences. In Paper I and II we confirmed trans- lation of the overexpressed CI2c into a functional protein by studying the inhibitory effects of CI2c against added chymotrypsin in enzyme extracts in vitro. Similar protocols using fluorogenic protease substrates have been previously used to identify plant protease inhibitors (Carrillo et al., 2011a;

50 Carrillo et al., 2011b). More direct methods to detect proteins are the commonly used Western-blotting or ELISA techniques, however they require specific antibodies (Bass et al., 2017). In Paper III we attempted to directly confirm the presence of LOX2.2 protein by Western-blotting. We tested an antibody directed against the wheat LOX2 protein (anti-LOX2, AS08 340, Agrisera), with 42% similarity to barley LOX2.2, since antibodies for barley LOX2.2 were not commercially available. Unfortunately, no barley LOX protein could be detected with this method, which could be explained firstly by low specificity of the wheat anti-LOX2 antibody towards antigens present on barley LOX and secondly due to low concentrations of LOX protein in protein extracts from barley. This problem could be solved by recombinant expression of the target protein, followed by purification and antibody preparation.

Two indirect methods were employed additionally in Paper III to confirm transformation. Firstly, we carried out the analysis of differential expression of JA-regulated genes in control, overexpressing and antisense LOX2.2 lines.

Secondly, the analysis of volatiles released from the control and overexpressing LOX2.2 lines, without and with BCA infestation was performed. The method of collecting plant volatiles by air-entrainment followed by GC/MS has been previously shown as suitable for barley (Glinwood and Pettersson,

2000b; Ninkovic et al., 2001; Glinwood et al., 2007). Our analysis was carried out after five days of aphid infestation. At this time point we observed effects of LOX2.2 overexpression on BCA and GPA fecundity (Figure 3 in Paper III), suggesting that during five days the activity of LOX2.2 was sufficient to affect aphids. It was also shown before, that collecting volatiles after

72 h of aphid infestation on barley was effective to induce responses in C. septempunctata, suggesting that changes in emitted volatiles occurred

(Ninkovic et al., 2001).

51 Plant phenotype

While assessing the effects of transformation and treatment on aphids, it is important to consider possible effects on plant phenotype. Adverse phenotype affecting the nutritional value of plants could influence the interaction between plants and aphids and introduce false information into the research.

For example, plant PIs which constitute an important component of the protein turnover machinery, can also be a part of the plant defense system against insects. It was shown that overexpression of the rice PI protein OCPI2 in Ara- bidopsis affected the plant’s phenotype by improving biomass accumulation and reproductive potential, features important when studying interaction between plants and aphids (Tiwari et al., 2015). A negative effect on GPA in choice tests was observed in Arabidopsis plants expressing the FaNES1 gene from cultivated strawberry (Fragaria × anannasa cv Elsanta) encoding S-linalool/(3S)-E-nerolidol synthase. Those plants however showed retarded growth

(to a varying degree) when compared to control plants, possibly due to the reduction in the availability of substrates for other metabolites produced in the plastids, or the toxic effect of the high levels of linalool produced, compro- mising the results from aphid bioassays (Aharoni et al., 2003). A careful investigation of the effects of transformation or treatments was carried out in our studies, with regards to effects on seed germination, flowering and leaf area

(Paper I), as well as plant growth and biomass accumulation (Papers II, III and

IV). It is also known that the interaction between plants and aphids differs depending on the growth stage of plants (de Vos et al., 2007; Louis et al., 2012), and thus in Paper I we used both bolting and non-bolting plants for tests with aphids.

52 Aphid performance

Antixenosis

Antixenosis describes the inability of a plant to serve as a host, and antixenosis-based resistance pressures the insect pest to select an alternate host plant (Smith, 2005a). To measure aphid host acceptance in barley, tests based on observations of aphid settling in no-choice and choice conditions have been carried out previously (Pettersson et al., 1996; Åhman et al., 2000;

Glinwood et al., 2007; Ninkovic and Åhman, 2009). Aphid settling is usually measured in time scales of hours; a period of 2 h has been reported to be sufficient for aphids to accept the host and reach the phloem (Prado and

Tjallingii, 1997). The preference behavior can be also measured in an olfactometer, where aphids are presented with different olfactory cues (Pettersson et al., 1994; Slesak et al., 2001; Glinwood et al., 2004), or with the EPG technique, enabling monitoring aberrances in aphid feeding behavior, by record- ing salivation, probing and phloem ingestion activities (Tjallingii and Esch,

1993).

In our work we carried out tests monitoring aphid behavior in no-choice and choice conditions. Those tests provided us with information about GPA settling on control and transgenic Arabidopsis plants overexpressing barley

CI2c gene (Paper I). Aphid acceptance in no-choice conditions was measured after 2, 4 and 6 h (Figure 2 in Paper I). Aphid preference for control or transgenic was measured after 24 h (Figure 3 in Paper I). Tests with GPA were ad- justed, since barley is a poor host for this species and GPA showed general avoidance towards barley (Papers II and III).

53 Antibiosis

Antibiosis resistance occurs when the negative effects of a resistant plant affect the biology of an insect pest attempting to use that plant as a host; the effects might be mild to lethal, chronic or acute (Smith, 2005b). Antibiosis resistance may affect different aspects of aphid biology such as growth, fecundity or longevity.

Analysis of aphid growth (i.e. weight) was previously used to identify aphid-resistant barley genotypes (Åhman et al., 2000; Ninkovic and Åhman,

2009). We measured the effects on fecundity in short-term tests, which provided us with information on possible resistance factors in the peripheral plant tissues encountered by aphids at the time of penetration and probing

(Powell et al., 2006). Analyzing aphid numbers instead of weight of individual insects is an easier approach, introducing less error into the results. Another approach could be comparing the total weight of counted aphids between experimental plants.

In our tests, we used both synchronous (Figure 5 in Paper I) and mixed populations of adult aphids (other tests in Papers I, II, III and IV). Using the uniform, calibrated populations in bioassays requires more time and labor in- vestment, they are however more suitable for time-scale experiments, since possible effects on aphids would be pronounced at the same time in all replicates, allowing to determine the moment of emergence and duration of those effects.

In the long term life span test, the reproduction of individual nymphs was monitored during their total life and since it follows aphids that have established feeding, it mainly gives indication of resistance factors present in the phloem (Nam et al., 2013; Will et al., 2013).

Both antixenosis- and antibiosis-based resistance can be measured not only on intact plants but also on excised plant leaves, leaf discs or with the use of

54 artificial diets (Forslund et al., 1998; Slesak et al., 2001; Bos et al., 2010; Pitino et al., 2011). For example, a functional leaf disc-based assay was used to determine whether aphid virulence was affected by overexpression of aphid candidate effectors (Bos et al., 2010). The results from assays performed on excised leaves should however be interpreted with caution as they may differ significantly from those obtained from intact plants (Schmelz et al., 2001). The same caution applies for tests using artificial diets, where in general, aphid performance is decreased when compared to plant-reared aphids (Douglas and van Emden, 2007). It should also be noted that results obtained under controlled laboratory conditions may not be observed at lager scales, i.e. in the field, where additional factors and interactions apply.

Another important aspect is the selection of rearing plants for aphid colonies, which should preferably be a different species than the plants used for bioassays with aphids. Insects may become adapted to a specific species or cultivar prior to experiment, which may affect the results. The aspect of rearing plants was relevant for consideration in our studies with BCA and GPA and addressed in Papers II and IV.

56 Main results

Effects of transformation or plant treatments with volatiles

Arabidopsis transformation with the barley CI2c sequence was confirmed at transcript and enzyme activity level (Figure 1 in Paper I). Transcript accumulation was much higher in lines with constitutive transgene expression driven by CaMV 35S promoter, than in phloem specific (ps) lines with expression restricted to phloem tissue, controlled by rolC promoter (Figure 1 in Pa- per I). We did not observe any adverse effects on plant shoot phenotype at the age of the aphid tests (Table S1, Figures S2 and S3 in Paper I).

CI2c expression in barley was confirmed at gene expression and enzyme activity level (Figure 1 in Paper II), with no effects on plant phenotype (Figure

S2 in Paper II). CI2c accumulation was also studied in lines with expression restricted to phloem tissues (under control of rolC promoter). As in the case of

Arabidopsis transformants, transcript accumulation was to minimal levels when compared to lines with expression under Ubi-1 promoter and did not differ significantly from the expression in control lines (data not shown).

Thus, the transgenic status of barley plants expressing CI2c under the rolC promoter could not be confirmed. This issue could be solved by carrying out transformation using constructs containing a marker gene, encoding for example for green fluorescent protein (Stewart Jr, 2001).

In Paper III, LOX2.2 overexpression (OeLOX2.2 lines) was confirmed at transcript level as a significantly higher expression of LOX.2.2 and a number of other JA-regulated genes (Figure 7; Figures 4 and 5 in Paper III). LOX2.2 was expressed at low levels both in the control and antisense (antiLOX2.2)

57 lines. The antisense status was confirmed indirectly, by showing a lower expression of three other JA-regulated genes, i.e. LOX2.1, THIO1567 and CI2c

(Figure 7), and no significant upregulation of LOX2.2 by aphids (Figures 4 and

5 in Paper III) in antiLOX2.2 line.

Figure 7. Relative transcript abundance of LOX2.2 and a selection of other jasmonic acid (JA)-regulated genes in OeLOX2.2 and antiLOX2.2 lines and their respective controls; bars indicate SE. Letters indicate significant difference between lines for a particular gene (Kruskal-Wallis test at p < 0.05). Samples were from leaves of 14-day old plants (second leaf). Reference genes: Hsp70 and Tubulin.

No barley LOX protein could be detected with Western-blotting, for possible reasons discussed in section “Confirmation of transformation”. Another possible way to indirectly detect active LOX2.2 protein would be to determine the concentration of JA and its conjugates in control and transgenic plants, assuming LOX2.2 involvement in JA biosynthesis (Zhou et al., 2009; Zhou et al.,

2014).

Similarly to LOX2.2, HPL was shown previously to be localized in plastids

(Froehlich, 2001; Koeduka et al., 2003), and thus possibly supplied by products from the overexpressed LOX2.2 to produce GLVs. Analysis of volatiles

58 showed however no differences in the amounts or composition between uninfested or BCA-infested control and OeLOX2.2 line. LOX-derived volatiles however, especially (Z)-3-hexen-1-ol, were reduced after aphid infestation in the control line but not in the overexpressing line (Table 1 in Paper III). This is possibly due to LOX2.2 not being involved in production of GLVs, or to the fact that the amounts of other volatiles were at low concentrations and not detected in our analysis. We cannot exclude the possibility that we missed time points with differences in volatile composition. For a more comprehen- sive study, it would be interesting to include also the antisense lines and more time points, possibly within hours of the start of aphid infestation. It was shown in maize that the most pronounced changes in transcriptome and metabolome occur in the first few hours of R. maidis feeding. After four days, gene expression and metabolomic changes caused by aphid infestation were again similar to those of control plants (Tzin et al., 2015). In view of the fact that both MeJA and MeSA treatments were effective against aphids during daylight (Glinwood et al., 2007) and that HPL expression shows upregulation during the day in barley (unpublished results), it would also be of interest to use more plants and compare samples collected during the light period only.

In Paper IV, we studied the effects of volatile treatments on plants. There were no effects of MeSA, (Z)-3-hexen-1-ol or low concentrations (45 μM in

70% ethanol, 25 μl on filter paper in each cage) of MeJA on the plant phenotype, however high concentrations (440 μM in 99% ethanol, 20 μl on filter paper in each cage) of MeJA during life span tests with daily treatments caused obvious changes in plant metabolism resulting in stunted growth (Figure 5 in

Paper IV). Reduction in plant fitness could be expected however, since constant induction of defense is a cost for the plant. MeJA treatments of tomato

(Solanum lycopersicum cv. Micro-Tom) seeds were shown previously to induce defenses against tomato fruit worm (Helicoverpa zea Boddie) larvae, however

59 a reduction in seedling germination was observed with higher (0.2, 0.4, 1 mM) doses of MeJA (Paudel et al., 2014).

Effects of transformation or treatments on aphid performance

CI2c overexpression

Effects on GPA

The no-choice test showed no difference between control and transgenic

Arabidopsis in the proportion of aphids settled (Figure 2 in Paper I). Results from choice tests showed a significant difference in aphid settling in one com- bination: with control and CI2c p:7 bolting plants (Figure 3 in Paper I). The results from short term fecundity tests showed lower numbers of aphids on both CI2c transgenic lines and on psCI2c p:3, as compared to on control plants

(Figure 4 in Paper I). More detailed investigation of the time scale of this effect was studied over a period of eight days with control and CI2c p:8 line. The difference in number of nymphs between the two lines was significant at day

5-7 (Figure 8).

Figure 9. Numbers of GPA adults and nymphs with time in choice tests. White bars represent control plants and black bars transgenic plants. Ten adult apterous GPAs were placed within small cages on control and CI2c 6- 4 plants in the same pot. After 24 h, the cages were opened and the aphids were free to move. Adults and nymphs were counted on a subset of plants each day after the release. Bars show average numbers ± SE. Dotted lines indicate trends. Asterisks indicate significant differences between control and the overexpressing CI2c line for a certain day at p≤0.05 (Wilcoxon matched pair test). n=6, except for day 5 where n=9.

Effects on BCA

The results from a fecundity test, where aphids were feeding on either control or CI2c-overexpressing barley growing in separate pots showed no differences in aphid numbers. Similarly, there were no negative effects of CI2c overexpression on aphid settling in the prolonged five-day choice test (Figure 2 in

Paper II) or life span (Figure S3 in Paper II).

With regard to defense against aphids, it has been shown that PIs can inhibit proteases in aphid saliva or gut during probing, feeding establishment and digestion (Carolan et al., 2009; Carolan et al., 2011; Pyati et al., 2011; Furch et al., 2015; van Bel and Will, 2016), causing reduction in their growth and reproduction (Ryan, 1990). The negative effect on aphid biology was tested both in transgenic plants (Rahbé et al., 2003; Ribeiro et al., 2006; Alvarez-

Alfageme et al., 2011; Carrillo et al., 2011b) and in artificial diets (Tran et al.,

1997; Azzouz et al., 2005; Pyati et al., 2011). Whether the PIs affect aphids by

62 binding to digestive proteases is still under investigation, but evidence exists for the presence of digestive proteases in aphid’s gut, of the cysteine and serine type, which are inhibited by plant PIs (Pyati et al., 2011; Carrillo et al.,

2011b). It is unlikely that CI2c targets aphid’s digestive proteases, since we did not observe any long-term negative effects on aphid biology. It is possible that the inhibitor act on functions related to aphid reproduction or interaction with bacterial symbionts. This idea was suggested by Rahbé et al. (2003), who demonstrated that protease inhibitor oryzacystatin OC-I expressed in oilseed rape (Brassica napus L.) and fed to GPA was localized within the bacteriocytes of the aphids (Rahbé et al., 2003). Another possibility is that CI2c regulates function of plant endogenous proteases involved in plant stress responses

(Solomon et al., 1999; Luo et al., 2009).

LOX2.2 overexpression and down-regulation

LOX2.2-overexpressing plants supported significantly lower BCA and

GPA numbers, and antiLOX2.2 plants higher aphid numbers, when compared to respective controls (Figure 10).

Figure 10. Aphid numbers on control and transgenic lines, five days after infestation with twenty adult apterous aphids. (a) Numbers of bird cherry- oat aphid (BCA), (b) numbers of green peach aphid (GPA). White and black bars indicate, respectively, control and transgenic lines. Bars indicate the average (±SE). n = 12 for BCA and n = 22 for GPA. Asterisks indicate significant differences after aphid infestation between control and transgenic plants (t-test, * p ≤ 0.05; ** p ≤ 0.01)

63 Overexpression or down-regulation of LOX2.2 did not affect BCA or GPA settling in no-choice conditions (Figure S3 in Paper III), or the life span param- eters, when control and transgenic plants were compared (Table 2, Paper III).

The results point to a role of LOX2.2 in basic plant defense. The observed effects on aphid fecundity may be caused by proteins, such as thionins, or protein inhibitors, present at higher or lower levels respectively in the over-expressing and antisense LOX2.2 transformants, and targeting aphid metabolism and/or reproductive functions (Delp et al., 2009; Mehrabi et al., 2014).

Volatile treatments

The results from aphid tests showed that treatment with MeJA had a negative effect on BCA fecundity (Figure 11), whereas exposure to MeSA prolonged the pre-reproductive period, as well as decreased the intrinsic population increase rate, without any effect on aphid reproduction (Table 2 in Pa- per IV, Figure 11).

Figure 11. Aphid numbers on control and volatile-treated barley cultivars seven days after infestation with ten adult apterous BCAs. White and black bars indicate respectively control and volatile-exposed plants. Volatiles used were: MeSA (three repetitions with 5-12 plants in each), MeJA (one experiment with indicated umber of plants for each cultivar) and (Z)-3- hexen-1-ol (three repetitions with 7-10 plants in each). Bars indicate means (±SE). Aphid numbers were normalized to average aphid numbers on control plants as 100 (these numbers were for cv. Scandium 82 and 41, for cv. Barke 101 and 183 and for cv. Lina 102 and 100 in the experiment with MeSa and MeJA, respectively and 76 for cv. Lina in the experiment with (Z)-3- hexen-1-ol). Asterisks indicate significant differences after aphid infestation between control and volatile-treated plants (t-test, *p ≤ 0.05)

Treatment with (Z)-3-hexen-1-ol did not affect aphid performance on exposed plants, when compared to controls (Figure 11). Treatment with MeJA affected aphid fecundity on barley cultivars Scandium and Barke, but not on

Lina, which was used as a rearing plant and thus aphids became adapted (Fig- ure 11).

For volatile treatment, single compounds or complex blends may cause different responses in plants and aphids (Glinwood et al., 2007; Webster et al.,

2010; Webster, 2012). Important aspects to consider are the timing and length of the exposure, as well as the concentration of the compound. It was shown

65 that aphid acceptance was negatively affected by exposures to volatiles from undamaged plants of barley cv. Alva or thistle (Cirsium vulgare (Savi) Ten.), only when plants were exposed during darkness, whereas acceptance of plants treated with MeSA or MeJA was significantly reduced only after exposures during daylight. Interestingly, marker genes for treatment with MeSA and MeJA, BCI-4 and AOS2, respectively, were induced both during daylight and darkness (Glinwood et al., 2007). The effect of treatment on pathogen resistance was shown to depend on volatile dose and exposure time. Low concentration of MeSA over 6 h did not reduce Pseudomonas syringae infections in lima bean (Phaseolus lunatus L.), whereas exposure to the same concentration over 24 h, or higher concentration for shorter period, significantly enhanced resistance (Girón-Calva et al., 2012). Analyzing the effects of volatile exposures on aphids, it is important to consider whether the observed effect comes from the volatile itself or from the changes in host plant status. For aphid behavior, this aspect can be tested in an olfactometer, where aphid responses are measured after presenting the insects with cues coming either from inducing volatiles or exposed plants (Glinwood et al., 2004). Further, the issue can be solved by separating the treatments from the bioassays, both spatially and temporary. In our 7-day fecundity tests treated plants were kept in a growth chamber separate from controls. In the case of MeSA and MeJA, the inducing agent was removed, and the plants left overnight before introducing aphids.

After exposure with (Z)-3-hexen-1-ol, the aphids were introduced immedi- ately, since this treatment was shorter than with the above-mentioned compounds, and the effects observed one gene expression were mainly within the first few hours after the beginning of the exposure (Figure 2 in Paper IV).

66 Other effects of transformation or treatments

In Paper II we showed that a number of defense-related genes (for example

CI2c, TPPE and hordolisin), which were upregulated by aphids in control and moderately resistant barley genotypes (Hsp5 and DH28:4), were expressed at the same or lower levels in CI2c-overexpressing plants after aphid infestation

(Figure 12).

67 Figure 12. Transcript abundance in barley leaves with and without BCA. Primary leaves were infested during 48 h with twenty adult apterous BCA. White bars represent uninfested plants and black bars aphid-infested plants (±SE). The transcript abundance was calculated relative to the reference genes: Hsp70 and SF427 and normalized to uninfested control line set as 1.00. Different letters indicate significant differences between the lines (Kruskal-Wallis test, p ≤ 0.05), asterisks indicate significant difference in one genotype with or without aphids (Mann-Whitney test, *p ≤ 0.05, **p ≤ 0.01). Six biological replicates (with two plants each) and three technical replicates.

68 Although CI2c was suppressed in aphid infested samples from transgenic plants, the chymotrypsin inhibitory activity was higher after aphid infestation when compared to un-infested plants, with a significant difference for line

CI2c 6-4 (Fig 5A in Paper II). In addition, the transgenic lines showed higher endogenous proteolytic activity than control or moderately-resistant lines

(Figure 5B in Paper II), possibly due to induction of other, inhibitor-insensitive proteases in transgenic plants. The indirect method to detect CI2c employed in our studies has one weakness however. In the tests we measured the com- bination of the inhibitory activity of CI2c as well as the activity of added chymotrypsin and plant endogenous proteases. This makes it difficult to exactly identify the level of CI2c under conditions where both endogenous protease and CI2c were induced. Thus, although we could clearly confirm the overexpression of CI2c in Arabidopsis and barley, the contribution of CI2c upon aphid infestation in barley was more difficult to compare between the genotypes (Figure 5A in Paper II). These problems can be solved by applying direct methods of protein detection such as Western-blotting or ELISA techniques

(Bass et al., 2017)

In Paper III, LOX2.2 overexpressing lines showed up-regulation of several

JA-regulated genes, such as LOX2.1, AOS1, LOX1 and CI2c (Figure 7). Anti- sense lines showed down-regulation of such genes (LOX2.1, THIO1567 and

CI2c) (Figure 7). Aphid infestation led to further up-regulation of the studied

JA-regulated genes (i.e. LOX2.1, AOS1, AOS2, CI2c), the expression pattern was shown similar for both studied aphid species (Figure 4 and 5 in Paper III).

This suggests that LOX2.2 plays a role in the activation of JA-mediated defense responses in plants.

In the study presented in Paper IV we found genes encoding CI2c, LOX2.2 and BCI-4 induced by exposure to MeSA. The effect was transient, observed at 6, 12 and 24 h after start of the treatment and decreasing with time (Figure

69 1 in Paper IV). Both MeJA and (Z)-3-hexen-1-ol treatments up-regulated

LOX2.1 and AOS2, the pattern of up-regulation by MeJA varied however from the induction observed for (Z)-3-hexen-1-ol, probably due to the different experimental setup (Figures 2 and 3 in Paper IV). The analysis of induction of gene expression by the three volatiles was based on few selected genes, especially in the case of MeJA treatment of Lina, and it would be of importance to complement the study by analyzing additional gene sequences and cultivars, as well as to carry out a careful investigation with regard to exposure time and volatile concentrations.

Differences in aphid performance

Between aphid species: BCA and GPA on barley

BCA and GPA performance on barley was studied in Papers II and III.

BCA, which is a specialist on grasses, did not show any type of avoidance or restlessness while settling on barley in no-choice (Figure S3 in Paper III) and choice conditions (Figure 2 in Paper II). In the choice tests, BCAs were released on a filter paper placed between two plants (Figure S1 in Paper II). GPA did not settle on barley when non-confined and thus aphids were added within a small cage mounted on the second leaf of each of a control and transgenic plant in the same pot (Figure S1 in Paper II). Only when forced to stay on plants, did the aphids stay, establish feeding and produced nymphs, with numbers in the same order of magnitude as in the case of BCA (60 vs 100)

(Paper II). However, when confined in small cages mounted around the base part of the plant, GPA did not survive on barley, whereas BCA performed equally well on the base as on the leaf blade (Figure 13). This indicates that compounds or macromolecules with antibiosis activities that provide resistance against GPA are present at higher relative levels in the base of barley

70 plants. In the choice tests with aphids, additional tests with alataes would be of value since it is known that the winged migrants respond differently to olfaction cues than do apterous forms (Webster, 2012), and are responsible for deciding whether to leave or land on plants, whereas wingless females are mainly responsible for sustaining colony growth.

20 Number of aphids Number of 10

0 GPA BCA

Figure 13. Numbers of GPAs and BCAs five days after application of ten adult aphids in the cage mounted at the base part of the plant. Error bars indicate SE, n=8.

During long-term life span tests, GPA showed a delayed onset of reproduction and lower fecundity, compared to BCA (Table 2 in Papers II and III), however other aspects of aphid life history (length of reproduction and life span) were comparable between the two species (Table 2 and Figure S3 in Pa- per II, Table 2 in Paper III).

The production of nymphs per day had a broad optimum and declined with time for BCA, whereas for GPA it was low throughout the reproductive life, which suggests that barley, although it secures survival, is a poor host for

GPA (Figure S3 in Paper II).

71 Between host plants: GPA on Arabidopsis and barley

GPA performed better on Arabidopsis when compared to barley. The pre- reproductive period on Arabidopsis was shorter, 8.7 ± 0.2 (SE) on control plants (Table 1 in Paper I), when compared to 12.0 ± 0.6 (SE) on control barley

(Table 2 in Paper III). The number of nymphs per individual or per reproductive day, as well as the intrinsic rate of population increase were higher on

Arabidopsis, when compared to aphids feeding on barley. The reproductive life and life span were also longer for GPA on Arabidopsis than barley (Table

1 in Paper I vs Table 2 in Paper II and III). Some similarities between the two hosts were however observed in 5-day fecundity tests. In non-confined conditions on Arabidopsis aphid numbers reached 55 ± 4.1 (Figure 4 in Paper I).

For barley, in experiments starting with twenty adult aphids, total aphid numbers after five days in confined and non-confined conditions were 60 ± 5.2 (SE)

(Paper II) and 32.2 ± 4.3 (SE), respectively (Figure 10). Those results suggest that GPA avoids using barley as a host when an opportunity to escape exists.

Aphids survive and reproduce when forced to stay on the plants, which indicates that factors encountered during penetration rather than the phloem composition are responsible for the observed avoidance (Figure 2 in Paper II;

Figure 9). Another aspect when analyzing aphid responses to host plants is the selection of suitable rearing plant for aphid colonies. It was shown that

GPAs reared on potato did not survive to reproduce when added to barley, but survived when reared on barley (Davis and Radcliffe, 2008), or as in our studies, on kohlrabi (Paper II and III).

72 Conclusions

This thesis provides new knowledge important to explain plant-aphid interactions, by characterisation of plant resistance genes against aphids, as well as explanation of induced plant defense responses. The main results can be summarized as follows:

1. The barley gene CI2c when expressed in Arabidopsis resulted in a

gene product that was taken up by GPA from the tissue during

probing and transiently reduced GPA fecundity on transgenic Ar-

abidopsis. CI2c overexpression in barley did not affect the special-

ist BCA, but decreased avoidance of the specialist GPA due to sec-

ondary effects on defense induction.

2. LOX2.2 was shown to be involved in activation of other JA-regu-

lated genes related to defense. Overexpression of LOX2.2 de-

creased BCA and GPA performance on barley, suggesting a role of

the gene in basic plant defense, most likely due to increased syn-

thesis of JA or GLVs.

3. MeSA, MeJA and (Z)-3-hexen-1-ol treatment caused up-regulation

of defense-related genes. From the three volatiles tested, only the

exposure to MeJA reduced aphid populations. Exposure to high

concentrations of MeJA had negative consequences for plant

growth, but even the stunted plants served as good hosts for the

aphids in life span tests, i.e. the induced defense was only active

during probing and penetration and the phloem composition had

no negative effect on aphid performance.

73 The results presented in the thesis show that products of certain genes, such as CI2c and LOX2.2, can have a negative effect on aphid biology (Papers

I and III), as well as act indirectly by influencing other genes involved in plant defense responses (Papers II and III). The resistant plants used in our studies were shown in the background study to have a moderate resistance (up to

40% reduction in growth), when compared to susceptible cultivars. Knowing this, we could not expect each resistant gene identified in the resistant genotypes to provide a large difference, but rather contribute to the observed effect. Testing gene induction in the resistant plants as well as volatile treatments proved to be a useful approach in finding answers regarding compatible and non-compatible plant-aphid interactions and provided new research questions.

74 Future perspectives

The studies of CI2c indicated that the CI2c protein had a negative in-

fluence on GPA. This aspect could be further explored by purifying

the protein and adding it to artificial diets. There is a limited value of

this in barley breeding, since GPA is not an important pest on this

crop, but it may be of interest for improving other host crops by trans-

genic approaches. The NPBTs would be the methods of choice. It is

not however clear yet whether those techniques or organisms pro-

duced with the use of NPBTs are subjects of the current GMO legisla-

tion, and thus whether they will avoid the problems of GMO ap-

proval.

More detailed analysis of the gene up-regulation by aphids in Papers

II and III including early time points would help to gain knowledge

about time-scale of aphid-induced responses. Knowing which re-

sponses are activated and when would help to detect additional fac-

tors acting in plant-aphid interactions and be useful in finding new

targets for inducing resistance.

The effects of overexpression and silencing of LOX2.2 were analysed

with regard to gene-expression, volatile emissions and aphid perfor-

mance. We have shown that JA/GLV-related induction helps to pro-

tect against both BCA and GPA, however whether this level of re-

sistance will be sufficient to protect against aphids in the field would

need further investigation. Assuming that lower numbers of aphids

would be observed also in the field, one could expect decreasing num-

ber of offspring in each generation and fewer occasions where

75 thresholds for insecticide treatments would be reached. The next step

would thus be the evaluation of the LOX2.2-transgenic plants in field

trials.

Analysis of volatiles would benefit from testing plants infested with

aphids for different time periods as well as including the LOX2.2 an-

tisense line into the investigation. An important question left unan-

swered in is whether GLVs or JA are inducing the other JA-regulated

genes. An experiment designed to answer this question would be to

carry out tests on transgenic plants with knocked-out HPL gene and

hindered production of GLVs.

The effects of volatile treatments were studied on only a few selected

barley cultivars. To get a more detailed information of induced plant

defense responses, it would be of importance to include more de-

fense-related gene sequences in the analysis, extend the study by in-

cluding more cultivars and carry out a careful investigation with re-

gard to exposure time and volatile concentrations. This would help to

characterize the inducible factors acting in plant-aphid interaction

and assess how they depend on the strength (concentration, time of

exposure) of the inducing agent. Applications of volatiles could be a

way forward to less insecticide use, however there is still a gap in our

understanding of their action on a large scale in the field, where many

other factors are present, such as additional biotic and abiotic interac-

tions.

Our suggestion of barley as a non-host for GPA needs further inves-

tigation. To provide more knowledge about this interaction, one

could study plant responses in compatible and incompatible interac-

tions on molecular level on a broader scale (microarray, RNA se-

quencing) and with regard to the responses activated in different

76 plant tissues. Non-host resistance is often described as multi-layered

and it would be of interest to answer questions on the localisation of

the resistance, for example by monitoring aphid feeding behaviour

using EPG.

In Paper IV it has been suggested that the results from aphid experi-

ments depend on the possibility for aphid adaptation. This aspect is

not well studied in plant-aphid interactions and it would be interest-

ing to test for such possibilities. This could be done for example by

rearing aphids on different cultivars prior to experiments. Addition-

ally, this approach could provide additional insights to the barley-

GPA interaction, by analysing the possibility of GPA to adapt to feed-

ing on barley cultivars over several generations.

78 Acknowledgements

I would like to take this opportunity and thank everyone who made this

PhD thesis possible.

Firstly, I would like to express my endless gratitude to my supervisor Pro- fessor Lisbeth Jonsson. Thank you for giving me the opportunity to work with you and for sharing your knowledge. I am grateful for your guidance, pa- tience and constant support. You have helped me to develop as a scientist and as a person.

I am thankful to my co-supervisors, Docents Ulla Rasmussen and Robert

Glinwood for their assistance and encouragement during my studies and support with writing the thesis. I am obliged to Professor Katharina Pawlowski and Docent Edouard Pesquet for careful revision of my thesis, valuable advice and constructive criticism.

I would like to express my appreciation to Professor Inger Åhman for help- ing with barley cultivation and collection of seeds used in my experiments.

Many thanks to the previous group members, to Lisa Beste for her scientific input, to Sara Mehrabi and Professor Afaf Hamada for sharing their experi- ence and making the “lab time” a “nice time”. I am thankful to Anneli Björk for all her ideas and incredible involvement in the experimental work during her Master’s thesis project. I wish to acknowledge all students involved in developing and carrying out tests with aphids, especially François, Thomas, Ma- rie and Cyrielle. It was a pleasure to work with you in the lab.

I want to say thank you to my Uni friends, present and past. To Marco,

Eric, Sandra, Lina, Denis, Paul and Van. To my office neighbours from

79 Ecology, especially to Pil and Beate, the pub-party girls. Thank you all for your friendship and kindness.

I am grateful to my parents for all the effort they put in my development and education, to my sisters, Kasia, Marta and Magdalena, for their interest in my work, and to Elżbieta and Jens Losvik, for always believing in me.

Dziękuję!

And lastly, I would like to thank my dearest husband Johan. Without you,

I would never accomplish this much. Thank you for making me brave.

To all people who made it possible for me to be where I am in my life,

Thank you!

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