Induction of Indirect Plant Defense in the Context of Multiple Herbivory

Induction of indirect plant defense in the context of multiple herbivory

Gene transcription, volatile emission, and predator behavior

Tila R. Menzel Thesis committee

Promotors Prof. Dr M. Dicke Professor of Entomology Wageningen University

Prof. Dr J.J.A. van Loon Personal chair at the Laboratory of Entomology Wageningen University

Other members Prof. Dr R.G.F. Visser, Wageningen University Dr M.A. Jongsma, Wageningen University Dr R.C. Schuurink, University of Amsterdam Dr S.C.M. van Wees, Utrecht University

This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences. Induction of indirect plant defense in the context of multiple herbivory

Gene transcription, volatile emission and predator behavior

Tila R. Menzel

Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday19 November 2014 at 11 a.m. in the Aula. Tila R. Menzel Induction of indirect plant defense in the context of multiple herbivory: Gene transcription, volatile emission, and predator behavior 158 pages.

PhD thesis, Wageningen University, Wageningen, NL (2014) With references, with summaries in Dutch and English

ISBN 978-94-6257-129-7

Abstract

Plants live in complex environments and are under constant threat of being attacked by herbivorous arthropods. Consequently plants possess an arsenal of sophisticated mechanisms in order to defend themselves against their ubiquitous attackers. Induced indirect defenses involve the attraction of natural enemies of herbivores, such as predators and parasitoids. Predators and parasitoids use odors emitted by damaged plants that serve as a “cry for help” to find their respective prey or host herbivore. The aim of this thesis was to use a multidisciplinary approach, with focus on molecular and chemical methods, combined with behavioral investigations, to elucidate the mechanisms of plant responses to multiple herbivory that affect a tritrophic system consisting of a plant, an herbivore and a natural enemy.

Induced plant defenses are regulated by a network of defense signaling pathways in which phytohormones act as signaling molecules. Accordingly, simulation of herbivory by exogenous application of phytohormones and actual herbivory by the two-spotted spider mite Tetranychus urticae affected transcript levels of a defense gene involved in indirect defense in Lima bean. However, two other genes involved in defense were not affected at the time point investigated. Moreover, application of a low dose of JA followed by minor herbivory by T. urticae spider mites affected gene transcript levels and emissions of plant volatiles commonly associated with herbivory. Only endogenous phytohormone levels of jasmonic acid (JA), but not salicylic acid (SA), were affected by treatments. Nevertheless, the low-dose JA application resulted in a synergistic effect on gene transcription and an increased emission of a volatile compound involved in indirect defense after herbivore infestation.

Caterpillar feeding as well as application of caterpillar oral secretion on mechanically inflicted wounds are frequently used to induce plant defense against biting-chewing insects, which is JA-related. Feeding damage by two caterpillar species caused mostly identical induction of gene transcription, but combination of mechanical damage and oral secretions of caterpillars caused differential induction of the transcription of defense genes. Nevertheless, gene transcript levels for plants that subsequently experienced an infestation by T. urticae were only different for a gene potentially involved in direct defense of plants that experienced a single event of herbivory by T. urticae. Indirect defense was not affected. Also sequential induction of plant defense by caterpillar oral secretion and an infestation by T. urticae spider mites did not interfere with attraction of the specialist predatory mite P. persimilis in olfactometer assays. The predator did distinguish between plants induced by spider mites and plants induced by the combination of mechanical damage and caterpillar oral secretion but not between plants with single spider mite infestation and plants induced by caterpillar oral secretion prior to spider mite infestation. The composition of the volatile blends emitted by plants induced by spider mites only or by the sequential induction treatment of caterpillar oral secretion followed by spider mite infestation were similar. Consequently, the induction of plant indirect defense as applied in these experiments was not affected by previous treatment with oral secretion of caterpillars. Moreover, herbivory by conspecific T. urticae mites did not affect gene transcript

vii levels or emission of volatiles of plants that experienced two bouts of herbivore attack by conspecific spider mites compared to plants that experienced only one bout of spider mite attack. This suggests that Lima bean plants do no increase defense in response to sequential herbivory by T. urticae.

In conclusion, using a multidisciplinary approach new insights were obtained in the mechanisms of induction of indirect plant defense and tritrophic interactions in a multiple herbivore context, providing helpful leads for future research on plant responses to multiple stresses.

viii ix x Table of contents

Abstract ...... vii Chapter 1...... 1 General Introduction

Chapter 2...... 21 Defense gene induction in Lima bean in response to phytohormone application and spider-mite feeding

Chapter 3...... 33 Synergism in the effect of prior jasmonic acid application on herbivore-induced volatile emission by Lima bean plants: transcription of a monoterpene synthase gene and volatile emission

Chapter 4...... 55 Transcriptional responses of Lima bean defense genes to feeding or oral secretions of Mamestra brassicae L. and Spodoptera exigua Hübner caterpillars

Chapter 5...... 71 Effect of sequential induction byMamestra brassicae L. and Tetranychus urticae Koch on Lima bean plant indirect defense

Chapter 6...... 89 Effect of sequential herbivory by conspecific herbivorous spider mites on gene transcription and volatile emission in Lima bean plants

Chapter 7...... 105 General discussion

Summary...... 119 Samenvatting...... 123 Acknowledgements...... 129 Curriculum vitae...... 133 List of publications...... 135 Education statment...... 136 Supplemental data...... 138

Chapter 1 General Introduction Tila R. Menzel

1 Chapter 1

General Introduction

The green leaf material of plants is exploited as a source of nutrients by other organisms. Arthropods, including insects, are the most species-rich group of organisms with an estimated six million species, of which approximately 50% are herbivorous (Schoonhoven et al., 2005). Plants and insects co-exist since the Devonian Era from 416 million years ago to 359 million years ago (Labandeira, 2007). Despite extensive tissue damage and complete defoliation at times, plants are evidently able to persist and thrive. Their inability to escape their ubiquitous herbivorous attackers requires plants to rely on a combination of constitutive and induced defenses to protect them against insect herbivores (Schoonhoven et al., 2005). Constitutive defenses comprise defensive structure such as thorns, waxy layers on plant tissues, and trichomes, and often provide a first line of defense. Induced defenses are a second line of defense, and are a highly plastic trait allowing plants to change their phenotype according to external stimuli in their environment that indicate the presence of herbivores, including the touch by an attacker (Bown et al., 2002). Induced defenses include changes in morphology or synthesis of defense-associated proteins and secondary metabolites. The latter include for instance toxic compounds, proteinase inhibitors that interfere with digestion of plant material, and volatile organic compounds that repel herbivores or attract the natural enemies of herbivores (Mithöfer and Boland, 2012). Since defenses come at metabolic costs and ecological costs, plants have developed a sophisticated network of regulatory mechanisms to optimize their defense for maximum selectivity and efficiency (Karbanet al., 1997; Baldwin, 1998; Heil and Baldwin, 2002; Strauss et al., 2002).

Early signaling steps in herbivore recognition During the past years, advances in molecular, genetic, and biochemical methods have made it possible to identify some of the key players in signaling pathways leading to plant defense responses. The first step in the activation of any type of induced plant defense involves the sensing of the invader and recognition of an attack. Plants possess a complex sensory system which responds to mere touch by a herbivore, deposition of non-feeding stages such as eggs, and tissue damage caused and insect-derived elicitors introduced by feeding herbivores (reviewed in Hilker and Meiners, 2010). Cell membrane depolarization and increase in cytosolic Ca2+ which occurs within seconds, is an early event in defense signaling and appears to be a master regulator required for many subsequent signaling steps (Fig. 1) (Scheel, 1998; Maffei et al., 2007). Next to Ca2+ and H+ influx, Cl– efflux, membrane depolarization and production of reactive oxygen species (ROS), such as H2O2 and nitric oxide (NO), also belong to the early events in generating plant defense (Levine et al., 1994; Pugin, 1997; Blume et al., 2000). Moreover, ROS, which accumulate in local and systemic leaves of attacked plants act thereby not only as signaling molecules but also as defense mechanism (Orozco-Cárdenas et al., 2001; Foyer and Noctor, 2005).

2 Chapter 1 , 3 et al. , 2007; Pieterse , et al. et General Introduction General , 2006; Browse, 2009; Pieterse , 2012). Chemically diverse compounds et al. et al. (2007). Abbreviations: Membrane potential (Vm), jasmonic acid (JA), (JA), acid jasmonic (Vm), potential Membrane (2007). Abbreviations: et al. , 2012). Fig. 1. Overview of events involved in the generation of an induced plant defense response in chronological in chronological response defense induced an plant of Fig. 1. Overview in the generation involved events of Maffei Modifiedafter order. speciessalicylic (ROS) oxygen (SA), reactive acid to This stress. so-called phytohormone crosstalk allows plants to regulate and fine-tune their the two major defense hormones (Van Loon the two major defense hormones (Van Plant hormones, also known as phytohormones, act as central players in activating the actual with a possible defensive function are produced by plants and belong to various chemical pathways induced can determine whether plant tissues become more susceptible or resistant resistant pathways induced can determine or whether plant tissues become more susceptible pathways can be differentially activated, whereby composition and timing of the signaling activated, whereby composition and timing of the signaling pathways can be differentially molecular recognition events. The two phytohormones jasmonic acid (JA) and salicylic acid phytohormones jasmonic acid (JA) The two molecular recognition events. plant defense signaling network leading of to synthesis of defensive metabolites downstream such as ethylene function rather as modulators (ET), of defense responses mediated by of plant hormones, as well as (a)biotic stresses, and pathogens (Maffei and pathogens (Maffei (a)biotic stresses, well as hormones, as plant of defense genes and production of defensive chemical compounds (Reymond and Farmer, defense genes and production of defensive chemical compounds (Reymond and Farmer, defense responses in an attacker-specific manner in terms of activation of specific sets of classes. The main chemical classes include for instance terpenoids, N-containing alkaloids, classes. cellular responses, and MAPK cascades are important pathways that play a role in signaling Phytohormone signaling and defense activation defense and signaling Phytohormone Subsequently, Mitogen-activated protein kinases (MAPK) transfer information from sensors to sensors from information transfer (MAPK) kinases protein Mitogen-activated Subsequently, 2012). Depending on the identity of an attacker, antagonistic and synergistic phytohormone antagonistic and synergistic an attacker, 2012). Depending of identity on the (SA) are widely recognized as the major defense hormones, whereas other phytohormones, major defense hormones, whereas other phytohormones, the widely recognized as are (SA) et al. 1998; Koornneef and Pieterse, 2008; Thaler 1998; Koornneef and Pieterse, 2008; Chapter 1 and phenolic compounds including flavonoids (Mithöfer and Boland, 2012). However, the majority of defense compounds are terpenoids, which comprise a large and diverse class of organic compounds, of which some 30,000 molecular structures have been identified and characterized. Whereas terpenoids are produced ubiquitously among plants, some defense compounds are produced only by certain plant taxa. Many defense compounds need to be stored in specialized compartments, such as vacuoles or the apoplast in order to avoid autotoxicity. The general mode of action of defensive compounds includes membrane disruption, inhibition of nutrient and ion transport, inhibition of signal-transduction processes, inhibition of metabolism, or disruption of hormonal control of physiological processes (Wittstock and Gershenzon, 2002; Mumm and Hilker, 2006; Mithöfer and Boland, 2012). However, not all chemical compounds involved in plant defense affect herbivorous attackers directly, and are thus part of the so-called plant indirect defense.

Plant indirect defense and involved compounds Some volatile organic compounds are released by plants and attract natural enemies of herbivores, such as predators and parasitoids. Since herbivorous arthropods are often small organims compared to the host plant they consume and try to avoid betraying their presence, their natural enemies often rely on chemical information provided by plants, such as volatile compounds, to serve as reliable and detectable host-location cues. Dicke and colleagues (Dicke and Sabelis, 1988; Dicke et al., 1990) were the first to provide evidence for an active release of VOCs in response to herbivory in order to attract natural enemies. Since then the actual complexity of these tritrophic interactions between plant, herbivore, and natural enemy has been the subject of many studies. Volatile blends released after herbivory may consist of up to 200 VOCs, also known as herbivore-induced plants volatiles (HIPVs), which can be roughly divided into 3 groups, namely terpenoids, fatty acid derivatives and phenylpropanoids or benzenoids (Fig. 2) (Mumm and Dicke, 2010).

The key players among terpenoid volatiles consist of monoterpenes (C10), sesquiterpenes (C15), and homoterpenes (C11 or C16), which all significantly contribute to any blend of plant-derived volatiles. All terpenoids are synthesized via one of two pathways, known as the cytosolic mevalonate (MVA) pathway and the methylerythritol 4-phosphate (MEP) pathway (Chappell, 1995; Aharoni et al., 2005; Cheng et al., 2007). Antagonistic or synergistic crosstalk between JA, SA, and ET signaling pathways has been suggested to regulate the characteristic blend of terpenoids in response to herbivory (Ozawa et al., 2000; Engelberth et al., 2001; Horiuchi et al., 2001; Schmelz et al., 2003). Moreover, it has been shown that terpenoids can be attractive to natural enemies themselves or act synergistically with other plant volatiles or herbivore pheromones (Dicke et al., 1990; Erbilgin and Raffa, 2001; Pettersson, 2001; De Boer and Dicke, 2004; Mumm and Hilker, 2005).

Volatile fatty acid derivatives are often associated with the green leaf odor emitted after tissue damage and are also known as green leaf volatiles (GLVs). GLVs originate from C18 unsaturated fatty acids, such as linoleic acid and linolenic acid, and are synthesized via the octadecanoid pathway through which also the phytohormone JA is produced.

4 Chapter 1 5 General Introduction General Fig. 2. Representative compounds of the three major classes of compounds found in the headspace found compounds classes of the three major of compounds Fig. 2. Representative (2010). Dicke and Mumm from Adapted egg or herbivory induced deposition. by plants of Chapter 1

In plant indirect defense they act as attractants for natural enemies of herbivores or take part as volatile signal involved in systemic induction or priming of changes in plant phenotype (Reddy, 2002; Shiojiri et al., 2006; Frost et al., 2007; Heil and Bueno, 2007; Van Wijk et al., 2008; Wei and Kang, 2011).

Phenylpropanoids and benzenoids originate from the amino acid L-phenylalanine, but comparatively little is known about the detailed biosynthesis of these compounds (Dudareva et al., 2006). The benzenoid ester methyl salicylate (MeSA) is the best studied of this class of compounds because it is frequently emitted by herbivore-infested plants and functions in plant indirect defense (Dicke et al., 1990; Van Poecke and Dicke, 2002; James, 2003; Ament et al., 2004; De Boer et al., 2004).

Generally, plant volatiles, including the major classes of HIPVs, are low-molecular-weight compounds (below 300 Da) that are lipophilic liquids with high vapor pressure (Pichersky et al., 2006). This way they are able to cross membranes freely to be released into the atmosphere or soil. Thereby they are not only released from attacked sites, but also from systemic tissues, and can be perceived by herbivores, natural enemies of herbivores, and neighboring plants. Herbivores may be attracted or repelled depending on the blend and neighboring plants may respond to the information by entering a state of increased readiness for a herbivore attack which is called the “primed state” (Bruin et al., 1992; Bolter et al., 1997; Arimura et al., 2000; Kalberer et al., 2001; Karban et al., 2003; Conrath, 2009). Composition of volatile blends is influenced by underlying defense pathways induced by a herbivore, and a certain blend can convey information to other members of the surrounding ecological community. Information contained in a volatile blend includes, for instance, information on plant species, herbivore species, density, and developmental stage (Takabayashi et al., 1995; De Moraes et al., 1998; Gols et al., 2003).

Specific activation of defense signaling pathways seems to be important in transmitting all this information. Generally, plant defense responses to wounding, biting-chewing herbivores, and certain cell-content feeding mites, are regulated by the JA signaling pathway (McConn et al., 1997; Ament et al., 2004). In contrast, plant defense responses against piercing-sucking herbivores and the generation of systemic acquired resistance (SAR) are regulated by the SA signaling pathway (Conrath et al., 2001; Kempema et al., 2007). However, cross-talk between the JA and SA signaling pathways, may take the form of antagonism and occur via MAPKs, transcription factors, or phytohormones, and thus allow for fine-tuning of plant defense responses against specific herbivores (Pieterse and Dicke, 2007; Thaleret al., 2012; Stam et al., 2014). Even though JA, SA, and ET are evidently important signaling molecules, additional layers of regulation seem to shape the outcome of defense responses. Yet, unidentified regulatory factors or cross-talk between the signaling pathways and the resulting differences in quantity, composition and timing of phytohormones might act in fine-tuning of plant defense responses and activate distinct sets of defense-related genes (De Vos et al., 2005). Herbivore-associated elicitors (HAE) are likely to be involved in increasing specificity of plant defense responses (Bonaventure et al., 2011). Exposure of plants to the combination of artificial wounding and HAE, as present for example in caterpillar oral secretions, has

6 Chapter 1 7 ,

plants) plants)

General Introduction General

ground ground ground - Neighboring Neighboring (( herbivores

Above Predators

Pollinators

Hyperparasitoids ground - et al., 1999; Schmelz et al., et al., 1997; Bergey 2006; Alborn

Parasitoids

herbivores

Below Plants level trophic level trophic

Second Second level trophic Third Fourth Fourth

First trophic level trophic First et al., 1995; Alborn (2011) showed that even herbivores that even herbivores showed et al. (2011) Bidart-Bouzat et al., 1990). Moreover, www.bugsinthepicture.com Fig. 3. Simplifi ed overview of the members of the community associated with a plant. Adapted Adapted overview ed associated plant. a with the of community members the of Simplifi 3. Fig. Fatouros Nina and Smid Hans of courtesy are (2014). Insect et al. pictures Stam from transcription, and defense metabolite induction. The HAE identifi ed to date comprise a large transcription, and defense metabolite induction. The HAE identifi herbivores and thus comprise traits that are not easily lost over evolutionary time, despite the comprise traits that are not easily lost over herbivores and thus been shown to induce defense responses that closely resemble herbivore feeding in plants in feeding herbivore resemble closely that responses defense induce to shown been herbivore feeding are thus able to affect the other members of the community directly or directly community the of members other the affect to able thus are feeding herbivore have been extensively studied for many plant-herbivore and tritrophic interactions. However, However, interactions. tritrophic and plant-herbivore many for studied extensively been have indirectly. in their natural environment, plants live in complex communities consisting of neighboring plants live in complex communities in their natural environment, Mattiacci Plant defense responses to a single biotic stress, such as attack by a single herbivore species, to a single biotic stress, such as attack Plant defense responses with the same feeding mode can induce species-specifi c defense responses in terms of gene of terms in responses defense c species-specifi induce can mode feeding same the with plants and various arthropods from different trophic levels (Fig. 3). Changes caused in a in caused Changes (Fig. 3). trophic levels different from various arthropods plants and the underlying transcriptomic changes induced by plant phenotype by phytohormones and disadvantages they pose (Yoshinaga et al., 2008). pose (Yoshinaga disadvantages they acids, fragments of cell walls, and peptides from digested plant proteins (Doares et al., 1995; cell walls, and peptides from digested plant acids, fragments of variety of molecules, including ed enzymes, forms modifi of lipids, sulphur-containing fatty (Turlings (Turlings Multiple herbivores and plant defense plant and herbivores Multiple , 2007). It has been suggested that HAE are essential for growth and development of suggested that HAE are essential for et al., 2007). It has been Chapter 1

Indeed several studies have shown that herbivory by one species can affect the performance of a second herbivore species (e.g. Van Zandt and Agrawal, 2004; Viswanathan et al., 2007; Poelman et al., 2008; Erb et al., 2011). Moreover, multiple herbivory can cause temporal changes in defense phytohormone signaling and the underlying gene regulatory network that subsequently lead to distinct defense responses. Some of these latter defense responses have shown to be in fact different than the combined response to either individual attacker (De Boer et al., 2008; Rodriguez-Saona et al., 2010). Higher trophic levels such as natural enemies of herbivores can be affected by phenotypic changes caused by interactions of multiple herbivores as well (Dicke et al., 2009). Generally, herbivores which induce the same defense signaling pathways seem to increase the level of plant defense, including the attraction of natural enemies (De Boer et al., 2008). In contrast, herbivores which induce antagonistic signaling pathways seem to interfere with plant indirect defenses (Zhang et al., 2009). However, the effect of multiple herbivores on plant defense also depends on factors such as length of the time interval after which a second herbivore species attacks the same plant, on the sequence of attack and herbivore densities. Time intervals are important because plants are able to form a sort of memory, also known as “priming”, in response to herbivory, which can lead to increased defenses against subsequent herbivores (Frost et al., 2008). Even plants that have not experienced herbivory themselves, have been shown to increase direct and indirect defenses against subsequent herbivores in response to exposure to HIPVs from attacked neighboring plants (Kost and Heil, 2006; Yi et al., 2009; Peng et al., 2011). It is still unclear how this mechanism of memory formation works, but involvement of phytohormones and accumulation of signaling proteins in their inactive form have been proposed (Conrath et al., 2001; Beckers et al., 2009).

Studies that probe the dynamics underlying regulatory modules of plant defense, including phytohormones and gene transcription, are necessary for a better understanding of how plants deal with complex interactions within their associated communities. The objective of this thesis is to study the underlying mechanisms of plant indirect defense in response to damage by different herbivores and multiple herbivory. The following research questions describe three existing gaps in our knowledge about tritrophic interactions in a multiple- herbivore environment, which will be addressed in this thesis:

Question I: Can plant indirect defense genes and metabolites be primed with low doses of phytohormones?

Question II: Does minor herbivory change or prime plant genes and metabolites for enhanced induction of indirect defense by subsequent herbivory?

Question III: Do herbivores that differ in feeding mode differentially influence plant indirect defense in terms of gene transcription and metabolites?

T.u Chapter 1

9 gene (predatory mite) (predatory

spin mite, mite, spin (caterpillar) (

et al., 2004; Reddall General Introduction General

persimilis urticae

, predator colonies urticae, T.

brassicae Phytoseiulus persimilis : Synthase (OS) Synthase plant 4 gene 4 gene genes

- MamestraTetranychus Phytoseiulus Ocimene PR mite may consume up to 30 eggs per day (Sabelis, mite may consume up to 30 eggs per    Two   Lima bean Lima herbivores: Two Predator: One Phaseolus lunatus , 1975). The mites use their needle-like The mites use stylets to feed on the cell contents et al., 1975). females approximately 0.5 approximately females persimilis P. for 0.4 mm and is approximately females , 1975). In the absence of natural enemies or other control measures, the quick the measures, control other or enemies natural of absence the In 1975). al., et Tetranychus urticae Tetranychus Fig. 4. Th ree species involved in the principal tritrophic interaction studied in this thesis interaction ree species principaltritrophic in the involved 4. Th Fig. T. size of Average www.bugsinthepicture.com). Smid, Hans of courtesy pictures (mite urticae mm. (Fig. 4). This (Fig. 4). urticae is successfully used worldwide as biological control agent against T. is distributed globally and is able to feed on a wide variety of over 960 different plant species 960 different over feed on a wide variety of and is able to is distributed globally from egg to adult is temperature dependent may take less than a week under favorable and up to 2 - 3 eggs per day on bean plants and have a lifespan of up to 30 days. The rate of up to 2 - 3 eggs per day on bean plants and have a lifespan of up to 30 days. population growth of spider mites results in rapid overexploitation of the host. Afterwards the population growth of spider mites results in rapid overexploitation of the host. of silk. mites disperse through the air using strands photosynthetic capability photosynthetic capability changes of plants, and causes in essential physiological processes mites was used as the basis for studying the research questions of this thesis (Fig. 4). questions of this studying the research used as the basis for mites was Study system Study such as transpiration, such as transpiration, stomatal conductance and respiration (Landeros P. persimilis One P. on eggs of their prey. of plant parenchyma cells by piercing through cell walls. This feeding in decreases results in of plant parenchyma cells by piercing through cell walls. only on passive transport by wind currents for long-range dispersal (Johnson and Croft, 1976, long-range dispersal (Johnson and Croft, currents for windonly on passive transport by development and oviposition are dependent On bean on host plant and temperature. plants, are often founded by a single female, which can lay up to fi ve eggs per day. Developmental time Developmental day. per eggs ve fi to up lay can which female, single a by founded often are conditions. Once a prey populationconditions. Once is eradicated minute winglessphytoseiid predators rely , and prefers to feed , voracious carnivore specializes on spider mites in the genus Tetranychus is a specialist predatory mite originating from South America, which South originating specialist predatory mite from a Phytoseiulus persimilis is (Jeppson (Fig. 4) (Jeppson , 1990). Just as for 1990). Just al., Dicke et De Baan, 1983; (Sabelis and Van , 2004). Fast growing populations single lay female which can often founded by a are 2004). Fast al., et A tritrophic system consisting of Lima bean plants, two-spotted spider mites and predatory spider mites bean plants, two-spotted consisting of Lima tritrophic system A 1981; Hoy, 1982). 1981; Hoy, 1981). The blind predators use chemical cues, including HIPVs, as main prey-locating cues 1981). completes its four-stage development from egg to adult in 13 - 21 days at 22 °C 22 °C at 21 days - adult in 13 to egg from four-stage development completes its urticae T. , the two-spotted spider mite, is a highly polyphagous highly polyphagous that a pest agricultural is spider mite, two-spotted the , urticae Tetranychus Chapter 1

The tritrophic interaction between Lima bean plants, the herbivores and natural enemies has been well studied. It is frequently used as a model system to study induced indirect defenses (e.g. Dicke et al., 1999; Dicke et al., 2003; Horiuchi et al., 2003; Mithöfer et al., 2005; Heil and Bueno, 2007; Mumm et al., 2008). Induced indirect defense by Lima bean plants includes, for example, the secretion of extrafloral nectar (EFN) and the release of HIPVs. Both EFN and HIPVs are an inducible defense mechanism in the sense that their production rate increases in response to herbivory and their response is regulated by the herbivore- induced phytohormone signaling pathways (Hopke et al., 1994; Dicke et al., 1999; Heil et al., 2001). Moreover, both defense mechanisms can be induced by exogenous JA application (Dicke et al., 1999; Heil, 2004). EFN is produced by specialized nectar-producing glands which are located at the base of the petioles and thus physically apart from the flower. They provide a carbohydrate-rich exudate that is used as food by natural enemies of herbivores (Kost and Heil, 2005). In contrast, HIPVs are produced and released locally and systemically from herbivore-damaged sites and provide natural enemies and other plants with valuable information on host or prey presence. Consequently, EFN-related indirect defense in Lima bean has been mostly studied in the context of ants as bodyguards (e.g. Kost and Heil, 2005, 2006; Ballhorn et al., 2014), whereas HIPVs have been investigated for their interaction with P. persimilis (e.g. Dicke et al., 1990; Gols et al., 2003; De Boer and Dicke, 2004; De Boer et al., 2004; De Boer et al., 2008; Zhang et al., 2009) and other plants (e.g. Arimura et al., 2000; Choh et al., 2004; Heil and Bueno, 2007; Muroi et al., 2011).

Herbivory by T. urticae is known to induce de novo synthesis of Lima bean plant volatiles which attract the predator P. persimilis (Dicke et al., 1999). Moreover, neighboring plants have been shown to perceive these volatile signals and can become “primed” and in case of infestation respond with enhanced indirect defense (Choh et al., 2004; Heil and Kost, 2006). Several studies have outlined the crucial role of the JA and SA signaling pathway in shaping indirect defense and the composition of herbivore-specific induced HIPV blends of different herbivores in Lima bean (Ozawa et al., 2000; Zhang et al., 2009; Wei et al., 2014). Dicke et al. (1999) found that exogenous application of JA induces a volatile blend that is attractive to P. persimilis as it closely resembles the T. urticae-induced blend. Moreover, Gols et al. (2003) found that concomitant application of JA and T. urticae potentiated indirect defense. To date it is one of the few tritrophic interactions for which the principal attractive compounds that mediate the interaction with a natural enemy have been identified (Dicke et al., 1990; De Boer and Dicke, 2004; De Boer et al., 2004). Despite the fact that the Lima bean genome has not been sequenced yet, sequences of a limited number of genes potentially involved in Lima bean defense have been identified (Arimura et al., 2000; Arimura et al., 2008). Of particular interest in this context is the information about the Phaseolus lunatus ocimene synthase (PlOS) gene. Ocimene synthase is the enzyme that catalyzes the last dedicated step in the synthesis of (E)-β-ocimene. The monoterpene (E)-β-ocimene is an HIPV produced by many plant species in response to herbivory and has been identified as one of the principal attractants of P. persimilis (Dicke et al., 1990). Other genes that have been identified include pathogenesis-related (PR) genes, such as the β-1,3-glucanase PR-2 and the chitinases PR-3 and PR-4, a lipoxygenase (LOX), which catalyzes an early step in the JA signaling pathway, phenylalanine ammonia-lyase (PAL) in the phenylpropanoid pathway (as part of

10 Chapter 1 et 11 , 2000; Arimura General Introduction General et al. , 2009). Here, I will integrate et al. , 2000; Ozawa , et al. , 2009; Zhang , 2009; Zhang . et al. that were inoculated subsequently. T. urticae T. T. urticae T. , 2008; Ozawa , 2008; Ozawa et al. , 2002; Arimura levels of relevant defense genes, and the resulting blend of volatile metabolites. levels of relevant defense genes, and the thesis. terms of indirect defense against the other chapters to monitor plant defense responses, are affected by phytohormones and affected monitor plant defense responses, are the other chapters to tritrophic interactions through phenotypic changes of the plant. tritrophic interactions the SA signaling signaling pyrophosphate farnesyl and pathway), to isoprene (FPS) related synthetase the SA herbivory. The effects of phytohormone dose and herbivore density were assessed. dose and herbivore density were of phytohormone The effects herbivory. biosynthesis biosynthesis which is the precursor terpenoid of biosynthesis Nevertheless, . of the number indirect defense against molecular, chemical and behavioral approaches to elucidate effects of multiple herbivory on of multiple herbivory chemical and behavioral approaches to elucidate effects molecular, studies aimed at relating indirect defense in terms of behavior, metabolites, metabolites, phytohormones studies aimed at relating indirect defense in terms of behavior, of this introduction. on the gene transcription of several defense genes in Lima bean. Moreover, it explores the it Moreover, several defense genes in Lima bean. gene transcription of on the I thank Marcel Dicke and Joop J.A. van Loon for helpful comments on the previous version helpful comments on the previous van Loon for thank Marcel Dicke and Joop J.A. I In Chapter 3 the question whether a low-dose JA application can induce priming of plant indirect plant of priming induce can application JA low-dose a whether question the 3 Chapter In defense, and thus memory formation, was addressed. This was done in a multidisciplinary This formation, was addressed. defense, and thus memory effects on defense gene induction by a subsequently arriving herbivore. on effects approach that included the quantification of endogenous phytohormone levels, gene transcript gene levels, phytohormone endogenous of quantification the included that approach and genes for this system is quite limited (Arimura (Arimura limited quite is system this for genes and Chapter 6 investigates the effect of conspecific herbivores on plant memory formation in Chapter 5 investigates how defense induction by a heterospecific non-prey herbivore affects Chapter 4 addresses the effects of feeding and oral secretions of two caterpillar species Chapter 4 addresses the effects Chapter 2 explores of relevant how gene transcript levels which defense genes were used in al. Thesis outline Thesis The last chapter of this thesis, Chapter 7, summarizes and discusses the main results of this Acknowledgments Chapter 1

References Aharoni A, Jongsma MA, Bouwmeester HJ. 2005. Volatile science? Metabolic engineering of terpenoids in plants. Trends in Plant Science 10, 594-602.

Alborn HT, Hansen TV, Jones TH, Bennett DC, Tumlinson JH, Schmelz EA, Teal PEA. 2007. Disulfooxy fatty acids from the American bird grasshopper Schistocerca americana, elicitors of plant volatiles. Proceedings of the National Academy of Sciences of the United States of America 104, 12976-12981.

Alborn HT, Turlings TCJ, Jones TH, Stenhagen G, Loughrin JH, Tumlinson JH. 1997. An elicitor of plant volatiles from beet armyworm oral secretion. Science 276, 945-949.

Ament K, Kant MR, Sabelis MW, Haring MA, Schuurink RC. 2004. Jasmonic acid is a key regulator of spider mite-induced volatile terpenoid and methyl salicylate emission in tomato. Plant Physiology 135, 2025-2037.

Arimura GI, Köpke S, Kunert M, Volpe V, David A, Brand P, Dabrowska P, Maffei ME, Boland W. 2008. Effects of feeding Spodoptera littoralis on Lima bean leaves: IV. Diurnal and nocturnal damage differentially initiate plant volatile emission. Plant Physiology 146, 965-973.

Arimura GI, Ozawa R, Nishioka T, Boland W, Koch T, Kühnemann F, Takabayashi J. 2002. Herbivore-induced volatiles induce the emission of ethylene in neighboring Lima bean plants. Plant Journal 29, 87-98.

Arimura GI, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J. 2000. Herbivory- induced volatiles elicit defence genes in Lima bean leaves. Nature 406, 512-515.

Baldwin IT. 1998. Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proceedings of the National Academy of Sciences of the United States of America 95, 8113-8118.

Ballhorn DJ, Kay J, Kautz S. 2014. Quantitative effects of leaf area removal on indirect defense of Lima bean (Phaseolus lunatus) in nature. Journal of Chemical Ecology 40, 294- 296.

Beckers GJM, Jaskiewicz M, Liu Y, Underwood WR, He SY, Zhang S, Conrath U. 2009. Mitogen-activated protein kinases 3 and 6 are required for full priming of stress responses in Arabidopsis thaliana. Plant Cell 21, 944-953.

Bergey DR, Orozco-Cardenas M, De Moura DS, Ryan CA. 1999. A wound- and systemin- inducible polygalacturonase in tomato leaves. Proceedings of the National Academy of Sciences of the United States of America 96, 1756-1760.

Bidart-Bouzat MG, Kliebenstein D. 2011. An ecological genomic approach challenging the paradigm of differential plant responses to specialist versus generalist insect herbivores. Oecologia 167, 677-689.

Blume B, Nurnberger T, Nass N, Scheel D. 2000. Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley. Plant Cell 12, 1425-1440.

Bolter CJ, Dicke M, Van Loon JJA, Visser JH, Posthumus MA. 1997. Attraction of Colorado potato beetle to herbivore-damaged plants during herbivory and after its termination. Journal of Chemical Ecology 23, 1003-1023.

Bonaventure G, VanDoorn A, Baldwin IT. 2011. Herbivore-associated elicitors: FAC signaling and metabolism. Trends in Plant Science 16, 294-299.

12 Chapter 1 13 1990. General Introduction General 1998. Herbivore-infested 2005. Signal signature and 2004. Exposure of Lima bean 2004. Exposure of 2001. Priming as a mechanism 2003. Mixed blends of herbivore- 2007. Plant terpenoids: Biosynthesis 2008. Prey and non-prey arthropods 1999. Jasmonic acid and herbivory 2004. Identification of volatiles that are used in 2002. Insect footsteps on leaves stimulate the stimulate leaves on footsteps Insect 2002. . Journal of Chemical Ecology 30, 255-271. 1992. Plants are better protected against 1992. Plants are better spider-mites after 2004. The role of methyl salicylate in prey searching The behavior of the 2004. 1988. How plants obtain predatory mites as bodyguards. Netherlands Phytoseiulus persimilis 2009. Priming of induced plant defense responses. Plant Innate Immunity 51, Immunity induced plant defense responses.Plant Innate Priming of 2009. 1995. Biochemistry 1995. Biochemistry and molecular biology isoprenoid of the biosynthetic pathway 2009. Jasmonate passes muster: A receptor and targets for the defense hormone. for the defense hormone. and targets receptor A passes muster: 2009.Jasmonate leaves to volatiles from herbivore-induced conspecific plants results in emission of carnivore transcriptome changes of Arabidopsis during pathogen Molecular and insect attack. Plant- transcriptome changes of induced plant volatiles and foraging success of carnivorous arthropods. Oikos 101, 38-48. induced plant volatiles and foraging success in induced systemic resistance of plants. European Journal of Plant Pathology 107, 113-119. in induced systemic resistance of plants. in plants. Review of Plant Physiology and Plant Molecular Biology 46, 521-547. Plant Physiology and Plant Molecular Biology in plants. Review of Dicke M, Van Beek TA, Posthumus MA, Ben Dom N, Van Bokhoven H, De Groot A. Bokhoven H, De Groot Posthumus MA, Ben Dom N, Van Beek TA, Dicke M, Van Dicke M, Sabelis MW. Dicke M, Dicke D, Posthumus MA. M, Gols R, Ludeking Dicke M, De Boer JG, Höfte M, Rocha-Granados MC. Höfte M, De Boer JG, Dicke M, De Vos M, Van Oosten VR, Van Poecke RMP, Van Pelt JA, Pozo MJ, Mueller MJ, Buchala Pelt JA, Pozo Van Poecke RMP, Oosten VR, Van M, Van De Vos De Moraes CM, Lewis WJ, Paré PW, Alborn HT, Tumlinson JH. Tumlinson HT, Alborn Paré PW, WJ, Lewis De Moraes CM, De Boer JG, Posthumus MA, Dicke M. MA, Posthumus De Boer JG, De Boer JG, Dicke M. De Boer JG, MA, Dicke De Boer JG, Hordijk CA, Posthumus M. Bruin J, Dicke M, Sabelis MW. MW. Bruin J, Dicke M, Sabelis Browse J. Bown AW, Hall DE, MacGregor KB. MacGregor DE, Hall AW, Bown Microbe Interactions 18, 923-937. plants selectively attract parasitoids. Nature 393, 570-573. plants selectively attract parasitoids. Nature predatory mite sharing a host plant: Effects on induced volatile emission and predator attraction. Journal of on induced volatile emission and predator attraction. sharing a host plant: Effects Isolation and identification of volatile kairomone that affects acarine predator-prey interactions predator-prey acarine affects that kairomone volatile of identification and Isolation Involvement of host plant in its production. Journal of Chemical Ecology 16, 381-396. differentially induce carnivore-attracting plant volatiles in Lima bean plants. Journal of differentially discrimination between plants infested with prey or nonprey herbivores by a predatory mite. attractants: Active or passive process? Journal of Chemical Ecology 30, 1305-1317. Active or passive process? attractants: and ecological functions. Journal of Integrative Plant Biology 49, 179-186. Journal of Integrative Plant Biology and ecological functions. exposure to volatiles from infested conspecifics. Experientia 48, 525-529. from infested conspecifics. Experientia exposure to volatiles accumulation accumulation of 4-aminobutyrate visualized and can be increased through chlorophyll 361-395. fluorescence and superoxide production. Plant Physiology 129, 1430-1434. production. Plant and superoxide fluorescence Conrath U, Thulke O, Katz V, Schwindling S, Kohler A. S, Kohler Schwindling Conrath U, Thulke O, Katz V, Conrath U. Conrath Choh Y, Shimoda T, Ozawa R, Dicke M, Takabayashi J. Ozawa R, Dicke M, Takabayashi Shimoda T, Y, Choh Chappell J. LJ, Chen XY. YB, Lu S, Wang YG, Mao AX, Lou Cheng Chemical Ecology 25, 1907-1922. Chemical Ecology 34, 281-290. Annual Review of Plant Biology, 60, 183-205. of Plant Biology, Annual Review AJ, Métraux JP, Van Loon LC, Dicke M, Pieterse CMJ. Loon Van AJ, Métraux JP, Journal of Zoology 38, 148-165. Journal of Chemical Ecology 30, 2215-2230. Chapter 1

Dicke M, Van Loon JJA, Soler R. 2009. Chemical complexity of volatiles from plants induced by multiple attack. Nature Chemical Biology 5, 317-324.

Doares SH, Syrovets T, Weiler EW, Ryan CA. 1995. Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoid pathway. Proceedings of the National Academy of Sciences of the United States of America 92, 4095-4098.

Dudareva N, Negre F, Nagegowda DA, Orlova I. 2006. Plant volatiles: Recent advances and future perspectives. Critical Reviews in Plant Sciences 25, 417-440.

Engelberth J, Koch T, Schüler G, Bachmann N, Rechtenbach J, Boland W. 2001. Ion channel-forming alamethicin is a potent elicitor of volatile biosynthesis and tendril coiling. Cross talk between jasmonate and salicylate signaling in Lima bean. Plant Physiology 125, 369-377.

Erb M, Robert CA, Hibbard BE, Turlings TC. 2011. Sequence of arrival determines plant- mediated interactions between herbivores. Journal of Ecology 99, 7-15.

Erbilgin N, Raffa KF. 2001. Kairomonal range of generalist predators in specialized habitats: Responses to multiple phloeophagous species emitting pheromones vs. host odors. Entomologia Experimentalis et Applicata 99, 205-210.

Foyer CH, Noctor G. 2005. Oxidant and antioxidant signaling in plants: A re-evaluation of the concept of oxidative stress in a physiological context. Plant, Cell and Environment 28, 1056-1071.

Frost CJ, Appel HM, Carlson JE, De Moraes CM, Mescher MC, Schultz JC. 2007. Within- plant signaling via volatiles overcomes vascular constraints on systemic signaling and primes responses against herbivores. Ecology Letters 10, 490-498.

Frost CJ, Mescher MC, Carlson JE, De Moraes CM. 2008. Plant defense priming against herbivores: Getting ready for a different battle. Plant Physiology 146, 818-824.

Gols R, Roosjen M, Dijkman H, Dicke M. 2003. Induction of direct and indirect plant responses by jasmonic acid, low spider mite densities, or a combination of jasmonic acid treatment and spider mite infestation. Journal of Chemical Ecology 29, 2651-2666.

Heil M. 2004. Induction of two indirect defences benefits Lima beanPhaseolus ( lunatus, Fabaceae) in nature. Journal of Ecology 92, 527-536.

Heil M, Baldwin IT. 2002. Fitness costs of induced resistance: Emerging experimental support for a slippery concept. Trends in Plant Science 7, 61-67.

Heil M, Bueno JCS. 2007. Within-plant signaling by volatiles leads to induction and priming of an indirect plant defense in nature. Proceedings of the National Academy of Sciences of the United States of America 104, 5467-5472.

Heil M, Koch T, Hilpert A, Fiala B, Boland W, Linsenmair KE. 2001. Extrafloral nectar production of the ant-associated plant, Macaranga tanarius, is an induced, indirect, defensive response elicited by jasmonic acid. Proceedings of the National Academy of Sciences of the United States of America 98, 1083-1088.

Heil M, Kost C. 2006. Priming of indirect defences. Ecology Letters 9, 813-817.

Hilker M, Meiners T. 2010. How do plants “notice” attack by herbivorous arthropods? Biological Reviews 85, 267-280.

14 Chapter 1 15 ) to 2003. 2003. . Journal of Amblyseius 2001. Exogenous Oreina cacaliae can be triggered can be triggered by General Introduction General 2004. Effect of different of different 2004. Effect assimilation, transpiration, 2 2003. Herbivore damage to Zea mays (Acarina: Phytoseiidae) in an Chrysopa nigricornis on CO and 2007. Arabidopsis changes transcriptome 2007. 1994. Herbivore-induced volatiles: The emission emission The Herbivore-induced 1994. volatiles: Amblyseius fallacis 2001. Attraction of a leaf beetle ( 2001. Phaseolus Phaseolus lunatus 1997. The benefits of induced defenses against Tetranychus urticae Tetranychus . Entomologia Experimentalis et Applicata 32, 205-212. 32, Applicata Entomologia Experimentalis. et 1975. Mites injurious to economy plants. Berkeley, CA: Berkeley, injurious economy plants. 1975. Mites to , Fabaceae). Basic and Applied Ecology 6, 237-248. , Fabaceae). Basic and 2008. Cross talk in defense signaling. Plant Physiology 146, 2008. Cross talk 1976. Laboratory the dispersal study of behaviour of 2006. Herbivore-induced an indirect defence in plant volatiles induce 2005. Increased availability of extrafloral nectar reduces herbivory in Lima 2007. The originherbivory on land: Initial patterns of plant tissue consumption of 2007. Metaseiulus occidentalis Croft BA. 1981. Dispersal of BA. Croft Phaseolus lunatus 2003. Field evaluation herbivore-induced 2003. of for plant volatiles as attractants 1982. Aerial dispersal and field efficacy of a genetically improved strain of the spider the of strain improved a genetically of efficacy field and dispersal Aerial 1982. (Acarina: Phytoseiidae). Annals of the Entomological Society of America 69, 1019- Annals of the Entomological Society of (Acarina: Phytoseiidae). -glucosidase and jasmonic acid. FEBS Letters 352, 146-150. FEBS Letters 352, and jasmonic acid. -glucosidase β by arthropods. Insect Science 14, 259-275. bean plants ( herbivores. Ecology 78, 1351-1355. beneficial insects: Methyl salicylate andthe green lacewing, in response to phloem-feeding silverleaf whitefly nymphs. Similarities and distinctions in in addition to carnivores. Applied Entomology and Zoology 38, 365-368. and Zoology Applied Entomology to carnivores. in addition Hoy MA. Horiuchi JI, Arimura GI, Ozawa R, Shimoda T, Takabayashi J, Nishioka T. J, Nishioka Takabayashi T, R, Shimoda Ozawa Arimura GI, JI, Horiuchi Letters 509, 332-336. Horiuchi JI, Arimura GI, Ozawa R, Shimoda T, Dicke M, Takabayashi J, Nishioka T. Nishioka J, Takabayashi Dicke M, T, Shimoda R, Ozawa Arimura GI, JI, Horiuchi herbivores attract volatiles plant conspecific herbivore-induced to exposed leaves bean Lima Hopke J, Donath J, Blechert S, Boland W. S, Boland J, Blechert J, Donath Hopke Kost C, Heil M. M. Kost C, Heil Koornneef A, Pieterse CMJ. A, Koornneef Kempema LA, Cui X, Holzer FM, Walling LL. Walling Holzer FM, Cui X, Kempema LA, Karban R, Maron J, Felton GW, Ervin G, Eichenseer H. GW, Karban R, Maron J, Felton Karban R, Agrawal AA, Mangel M. AA, Mangel Agrawal Karban R, Kalberer NM, Turlings TCJ, Rahier M. Kalberer NM, Turlings Landeros J, Guevara LP, Badii MH, Flores AE, Pámanes A. AE, Pámanes MH, Flores Badii Landeros J, Guevara LP, Labandeira C. responses to aphids. Plant Physiology 143, 849-865. responses to aphids. Plant Physiology 143, neighbouring plants. Journal of Ecology 94, 619-628. mite predator 839-844. fallacis sagebrush induces resistance in wild tobacco: Evidence for eavesdropping between plants. University of California Press. University of California of acyclic homoterpenes of acyclic of from leaves densities of the twospotted spiderdensities of mite damaged host plants. Journal of Chemical Ecology 27, 647-661. damaged host plants. Journal of Chemical and stomatal behaviour in rose leaves. Experimental and Applied Acarology 32, 187-198. Applied and stomatal behaviour in rose leaves. Experimental and apple ecosystem. Environmental Entomology 10, 313-319. apple ecosystem. Environmental Entomology a Oikos 100, 325-332. Chemical Ecology 29, 1601-1609. Chemical Ecology 29, ACC enhances volatiles production ACC enhances volatiles FEBS by jasmonic acid in Lima bean leaves. mediated Johnson DT, Johnson Johnson DT, Croft BA. DT, Johnson Jeppson LR, Keifer HH, BakerEW. Keifer HH, LR, Jeppson James DG. 1023. Chapter 1

Levine A, Tenhaken R, Dixon R, Lamb C. 1994. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79, 583-593.

Maffei ME, Mithöfer A, Boland W. 2007. Before gene expression: early events in plant-insect interaction. Trends in Plant Science 12, 310-316.

Mattiacci L, Dicke M, Posthumus MA. 1995. β-Glucosidase: An elicitor of herbivore-induced plant odor that attracts host-searching parasitic wasps. Proceedings of the National Academy of Sciences of the United States of America 92, 2036-2040.

McConn M, Creelman RA, Bell E, Mullet JE, Browse J. 1997. Jasmonate is essential for insect defense in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 94, 5473-5477.

Mithöfer A, Boland W. 2012. Plant defense against herbivores: Chemical aspects. Annual Review of Plant Biology 63, 431-450.

Mithöfer A, Wanner G, Boland W. 2005. Effects of feeding Spodoptera littoralis on Lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related volatile emission. Plant Physiology 137, 1160-1168.

Mumm R, Dicke M. 2010. Variation in natural plant products and the attraction of bodyguards involved in indirect plant defense. Canadian Journal of Zoology 88, 628-667.

Mumm R, Hilker M. 2005. The significance of background odour for an egg parasitoid to detect plants with host eggs. Chemical Senses 30, 337-343.

Mumm R, Hilker M. 2006. Direct and indirect chemical defence of pine against folivorous insects. Plant Science 11, 351-358.

Mumm R, Posthumus MA, Dicke M. 2008. Significance of terpenoids in induced indirect plant defence against herbivorous arthropods. Plant, Cell and Environment 31, 575-585.

Muroi A, Ramadan A, Nishihara M, Yamamoto M, Ozawa R, Takabayashi J, Arimura GI. 2011. The composite effect of transgenic plant volatiles for acquired immunity to herbivory caused by inter-plant communications. PLOS ONE 6.

Orozco-Cárdenas ML, Narváez-Vásquez J, Ryan CA. 2001. Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 13, 179-191.

Ozawa R, Arimura GI, Takabayashi J, Shimoda T, Nishioka T. 2000. Involvement of jasmonate- and salicylate-related signaling pathways for the production of specific herbivore- induced volatiles in plants. Plant and Cell Physiology 41, 391-398.

Ozawa R, Bertea CM, Foti M, Narayana R, Arimura GI, Muroi A, Horiuchi JI, Nishioka T, Maffei ME, Takabayashi J. 2009. Exogenous polyamines elicit herbivore-induced volatiles in Lima bean leaves: Involvement of calcium, H2O2 and jasmonic acid. Plant and Cell Physiology 50, 2183-2199.

Peng J, Van Loon JJA, Zheng S, Dicke M. 2011. Herbivore-induced volatiles of cabbage (Brassica oleracea) prime defence responses in neighbouring intact plants. Plant Biology 13, 276-284.

Pettersson EM. 2001. Volatile attractants for three Pteromalid parasitoids attacking concealed spruce bark beetles. Chemoecology 11, 89-95.

Pichersky E, Noel JP, Dudareva N. 2006. Biosynthesis of plant volatiles: Nature’s diversity and ingenuity. Science 311, 808-811.

16 Chapter 1 17 and 2012. . Physiologia . 2014. Plant 2010. Molecular, 2010. Molecular, Zea mays General Introduction General Tetranychus urticae Tetranychus 2008. Early season herbivore 2008. Early season 2005. Insect-Plant Biology. Oxford: Oxford Oxford: 2005. Insect-Plant Biology. 2004. Physiological to responses of cotton 2003. Synergistic interactions between volicitin, 2003. Synergistic interactions between 2006. Changing green leaf volatile biosynthesis in 1983. Location of distant spider mite colonies by phytoseiid 1998. Jasmonate and salicylate as global signals for defense gene 2007. Plant interactions Plant interactions 2007. microbes with molecular from and insects: Stephens (Neuroptera: Chrysopidae). Biological Control 25, 49-55. Chrysopidae). Biological Control 25, Stephens (Neuroptera: . Entomologia Experimentalis et Applicata 33, 303-314. . Entomologia Experimentalis et 1981. Biological spider mites using Phytoseiid predators. two-spotted control of 2002. Plant volatiles mediate orientation and plant preference by the predator 1998. Resistance response physiology 1998. Resistance response physiology and signal transduction. Current Opinion in 1997. Early events induced 1997. Early events by the elicitor cryptogein cells: Involvement of in tobacco 2006. Fragments of ATP synthase mediate plant perception of insect attack. Proceedings attack. insect mediate plant perception of synthase ATP of 2006. Fragments two-spotted spider mite damage. Crop Science 44, 835-846. two-spotted spider mite jasmonic acid and ethylene mediate insect-induced jasmonic acid and ethylene mediate insect-induced volatile emission in biochemical, and organismal analyses of tomato plants simultaneously plants simultaneously herbivores tomato attacked by biochemical, and organismal analyses of interactions with multiple insect herbivores: From community to genes. Annual Review of interactions with multiple insect herbivores: From community to genes. from two feeding guilds. Journal of Chemical Ecology 36, 1043-1057. from two feeding guilds. Journal of Chemical PEA. Pugin A. Pugin Poelman EH, Broekgaarden C, Van Loon JJA, Dicke M. JJA, Loon C, Van Poelman EH, Broekgaarden Pieterse CMJ, Van Der Does D, Zamioudis C, Leon-Reyes A, Van Wees SCM. Wees Van A, Leon-Reyes C, Zamioudis Does D, Der Van CMJ, Pieterse Pieterse Pieterse M. Dicke CMJ, Rodriguez-Saona CR, Musser RO, Vogel H, Hum-Musser SM, Thaler JS. Rodriguez-Saona CR, Musser RO, Vogel Reddy GVP. Farmer EE. Reymond P, Reddall A, Sadras VO, Wilson LJ, Gregg PC. Gregg LJ, Wilson Sadras VO, A, Reddall Nishioka T, Matsui K, Takabayashi J. Takabayashi K, Matsui T, Nishioka Plant Biology 65, 689–713. Proceedings of the National Academy of Sciences of the United States of America 103, of States United the Sciences of Academy of National the Proceedings of Plantarum 117, 403-412. Plantarum 117, Plant Biology 1, 305-310. Part I: Modelling the predator-prey interaction at the individual level. Agricultural Research Modellingthe predator-preythe individual I: Part at interaction level. Reports 910. plants: An approach for improving plant resistance against both herbivores and pathogens. approach for An plants: predators: Demonstration of specific kairomones emitted by pathway. Plant Cell 9, 2077-2091. pathway. mechanisms to ecology. Trends in Plant Science 12, 564-569. in Plant Science Trends to ecology. mechanisms Hormonal modulation of plant immunity. Annual Review of Cell and Developmental Cell and Developmental Annual Review of Biology plant immunity. modulation of Hormonal University Press. of the National Academy of Sciences of the United States of America 103, 8894-8899. of the United States of Academy of Sciences of the National differentially affects plant defence affects responses differentially to subsequently colonizing herbivores and their Stam JM, Kroes A, Li Y, Gols R, Van Loon JJA, Poelman EH, Dicke M. Loon R, Van Gols Y, A, Li Kroes Stam JM, Shiojiri K, Kishimoto K, Ozawa R, Kugimiya S, Urashimo S, Arimura G, Horiuchi J, Horiuchi G, Arimura K, Ozawa R, Kugimiya S, Urashimo S, K, Kishimoto Shiojiri Schoonhoven LM, Van Loon JJA, Dicke M. Loon Van LM, Schoonhoven Schmelz EA, Carroll MJ, LeClere S, Phipps SM, Meredith J, Chourey PS, Alborn HT, Teal Teal HT, Alborn J, Chourey PS, SM, Meredith Schmelz EA, Carroll MJ, LeClere S, Phipps Schmelz EA, Alborn HT, Tumlinson JH. Tumlinson HT, Alborn Schmelz EA, Scheel D. Sabelis MW, Van De Baan HE. Van Sabelis MW, Sabelis MW. Sabelis expression. Current Opinion in Plant Biology 1, 404-411. expression. Current Opinion in Plant Biology a plasma membrane NADPH oxidase activation and of glycolysis and the pentose phosphate abundance in the field. Molecular Ecology 17, 3352-3365. abundance in the field. 28, 489-521. Panonychus ulmi Chrysoperla carnea 16672-16676. Chapter 1

Strauss SY, Rudgers JA, Lau JA, Irwin RE. 2002. Direct and ecological costs of resistance to herbivory. Trends in Ecology and Evolution 17, 278-285.

Takabayashi J, Takahashi S, Dicke M, Posthumus MA. 1995. Developmental stage of herbivore Pseudaletia separata affects production of herbivore-induced synomone by corn plants. Journal of Chemical Ecology 21, 273-287.

Thaler JS, Humphrey PT, Whiteman NK. 2012. Evolution of jasmonate and salicylate signal crosstalk. Trends in Plant Science 17, 260-270.

Turlings TCJ, Tumlinson JH, Lewis WJ. 1990. Exploitation of herbivore-induced plant odors by host-seeking parasitic wasps. Science 250, 1251-1253.

Van Loon LC, Geraats BPJ, Linthorst HJM. 2006. Ethylene as a modulator of disease resistance in plants. Trends in Plant Science 11, 184-191.

Van Poecke RMP, Dicke M. 2002. Induced parasitoid attraction by Arabidopsis thaliana: Involvement of the octadecanoid and the salicylic acid pathway. Journal of Experimental Botany 53, 1793-1799.

Van Wijk M, De Bruijn PJA, Sabelis MW. 2008. Predatory mite attraction to herbivore- induced plant odors is not a consequence of attraction to individual herbivore-induced plant volatiles. Journal of Chemical Ecology 34, 791-803.

Van Zandt PA, Agrawal AA. 2004. Community-wide impacts of herbivore-induced plant responses in milkweed (Asclepias syriaca). Ecology 85, 2616-2629.

Viswanathan DV, Lifchits OA, Thaler JS. 2007. Consequences of sequential attack for resistance to herbivores when plants have specific induced responses. Oikos 116, 1389-1399.

Wei J, Van Loon JJA, Gols R, Menzel TR, Li N, Kang L, Dicke M. 2014. Reciprocal antagonism between two defense-signaling pathways in Lima bean modulates volatile emission and herbivore behavior. Journal of Experimental Botany 65, 3289-3298.

Wei J, Kang L. 2011. Roles of (Z)-3-hexenol in plant-insect interactions. Plant Signaling and Behavior 6, 369-371.

Wittstock U, Gershenzon J. 2002. Constitutive plant toxins and their role in defense against herbivores and pathogens. Current Opinion in Plant Biology 5, 300-307.

Yi HS, Heil M, Adame-Alvarez RM, Ballhorn DJ, Ryu CM. 2009. Airborne induction and priming of plant defenses against a bacterial pathogen. Plant Physiology 151, 2152-2161.

Yoshinaga N, Aboshi T, Abe H, Nishida R, Alborn HT, Tumlinson JH, Mori N. 2008. Active role of fatty acid amino acid conjugates in nitrogen metabolism in Spodoptera litura larvae. Proceedings of the National Academy of Sciences of the United States of America 105, 18058-18063.

Zhang PJ, Zheng SJ, Van Loon JJA, Boland W, David A, Mumm R, Dicke M. 2009. Whiteflies interfere with indirect plant defense against spider mites in Lima bean. Proceedings of the National Academy of Sciences of the United States of America 106, 21202-21207.

18 Chapter 1 19 General Introduction General 20 Chapter 2 Defense gene induction in Lima bean in response to phytohormone application and spider-mite feeding Tila R. Menzel, Joop J.A. van Loon, Marcel Dicke

21 Chapter 2

Abstract Phytohormones play an important role in plant defense by regulating gene transcription of defense-related genes. Exogenous application of phytohormones can be used to simulate herbivory and induce gene transcription. Here we studied the response of three defense- related genes, Phaseolus lunatus lipoxygenase (PlLOX), Phaseolus lunatus β-ocimene synthase (PlOS) and Phaseolus lunatus Pathogenesis-related protein 4 (PlPR-4) to different doses of phytohormones and different densities of the herbivore Tetranychus urticae. Transcript levels of the three defense genes of Lima bean plants were quantified at 48 hours after initiation of treatment. They differently correlated to phytohormone dose and herbivore density. The jasmonic acid (JA)-responsive gene PlOS responds to JA and salicylic acid (SA) and reflected the antagonistic interactions between the JA and SA phytohormone signaling pathways. Moreover, PlOS transcript positively correlated to herbivory by to JA-inducing T. urticae mites. The JA-responsive gene PlLOX, did not show a correlation to phytohormone doses or herbivore densities at the time point investigated here. The PlPR-4, previously reported to respond to methyl salicylate-responsive gene, did not respond to treatment with SA.

Chapter 2 , , 23 et al. et et al. , 2012). , 1990). , PR-protein

mites have et al. et al. , 2012). The two , 2012). Phaseolus lunatus , 1999; Ament , 2014). (Dicke T. urticae T. et al. , 2005). The signaling signaling The 2005). , Phytoseiulus persimilis -ocimene (Ament et al. et β et al. - et al. et (E) Phaseolus lunatus , 1999; Gols , , 2009; Wei , 2009; Wei , 2007; Pieterse Tetranychus urticae Tetranychus et al. et et al. et al. , is expected to be induced by increased JA , is expected to be induced by increased JA ) gene is induced through the JA pathway and ) gene is induced through the JA Phytohormone application and mite feeding feeding mite and application Phytohormone PlOS PlOS ( , 1995; Dicke , and , 2008; Zhang et al. et et al. , 2002; Kempema PlLOX , 2000). Consequently, we expected , 2000). Consequently, et al. , 2012). Moreover, the ethylene pathway plays an important role in plays an important role pathwayethylene the 2012). Moreover, , ) gene is involved in the JA signaling pathway. Transcription of the Transcription signaling pathway. ) gene is involved in the JA ocimene synthase et al. - , 2008). The latter compound serves an important function in plant indirect , 2008). et al. et β

, 2002). In contrast, chitinases, such as the pathogenesis-related contrast, 2002). In (PR) protein , , 2005; Ozawa PlLOX , 1997; Li et al. ( et al. et , 2014). et al. et al. on transcript levels of three genes involved in plant defense. The on transcript levels of three genes involved in plant defense. et al. , 1991; Arimura to respond to SA treatment. to respond to SA levels caused by exogenous application of the phytohormone itself, and to be suppressed by to Lima bean plants infested with the spider mite to Lima bean plants infested with the the induction mechanisms of defense regulated by the other pathway (Thaler is involved in the production of the monoterpene plant volatile in comparison to the static constitutive defenses (Schoonhoven constitutive defenses (Schoonhoven static the in comparison to feeders induce the JA pathway, whereas phloem-feeding herbivores activate the SA pathway pathway whereas phloem-feeding SA herbivoresactivate the pathway, JA feeders induce the Depending on the type of herbivore that attacks a plant, different signal-transduction signal-transduction pathways Depending on the type of herbivore that attacks a plant, different Plants rely on a sophisticated network of defense signaling pathways and their fine-tuned Defense signal-transduction induced plant defenses. part of so-called pathways are the central which in turn results in production of secondary plant metabolites that mediate plant defense plant metabolites that secondary results in productionof which in turn regulation to generate an effective defense response defense herbivorous against attacking arthropods. regulation generate an effective to pathways act antagonistically and thus induction of one signaling pathway interfere may with pathway (Pieterse biting-chewing herbivores and certain cell content- Generally, pathway. potentiating the JA pathways ultimately result in the activation of transcription of genes involved result in the activation of transcription of pathways ultimately defense, in plant lipoxygenase signal transduction (e.g. Boland signal transduction (e.g. In this study we investigated the effects of phytohormone application and actual herbivory by In this study we investigated the effects damage (Li defense, by attracting natural enemies defense, by attracting natural enemies such as the predatory mite also been shown to induce JA in Lima bean plants, spider mite feeding is expected to exert also been shown to induce JA an influence on JA-responsive genes by naturally increasing endogenous levels via feeding exogenous application of the antagonistic phytohormone Because SA. as octadecanoid pathway, and the salicylic acid (SA) pathway, also known as shikimate also known as shikimate pathway, salicylic acid (SA) and the octadecanoidas pathway, can be induced. The main signaling pathways are the jasmonic acid (JA) pathway, also known pathway, jasmonic acid (JA) main signaling pathways are the The can be induced. 2004; Arimura 2004; Lou Phaseolus lunatus (McConn (Stam et al. 4, are commonly induced in plant tissues in response to SA-related signal transduction (Ward SA-related signal transduction (Ward are commonlyinduced in planttissues in response to 4, Introduction Transcription of both genes, Transcription The application of phytohormones can be used to effectively mimic herbivory in initiating effectively phytohormonescan be used to application of The This type of plantThis type plastic and allows defense is highly in defense to vary investment plants 4 T. urticae T. Chapter 2

Materials and Methods

Plants and mites Lima bean plants (Phaseolus lunatus L., cv. Wonderbush, De Bruyn Seed Company, Michigan, USA) were cultivated in a greenhouse compartment at 23 ± 2 °C, 60 ± 10 % relative humidity (RH) and a 16 : 8 h light : dark (L : D) photoregime. Plants were grown in 11 x 11 cm plastic pots. After 12 - 14 days plants with two expanded primary leaves were transferred to a climate chamber and incubated at 25 ± 1 °C, 60 ± 10 % R.H. and 16 : 8 h L : D. A colony of two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae), was maintained on Lima bean plants in a greenhouse compartment at 25 ± 5 °C, 50 – 70 % R.H., 16 : 8 h L : D. Adult female spider mites that were used in the experiments were selected randomly from the colony.

Plant treatments

Phytohormone treatment To determine the effect of phytohormones on gene transcription, a dose-response experiment was performed. Primary leaves of Lima bean plants were sprayed with 1 ml per leaf of JA or SA solutions at different concentrations (see below) in water or with 1 ml of water as a control. JA and SA were purchased from Sigma-Aldrich. The plants were left to dry for 30 - 60 min. After phytohormone or control treatment, plants were transferred to a climate chamber and incubated in cages (metal frame 90 x 90 x 60 cm, walls of polyethylene sheet) separated by treatment at 23 ± 2 °C, 60 ± 10 % RH and 16L : 8D. Each cage contained 6-9 plants per treatment per experiment. The building’s vacuum system was connected to the top of each cage with a suction of approximately 7 L/min. The four treatments for the JA dose response curve were: i) water, ii) 0.01 mM JA, iii) 0.1 mM JA, and iv) 1 mM JA. The five treatments for the SA dose response curve were: i) water, ii) 0.001 mM SA, iii) 0.01 mM SA, iv) 0.1 mM SA, and v) 1 mM SA. Plants were incubated for 48 h after their respective treatment.

Spider mite density To investigate the effects of spider-mite density on gene transcription, adult female mites were evenly distributed over the two primary leaves of plants from the respective treatments using a fine paint brush. Treatments consisted of i) no mites (control), ii) eightT. urticae mites, iii) 20 T. urticae mites, and iv) 50 T. urticae mites. After 48 h of incubation, the mites and their products (webbing, eggs) were removed using a fine paint brush.

RNA extraction and cDNA synthesis After 48 h of incubation, plant material was collected and processed as previously described in Menzel et al. (2014). In short, 2-3 biological replicates were collected for each treatment per experiment. Each biological replicate consisted of plant leaf material pooled from primary leaves of three plants. Experiments were repeated up to two times.

Chapter 2

25 PlPR-4 PlACT1 P. lunatus P. ; GenBank ; GenBank primers were PlACT1 PlOS ( ; GenBank accession ; GenBank (2014). Relative gene et al. et acidic pathogenesis-related acidic pathogenesis-related PlNMP1 ( 5’-TGCTGCTTCCCCTCTCTCTA-3’, 5’-TGCTGCTTCCCCTCTCTCTA-3’, primers were F- primers were 5’-AGCCAGATCAAGACGAAGGA-3’, 5’-AGCCAGATCAAGACGAAGGA-3’, P. lunatus Actin1 P. PlOS PlACT1 PlACT1 Phytohormone application and mite feeding feeding mite and application Phytohormone 5’-CCGGAATGGAGTGTTGACGAGCA-3’ and 5’-CCGGAATGGAGTGTTGACGAGCA-3’ PlNMP1 5’-ACGCTTTCCTCAGTGCTCTC-3’ and R- and 5’-ACGCTTTCCTCAGTGCTCTC-3’ ; GenBank accession EU194553),GenBank accession ; P. lunatus Nuclear lunatus matrix protein 1 P. PlPR-4 PlOS ( ), and the two reference ), and the genes , simply omitting the calculation , simply omitting the of a geometric average. primers were F- primers were PlPR-4 5’-CCAGCTCAGAAACATCTGGCAATGG-3’. PlAct1 ( 5’-TGCATGGGTCTCAGTCTCTG-3’ and R- 5’-TGCATGGGTCTCAGTCTCTG-3’ were F- PlNMP1 PlNMP1 PlOS ocimene synthase ocimene synthase - levels and phytohormone doses or spider mite density, were analyzed using Pearson’s were analyzed using Pearson’s levels and phytohormone doses or spider mite density, transcripts for assessing the dose-response curve with JA and mite density were calculated and mite transcripts for assessing the dose-response curve with JA Log transformation was applied to data from gene transcription experiments in order to meet Data that violated assumptions on normality and equal variance after log transformation were transformation log after variance equal and normality on assumptions violated that Data R- Research) with a 72-well rotor; for a detailed description see Menzel a for 72-well rotor; Research) with a with two reference genes, while relative gene transcripts for SA dose response were calculated while with two reference genes, for SA relative gene transcripts F- 5’-CCAAGGCTAACCGTGAAAAG-3’ and R- 5’-CCAAGGCTAACCGTGAAAAG-3’ 5’-TCCTCGTCGTCGCAGTAATCCTT-3’, 5’-TCCTCGTCGTCGCAGTAATCCTT-3’, Statistical analysis Statistical only with analyzed by Spearman’s rank correlation test. analyzed by Spearman’s assumptions and homogeneity of normality of variances. Correlations between gene transcript and accession DQ159907) accession and β correlation Chicago, statistical software SPSS version 19 (SPSS Inc., tests in the IL, USA). protein 4 PlPR-4 Quantitative RT-PCR Quantitative AF289260.1). Quantitative RT-PCR was performed in a Rotor-Gene 6000 machine (Corbett AF289260.1). Quantitative RT-PCR A A real-time quantitative RT-PCR was used to quantify gene transcript levels of Chapter 2

Results In response to exogenous application of different doses of JA, a positive correlation with PlOS transcript levels was found (Fig 1A, Spearman’s correlation, r = 0.76, P < 0.05). Furthermore, transcript levels of PlOS showed a negative correlation with increasing SA doses (Fig 1B, Pearson’s correlation, r = - 0.81, P < 0.001). The density of T. urticae mites was also positively correlated with PlOS transcript levels (Fig. 1C, Spearman’s, r = 0.74, P < 0.001).

A B r = -0.81

r = 0.76 6 1.5 P = 0.05 P < 0.001 PlOS PlOS 5

4 1.0

2 0.5

1 Relative gene transcript levels of levels transcript gene Relative Relative gene transcript levels of levels transcript gene Relative 0 0 0.01 0.1 1 water 0.001 0.01 0.1 1 Jasmonic acid (mM) Salicylic acid (mM)

Fig. 1. Relative gene transcript levels of PlOS C quantifi ed in P. lunatus plants 48 h aft er treatment with diff erent doses of (A) JA, (B) 12 r = 0.74 SA, (C) infestation with diff erent densities

PlOS P < 0.001 of T. urticae for 48 h. Each dot represents 10 one biological replicate. For the JA dose- response curve and mite density experiment 8 PlLOX transcript levels were normalized to the normalization factor obtained from 6 geometrically averaging the Ct values of the two reference genes PlACT1 and PlNMP1 for 4 es were normalized only to the averaged Ct ach sample. For SA dose-response curve transcript 2 levelvalue of the reference genes PlACT1 for

Relative gene transcript levels of levels transcript gene Relative each sample. Correlation coeﬃ cient, r, and 0 signifi cances, P, are indicated in the right Ctrl 8 10 20 30 40 50 corner in each fi gure (Pearson’s and Spearman’s T. urticae (adult females/plant) correlation tests respectively, α = 0.05).

Lipoxygenase is a central enzyme in the wound-induced biosynthesis of JA via the octadecanoid pathway (Bell et al., 1995). However, transcript levels of PlLOX showed no correlation with the dose of the phytohormones JA or SA at 48 h after treatment (Fig 2 A, B, Pearson’s correlation, both P > 0.05). PlLOX transcript levels also did not correlate with the density of T. urticae mites (Fig. 2 C, Spearman’s, P > 0.05).

Chapter 2 27 1 transcript transcript r = 0.14 P > 0.05 for 48 h. for

0.1 transcript levels transcript for each sample. each sample. for 0.01 Salicylic (mM) Salicylic acid 0.001 PlNMP1

and and water for each sample. For SA dose-response For each sample. for

0 3 2 4 1 PlLOX Relative gene transcript levels of of levels transcript gene Relative

levels PlPR-4 transcript gene of 3. Relative Fig. treatment er lunatus 48 h aft plants ed in P. quantifi SA. of doses represents erent Each dot withdiff biological one PlPR-4 replicate. normalizedlevels the normalization were to geometrically from obtained factor averaging genes reference the two values of the Ct PlACT1 B PlLOX levels PlLOX transcript gene of 2. Relative Fig. er edquantifi lunatus 48 h aft plants in P. phytohormones of doses erent with diff treatment doses erent (A) Diff mites. spider of densities or Inoculation of SA. (C) doses erent (B) Diff JA. of urticae of T. densities erent with diff Each dot represents one biological one Each replicate. represents dot dose-response curve mite the JA and For PlLOX experiment density were normalized to the normalization factor factor normalized normalization the were to geometrically from Ct the obtained averaging and PlACT1 genes reference the two values of PlNMP1 curve normalized levels transcript were only genes the reference value of Ct the averaged to cient, coeﬃ Correlation each sample. for PlACT1 indicated in the right P, are cances, signifi and r, Spearman’s and (Pearson’s gure in each fi corner α = 0.05). respectively, tests correlation Phytohormone application and mite feeding feeding mite and application Phytohormone 50 1

1 0 r = - 0.27 P > 0.05 4

) r = 0.27 P > 0.05 r = 0.56 P > 0.05

0.1

0.01 0.1 (adult females/plant (adult Salicylic (mM) acid Salicylic 20

Jasmonic acid (mM) acid Jasmonic did not show a correlation with the dose of the phytohormone SA the phytohormone SA show a correlation with the dose of did not PlPR-4 0.001 T. urticae T.

8 0.01 water

Ctrl

4 3

1 0 2

Relative gene transcript levels of of levels transcript gene Relative

PlLOX 0.5 2.0 1.0 1.5

Relative gene transcript levels of of levels transcript gene Relative 4 - 2.0 PlPR 1.5 1.0 0.5 Relative gene transcript levels of of levels transcript gene Relative

PlLOX

A C (Fig. 3, Pearson’s correlation, r = - 0.27, P > 0.05). (Fig. 3, Pearson’s Transcript levelsof Transcript

Chapter 2

For the JA dose-response curve and mite density experiment, PlOS transcript levels were normalized to the normalization factor obtained from geometrically averaging the Ct values of the two reference genes PlACT1 and PlNMP1 for each sample. For the SA dose-response curve transcript levels were normalized only to the averaged Ct value of the reference genes PlACT1 for each sample. Correlation coefficient, r, and significances, P, are indicated in the right corner in each figure (Pearson’s and Spearman’s correlation tests respectively, α = 0.05).

Chapter 2

29 PlOS PlLOX mites and mites transcript levels , in Lima bean plants. PlOS , 2004). Moreover, we , 2004). Moreover, T. urticae T. (2009) found a negative et al. (2000) found an induction of PlPR-4 transcript levels. It has been transcript levels. It when plants were co-infested et al. mites correlated positively with mites correlated positively , 2009). The down-regulation of , 2009). et al. PlOS (2008) who demonstrated for Lima (2008) who demonstrated , and PlLOX et al. et al. T. urticae T. , on , PlLOX , , 2002; Ament expression is upregulated earlier than 12 h expression is , 1995). Zhang (2000) found that an incubation of Lima bean Phytohormone application and mite feeding feeding mite and application Phytohormone PlOS are known to induce the octadecanoid induce the octadecanoid are known to pathway et al. PlLOX induction was demonstrated, the peak of et al. et al. PlOS Bemisia tabaci and SA was found at the investigated time point. and SA , 2000; Li 2009). PlPR-4 et al. et al. Tetranychus urticae Tetranychus is only active during the light period, it follows is only active during the pattern of endogenous , did not show correlation between phytohormone doses or herbivore density herbivore density show correlation between phytohormone doses or did not , PlOS , 1991). In fact, Arimura 1991). In fact, , and whiteflies after 12 h. Moreover, Arimura Moreover, h. 12 after whiteflies and PlLOX et al. in Lima bean plants 24 h after application. However, in our experiment no correlation in our experiment in Lima bean plants 24 h after application. However, 24 h after exogenous application of JA. The apparent discrepancy might be explained 24 h after exogenous JA. of application transcription is thus probably caused by the down-regulation of endogenous JA levels transcription is thus probably the down-regulation caused by endogenous of JA transcript levels. T. urticae T. hytohormonal hytohormonal signaling the regulation plant defense. Here, induced for pathways are vital of levels defense-related of three genes, i.e. that while at 48 h since treatment mite infestation JA spider of the initiation already passed. Indeed, 12 h after transcription has to be induced early in response to herbivory mean and LOX is upstream of OS which may these herbivores (Ozawa transcript levels. This is in accordance with Arimura accordance This is in with transcript levels. three defense genes correlated with phytohormone doses and herbivore density. A positive A and herbivore density. correlated with phytohormone doses three defense genes between transcript levels of by the different sampling LOX is known time points compared to the current experiments. by the different because of the negative cross-talk between the JA and the SA signaling (Koornneef pathways and the SA because of the negative cross-talk between the JA bean that while bean that is induced in Lima bean, suggesting that induced in Lima is found a negative correlation between SA application application and found a negative correlation between SA Pathogenesis-related genes are commonly associated with SA-mediated defense responses P with we investigated the effect of phytohormone application and herbivory gene transcript on we investigated effect the plants with the methylated form of SA, methyl salicylate, results in the induction of acidic methyl salicylate, results in the induction of SA, of plants with the methylated form pathway in response to wounding (Bell pathway, pathway, since treatment (Zhang shown that herbivores that induce SA, such as whiteflies, decrease and exogenous application of SA usually activates the expression usually activates these defense genes of and exogenous application SA of effect of the SA-inducing herbivore,the of effect and thus interfere with JA-induced defenses (Zhang and Pieterse, 2008). Nevertheless, the transcript levels of a genelocated in the octadecanoid and Pieterse, 2008). Nevertheless,the transcript levels of signaling The LOX gene catalyzes an JA early step of the at the time point investigated. and an intact JA-related signaling is required to induce direct and indirect defense against correlation was found between exogenous JA doses and density of between exogenous JA correlation was found PlPR-4 PlLOX PlOS PlOS (Yalpani (Yalpani Discussion JA levels. In our study, also the infestation level of also the infestation level In our study, levels. JA The results show that at 48 h after initiation of treatment transcript levels of one of the Chapter 2

References Ament K, Kant MR, Sabelis MW, Haring MA, Schuurink RC. 2004. Jasmonic acid is a key regulator of spider mite-induced volatile terpenoid and methyl salicylate emission in tomato. Plant Physiology 135, 2025-2037.

Arimura GI, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J. 2000. Herbivory- induced volatiles elicit defence genes in Lima bean leaves. Nature 406, 512-515.

Bell E, Creelman RA, Mullet JE. 1995. A chloroplast lipoxygenase is required for wound- induced jasmonic acid accumulation in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 92, 8675-8679.

Boland W, Hopke J, Donath J, Nuske J, Bublitz F. 1995. Jasmonic acid and coronatin induce odor production in plants. Angewandte Chemie - International Edition in English 34, 1600-1602.

Dicke M, Gols R, Ludeking D, Posthumus MA. 1999. Jasmonic acid and herbivory differentially induce carnivore-attracting plant volatiles in Lima bean plants. Journal of Chemical Ecology 25, 1907-1922.

Dicke M, Van Beek TA, Posthumus MA, Ben Dom N, Van Bokhoven H, De Groot A. 1990. Isolation and identification of volatile kairomone that affects acarine predator-prey interactions Involvement of host plant in its production. Journal of Chemical Ecology 16, 381-396.

Gols R, Posthumus MA, Dicke M. 1999. Jasmonic acid induces the production of gerbera volatiles that attract the biological control agent Phytoseiulus persimilis. Entomologia Experimentalis et Applicata 93, 77-86.

Kempema LA, Cui X, Holzer FM, Walling LL. 2007. Arabidopsis transcriptome changes in response to phloem-feeding silverleaf whitefly nymphs. Similarities and distinctions in responses to aphids. Plant Physiology 143, 849-865.

Koornneef A, Pieterse CMJ. 2008. Cross talk in defense signaling. Plant Physiology 146, 839-844.

Li C, Williams MM, Loh YT, Gyu IL, Howe GA. 2002. Resistance of cultivated tomato to cell content-feeding herbivores is regulated by the octadecanoid-signaling pathway. Plant Physiology 130, 494-503.

Lou YG, Du MH, Turlings TCJ, Cheng JA, Shan WF. 2005. Exogenous application of jasmonic acid induces volatile emissions in rice and enhances parasitism of Nilaparvata lugens eggs by the parasitoid Anagrus nilaparvatae. Journal of Chemical Ecology 31, 1985- 2002.

Menzel TR, Weldegergis BT, David A, Boland W, Gols R, Van Loon JJA, Dicke M. 2014. Synergism in the effect of prior jasmonic acid application on herbivore-induced volatile emission by Lima bean plants: transcription of a monoterpene synthase gene and volatile emission. Journal of Experimental Botany 65, 4821-4831.

Chapter 2 31 2012. 2009. 2014. Plant 2014. Reciprocal 2000. 2000. Involvement of 1991. Salicylic acid is a systemic Salicylic acid is 1991. 2008. Maize plants sprayed with either sprayed with either 2008. Maize plants 2005. Insect-Plant Biology. Oxford: Oxford Oxford: Biology. 2005. Insect-Plant Phytohormone application and mite feeding feeding mite and application Phytohormone 2012. Evolution of jasmonate and salicylate signal 1991. Coordinate gene activity in response to agents that induce agents that 1991. Coordinate gene activity in response to jasmonic acid or its precursor, methyl linolenate, attract armyworm parasitoids, but the parasitoids, but the armyworm linolenate, attract methyl precursor, its acid or jasmonic jasmonate- jasmonate- and salicylate-related signaling pathways for the production of specific herbivore- interactions with multiple insect herbivores: From community to genes. Annual Review of interactions with multiple herbivores: insect to genes. From community induced volatiles in plants. Plant and Cell Physiology 41, 391-398. and Cell Physiology in plants. Plant induced volatiles P, Métraux JP, Ryals JA. JP, Métraux P, Pieterse CMJ, Van Der Does D, Zamioudis C, Leon-Reyes A, Van Wees SCM. Wees Van A, Leon-Reyes C, Zamioudis Der Does D, Van Pieterse CMJ, Plant Biology 65, 689–713. Wei J, Van Loon JJA, Gols R, Menzel TR, Li N, Kang L, Dicke M. R, Menzel JJA, Gols Loon J, Van Wei Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, Alexander DC, Ahl-Goy Alexander DC, DL, Wiederhold SS, Dincher SC, Williams SJ, Uknes ER, Ward Hormonal modulation of plant immunity. Annual Review of Cell and Developmental Cell of Annual Review Biology plant immunity. of Hormonal modulation Zhang PJ, Zheng SJ, Van Loon JJA, Boland W, David A, Mumm R, Dicke M. R, Mumm A, David W, JJA, Boland Loon Van SJ, Zheng PJ, Zhang Whiteflies interfere with indirect plant defense against spider mites in Lima bean. Proceedings bean. Lima in mites spider against defense plant indirect with interfere Whiteflies signal and an inducer of pathogenesis-related signal and an inducer of proteins in virus-infected tobacco. Plant Cell systemic acquired resistance. Plant Cell 3, 1085-1094. systemic acquired resistance. Plant Cell University Press. of the National Academy of Sciences of the United States of America 106, 21202-21207. of the United States of Academy of Sciences of the National antagonism between two defense-signaling antagonism between two pathways in Lima bean modulates volatile Journal of Experimental Botany 65, 3289-3298. emission and herbivore behavior. Stam JM, Kroes A, Li Y, Gols R, Van Loon JJA, Poelman EH, Dicke M. Loon R, Van Gols Y, A, Li Kroes Stam JM, Schoonhoven LM, Van Loon JJA, Dicke M. JJA, Dicke Loon Van LM, Schoonhoven crosstalk. Trends in Plant Science 17, 260-270. in Plant Science Trends crosstalk. composition of attractants differs. Entomologia Experimentalis et Applicata 129, 189-199. Applicata et Entomologia Experimentalis of attractants differs. composition 3, 809-818. Ozawa R, Shiojiri K, Sabelis MW, Takabayashi J. Takabayashi MW, Sabelis K, R, Shiojiri Ozawa Ozawa R, Arimura GI, Takabayashi J, Shimoda T, Nishioka T. T. Nishioka T, J, Shimoda Takabayashi GI, Arimura R, Ozawa 28, 489-521.

Thaler JS, Humphrey PT, Whiteman NK. PT, Thaler JS, Humphrey Yalpani N, Silverman P, Wilson TMA, Kleier DA, Raskin I. TMA, Wilson Silverman P, N, Yalpani 32 Chapter 3 Synergism in the effect of prior jasmonic acid application on herbivore-induced volatile emission by Lima bean plants: transcription of a monoterpene synthase gene and volatile emission Tila R. Menzel, Berhane T. Weldegergis, Anja David, Wilhelm Boland, Rieta Gols, Joop J.A. van Loon, Marcel Dicke

Published in Journal of Experimental Botany (2014) 65, 4821-4831.

33 Chapter 3

Abstract Jasmonic acid (JA) plays a central role in induced plant defense e.g. by regulating the biosynthesis of herbivore-induced plant volatiles which mediate the attraction of natural enemies of herbivores. Moreover, exogenous application of JA can be used to elicit plant defense responses similar to those induced by biting-chewing herbivores and mites that pierce cells and consume their contents. In the present study, we used Lima bean (Phaseolus lunatus) plants to explore how application of a low dose of JA followed by minor herbivory by spider mites (Tetranychus urticae) affects transcript levels of P. lunatus (E)-β- ocimene synthase (PlOS), emission of (E)-β-ocimene and 9 other plant volatiles commonly associated with herbivory. Furthermore, we investigated the plant’s phytohormonal response. Application of a low dose of JA increased PlOS transcript levels in a synergistic manner when followed by minor herbivory for both simultaneous and sequential infestation. Emission of (E)- β-ocimene was also increased, and only JA, but not SA, levels were affected by treatments. Projection to Latent Structures-Discriminant Analysis (PLS-DA) of other volatiles showed overlap between treatments. Thus, a low-dose JA application results in a synergistic effect on gene transcription and an increased emission of a volatile compound involved in indirect defense after herbivore infestation.

Chapter 3 , et 35 et al. , 2009), , , 1995; De , et al. et et al. , 2004; Lorenzo , 2009; Bruinsma et al. et al. , 2007; Dicke , or in increased amounts by or , 1997; Kessler and Baldwin, , 1997; Kessler and et al. et et al. de novo , 2009a; Snoeren , 2005), ontogenetic stage of the attacked 2005), ontogenetic stage of , , 2009). While many processes within the , et al. et al. et et al. et , 2007). Activation of these JA-responsive genes , 2007). , 2003; Wentzell and Kliebenstein, 2008; Kegge , 2003; Wentzell et al. et al. , 1998; De Vos 1998; De Vos , , 1999; Bruinsma et al. et et al. , 2007; Thines Effects of JA application on herbivore-induced volatile emission volatile herbivore-induced on application JA of Effects , 2012). Both pathways regulate large-scale , 2012). Both pathways changes in defense-related et al. et al. , 1998; Stout , , 1999; Koch et al. et et al. , 2010). Volatile compounds that are synthesized compounds that 2010). Volatile , , 2004; Chini , 2013). Moreover, plant defenses are further modulated by the simultaneous presence of , 2013). Moreover, the ecological context. Timing, intensity, and other characteristics of the defense response are intensity, Timing, the ecological context. that account for JA-mediated resistance are still unknown in most non-model plant species for non-model plant species for JA-mediated resistance are still unknown in most for account that through the plant and induce JA responses distant organs, in where they provide protection through the plant and induce JA then leads to the production of metabolites involved in plant resistance. Local activation of JA JA resistance. Local activation of metabolites involved in plant the production of then leadsto by the fact that the exact expression of the defense response by a plant is often modulated that the exact expression of the defense response by a plant is often modulated by the fact by herbivores herbivores and foliar wounding is elicitation of the jasmonic (JA) signaling acid pathway in influenced by factors such as the specific nature of the attacker (Takabayashi attacker the of nature specific the as such factors by influenced indirect resistance mechanisms (Kessler and Baldwin, 2002; Pieterse and Dicke, 2007). (Kessler and Baldwin, 2002; indirect resistance mechanisms Biosynthesis of JA is initiated by the perception of herbivore- and damage-associated Biosynthesis of JA Moraes Plants possess a whole Plants possess arsenal mechanisms of attacks by pathogens to resist and herbivorous use plant volatiles to locate their herbivorous as cues 2010). host or prey (Mumm and Dicke, which genomic sequence information is not yet available. which genomic sequence information is which the phytohormone JA plays a central role (McConn plays a central which the phytohormone JA relevant transcription factors and defense-related genes in the plant (Boter plants and density of attackers (Gols multiple herbivoressame plant (Moayeri and pathogenson the plant (Hare, 2010) and plant tissue (Wentzell and Kliebenstein, 2008), population density of plant (Hare, 2010) and plant tissue (Wentzell particularly involved in mediating tritrophic interactions, in which natural enemies of herbivores molecular patterns (HAMPs and DAMPs, respectively), which accompany herbivore attack which accompany herbivore respectively), DAMPs, and (HAMPs molecular patterns parts of the plant transcriptome, proteome, and metabolome, which underlie proteome, and metabolome, which underlie the plant transcriptome, plant direct and parts of While many of these compounds have been identified, another level of complexity is posed synthesis of volatiles, which serve an important function in plant interactions with arthropods studied. Early and late intermediates of the JA pathway as well as the final product, JA, induce JA, product, final the as well as pathway JA the of intermediates late and Early studied. signaling signaling can spread systemically production of also results in the molecules that salicylic acid (SA) signaling pathway, which antagonizes the JA pathway (Kempema which antagonizes the JA salicylic acid (SA) signaling pathway, attacked plants are called herbivore-induced plant volatiles (HIPVs). These compounds are These attacked plants are called herbivore-induced plant volatiles (HIPVs). against imminent attackers (Ryan, 2000; Koo against imminent attackers (Ryan, and mechanical damage of plant tissue (Mithöfer & Boland 2008). The synthesis and The synthesis and and mechanical damage of plant tissue (Mithöfer & Boland 2008). derepression causes a generally of JA-isoleucine the conjugate, JA-Ile, accumulation of arthropods. The basis of inducedThe basis of plant resistance against herbivory insect of a consists arthropods. complex network of phytohormonal signaling. A general general component of the response to chewing A complex of phytohormonalnetwork signaling. 2007; Thaler 2002). In contrast, piercing-suckinginsects and biotrophic pathogens commonly induce the (Dicke et al. et et al. al. Introduction JA pathway have JA been widely studied, the identity of specific gene products and metabolites The role of the JA pathway in the regulation of induced plant volatile synthesis has been well The role of the JA Chapter 3 as well as previous infestations (Stout et al., 1998; Jung et al., 2009; Ponzio et al., 2013).

Exogenous application of key phytohormones in defense signaling pathways can be used to elicit plant defense responses similar to those induced by arthropod herbivores or pathogens (Dicke et al., 1999; Gols et al., 1999; Koornneef et al., 2008). Treatment of plants with JA, or its volatile derivative methyl jasmonate (MeJA), has been shown to confer broad resistance against plant attackers such as nematodes (Cooper et al., 2005), biting-chewing insects (Omer et al., 2000; Tierranegra-García et al., 2011), and necrotrophic pathogens (Brader et al., 2001; Yamada et al., 2012). Even plants grown from seeds previously exposed to JA, have been found to be more resistant to herbivory (Worrall et al., 2012). Observed JA-mediated resistance is attributed to enhanced induction of direct resistance mechanisms, such as accumulation of plant toxins or proteinase inhibitors, or indirect resistance mechanisms, that promote the effectiveness of natural enemies of plant attackers. Generally, application of JA induces volatile blends that are similar to those induced by herbivory (Dicke et al., 1999; Gols et al., 1999; Kessler and Baldwin, 2001). These volatile blends consist of compounds that can be exploited by natural enemies as cues to locate their herbivorous prey or host. Several studies have investigated the effect of phytohormonal induction on indirect resistance (e.g. Dicke and Vet, 1999; Gols et al., 1999; Ozawa et al., 2000; Bruinsma et al., 2008; Bruinsma et al., 2009b). Phytohormone application allows for manipulation of defined steps in signal- transduction pathways and to induce plants in a dose-controlled manner without removal of plant tissue.

In the present study, we have explored how a low JA-dose affects Lima bean indirect defense against the generalist herbivorous mite Tetranychus urticae. JA is a key regulator of the induction of volatiles emitted in response to T. urticae infestation such as (E)-β-ocimene (Dicke et al., 1999; Ament et al., 2004). The monoterpene (E)-β-ocimene is an HIPV released in response to herbivory by a range of plant species including cucumber, apple, Lima bean, cotton, corn, and tobacco (Paré and Tumlinson, 1999). Moreover, (E)-β-ocimene is one of the five principle compounds that mediate the attraction of the specialist predator Phytoseiulus persimilis to T. urticae-infested plants (Dicke et al., 1990; De Boer and Dicke, 2004).

Gols et al. (2003) found that treatment of Lima bean plants with a low dose of JA, which in itself did not result in attraction of the predatory mite P. persimilis, resulted in an enhanced attraction of P. persimilis in response to herbivory by a low density of spider mites. Enhanced predator attraction was still found when a time lapse of 7 days was introduced between the treatment with JA and the infestation of spider mites. Here, we investigated the underlying mechanism. We hypothesized that exogenous application of a low dose of JA to Lima bean would induce JA-responsive gene transcription and subsequent terpene emissions with a priming or additive effect when followed by minor herbivory. We have focused on the transcription of the Phaseolus lunatus β-ocimene synthase (PlOS) gene. PlOS codes for the enzyme ocimene synthase that mediates the rate-limiting step in the biosynthesis of (E)-β- ocimene (Ament et al., 2004; Arimura et al., 2004).

Chapter 3 37 Koch (Acari: Tetranychidae), Tetranychidae), Koch (Acari: Tetranychus urticae Tetranychus L., cv Wonderbush) in a sown and grown were L., cv Wonderbush) Effects of JA application on herbivore-induced volatile emission volatile herbivore-induced on application JA of Effects Phaseolus Phaseolus lunatus

lanolin paste was applied around the petioles of both primary leaves of each plant to confine the mites to the leaves. After a seven day incubation a seven day incubation After period, leaf material from plants of the mites to the leaves. i.e. ii) water The two other treatments, and mites, was collected. treatments i) water and iii) JA the spider-mite culture. After two days of incubation, the mites and their products (webbing, incubation, the mites and their products two days of After the spider-mite culture. treatment for volatile trapping experiments. The building’s vacuum system was connected vacuum system was connected The building’s treatment for volatile trapping experiments. approximately to the top of each cage with a suction of 7 L/min to avoid interactions through incubated for another two days, after which leaf material was collected. from the respective treatments using a fine paint brush. Mites were randomly selected from Lima bean plantsLima bean ( Primary leaves of Lima bean plants were sprayed with 1 ml per leaf of 0.1 mM JA solution JA mM 0.1 leaf of per ml 1 were sprayed with Lima bean plants Primary leaves of were dry. Four adult female mites were evenly distributed over the two primary leaves of plants Four adult female mites were evenly distributed were dry. were reared on Lima bean plants in a different greenhouse greenhouse under the same compartment bean plantswere reared on Lima in a different plants per treatment for gene transcription and phytohormone gene transcription and plants per treatment for analysis or four plants per 8D. Plants having two fully expanded primary leaves were used for experiments at 12 - 15 simultaneous infestations, spider mites were applied after plants sprayed with JA solutions simultaneous spiderinfestations, mites were applied with JA after plants sprayed greenhouse compartment at 23 ± 2 °C with 60 ± 10 % R.H., and a photoperiod of 16L : 16L photoperiod of and a R.H., 10 % with 60 ± °C 2 ± 23 greenhouse at compartment of polyethylene sheet) at 23 ± 2 °C, 60 ± 10 % RH and 16L : 8D. Each cage contained 16 of polyethylene at 23 ± 2 °C, 60 ± 10 % RH and sheet) 16L In subsequent experiments with sequential infestation, mites were inoculated seven days after days seven inoculated were mites infestation, sequential with experiments subsequent In days after sowing. Two-spotted spider mites, Two-spotted days after sowing. and iv) JA and mites, received treatment (four adult the mite females per plant) and were and iv) JA eggs) were removed using a fine paint brush. eggs) were removed using a fine paint chamber and incubated separated by treatment in cages (metal frame 90 x 90 x 60 cm, walls conditions as the Lima bean plants. Only adult female mites were used for experiments. bean plants. Only adult female mites conditions as the Lima 30 - 60 min. After phytohormone or control treatment, plants were transferred to a climate 30 - 60 min. Plants and mites and Plants volatiles between plants of different treatments. volatiles between plants of different transcription (Sigma-Aldrich) in water or with 1 ml of water as a control. The plants were left to dry for dry to The plants were left a control. water as (Sigma-Aldrich) 1 ml of with in water or Treatments Combined effects of JA and simultaneous or sequential spider-mite infestation on PlOS on infestation spider-mite or sequential simultaneous and JA of effects Combined Materials Methods and JA treatment and transferred to cages as described above. Two days before mite application, days before mite application, Two treatment and transferred to cages as described above. JA The four treatments were: i) water, ii) water and mites, iii) JA, and iv) JA and mites. For and mites, iii) JA, and iv) JA ii) water The four treatments were: i) water, Chapter 3

RNA extraction and cDNA synthesis Leaf material was collected by excising four leaf discs at 12:00 - 13:00h from a primary leaf using a cork borer (diameter 2 cm), and the leaf discs obtained from three plants were pooled to give one biological replicate. Upon collection, samples were immediately shock-frozen in liquid nitrogen and stored at -80 °C until processing. The leaf material was homogenized without thawing using a mortar and pestle. Total RNA was extracted and purified using the Qiagen RNeasy Plant Mini kit with integrated DNAse treatment, following the manufacturer’s instructions. Absence of genomic DNA contamination and RNA quality were assessed using Agilent 2100 Bioanalyzer with the RNA 6000 Nano Labchip® kit (all from Agilent Technologies). RNA was quantified with a NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Only RNA samples with 260 / 280 wavelength ratio > 2 and a RIN value > 7 were used for cDNA synthesis. cDNA was generated from total RNA by using the Bio-Rad iScript cDNA synthesis kit (Bio-Rad), following the manufacturer’s instructions.

Quantitative RT-PCR Transcript levels of P. lunatus β-ocimene synthase (PlOS; GenBank accession EU194553) and the two reference genes P. lunatus Actin1 (PlACT1; GenBank accession DQ159907) and P. lunatus Nuclear matrix protein 1 (PlNMP1; GenBank accession AF289260.1) were quantified by performing a real-time quantitative RT-PCR in a Rotor-Gene 6000 machine (Corbett Research) with a 72-well rotor. Reactions were performed in a final volume of 25 µl, that included 12 µl iQTM SYBR® Green Supermix (Bio-Rad), 1 µl forward primer (4 µM) and reverse primer (4 µM) pairs (final primer concentration: 160 nM), and 5 µl cDNA (4 ng/µl) first strand template. The PCR program for PlOS and the reference gene PlACT1 was the same as described by Zheng et al. (2007). The PlOS primers were F-PlOS5’-TGCATGGGTCTCAGTCTCTG-3’ and R-PlOS5’- TGCTGCTTCCCCTCTCTCTA-3’ with a predicted product length of 189 bp. PlACT1 primers were F-PlACT1 5’-CCAAGGCTAACCGTGAAAAG-3’ and R-PlACT1 5’-AGCCAGATCAAGACGAAGGA-3’ with predicted product length of 208 bp. The second reference gene, PlNMP1, was designed with the Geneious software version 4.8.3 under default parameters except that the annealing temperature was set to 56 °C. Predicted product length of the PlNMP1 primers F-PlNMP1 5’-CCGGAATGGAGTGTTGACGAGCA-3’ and R-PlNMP1 5’-CCAGCTCAGAAACATCTGGCAATGG-3’ was 157 bp. The PCR program for PlNMP1 was adapted from Zheng et al. (2007), whereby the extension time was increased from 45 s to 48 s. Specificity of amplicons was verified for each primer pair by melt-curve analysis to assure absence of nonspecific products as well as primer-dimer formation. Relative quantification of PlOS transcription was calculated with the 2(-Delta Delta C(T)) method (Livak and Schmittgen, 2001), using a normalization factor (Vandesompele et al., 2002). The normalization factor was calculated by geometrically averaging the threshold cycle (Ct) values from the two reference genes PlACT1 and PlNMP1 (M < 0.03, GeNorm). Subtraction of the normalization factor from PlOS Ct values normalizes for differences in cDNA synthesis.

Chapter 3 39 (2006). Samples were analyzed on a Finnigan ITQ Instrument Instrument Finnigan ITQ a were analyzedSamples on (2006). et al. et Effects of JA application on herbivore-induced volatile emission volatile herbivore-induced on application JA of Effects libraries. Experimentally calculated linear retention indices (LRI) were also used as additional lid with an inlet and outlet. Compressed air was filtered by passing through charcoal before those in the NIST 2005 and Wageningen Mass Spectral Database of Natural Products MS Spectral Database of Mass 2005 and Wageningen NIST the those in transfer line and ion source were set at 275 and 250 °C, respectively. Compound identification Compound respectively. °C, 250 and 275 at set were source ion and line transfer treatment i) water, ii) water and mites, iii) JA, and iv) JA and mites. Fresh weight of above- Fresh weight of and mites. and iv) JA iii) JA, ii) water and mites, treatment i) water, the protocol of Schulze Schulze protocol of the jars containing the plants were flushed for an additional 30 min before connecting stainless held at 40 °C for 2 min and was raised at 10 °C/min to a final temperature of 280 °C, where it where °C, 280 of temperature final a to °C/min 10 at raised was and min 2 for °C 40 at held helium flow of 20 ml/min, while re-collecting the volatiles in a thermally cooled universal balance (NewClassic ML, Mettler Toledo, Switzerland). Toledo, balance (NewClassic ML, Mettler was based on retention time of authentic standards and comparison of mass spectra with was kept for 4 min under a helium flow of 1 ml/min in a constant flow mode. The DSQ mass with compressed air for 1 h. Pots in which the plants had grown were removed, roots and soil The glass were carefully wrapped in aluminum the plant foil, then was placed in glass a jar. measure to confirm the identity of compounds. min) for 10 min at ambient temperature in order to remove moisture and the organic solvent ambient temperature in order to min) for 10 min at the were released from The collected volatiles and I.S. prepare the I.S. methanol used to plant volatiles. Prior to release of the volatiles, each sample was spiked with 10 ng/µl of scans/s and spectra were recorded in electron impact ionization (EI) mode at 70 eV. MS scans/s and spectra were recorded in electron impact ionization (EI) mode at 70 eV. spectrometer (MS) was operated of 35 – 350 amu at 5.38 in a scan mode with a mass range solvent trap at 10 °C using Unity (Markes). Once the desorptionsolvent trap at 10 °C using Unity (Markes). process was completed, steel tubes filled with Tenax TA. Plantsteel Tenax volatiles tubes werefilled collected with from seven replicates of each ground immediately plant tissue was determined after volatile collection using an analytical glass jar at a constant rate of 200 ml/min through a stainless steel tube filled with 200 mg a ZB-5MSi analytical column [30 m x 0.25 mm I.D. x 1.00 (Phenomenex, Torrance, µm F.T. entering the glass jar containing the plant. Volatiles were collected by sucking air out of the were collected by sucking air out containingentering the glass jar plant. Volatiles the Phytohormone quantification Phytohormone Quantification of JA and SA levels in samples used for gene transcription analysis followed volatile compounds were released from the cold trap by ballistic heating at 40 °C/s to 280 to 40 °C/s by ballisticheating at trap cold the volatile compounds were released from CA, USA)], in a splitless mode for further separation. The GC oven temperature was initially CA, USA)], in a splitless mode for further separation. Collection of plant volatiles was carried out in 20-L glass jars sealed with a viton-lined glass jars sealed glass was carried out in 20-L volatiles Collection plant of Dynamic headspace collection volatiles plant of (Thermo Fisher Scientific, Waltham, USA)was used for separation and detection of (Thermo Electron, Bremen, Germany) running in a CI-negative ion mode. running in Bremen, Germany) (Thermo Electron, Tenax TA using the Ultra 50 : 50 thermodesorption using the Ultra 50 : 50 thermodesorption unit (Markes) at 250 °C for 10 min under TA Tenax Thermo Trace GC Ultra coupled with Thermo Trace DSQ quadrupole mass spectrometer Tenax TA (Markes, Llantrisant, (Markes, Llantrisant, sampling, UK) for 2 h. Before glass empty jars were purged TA Tenax 1-bromodecane as internal standard (I.S.) and dry-purged under a stream of nitrogen (50 ml/ of dry-purged undera stream and 1-bromodecane standard (I.S.) as internal Analysis of plant volatiles plant of Analysis °C. The temperature was kept at 280 °C for 10 min, while the volatiles were transferred to The temperature °C. Chapter 3

Standards of (E)-2-hexenal, (Z)-3-hexen-1-ol, (Z)-3-hexen-1-ol acetate, (E)-β-ocimene, linalool, methyl salicylate (MeSA), indole, caryophyllene as well as the internal standard (I.S.) 1-bromodecane, a series of alkane mixtures (C8 – C20) and the solvent methanol (GC grade) were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Additional standards (E)-4,8-dimethylnona-1,3,7-triene [(E)-DMNT] and (E,E)-4,8,12-trimethyltrideca-1,3,7,11- tetraene [(E,E)-TMTT] were synthesized at the Max Planck Institute of Chemical Ecology (Jena, Germany) following the procedure by Boland and Gäbler (1989). For quantification, calibration lines were constructed for each compound using seven data points at different concentrations (two replicates of each data point) and was carried out using a single (target) ion, in selected ion monitoring (SIM) mode.

Statistical analysis Univariate data, i.e. gene transcription and plant volatile data were log-transformed to meet the test assumptions of normality and homogeneity of variances. Phytohormone data were analyzed without transformation. Analyses were performed using one-way ANOVA followed by Fisher’s least significant difference (LSD) post-hoc tests for pairwise comparisons between treatments in the statistical software SPSS version 19 (SPSS Inc., Chicago, IL, USA). If assumptions on normality and equal variance were violated, Kruskal-Wallis tests followed by Mann-Whitney U tests with a Bonferroni correction as post-hoc tests were used. Assumption of synergism was tested by subtraction of baseline levels of both single treatments and subsequent summation. If the resulting value was outside the 95 % confidence interval of the mean from a combination treatment, the interaction between the single treatments was considered significantly different.

Effects of treatments, time of trapping and the interaction on (E)-β-ocimene emission were analyzed by general linear model (GLM) with LSD post-hoc tests. Evaluation of differences between treatments of morning trapping and afternoon trapping were done by an one-way ANOVA followed by Fisher’s least significant difference (LSD) post-hoc tests for pairwise comparisons.

The multivariate data analysis of plant volatiles corrected by fresh weight using Projection to Latent Structures-Discriminant Analysis (PLS-DA) was performed to test for differences in volatile profiles among different treatments. The analysis was carried out using the software SIMCA P+ version 12 (Umetrics, Umeå, Sweden). Data were log-transformed and univariate- scaled prior to PLS-DA analysis.

Results

Transcriptional changes in PlOS levels in response to JA and spider-mite treatment Transcript levels of PlOS in response to the treatments, i.e. i) water (control), ii) 0.1 mM JA, iii) four T. urticae, and the combined treatment iv) 0.1 mM JA with simultaneous inoculation of four T. urticae showed significant differences (Fig. 1A).

Chapter 3

T. T. 41 PlOS mites mites T. urticae T. transcript PlOS < 0.05 for both (P plants treated with i) water with i) water treated plants on plants was done immediately immediately was done plants on levels after 48 h compared to control 48 h compared to levels after T. urticae T. with four iv) 0.1 mM JA ), or < 0.05) compared to 0.1 mM JA treatment < 0.05) compared to 0.1 mM JA PlOS alone showed higher level than would be obtained from additive (P levels compared to control, 0.1 mM JA, and four JA, control, 0.1 mM levels compared to PlOS P. lunatus in P. quantified < 0.01) < T. urticae T. (P PlOS (water + 4Tu (water transcript levels in plants treated with 0.1 mM JA were not transcript levels in plants treated with 0.1 mM JA was done seven days after the application of 0.1 mM JA or or JA mM 0.1 application the of done seven days after was < 0.05 for all comparisons). Compared to 0.1 mM JA or four < 0.05 for all comparisons). Compared to 0.1 mM JA PlOS (P and 0.1 mM JA, indicating a synergistic effect of the two treatments of indicating and 0.1 mM JA, a synergistic effect Effects of JA application on herbivore-induced volatile emission volatile herbivore-induced on application JA of Effects was done seven days after incubation with water or 0.1 mM JA and mites and mites JA mM or 0.1 water incubation with seven after was done days T. urticae T. also showed higher transcript levels after 48 h compared to control plants, but did not differ control plants, but did not differ transcript levels after 48 h compared to for each sample. Baseline represents transcript level in control plants. level transcript in control Baseline each represents sample. PlNMP1 for T. urticae T. adult female four of ). (A) Inoculation T. urticae T. PlOS and and for 2 days showedhigher for T. urticae T. < 0.05; Fig. 1B). transcript level was significantly higher significantly was level transcript treatment alone transcript levels. alone, the combination had a higher were additive, revealing a synergistic effect of the two treatments on were additive, revealing a synergistic effect (P PlACT1 PlACT1 T. urticae T. PlOS PlOS levels. the transcript level that is twice the level that would be obtained if the effects of JA and four JA of the effects obtained if would be transcript level that is twice the level that inoculated four from each other. Plants treated with the combination of 0.1 mM JA and four simultaneously and four simultaneously JA Plants treated with the combination 0.1 mM of each other. from Plants treated with 0.1 mM JA or four Plants treated with 0.1 mM JA were consistent with those presented Fig. 1. See Supplementaryin Fig. 1 and 2 for the results. were inoculated on water-treated plants at this time point and incubated for another two days, another two point and incubated for time this plants at were inoculated on water-treated which inoculationof water significantly from different control plants after seven days of incubation. When four on effects of four effects alone. After seven days of incubation, plants treated with the combination of 0.1 mM JA and After seven days of incubation, plants treated with the combination of 0.1 mM JA alone. and the single treatment with JA or mites. The combination resulted in a treatment mites. or and the single JA treatment with comparisons) Significant differences between treatments were also found in the second experiment in urticae urticae T. urticae T. iii) four mM JA, ii) 0.1 (control), Fig. 1. Relative gene transcript levels PlOS transcript gene of 1. Relative Fig. had since been feeding for 48 h. Values are the mean (± SE) of three to four biological four three to replicates, of the meanSE) (± are 48 h. Values beenhad since feeding for transcriptin levelstreatments significantbetween above indicate differences bars letters different normalized levels α = 0.05). PlOS the transcript test, were to LSD followed Fisher’s by (ANOVA geometrically reference the two values from of obtained the Ct factor averaging normalization genes (0.1 mM JA + 4Tu (0.1 mM JA four inoculation (B) 48 h, and been of had since mites feeding for and JA-treatment following urticae T. adult female This experiment has been repeatedrespectively two and three more times and the results T. urticae T. Chapter 3

Phytohormone levels We investigated the effects of single treatments i) water (control), ii) 0.1 mM JA, iii) four T. urticae, and iv) the combined treatment of 0.1 mM JA with simultaneous inoculation of four T. urticae on JA levels (Fig. 2). A significant treatment effect was found (P = 0.01; Fig 2A). Application of 0.1 mM JA resulted in higher JA levels at 48 h compared to control plants. Four T. urticae, however, did not increase JA levels in the plants compared to the control treatment. Plants treated with the combination of 0.1 mM JA and simultaneously four T. urticae also showed higher JA levels compared to control, but not different from 0.1 mM JA treatment alone.

Fig. 2. JA levels in ng JA/g FW in P. lunatus plants treated with i) water (control), ii) 0.1 mM JA, iii) four T. urticae (water + 4Tu), or iv) 0.1 mM JA with four T. urticae mites (0.1 mM JA + 4Tu). (A) Plants were inoculated with four adult female T. urticae immediately after JA treatment and incubated for 48 h, and (B) plants were inoculated with four adult female T. urticae 7 days after JA treatment and incubated for an additional 48 h. Values are the mean (± SE) of four biological replicates, and were analyzed by Kruskal-Wallis test (A) or ANOVA (B) respectively (α = 0.05).

Significant differences in JA levels were also found among treatments when mites had been inoculated seven days after JA or water application (P < 0.01; Fig 2B). After seven days of incubation with 0.1mM JA there is still an increase (P < 0.001) in JA level compared to control. The combination of 0.1 mM JA application and inoculation of T. urticae seven days later that had been feeding for two days resulted in JA levels after nine days that were similar to that of the control treatment. The introduction of four T. urticae alone did not affect JA levels.

No treatment effect was found for SA levels between control and other treatments for simultaneous (P = 0.81; Supplementary Fig. 3A) or sequential mite application (P = 0.33; Supplementary Fig. 3B).

Volatile emission Emission rates of the monoterpene (E)-β-ocimene were compared among treatments and time of trapping of the simultaneous T. urticae application experiment. There was a treatment effect (P < 0.05), however, although emission rates of plants treated with 0.1 mM JA, mites, or both, were higher than control treatment, the post-hoc test did not yield statistical differences among treatments (P > 0.05; Fig. 3A). However, the time of trapping (morning, i.e. ca. 11.00 -

Chapter 3 -3- 43

(Z) plants. plants. -3-hexen-1-ol, (Z) ), or iv) 0.1 mM JA with iv) 0.1 mM JA ), or = 0.20; Fig. 3B). In afternoon 3B). In afternoon = 0.20; Fig. (P -2-hexenal, mites (0.1 mM JA + mM JA (0.1 urticae mites T. -ocimene emission compared other to ) inoculated immediately after JA ) inoculated after immediately (E) β = 0.02; Fig. 3C), and plants treated with and plants treated with 3C), Fig. 0.02; = - -ocimene emission (E)-β-ocimene emission 3. Average Fig. different four FW/h after in ng/g rates lunatus P. of treatments (water), i) control were Treatments urticae T. iii) four ii) 0.1 mM JA, + 4Tu (water four 4Tu 48 h. for incubated and application and (A) depicts morning combined morning (B) trappings, afternoon (ca. only 11.00 - 13.00), and trappings only (ca. 14.00 trappings (C) afternoon of the mean (± SE) are - 16.00). Values seven (A), to six biological for replicates biological four three replicates to and + 4Tu water for (C), except (B) and for biologicalin (C) with two replicates. above indicate bars letters Different rates emission in significant differences tests, LSD (Fisher’s between treatments α = 0.05). (P (E) showed increased showed increased Effects of JA application on herbivore-induced volatile emission volatile herbivore-induced on application JA of Effects T. urticae T. < 0.05). P treatments (Fig. 4). These ten compounds were treatments (Fig. 4). trappings, however, a treatment effect was found was found effect treatment a however, trappings, ( treatments Emission of a total of the ten major volatile compounds was also compared among the Emission of a total of the ten major volatile compounds was also compared 0.1 mM JA and four and four 0.1 mM JA during mornings showed no overall effect of treatments during mornings effect showed no overall 13.00 or afternoon, i.e. ca. 14.00 - 16.00) may also have an effect. Volatile trappings executed Volatile an effect. also have 16.00) may 14.00 - i.e. ca. or afternoon, 13.00 Chapter 3 hexen-1-ol acetate, (E)-β-ocimene, linalool, methyl salicylate, indole, β-caryophyllene, (E)- DMNT, and (E,E)-TMTT. They constitute well-known herbivore-induced plant volatiles (HIPV) observed in T. urticae-infested Lima bean plants (Dicke et al., 1990; Dicke et al., 1999). PLS- DA including all four treatments resulted in a model with one significant component, whereby volatile blends emitted by control (water-treated) plants clearly differed from those emitted by plants exposed to the other three treatments. The volatile emission profiles of plants exposed to the combined 0.1 mM JA plus four T. urticae treatment overlapped to a large extent with those of plants exposed to 0.1 mM JA alone. Volatile blends emitted by plants exposed to four T. urticae exhibited similarities with those from control plants, but also with those from 0.1 mM JA-treated plants. Treatment of plants with JA, mites, or a JA-mite combination increased the emission of all ten volatiles (Fig. 4B). Compared to the control treatment, treatment of plants with JA (J and JTu, Fig. 4B) resulted in higher emissions of indole, the green leaf volatiles (Z)- 3-hexen-1-ol acetate and (Z)-3-hexen-1-ol, and to a lesser extent the terpenoids (E)-DMNT, (E)-β-ocimene, as well as β-caryophyllene. The emission rates of the latter three compounds were intermediate in plants exposed to mites alone.

3.0 A W 2.0 WTu

J 1.0 JTu 0.0

-1.0 PLS2 (11.78%) PLS2

-2.0

-3.0 -5 -4 -3 -2 -1 0 1 2 3 4 5 PLS1 (38.64%)

B 5 WTu 0.4 JTu 2 0.2 3 4 10 6 0.0

8 7 W

PLS2 (11.78%) PLS2 -0.2

-0.4 J 1 -0.6 9 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 PLS1 (38.64%)

Fig. 4. Multivariate data analysis by PLS-DA and corresponding loading plot of targeted volatiles M3of -P. PLS lunatus-DA Observations plants (N)=27, exposed Variables to (K)=14 i) water (X=10, Y=4)(control, W), ii) 0.1 mM JA (J), iii) water and four T. urticae Aspider mitesR2X (W Tu), orR2X(cum) combined Eigenvaluetreatment iv)R2Y 0.1 mM JAR2Y(cum) with immediateQ2 applicationLimit of fourQ2(cum) T. Significance 0urticae (JTuCent.). (A) PLS-DA score plot showingCent. the ordination of the samples according to the first 1two PLS components0.386 based0.386 on the3.86 quantitative 0.204 values of0.204 volatiles between0.117 different0.05 treatments.0.117 R1 2Explained variance0.118 by 0.504first and second1.18 PLS0.127 components 0.331 is given in-0.0882 brackets. 0.05 Loading plot0.0392 (B) NS shows the contribution of each volatile to the discrimination between treatments using the first two PLS components. Numbers represent: 1, (E)-2-hexenal; 2, (Z)-3-hexen-1-ol; 3, (Z)-3-hexen- 1-ol acetate; 4, (E)-β-ocimene; 5, linalool; 6 (E)-4,8-dimethyl-1,3,7-nonatriene [(E)-DMNT]; 7, methyl salicylate (MeSA); 8, indole; 9, β-caryophyllene; 10, (E,E)-4,8,12-trimethyltrideca-1,3,7,11- tetraene [(E,E)-TMTT]. Squares represent the four treatments (labelled W, WTu, J, and JTu).

Chapter 3

R1 NS Significance ). ) 45

0.408 0.349 Q2(cum and JTu and

5 Tu Tu Tu 0.5 J W W ) resulted in a ) resulted

Tu 4 0.4

0.05 0.05 Limit (J ) or the combination combination the ) or

0.3 3 (WTu

T. urticae T. 0.2 0.681

0.408 - Q2

2 10

0.1

1 5

0.0 0.616 0.682 R2Y(cum)

7 1

0 4 PLS1 (26.18%) PLS1 0.1 -

PLS1 (26.18%) PLS1

2 1 - 0.2 0.616 Cent. 0.0658 R2Y -

) 2 0.3 ). The score plot (A) visualizes the separation pattern of the (A) plot ). The visualizesscore the separation pattern - - Tu

(J 9 2.62

1.25 Eigenvalue 0.4

- 3 -

T. urticae T. four either subjected to plants

Tu J

0.5 - 3

Effects of JA application on herbivore-induced volatile emission volatile herbivore-induced on application JA of Effects 4 -

0.262 0.386 R2X(cum) 8 0.6 -

5 - ) and combined 0.1 mM JA treatment plus four treatment plus four ) and combined mM JA 0.1

Tu 0.2

0.6 0.4 0.2 0.0

1.0 2.0 3.0

3.0 2.0 1.0 0.0

infestation infestation resulted in a plant volatile profilewas separate that from the profile of (12.46%) PLS2 - - - PLS2 (12.46%) PLS2

(W 0.262 Cent. 0.125 R2X B A Observations (N)=13, (N)=13, Observations (K)=12 Variables (X=10, Y=2 DA - T. urticae T. pairwise comparison of volatile profiles from treatments including mites, i.e.water plus four PLS

- to plants without the JA treatment. treatment. the JA plants without significant PLS-DA model with one significant component (Fig. 5). Pre-treatment with JA prior JA with Pre-treatment 5). (Fig. component significant one with model PLS-DA significant T. urticae T. four and mM JA 0.1 of explained the with and classessecond the first PLS component their using to according samples the class separa to volatiles - of loading the (B) depicts in brackets and plot variance the contribution significantnot only and is was Thecomponent PLS second PLS components. two the first using tion 3, 2, (Z)-3-hexen-1-ol; 1, (E)-2-hexenal; represent: purposes. Numbers representational shown for (E)- [ 4, (E)-β-ocimene; 5, linalool; acetate; 6 (E)-4,8-dimethyl-1,3,7-nonatriene (Z)-3-hexen-1-ol salicylate 7, methyl 8, indole; 9, β-caryophyllene;DMNT]; (MeSA); - 10, (E,E)-4,8,12-trimethyltride (labelled WTu treatments the two ca-1,3,7,11-tetraene [(E,E)-TMTT]. represent Squares - com volatile loading of plot corresponding and PLS-DA using analysis data 5. Multivariate Fig. lunatus P. by pounds emitted A T. urticae T.

1 0 2 M7 A Chapter 3

Discussion In their natural environment plants are frequently exposed to multiple herbivory, whereby herbivores may arrive simultaneously or separated in time. Both types of infestations may influence the plant phenotype and therefore affect tritrophic interactions with natural enemies involved in plant indirect defense. Here, we used the phytohormone JA followed by herbivory by a low number of herbivores to study the effects of this phytohormone on transcript levels of β-ocimene synthase, emission of the corresponding volatile compound, and other volatiles commonly emitted from plants in response to simultaneous and sequential herbivory. The volatile organic compound (E)-β-ocimene plays an important role in plant indirect defense in many plant species, including Lima bean, by attracting natural enemies of herbivorous arthropods (Dicke et al., 1990; Arimura et al., 2000; Arimura et al., 2002; Zhang et al., 2009a; Muroi et al., 2011).

We found that Lima bean plants treated with a low dose of JA exhibited increased PlOS transcript levels in a synergistic manner when followed by minor herbivory, irrespective of the herbivory occurring simultaneously or sequentially. Accordingly, Gols et al. (2003) found that plants treated with a low dose of JA followed by simultaneous or sequential minor herbivory by T. urticae were highly attractive to the predatory mite P. persimilis: the predators preferred volatiles emitted from plants treated with 0.1 mM JA and infested with four T. urticae over volatiles from plants infested with only four T. urticae. Quantification of (E)-β-ocimene emission in the headspace of Lima bean plants shows that the emission rate of the volatile itself was also increased in combination treatments. The increase was only significant during the afternoon. The latter connects to findings of Arimura et al. (2008) that show that (E)-β- ocimene emission rates increase from the onset of light and peak during the afternoon after herbivory or leaf damage. Generally, (E)-β-ocimene seems to play an important role in the attraction of P. persimilis in plant interactions with multiple herbivores. For instance, De Boer et al. (2008) found that (E)-β-ocimene emission and predator attraction were increased in a synergistic manner in response to simultaneous infestation by prey and non-prey herbivores on a Lima bean plant. Moreover, Zhang et al. (2009b) showed that feeding by a non-prey herbivore, i.e. whiteflies, negatively affected (E)-β-ocimene emission and corresponding transcript levels of PlOS, which resulted in decreased attraction of P. persimilis to Lima bean plants simultaneously infested with spider-mites and whiteflies. The main underlying mechanism seems to be phytohormone induction and crosstalk among them. Whiteflies induce SA, which antagonizes the JA pathway, whereas caterpillars and spider mites mainly induce the JA pathway (Blechert et al., 1995; Arimura et al., 2002; Schmelz et al., 2003). In our study we found a synergistic effect of a low dose 0.1 mM JA and a low density infestation by four T. urticae on PlOS transcript levels after 48 h of spider-mite infestation. In the case of a 7-day delay between JA treatment and spider-mite inoculation JA did not induce PlOS transcription but in combination with spider mite feeding resulted in an enhanced transcription compared to spider-mite induction alone. Thus, in this case JA had primed the transcription of this gene. Yet JA levels were similar for JA-treated plants and plants induced with both JA and T. urticae. Interestingly, even when JA titers and PlOS transcripts levels returned to control levels, subsequent mite infestation still increased PlOS transcript levels to higher values than

Chapter 3

, , L. 47 et al. et al. et al. , 2005; -ocimene T. urticae T. β - et al. et (E) by JA seemed to by JA (2004) found that -TMTT, in order to -TMTT, -ocimene, are likely to , 2006; Walters , 2006; Walters . While , 2009b). Our targeted PlOS et al. β Petroselinum crispum - (E,E) , 1999). However, defense , 1999). However, , 2002). et al. (E) et al. , 1998; De Vos 1998; De Vos , et al. et al. et al. P. persimilis P. , 1999; Gols , 2008; Bruinsma , 1998; Stout et al. et al. , which was more strongly attracted to sequentially , whereas a low infestation density of four , et al. mites (Dicke P. persimilis P. P. persimilis P. , 2004; Bruinsma (Thulke and Conrath, 1998; Kohler Effects of JA application on herbivore-induced volatile emission volatile herbivore-induced on application JA of Effects , 2011). In our experiments, In our previous induction of , 2011). T. urticae T. et al. , 1995; De Moraes et al. et al. , 2008; Conrath, 2009). , 2008; Conrath, 2009). Maintenance of plant defense is thought to entail costs and -ocimene is also emitted in response to caterpillar feeding. Predators must therefore Arabidopsis thaliana β - the blend induced by that plants are able to form some sort of memory, sometimes called a “primed state”, which called a “primed state”, sometimes to form some sort of memory, that plants are able by actual hosts or prey over JA-induced plants (Van Poecke and Dicke, 2002; De Boer and Poecke and plants (Van by actual hosts or prey over JA-induced is ineffective in the absence of herbivores. Consequently, plants have developed defense induced induced than to plants only plants It has been by spider mites. previously suggested is known to be an important host location bean, De Boer cues in Lima in volatile blends must thus affect the behavior of the predatory mite. Volatile emission profiles emission Volatile mite. predatory the of behavior the affect thus must blends volatile in induction by JA seems to be more generic seems to be more and natural enemies often prefer HIPVs induced induction by JA Blends can carry information on e.g. herbivore developmental identity or herbivore stage Lima bean plants among treatments showed indeed a large overlap for JA- and mite-treated indeed a large overlap for Lima bean plants among treatments showed Mumm and Dicke, 2010). JA application is known to induce a volatile blend that is similar to is volatile blend that application induce a is known to 2010). JA Mumm and Dicke, Natural enemies of herbivores respond to mixtures of HIPV rather than to a single volatile. a rather than to HIPV mixtures of herbivores respond to Natural enemies of Dicke, 2004; Ozawa reported for e.g. SA and the SA-analogue and the SA-analogue benzothiadiazole (BTH) in reported for e.g. SA recorded after mite infestation alone. Introduction of a time lag between first induction of plant plant of induction first between lag time a of Introduction alone. infestation mite after recorded mechanisms that are inducible by herbivory (Heil and Baldwin, 2002). In the case of priming, case of the are inducible 2002). In herbivory (Heil and Baldwin, mechanisms that by plants and a clear separation from the blend emitted by control plants. However, Gols plants and a clear separation from the blend emitted by control plants. However, sensitize the gene in such a way that a second challenge sensitize the gene in of herbivores using a small number generate of enhanced a primed state in terms defense gene transcription has previously been gain additional information from other HIPVs, such as MeSA and gain additional information from other HIPVs, such as MeSA great overlap, but also demonstrated that other volatiles, besides of plants with herbivores with and without simultaneous JA treatment do not only show a treatment do not only of plants with herbivores with and without simultaneous JA defense induction. fact, this corresponds In with behavioral results reported by Gols defense by JA and a second induction and a plant ability for enhanced did not impair by herbivory JA defense by distinguish prey-infested plants from non-prey infested plants. determine attractiveness of the volatile blend attractive to and at a later time point resulted in increased transcript levels. The ability of phytohormones The ability of phytohormones to resulted in increased at a later time point transcript levels. enables them to accelerate enhance and/or to a second challenge defense responses (Frost and particularly the combination of treatments, does. Qualitative and quantitative differences considerably reducing the cost of this mechanism (Van Hulten considerably reducing the cost of this mechanism (Van costly defense metabolites are not produced immediately upon a minor challenge, are not produced immediately costly defense metabolites thereby chemical analysis comparing the volatile profiles of 10 well-known major HIPVs emittedby 2008; Perazzolli (Takabayashi (Takabayashi (2003) mite for the predatory (2003) found that volatiles emitted by Lima bean plants in response (2003) found that volatiles emitted by Lima to a low dose of 0.1 mM et al. (E) JA do not attract the predator do not attract JA Chapter 3

Conclusion Application of a low dose of the phytohormone JA results in augmented transcript levels of a terpene biosynthetic gene and emission of a volatile metabolite crucial in plant indirect defense, when followed by a minor infestation of herbivores. This synergistic effect is observed irrespective of whether phytohormone and infestation occur simultaneously or sequentially, and might lead to a memory effect of plant indirect defense. Phytohormone application has thus the potential to induce enhanced biological pest control against spider mites. Moreover, this study provides information that indirect defense is stable in case of simultaneous and sequential attack by herbivores that induce similar signal transduction pathways in plants and may even be enhanced in the presence of multiple herbivores. However, the effect on other tritrophic interactions, other plants species, and the persistence of this effect require further investigation.

Chapter 3 - . β - 49 (E) 2002. . Plant . Plant 2009b. 2000. Herbivory- Brassica oleracea Lotus japonicus 2004. Herbivore-induced 2004. Herbivore-induced in 2007. The JAZ family of 2007. Erwinia carotovora 2004. Jasmonic acid is a key 2004. Jasmonic 2009a. Comparing induction at on Lima bean leaves: IV. Diurnal Diurnal and leaves: IV. on Lima bean 2008. Differential effects of jasmonic effects 2008. Differential attract parasitoids: Effects of time and attract parasitoids: Effects ocimene synthase - β - 2004. Conserved MYC transcription factors play (E) Spodoptera Spodoptera littoralis Brassica oleracea on the attraction of pollinators, parasitoids, and butterflies. 2001. Jasmonate-dependent induction of indole glucosinolates Effects of JA application on herbivore-induced volatile emission volatile herbivore-induced on application JA of Effects 1989. Biosynthesis of homoterpenes in higher plants. Helvetica Chimica Brassica nigra 2008. Effects of feeding 2008. Effects 1995. The octadecanoic pathway: Signal molecules for the regulation of secondary 1995. 2010. Inhibition of lipoxygenase affects induction of both direct and indirect plant 2010. Inhibition of lipoxygenase affects in Arabidopsis by culture filtrates of the nonspecific pathogen induced volatiles elicit defence genes in Lima bean leaves. Nature 406, 512-515. defence genes in Lima bean leaves. induced volatiles elicit Dicke M. Bruinsma M, van Broekhoven S, Poelman EH, Posthumus MA, Müller MJ, van Loon JJA, MJ, van Loon Müller MA, Posthumus Poelman EH, van Broekhoven S, Bruinsma M, Bruinsma M, Posthumus MA, Mumm R, Mueller MJ, Van Loon JJA, Dicke M. Loon R, Mueller MJ, Van Bruinsma M, Posthumus MA, Mumm Bruinsma M, Pang B, Mumm R, Van Loon JJA, Dicke M. Loon B, Mumm R, Van Pang Bruinsma M, Bruinsma M, IJdema H, Van Loon JJA, Dicke M. Loon IJdema H, Van Bruinsma M, Brader G, Tas É, Palva ET. Brader G, Tas Boland W, Gäbler A. Gäbler Boland W, S. A, Prat Abdeen Boter M, Ruíz-Rivero O, Blechert S, Brodschelm W, Holder S, Kammerer L, Kutchan TM, Mueller MJ, Xia ZQ, Mueller Kammerer TM, Kutchan Holder S, L, Blechert S, Brodschelm W, Boland W. Boland López-Vidriero I, Lozano FM, Ponce MR, Micol JL, Solano R. JL, Solano Ponce MR, Micol FM, Lozano I, López-Vidriero Physiology 126, 849-860. Plant Journal 29, 87-98. Physiology 135, 1976-1983. Plant Physiology 135, 2025-2037. Plant Physiology repressors is the missing link in jasmonate signalling. Nature 448, 666-671. regulator of spider mite-induced volatile terpenoid and methyl salicylate emission in tomato. salicylate emission and methyl volatile terpenoid spider mite-induced regulator of Entomologia Experimentalis et Applicata 128, 109-116. Entomologia Experimentalis et pathways. Proceedings of the National Academy of Sciences of the United States of America of the United States Sciences of Academy of the National pathways. Proceedings of nocturnal damage differentially initiate plant volatile emission. Plant Physiology 146, 965-973. Plant initiate plant volatile emission. nocturnal damage differentially Herbivore-induced ethylene in neighboring volatiles induce the emission of Lima bean plants. 92, 4099-4105. Zenk MH. ocimene and transcript accumulation of defences against herbivorous insects. Oecologia 162, 393-404. dose, and comparison with induction by herbivores. Journal of Experimental Botany 60, 2575- defense response in a model legume. Two-spotted spider mites induce emission of spider mites Two-spotted a model legume. defense response in an early and late step in signal transduction mediating indirect defence in acid treatment of a key role in jasmonate signaling both in tomato and Arabidopsis. Genes and Development a key role in jasmonate signaling both in tomato and 2587. Chini A, Fonseca S, Fernández G, Adie B, Chico JM, Lorenzo O, García-Casado G, O, Lorenzo JM, Chico Adie B, Fernández G, A, Fonseca S, Chini Acta 72, 247-253. Arimura GI, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J. Takabayashi W, Boland Nishioka T, T, Ozawa R, Shimoda Arimura GI, Arimura GI, Ozawa R, Nishioka T, Boland W, Koch T, Kühnemann F, Takabayashi J. Takabayashi Kühnemann F, T, Koch W, Boland Arimura GI, Ozawa R, Nishioka T, Arimura GI, Ozawa R, Kugimiya S, Takabayashi J, Bohlmann J. J, Bohlmann Takabayashi S, Ozawa R, Kugimiya Arimura GI, Arimura GI, Köpke S, Kunert M, Volpe V, David A, Brand P, Dabrowska P, Maffei ME, P, Dabrowska P, A, Brand David V, S, Kunert M, Volpe Köpke Arimura GI, Ament K, Kant MR, Sabelis MW, Haring MA, Schuurink RC. Schuurink Haring MA, Sabelis MW, Kant MR, Ament K, References Jasmonic acid-induced volatiles of Journal of Experimental Botany 60, 2589-2599. 18, 1577-1591. Chapter 3

Conrath U. 2009. Priming of induced plant defense responses. Plant Innate Immunity 51, 361-395.

Cooper WR, Jia L, Goggin L. 2005. Effects of jasmonate-induced defenses on root-knot nematode infection of resistant and susceptible tomato cultivars. Journal of Chemical Ecology 31, 1953-1967.

De Boer JG, Dicke M. 2004. The role of methyl salicylate in prey searching behavior of the predatory mite Phytoseiulus persimilis. Journal of Chemical Ecology 30, 255-271.

De Boer JG, Hordijk CA, Posthumus MA, Dicke M. 2008. Prey and non-prey arthropods sharing a host plant: Effects on induced volatile emission and predator attraction. Journal of Chemical Ecology 34, 281-290.

De Boer JG, Posthumus MA, Dicke M. 2004. Identification of volatiles that are used in discrimination between plants infested with prey or nonprey herbivores by a predatory mite. Journal of Chemical Ecology 30, 2215-2230.

De Moraes CM, Lewis WJ, Paré PW, Alborn HT, Tumlinson JH. 1998. Herbivore-infested plants selectively attract parasitoids. Nature 393, 570-573.

De Vos M, Van Oosten VR, Van Poecke RMP, Van Pelt JA, Pozo MJ, Mueller MJ, Buchala AJ, Métraux JP, Van Loon LC, Dicke M, Pieterse CMJ. 2005. Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Molecular Plant- Microbe Interactions 18, 923-937.

Dicke M, Gols R, Ludeking D, Posthumus MA. 1999. Jasmonic acid and herbivory differentially induce carnivore-attracting plant volatiles in Lima bean plants. Journal of Chemical Ecology 25, 1907-1922.

Dicke M, Van Loon JJA, Soler R. 2009. Chemical complexity of volatiles from plants induced by multiple attack. Nature Chemical Biology 5, 317-324.

Dicke M, Vet LEM. 1999. Plant-carnivore interactions: Evolutionary and ecological consequences for plant, herbivore and carnivore. Herbivores: Between Plants and Predators, 483-520.

Frost CJ, Mescher MC, Carlson JE, De Moraes CM. 2008. Plant defense priming against herbivores: Getting ready for a different battle. Plant Physiology 146, 818-824.

Hare JD. 2010. Ontogeny and season constrain the production of herbivore-inducible plant volatiles in the field. Journal of Chemical Ecology 36, 1363-1374.

Heil M, Baldwin IT. 2002. Fitness costs of induced resistance: Emerging experimental support for a slippery concept. Trends in Plant Science 7, 61-67.

Chapter 3

51 2013. 2013. 2009. Priming in Priming 2009. JASMONATE-INSENSITIVE1 1997. Jasmonate is essential for 1999. Differential induction of plant induction of 1999. Differential 2004. 2007. Olfactory response of a predatory 2009. A rapid wound signal activates the A 2009. 2007. Arabidopsis transcriptome changes 2007. 2000. Jasmonic acid induced resistance in . New Phytologist 200, 861-874. . New Phytologist 2002. Benzothiadiazole-induced potentiated priming for 2001. Analysis of relative gene expression data using real-time Analysis of 2001. 2002. Plant responses to insect herbivory: The emerging The emerging molecular 2002. Plant responses insect herbivory:to 2008. Recognition herbivory-associatedof molecular patterns. Plant 2001. Defensive function of herbivore-induced2001. Defensive function plant volatile emissions Arabidopsis thaliana Arabidopsis Effects of JA application on herbivore-induced volatile emission volatile herbivore-induced on application JA of Effects 2010. Variation in natural plant products and the attraction of bodyguards 2010. Variation gene in Arabidopsis. Plant Physiology 128, 1046-1056. Arabidopsis. Plant Physiology gene in 2008. Kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role involved in indirect plant defense. Canadian Journal of Zoology 88, 628-667. insect defense in Arabidopsis. Proceedings of the National Academy of Sciences of the United the Sciences of of Academy National the Arabidopsis. Proceedings of insect defense in in nature. Science 291, 2141-2144. in nature. Science 291, in response to phloem-feeding silverleaf whitefly nymphs. Similarities and distinctions in for redox modulation. Plant Physiology 147, 1358-1368. for redox modulation. Plant Physiology Mumm R, Dicke M. Arimura GI. J, Ozawa R, Takabayashi M, Yamamoto A, Nishihara M, A, Ramadan Muroi Moayeri HRS, Ashouri A, Poll L, Enkegaard A. Enkegaard A, Poll L, Ashouri Moayeri HRS, Mithöfer A, Boland W. Mithöfer McConn M, Creelman RA, Bell E, Mullet JE, Browse J. Mullet Creelman RA, Bell E, M, McConn Koornneef A, Leon-Reyes A, Ritsema T, Verhage A, Den Otter FC, Van Loon LC, Pieterse Loon A, Den Otter FC, Van Verhage T, A, Ritsema A, Leon-Reyes Koornneef Koo AJK, Gao X, Daniel Jones A, Howe GA. A, AJK, Gao X, Daniel Jones Koo Kohler A, Schwindling S, Conrath U. Conrath S, Schwindling A, Kohler Kessler A, Baldwin IT. A, Baldwin IT. Kessler W. Boland J, Engelberth V, Jung T, Krumm T, Koch Kessler A, Baldwin IT. IT. A, Baldwin Kessler Kempema LA, Cui X, Holzer FM, Walling LL. Walling Holzer FM, Cui X, Kempema LA, Kegge W, Weldegergis BT, Soler R, Eijk MVV, Dicke M, Voesenek LACJ, Pierik R. Pierik LACJ, Voesenek Dicke M, MVV, Eijk R, Soler BT, Weldegergis W, Kegge Lorenzo O, Chico JM, Sánchez-Serrano JJ, Solano R. Sánchez-Serrano JJ, Solano JM, Chico O, Lorenzo Livak KJ, Schmittgen TD. Schmittgen KJ, Livak Physiology 146, 825-831. regulated defense responses in Arabidopsis. Plant Cell 16, 1938-1950. regulated defense responses in responses to pathogen infection, wounding, and infiltration of water into leaves requires the responses to aphids. Plant Physiology 143, 849-865. responses to aphids. mirid to herbivore induced plant volatiles: Multiple herbivory vs. single herbivory. Journal of mirid to herbivore induced plant volatiles: Multiple herbivory vs. single herbivory. systemic synthesis of bioactive jasmonates in Arabidopsis. Plant Journal 59, 974-986. in systemic synthesis of bioactive jasmonates signaling pathway. Plant Physiology 121, 153-162. Plant signaling pathway. systemic plant immunity. Science 324, 89-91. Science immunity. systemic plant grapevines to a root and leaf feeder. Journal of Economic Entomology 93, 840-845. Journal of Economic grapevines to a root and leaf feeder. organic compounds in organic compounds quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25, 402-408. quantitative PCR and the 2(T)(-Delta Delta encodes a MYC transcription factor essential to discriminate between different jasmonate- encodes a MYC transcription factor essential to discriminate between different analysis. Annual Review of Plant Biology, 53, 299-328. of Plant Biology, Annual Review analysis. caused by inter-plant communications. PLOS ONE 6. Omer AD, Thaler JS, Granett J, Karban R. AD, Thaler JS, Granett J, Karban Omer States of America 94, 5473-5477. States of volatile biosynthesis in the Lima bean by early and late intermediates of the octadecanoid- 2011. 2011. The composite effect of transgenic plant volatiles for acquired immunity to herbivory CMJ. Canopy light cues affect emission of constitutive and methyl jasmonate-induced and methyl jasmonate-induced constitutive of emission volatile cues affect Canopy light NPR1/NIM1 Applied Entomology 131, 326-332. Jung HW, Tschaplinski TJ, Wang L, Glazebrook J, Greenberg JT. J, Greenberg Glazebrook L, Wang TJ, Tschaplinski HW, Jung Chapter 3

Ozawa R, Shiojiri K, Sabelis MW, Arimura GI, Nishioka T, Takabayashi J. 2004. Corn plants treated with jasmonic acid attract more specialist parasitoids, thereby increasing parasitization of the common armyworm. Journal of Chemical Ecology 30, 1797-1808.

Paré PW, Tumlinson JH. 1999. Plant volatiles as a defense against insect herbivores. Plant Physiology 121, 325-331.

Perazzolli M, Roatti B, Bozza E, Pertot I. 2011. Trichoderma harzianum T39 induces resistance against downy mildew by priming for defense without costs for grapevine. Biological Control 58, 74-82.

Pieterse CMJ, Dicke M. 2007. Plant interactions with microbes and insects: from molecular mechanisms to ecology. Trends in Plant Science 12, 564-569.

Ponzio C, Gols R, Pieterse CMJ, Dicke M. 2013. Ecological and phytohormonal aspects of plant volatile emission in response to single and dual infestations with herbivores and phytopathogens. Functional Ecology 27, 587-598.

Ryan CA. 2000. The systemin signaling pathway: Differential activation of plant defensive genes. Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology 1477, 112-121.

Schmelz EA, Alborn HT, Banchio E, Tumlinson JH. 2003. Quantitative relationships between induced jasmonic acid levels and volatile emission in Zea mays during Spodoptera exigua herbivory. Planta 216, 665-673.

Schulze B, Lauchli R, Sonwa MM, Schmidt A, Boland W. 2006. Profiling of structurally labile oxylipins in plants by in situ derivatization with pentafluorobenzyl hydroxylamine. Analytical Biochemistry 348, 269-283.

Snoeren TAL, van Poecke RMP, Dicke M. 2009. Multidisciplinary approach to unravelling the relative contribution of different oxylipins in indirect defense of Arabidopsis thaliana. Journal of Chemical Ecology 35, 1021-1031.

Stout MJ, Workman KV, Bostock RM, Duffey SS. 1998. Specificity of induced resistance in the tomato, Lycopersicon esculentum. Oecologia 113, 74-81.

Thaler JS, Humphrey PT, Whiteman NK. 2012. Evolution of jasmonate and salicylate signal crosstalk. Trends in Plant Science 17, 260-270.

Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Nomura K, He SY, Howe GA, Browse J. 2007. JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature 448, 661-665.

Thulke O, Conrath U. 1998. Salicylic acid has a dual role in the activation of defence-related genes in parsley. Plant Journal 14, 35-42.

Chapter 3 : ). 53 2009a. Lactuca sativa Arabidopsis thaliana expression patterns during 2006. Costs and benefits of BoLOX 2009b. Riboflavin-induced priming for 2007. Sensitivity and speed of induced 2007. Sensitivity and speed of induced 2008. Priming for plant defense in barley 2011. Effect of foliar salicylic acid and methyl salicylic acid and foliar of Effect 2011. . Journal of Integrative Plant Biology 51, 167-174. . Journal of Integrative Plant Biology 51, L.): Dynamics of L.): 2008. Genotype, age, tissue, and environment regulate the and tissue, 2008. Genotype, age, 2002. Induced parasitoid 2002. Induced parasitoid attraction by 2012. Involvement of OsJAZ8 in jasmonate-induced OsJAZ8 2012. Involvement of resistance to Effects of JA application on herbivore-induced volatile emission volatile herbivore-induced on application JA of Effects Brassica oleracea Arabidopsis thaliana 2012. Treating seeds with activators of plant defence generates long-lasting priming of Treating 2012. jasmonate jasmonate applications against pill-bugs on protection ( in lettuce plants bacterial blight in rice. Plant and Cell Physiology 53, 2060-2072. bacterial blight in rice. Plant and Cell Physiology insect and pathogen attack. Molecular Plant-Microbe Interactions 20, 1332-1345. insect and pathogen attack. Molecular Plant-Microbe MR. M, Soto-Zarazúa GM, Guevara-González RG. Guevara-González GM, Soto-Zarazúa M, Botany 53, 1793-1799. Pathology 73, 95-100. Phytoparasitica 39, 137-144. Phytoparasitica resistance to pests and pathogens. New Phytologist 193, 770-778. resistance to pests and pathogens. New Worrall D, Holroyd GH, Moore JP, Glowacz M, Croft P, Taylor JE, Paul ND, Roberts Paul JE, Taylor Croft P, M, Glowacz JP, Moore GH, Holroyd D, Worrall Wentzell AM, Kliebenstein DJ. Kliebenstein AM, Wentzell Walters DR, Paterson L, Walsh DJ, Havis ND. Walsh L, DR, Paterson Walters pathogen defense in provides benefits only underhigh disease pressure. Physiological and Molecular Plant priming for defense in Arabidopsis. Proceedings of the National Academy of Sciences of the of Academy of Sciences Arabidopsis.Proceedings the National of defense in priming for Zheng SJ, Van Dijk JP, Bruinsma M, Dicke M. Dijk JP, SJ, Van Zheng Zhang S, Yang X, Sun M, Sun F, Deng S, Dong H. M, Sun F, X, Sun Yang Zhang S, Zhang PJ, Zheng SJ, Van Loon JJA, Boland W, David A, Mumm R, Dicke M. R, Mumm A, David W, JJA, Boland Loon Van SJ, Zheng PJ, Zhang Whiteflies interfere with indirect plant defense against spider mites in Lima bean. Proceedings bean. Lima in mites spider against defense plant indirect with interfere Whiteflies structural outcome of glucosinolate activation. Plant Physiology 147, 415-429. structural outcome of glucosinolate activation. United States of America 103, 5602-5607. America 103, United States of of the National Academy of Sciences of the United States of America 106, 21202-21207. of the United States of Academy of Sciences of the National of multiple internal control genes. Genome biology 3. of multiple internal control Involvement of the octadecanoid and the salicylic acid pathway. Journal of Experimental Journal of Experimental octadecanoid Involvement of the salicylic acid pathway. and the defense of cabbage ( defense of 2002. Accurate 2002. normalization of real-time quantitative data RT-PCR by geometric averaging García E, Mendoza-Diaz SO, Feregrino-Pérez AA, Mercado-Luna A, Vargas-Hernandez Vargas-Hernandez A, AA, Mercado-Luna Feregrino-Pérez SO, Mendoza-Diaz García E, Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Speleman F. A, De Paepe Roy N, Van B, Poppe Pattyn F, De Preter K, J, Vandesompele Van Hulten M, Pelser M, Van Loon LC, Pieterse CMJ, Ton J. Pieterse LC, Loon CMJ, Ton M, Van Hulten M, Pelser Van Dicke M. Poecke RMP, Van

Akimitsu K, Gomi K. Akimitsu K, Gomi Tierranegra-García N, Salinas-Soto P, Torres-Pacheco I, Ocampo-Velázquez RV, Rico- RV, Ocampo-Velázquez I, Torres-Pacheco P, N, Salinas-Soto Tierranegra-García Yamada S, Kano A, Tamaoki D, Miyamoto A, Shishido H, Miyoshi S, Taniguchi S, Taniguchi S, Miyoshi H, Shishido A, Miyamoto D, Tamaoki A, Kano S, Yamada 54 Chapter 4 Transcriptional responses of Lima bean defense genes to feeding or oral secretions of Mamestra brassicae L. and Spodoptera exigua Hübner caterpillars Tila R. Menzel, Joop J.A. van Loon, Marcel Dicke

55 Chapter 4

Abstract Recognition of herbivorous attackers comprises an integral part of plant defense and is essential for an adequate defense response to a specific attacker. Caterpillar feeding as well as application of caterpillar oral secretion on mechanical wounds, are frequently used to induce plant defense against biting-chewing insects. Here, we used feeding damage or the combination of mechanical damage and oral secretions of the generalist caterpillar species Mamestra brassicae and Spodoptera exigua to study the damage- and species-specific effects on transcript levels of three defense-related genes in Lima bean (Phaseolus lunatus), i.e. P. lunatus lipoxygenase (PlLOX), P. lunatus β-ocimene synthase (PlOS), P. lunatus acidic pathogenesis-related protein 4 (PlPR-4). Since induction of defense can affect subsequent herbivores, we also investigated how the induction of defense genes by feeding damage or mechanical damage plus oral secretion affected the defense response to subsequent herbivory by the spider mites Tetranychus urticae. While patterns of gene transcription were mostly identical in response to the two caterpillar species, feeding damage or mechanical damage plus caterpillar oral secretion caused differential induction of the transcription of defense genes. Nevertheless, compared to plants with single herbivory, plants with dual herbivory only showed differential gene induction for PlPR-4. Lima bean plants respond differently to caterpillar feeding than to mechanical damage plus caterpillar oral secretion, resulting in different effects on plant direct and indirect defense against subsequent herbivores.

Chapter 4 , et et 57 et al. et , 2014). et al. , 2014). It has , 2004; Schmelz et al. , 1998). et al. . Herbivory by caterpillars Herbivory by . , 2002; Stam et al. , 1990; Herms and Mattson, , 1990; Herms and et al. et al. , 2007). Plant defenses , 2007). Plant can be constitutive , 2011). The information conveyed can relate The information 2011). , , 1995; De Moraes et al. Tetranychus urticae Tetranychus et al. et al. , 2002). This is the result of a trade-off between of a trade-off This is the result , 2002). , 1997). Plants possess a sophisticated and well- a sophisticated 1997). Plants possess , Effect of feeding and oral secretions of caterpillars secretions oral and of feeding Effect et al. et al. et , 2005; Hanley , 2005; Hanley et al. , 2005). This system allows plants to mount an effective defense to mount an effective to allows plants This system 2005)., , 2011). Elicitors isolated from caterpillar oral secretions identified , 1995; McCloud and Baldwin, 1997; Roda , 2014). Plants can recognize attackers through elicitors released by et al. et et al. et al. et al. , 2007; Stam , 1990; Mattiacci , 2007). thus far are structurally diverse, comprising fatty acid amino acid conjugates (FACs) and thus far are structurally diverse, comprising fatty acid amino acid conjugates (FACs) to herbivore species and developmental stage, which suggests that plants can discriminate to constitutive defense (Karban constitutive defense (Karban to herbivores during activities such as feeding and oviposition, (reviewed in (Hilker and Meiners, feeding and oviposition, herbivores duringactivities such as been hypothesized that plants distinguish herbivores based on their feeding modes. Piercing- between the octadecanoid and the shikimate pathways, which is thought to play an important play an important thought to is shikimate pathways, which octadecanoid and the between the belonging to different feeding guilds. The main defense pathways are the octadecanoid feeding guilds. belonging to different between different attackers (Takabayashi attackers (Takabayashi between different herbivorous arthropods well as to other attackers such as pathogens. as Furthermore, it has such insects, beneficial to status attack the on information convey can plants that shown been Plants are frequently attacked by herbivorous arthropods, which can cause considerable with mounting defenses (Strauss role in fine tuning plant defense (Koornneef and Pieterse, 2008; Wei pathway, shikimate pathway, and ethylene pathway with jasmonic (JA), salicylic acid acid (SA), shikimate pathway, pathway, plant defense and plant growth and reproduction plant defense and plant (Baldwin Here we investigated the effect of feeding or a combination of standardized mechanical Here we investigated the effect same genes when induction by the caterpillar treatments was followed by infestation by a infestation by was followed by caterpillar treatments the same genes when induction by generalistspider mite second herbivore, the sucking herbivores, such as whiteflies, mainly induce SA and leaf-chewing herbivores and species (e.g. De Vos De Vos species (e.g. on the induction of Lima bean defense genes. Moreover, we investigated the effect on the effect we investigated the Lima bean defense genes. Moreover, inductionof on the orchestrated signaling network that results in responses that are specific for different attacker different for specific are that responses in results that network signaling orchestrated or induced, the latter only being activated in response to attack. Plant defense mechanisms attack. activated in response to only being or induced, the latter In fact plants are known to activate distinct defense pathways in response In fact plants are known to activate distinct to herbivores damage and application of oral secretions to the wounded tissue of two generalist caterpillars detrimental detrimental effects on plant fitness.Consequently, defense mechanisms ranging from morphological structures to the production of toxic plants have developedsophisticated and spider mites is known to induce JA-related defense (Li enzymes, and have been used to effectively mimic feeding of caterpillars (e.g. (Turlings feeding mimic of caterpillars (e.g. (Turlings enzymes, and have been used to effectively and ethylene (ET) as their respective signaling molecules. There is antagonistic crosstalk There their respective signaling as molecules. and ethylene (ET) as natural enemies of herbivores, and distant tissues or other plants (Rodriguez-Saona other plants (Rodriguez-Saona herbivores, and distant tissues or natural enemies of as are considered quite costly in the absence of an attacker due to allocation costs associated cell content feeders, such as caterpillars and spider mites, induce especially JA (Kempema cell content feeders, such as caterpillars and spider mites, induce especially JA compounds compounds (Schoonhoven 2010; Bonaventure 2009; Mumm and Dicke, 2010; Ramadan 2009; Mumm and Dicke, 2010; Ramadan et al. al. al. Introduction 1992). Induced defense can be very specific and provideadaptive advantage in comparison Chapter 4

Phaseolus lunatus lipoxygenase (PlLOX) and Phaseolus lunatus β-ocimene synthase (PlOS) are two JA-inducible genes (Arimura et al., 2000; Arimura et al., 2008). The gene PlLOX is located early in the octadecanoid pathway, while PlOS is located downstream and codes for a rate-limiting step in the biosynthesis of the inducible plant volatile (E)-β-ocimene, which is known to attract the predatory mite Phytoseiulus persimilis, a natural enemy of T. urticae (Dicke et al., 1990; Ament et al., 2004). The gene PlPR-4 codes for an acidic chitinase, which is methyl salicylate-responsive (Margis-Pinheiro et al., 1991; Arimura et al., 2000). We hypothesized that caterpillars would equally upregulate JA defenses, regardless of induction method and species, and that caterpillar pre-treatment would have a positive effect on plant defense against the second herbivore, T. urticae.

Materials and Methods

Plants and insects Lima bean plants (Phaseolus lunatus L., cv Wonderbush) were sown and grown in a greenhouse compartment at 23 ± 2 °C with 60 ± 10 % R.H., and a photoperiod of 16L : 8D. Plants were used for experiments 12 - 15 days after sowing, when their primary leaves had fully expanded. For the duration of experiments, plants were kept in a climate chamber and incubated at 25 ± 1 °C, 60 ± 10 % R.H. and 16L : 8D. In the climate chamber, plants of different treatments were kept separate in metal-frame cages with polyethylene sheet walls (90 x 90 x 60 cm). Each cage was connected to the house vacuum to prevent volatile transfer between plants with different treatments.

Two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae), were reared on Lima bean plants in a different greenhouse compartment under the same conditions as the Lima bean plants. Randomly selected adult female mites were used for experiments.

Cabbage moth, Mamestra brassicae L. (Lepidoptera: Noctuidae) and beet armyworm, Spodoptera exigua Hϋbner (Lepidoptera: Noctuidae), caterpillars were reared on cabbage plants (Brassica oleracea var. gemmifera cv. Cyrus, Syngenta seeds BV, Enkhuizen, The Netherlands) at 22 ± 1 °C and 50 - 70 % R.H., under the same photoregime as the Lima bean plants. Prior to experiments S. exigua, but not M. brassicae, caterpillars were transferred to feed on Lima bean plants for 24 h. Oral secretion of each caterpillar species was collected from 20 randomly selected caterpillars in the 5th instar.

For feeding treatments, eggs of S. exigua and M. brassicae were obtained from the general cultures in our laboratory (Smits et al., 1986; Menzel et al., 2014). Paper sheets containing the egg batches were then placed on Lima bean plants and kept in a climate chamber at 22 ± 1 and 50 - 70 % R.H. Once caterpillars hatched, Lima bean plants were added as food source if necessary and larvae in the 3rd instar were used for feeding experiments.

Chapter 4 , M. or 59 (2014). T. urticae T. . All plants et al. M. brassicae T. urticae T. were evenly distributed over the caterpillars that fed for 48 h, but 48 h, fed for caterpillars that , their webbing, feces were removed and T. urticae T. M. brassicae M. L3 caterpillar per clip cage was inoculated on that were evenly distributed over the two primary T. urticae T. Effect of feeding and oral secretions of caterpillars secretions oral and of feeding Effect T. urticae T. M. brassicae M. oral secretion as described above. After 48 h of incubation, 20 oral secretion as described above. infestation, iv) caterpillar feeding followed by infestation with iv) mechanical damage iv) mechanical plus caterpillar of oral secretion M. brassicae were evenly distributed over the primary leaves. Mites, their webbing, and Mites, primary leaves. were evenly distributed over the T. urticae T. S. exigua or respectively. Plants of all treatments received treatments received Plants of all one clip (diam. 2.5 cm) supported cage respectively. T. urticae T. leaves. lines ca. 7 cm length of leaves on the primary and then incubated until sample collection. For two primary leaves. After 48 h of infestation After 48 two primary leaves. the primary leaves. After 48 h caterpillars After 48 and their feces were carefully removed using a the primary leaves. treatment. For treatment ii) one treatment For treatment. treatment iii) two L3 caterpillars of either species were inoculated, each confined in a separate a in confined each inoculated, were species either of caterpillars L3 two iii) treatment biological replicate consisted of plant leaf material pooled from primary leaves of three plants. by sticks per primary leaf. Control by sticks per primary plants i) did not receive until further treatment sample immediately after removal of caterpillars, 20 female removal of immediately after infestation, iii) inflicted by the pattern wheel, and distributed using a fine paint brush. Plants of all treatments all of Plants brush. paint fine a using distributed and wheel, pattern the by inflicted female feces were removed with a fine paint brush after 48 h ofTreatment infestation. v) consisted Plant material was collected and processed as previously described in Menzel Plant treatments consisted of i) control, ii) caterpillar feeding followed by a period without Plant treatments consisted Plant treatments control of i) ii) mechanical damage iii) caterpillar feeding by with a fineTreatment paint iv) brush. was done by inflicting artificial damage and application were incubated for 48 h. Clip cages and caterpillars were removed shortly before sample received 2.5 cm) per primary leaf. Control plants i) did not receive further one clip cage (diam. Experiments were repeated up to seven times, whereby not all treatments were included whereby not all treatments in Experiments were repeated up to seven times, For treatment iii) plants were also exposed to For same manner as for treatment ii) and then applying caterpillar oral secretion onto the wounds. on ice before application. Using water and kept with tap was diluted 1:1 stock The species. of a 48 h infestation by 20 female of 10 µl of diluted of µl 10 of brassicae In short, four biological replicates were collected for each treatment per experiment. Each each experiment. a pipette, 10 µl of the diluted caterpillar oral secretion stock was applied onto the wounds RNA extraction and cDNA synthesis cDNA and extraction RNA collection and plants were carefully cleaned from feces with a fine paint brush. collection and plants were carefully cleaned clip cage on a primary leaf. Treatment iv) consisted of artificially damaging the plants in the collection. For treatment ii), plants were artificially damaged using a pattern wheel to draw 6 fine paint brush and plants were subsequently incubated for another 48 h without herbivores. v) incubation with caterpillar oral secretion followed by infestation with Oral secretion consisted Oral secretion consisted of a pooled stock collected from 20 caterpillar of either caterpillar Treatments Effects of M. brassicae feeding and oral secretion on subsequent herbivory subsequent on secretion oral and feeding brassicae of M. Effects Effects of feeding and mechanical damage plus oral secretion of the two caterpillar species caterpillar two the of secretion oral plus damage mechanical and feeding of Effects S. exigua Chapter 4

Quantitative RT-PCR To quantify transcript levels of P. lunatus β-ocimene synthase (PlOS; GenBank accession EU194553), P. lunatus acidic pathogenesis-related protein 4 (PlPR-4), P. lunatus lipoxygenase (PlLOX; GenBank accession X63521), and the two reference genes P. lunatus Actin1 (PlACT1; GenBank accession DQ159907) and P. lunatus Nuclear matrix protein 1 (PlNMP1; GenBank accession AF289260.1), real-time quantitative RT-PCR was performed in a Rotor- Gene 6000 machine (Corbett Research) with a 72-well rotor. For a detailed description refer to Menzel et al. (2014). PlOS primers were F-PlOS 5’-TGCATGGGTCTCAGTCTCTG-3’ and R-PlOS 5’-TGCTGCTTCCCCTCTCTCTA-3’, PlPR-4 were F-PlPR-4 5’-ACGCTTTCCTCAGTGCTCTC-3’ and R-PlPR-4 5’-TCCTCGTCGTCGCAGTAATCCTT-3’, PlLOX primers were F-PlLOX 5’-GGAATGGGACAGGGTTTATG-3’ and R-PlLOX 5’- CAAAGTCACTGGGCTTCTCA-3’, PlACT1 primers were F-PlACT1 5’-CCAAGGCTAACCGTGAAAAG-3’ and R-PlACT1 5’-AGCCAGATCAAGACGAAGGA-3’, and PlNMP1 primers F-PlNMP1 5’-CCGGAATGGAGTGTTGACGAGCA-3’ and R-PlNMP1 5’-CCAGCTCAGAAACATCTGGCAATGG-3’. Gene transcripts were quantified using the 2(-Delta Delta Ct) method (Livak and Schmittgen, 2001), using a normalization factor (Vandesompele et al., 2002). The latter was calculated by geometrically averaging the threshold cycle (Ct) values from the two reference genes PlACT1 and PlNMP1. Subtraction of the normalization factor from Ct values normalizes for differences in cDNA synthesis.

Statistical analysis Gene transcription data were log-transformed and analyzed by one-way ANOVA followed by Tukey’s Honestly Significant Difference (HSD) for pairwise comparisons between treatments in the statistical software SPSS version 19 (SPSS Inc., Chicago, IL, USA). If assumptions on normality and equal variance were violated, data were analyzed by Kruskal-Wallis tests followed by Mann-Whitney U tests with a Bonferroni correction as post-hoc tests.

Results

Effects of feeding or mechanical damage plus oral secretion of the two caterpillar species on gene transcription Relative quantification ofPlOS transcripts showed that treatments significantly affectedPlOS transcript levels (Kruskal Wallis test for both caterpillar species P < 0.01; Fig. 1). Plants treated with mechanical damage or mechanical damage plus caterpillar oral secretion did not show significant differences inPlOS transcript levels compared to control plants or when compared to each other. In contrast, feeding by two M. brassicae or S. exigua caterpillars resulted in significantly higher PlOS transcript levels compared to control plants and other treatments (Fig. 1).

Chapter 4 61 0.05). 0.05). . Plant . Plant P < P P <

. Plant treatments were were treatments . Plant Spodoptera exigua Spodoptera exigua Spodoptera < 0.001). However, the treatments the treatments < 0.001). However,

P. lunatus (E)-β-ocimene synthase (PlOS) (E)-β-ocimene lunatus P. were also significantlyaffected by different the P. lunatus lipoxygenase (PlLOX) lipoxygenase lunatus P. Effect of feeding and oral secretions of caterpillars secretions oral and of feeding Effect PlLOX transcript levels compared to the levels in control plants

PlLOX < 0.001 for both caterpillar species; Fig. 2). All three treatments, < 0.001 for both caterpillar species; Fig. 2). P Mamestra brassicae Mamestra brassicae Mamestra

(Kruskal Wallis test followed by Mann-Whitney U tests with Bonferroni correction, with Bonferroni U tests Mann-Whitney followed by test (Kruskal Wallis treatments were i) no treatment (control), ii) mechanical damage at 48 hours post treatment ii) mechanical post treatment 48 hours (control), at damage i) no treatment were treatments (mechanical 48 h (caterpillar(hpt) iv) feeding), and iii) caterpillar damage), feeding for (caterpillar oral 48 hpt caterpillaroral secretion of at mechanical application plus damage above letters 8-28 biological of (n). Different the mean (± SE) are replicates secretion). Values in transcript treatments barslevels within significantbetween a panel indicate differences Fig. 1. Relative gene transcript levels transcript gene of 1. Relative Fig. treatments (ANOVA,

Relative gene transcript levels of namely mechanical caterpillar damage, feeding, and mechanical caterpillar oral damage with showed no significant differences in transcript levels amongst each other. in transcript levels amongst showed no significant differences secretion led to increased (post hoc Tukey’s HSD for all pairwise comparisons Tukey’s (post hoc caterpillar oral secretion at 48 hpt (caterpillar oral secretion). Values are the mean (± SE) of 4-20 of the mean (± SE) are (caterpillar oral secretion). Values 48 hpt caterpillar oral secretion at abovesignificant bars indicate a differences within panel letters biological (n). Different replicates test, HSD post followed by hoc Tukey’s (ANOVA levelsin transcript between treatments Fig. 2. Relative gene transcript levels transcript gene of 2. Relative Fig. (mechanical (hpt) ii) mechanical post treatment 48 hours (control), at damage i) no treatment 48 h (caterpillar iv) mechanical feeding), and iii) caterpillardamage), feeding for with damage

Chapter 4

Transcript levels of PlPR-4 were significantly affected by M. brassicae and S. exigua treatments (Fig. 3, Kruskal Wallis test, P < 0.01). Whereas mechanical damage and caterpillar feeding did not significantly alter transcript levels compared to control levels, the application of M. brassicae and S. exigua caterpillar oral secretion resulted in an increase of PlPR-4 levels (Kruskal Wallis test followed by Mann-Whitney U tests with Bonferroni correction, P ≤ 0.001). The transcript level induced by mechanical damage plus M. brassicae oral secretion treatment was not significantly different from levels found in plants fed upon by M. brassicae or mechanical damage alone. In contrast, the PlPR-4 transcript level induced by S. exigua oral secretion treatment was significantly different from levels found in plants that had only been mechanically damaged, but also not different from plants fed upon by S. exigua. Mamestra brassicae Spodoptera exigua

Fig. 3. Relative gene transcript levels of P. lunatus pathogenesis related protein 4 (PlPR- 4). Plant treatments were i) no treatment (control), ii) mechanical damage at 48 hours post treatment (hpt) (mechanical damage), iii) caterpillar feeding for 48 h (caterpillar feeding), and iv) mechanical damage with caterpillar oral secretion at 48 hpt (caterpillar oral secretion). Values are the mean (± SE) of 4-28 biological replicates (n). Values within a panel having no letters in common above the bar were significantly different (Kruskal Wallis test followed by Mann-Whitney U tests with Bonferroni correction, P < 0.05).

Effects ofMamestra brassicae feeding or oral secretion application on gene induction by subsequent spider mite herbivory

Relative transcript levels of PlOS and PlPR-4 were significantly affected by treatments (Fig. 4, ANOVA, P < 0.001 for both genes). Plants infested for 48 h by T. urticae showed significantly increased PlOS transcript levels compared to control, irrespective of whether they had received a prior treatment with M. brassicae feeding or oral secretion (post hoc Tukey’s HSD, P < 0.001). However, there was no significant difference among T. urticae treatments, thus prior caterpillar treatments did not affect PlOS transcript levels compared to a T. urticae infestation alone (post hoc Tukey’s HSD, P > 0.05). Transcript levels of plants which had been fed upon by M. brassicae followed by 48 h without herbivory, showed transcript levels similar to those of control plants for PlOS and PlPR-4. Yet, prior treatment of plants with M. brassicae oral secretion resulted in higher PlPR-4 transcript levels after spider mite treatment compared to plants treated with T. urticae infestation alone (post hoc Tukey’s HSD, P < 0.01). Relative transcript levels of PlLOX were not significantly affected by treatments (Fig. 4 B).

Chapter 4 ), 63 feeding oral secretion feeding for 48 feeding for M. brassicae ), iv) mechanicaliv) ), damage oral secretion + T.u. (M.b. T.u. P. lunatus levels P. transcript gene of 4. Relative Fig. lunatus synthase (PlOS),(E)-β-ocimene P. lunatus pathogenesis P. , and (PlLOX) lipoxygenase were treatments . Plant 4 (PlPR-4) protein related ii) (control), no treatment i) for 48 h followed by 48 h without infestation infestation 48 h followed 48 h without by for feeding),M. iii) brassicae (M.b. urticae with 20 T. h followed 48 h infestation by feeding + (M.b. and application of M. brassicae of application and 48 h. Values urticae for with 20 T. iv) infestation in common no letters within having a panel significantly different barthe were above Mann-Whitney followed by test (Kruskal Wallis P < 0.05). correction, with Bonferroni U tests incubated for 48 h followed by 48 h infestation h infestation 48 h followed 48 by for incubated urticae with 20 T.

Effect of feeding and oral secretions of caterpillars secretions oral and of feeding Effect

Chapter 4

Discussion Plant defenses are fine-tuned to such an extent that plants respond differently depending on herbivore species, life stage, and condition. It has been previously shown that oral secretions of a broad range of lepidopterans contain FACs, which are known as potent inducers of plant direct and indirect defenses (Roda et al., 2004; Yoshinaga et al., 2010). Here we investigated the effects of caterpillar feeding and the application of oral secretion of two generalist caterpillar species on gene transcription of relevant defense genes in Lima bean plants that are involved in defense signaling and synthesis of defense metabolites. Our results showed that caterpillar species did not cause differential effects on gene transcription of PlOS, PlLOX or PlPR-4, suggesting that plants did not distinguish between species. Interestingly, caterpillar feeding and application of caterpillar oral secretion exerted significantly different effects on gene transcript levels. Whereas oral secretions did not increase PlOS transcripts above control levels, caterpillar feeding caused a strong increase in PlOS transcript levels. The latter is in accordance with findings by De Boer et al. (2008) who found that feeding by S. exigua causes an increase in the corresponding volatile (E)-β-ocimene in Lima bean plants. The monoterpene (E)-β-ocimene plays an important role in plant indirect defense in many plant species (Dicke et al., 1990; Arimura et al., 2000; Zhang et al., 2009; Muroi et al., 2011). It is likely that the observed difference in PlOS levels for these treatments were caused by the temporal differences in defense stimulation by mechanical damage plus oral secretion application and continuous feeding. Continuous feeding by caterpillars results in a repeated stimulation of plant defense via removal of plant tissue and thereby repeated application of oral secretion (Vadassery et al., 2012). In contrast, in our experiments mechanical damage with application of caterpillar oral secretion consisted of a single stimulation of defense which may subside quickly. Mithöfer et al. (2005) showed that rhythmic leaf removal was necessary to induce a volatile profile that closely resembles the volatile profile induced by S. littoralis feeding. Their study also showed that this was not the case for mechanical damage induced by one-time damaging with a pattern wheel; however, one-time mechanical damage was not combined with caterpillar oral secretion application in their study. Moreover, we can exclude the possibility that oral secretion was inactive or did not contain elicitors because PlPR-4 was significantly higher induced in plants that had received caterpillar oral secretion fromS. exigua compared to mechanical damage. Furthermore, caterpillar feeding and mechanical damage, either or not combined with application of oral secretions to the damaged tissue all induced PlLOX, a gene which is located early in the octadecanoid pathway and necessary for wound- induced JA accumulation (Bell et al., 1995). This suggests that JA and the octadecanoid pathway were indeed induced by all these treatments, leading to a generic wound response. However, PlOS, downstream the JA-signaling cascade, was activated only by caterpillar feeding at the time point investigated. It is less likely that the observed differences in our study were caused by the different diets of caterpillars used for feeding damage and oral secretion collection as it has been shown previously that the induction of defense and relative amounts of conjugates is diet and instar independent (Turlings et al., 1993; Pohnert et al., 1999).

Chapter 4

S. et et 65 et al. on Lima T. urticae T. . De Boer , 2009; Zhang 2009; , T. urticae T. et al. et PlPR-4 transcript levels were and PlPR-4 S. exigua , but only for , 2008; Dicke 2008; , et al. et PlLOX mites avoid leaves previously mites avoid leaves fed upon or did not show differences in defense response in defense response show differences did not PlOS T. urticae T. . Since lepidopterans, and certain cell-content feeders, Since lepidopterans, certain cell-content and . were not affected by the treatments, might be due to were not affected Effect of feeding and oral secretions of caterpillars secretions oral and of feeding Effect F. and mite oviposition was reduced on plants with F. , 2000). Chitinases digestiveare enzymes that degrade T. urticae T. PlLOX et al. T. urticae T. codes for a methyl salicylate-responsive codes for a methyl salicylate-responsive acidic chitinase (Margis- via direct defense mechanisms. In fact, we found an upregulation feeding or mechanical damage plus oral secretion application on mechanical on feeding or oral secretion plus damage application PlPR-4 T. urticae T. Spodoptera litura , mainly induce the octadecanoid pathway, and also volatile induction by and also volatile mainly induce the octadecanoid, pathway, , 1991; Arimura transcript levels when plants were treated with oral secretionbefore levels when transcript et al. M. brassicae T. urticae T. compared to control plants. This suggests that herbivory by caterpillars can also induce herbivory that This suggests control plants. compared to , 1986; Kramer and Muthukrishnan, 1997). Nevertheless, , 1986; Kramer and Muthukrishnan, 1997). , 2014). PlPR-4 , 2000). In our experiments, however, we used not only lower numbers of herbivores, but not only lower numbers of we used our experiments, however, 2000). In , , 2009). In our study, defense induction caterpillar by feeding or oral secretion which was , 2009). In our study, by the caterpillar both herbivores is regulated by this pathway, such an effect can be expected (Ozawa an effect such both herbivores is regulated by this pathway, bean indirect defense against bean indirect defense herbivore (e.g. (Kessler and Baldwin, 2004; De Boer and Baldwin, 2004; (Kessler (e.g. herbivore induction by caterpillar feeding and application of caterpillar oral secretion. include the activation of more specific defense gene induction and defense metabolites (Stam metabolites defense and induction gene defense specific more of activation the include its upstream location in the octadecanoid pathway. It has been suggested that early defense its upstream location in the octadecanoid pathway. infestation. The gene induction by the first herbivore can have a significant effect on the resistance against a second against resistance the on effect significant a have can herbivore first the by induction feeding. The latter confirms that there differencesare in defense gene induction between followed by herbivory by the mite the by followed by herbivory Plants differentially respond to caterpillar feeding and the application of caterpillar oral Plants differentially Pinheiro resistance against not significantly upregulated at the observed time point in plants that experienced caterpillar plants also received herbivore damage sequentially rather than simultaneously. Nevertheless, Nevertheless, simultaneously. than rather sequentially damage herbivore received also plants litura second herbivore gene expression appeared not to be affected by the differences in defense by the differences second herbivore gene expression appeared not to be affected secretion. The two caterpillar species had a similar effect on defense response in terms The two caterpillar species had a similar effect secretion. signaling includes phytohormone-mediated activation, while later responses defense pathway second herbivore. such as gene induction by subsequent spider mite herbivory mite spider subsequent by induction gene glycosidic bonds in chitin, which makes up the cell walls of plant pathogens and is a major of gene induction. Nevertheless, when defense induction was followed by infestation by a infestation by gene induction. Nevertheless,when defense induction was followed by of of In nature, plants are often attacked by more than one herbivore, by more plants are often attacked In nature, whereby the defense caterpillar oral secretions and caterpillar feeding, which influence the defense response to a component of the peritrophic membrane in the gut of herbivorous arthropods (Schlumbaum compared to single herbivory by the mite for Effect of Choh and Takabayashi that (2010) found Takabayashi Choh and (2008) found a synergistic effect of simultaneous feeding of (2008) found a synergistic effect et al. et al. al. al. Conclusion The fact that transcript levels of Chapter 4

Arimura GI, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J. 2000. Herbivory- induced volatiles elicit defence genes in Lima bean leaves. Nature 406, 512-515.

Baldwin IT, Sims CL, Kean SE. 1990. The reproductive consequences associated with inducible alkaloidal responses in wild tobacco. Ecology 71, 252-262.

Bonaventure G, VanDoorn A, Baldwin IT. 2011. Herbivore-associated elicitors: FAC signaling and metabolism. Trends in Plant Science 16, 294-299.

Choh Y, Takabayashi J. 2010. Predator avoidance by phytophagous mites is affected by the presence of herbivores in a neighboring patch. Journal of Chemical Ecology 36, 614-619.

De Moraes CM, Lewis WJ, Paré PW, Alborn HT, Tumlinson JH. 1998. Herbivore-infested plants selectively attract parasitoids. Nature 393, 570-573.

Dicke M, Van Loon JJA, Soler R. 2009. Chemical complexity of volatiles from plants induced by multiple attack. Nature Chemical Biology 5, 317-324.

Hanley ME, Lamont BB, Fairbanks MM, Rafferty CM. 2007. Plant structural traits and their role in anti-herbivore defence. Perspectives in Plant Ecology, Evolution and Systematics 8, 157-178.

Herms DA, Mattson WJ. 1992. The dilemma of plants: To grow or defend. Quarterly Review of Biology 67, 283-335.

Hilker M, Meiners T. 2010. How do plants “notice” attack by herbivorous arthropods? Biological Reviews 85, 267-280.

Karban R, Agrawal AA, Mangel M. 1997. The benefits of induced defenses against herbivores. Ecology 78, 1351-1355.

Chapter 4

67 1991. 2014. on Lima Nicotiana . Planta 203, 430- 2000. Involvement of Spodoptera littoralis 1999. New fatty acid amides from Nicotiana sylvestris 2002. Resistance of cultivated tomato to 2002. Resistance 2007. Arabidopsis changes transcriptome 2007. -Glucosidase: An elicitor of herbivore-induced An elicitor of herbivore-induced -Glucosidase: β 1995. Effect of feeding and oral secretions of caterpillars secretions oral and of feeding Effect 2005. Effects of feeding of 2005. Effects 1997. Insect chitinases: Molecular 1997. Insect chitinases: biology and potential use 2008. Cross talk in defense signaling. Plant Physiology 146, in defense signaling. Plant 2008. Cross talk 2001. Analysis of relative gene expression data using real-time relative gene Analysis of 2001. 1997. Herbivory and caterpillar regurgitants amplify the wound- 2004. Herbivore-induced plant vaccination. Part I. The orchestration The 2004. Herbivore-induced plant vaccination. I. Part 2010. Variation in natural plant products and the attraction of bodyguards in natural plant products and 2010. Variation . Plant Journal 38, 639-649. . Plant Journal jasmonate- and salicylate-related signaling pathways for the production of specific herbivore- bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to induced volatiles in plants. Plant and Cell Physiology 41, 391-398. involved in indirect plant defense. Canadian Journal of Zoology 88, 628-667. involved in indirect plant defense. Canadian induced increases in jasmonic acid but not nicotine in in response to phloem-feeding silverleaf whitefly nymphs. Similarities and distinctions in Muroi A, Ramadan A, Nishihara M, Yamamoto M, Ozawa R, Takabayashi J, Arimura GI. J, Ozawa R, Takabayashi M, Yamamoto A, Nishihara M, A, Ramadan Muroi Mumm R, Dicke M. Mithöfer A, Wanner G, Boland W. Boland G, Wanner A, Mithöfer Menzel TR, Weldegergis BT, David A, Boland W, Gols R, Van Loon JJA, Dicke Loon M. Gols R, Van A, Boland W, David BT, Menzel TR, Weldegergis McCloud ES, Baldwin IT. ES, Baldwin McCloud Mattiacci L, Dicke M, Posthumus MA. Mattiacci L, Margis-Pinheiro M, Metz-Boutigue MH, Awade A, de Tapia M, le Ret M, Burkard G. Burkard M, le Ret M, A, de Tapia Awade MH, Metz-Boutigue M, Margis-Pinheiro Pohnert G, Jung V, Haukioja E, Lempa K, Boland W. Haukioja E, Lempa Pohnert G, Jung V, Kramer KJ, Muthukrishnan S. Kramer KJ, Muthukrishnan Koornneef A, Pieterse CMJ. A, Koornneef Kessler A, Baldwin IT. IT. A, Baldwin Kessler Kempema LA, Cui X, Holzer FM, Walling LL. Walling FM, Holzer Cui X, LA, Kempema Livak KJ, Schmittgen TD. Schmittgen KJ, Livak Li C, Williams MM, Loh YT, Gyu IL, Howe GA. Howe Gyu IL, YT, MM, Loh C, Williams Li Physiology 130, 494-503. regurgitant of Lepidopteran (Noctuidae, Geometridae) caterpillars. Tetrahedron 55, 11275- Tetrahedron regurgitant of Lepidopteran (Noctuidae, Geometridae) caterpillars. responses to aphids. Plant Physiology 143, 849-865. to aphids. Plant Physiology responses plant odor that attracts host-searching parasitic wasps. Proceedings of the National Academy Proceedings host-searching parasitic wasps. the National plant odor that attracts of 839-844. of Sciences of the United States of America 92, 2036-2040. America 92, of Sciences of the United States of of plant defenses in nature and their fitness consequences in the tobacco wild Isolation of a complementary DNA encoding the bean an acidic PR4 chitinase: enzyme with Isolation of a complementary DNA quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25, 402-408. the 2(T)(-Delta Delta C) method. Methods quantitative PCR and elicit herbivory-related volatile emission. Plant Physiology 137, 1160-1168. elicit herbivory-related volatile emission. emission by Lima bean plants: transcription of a monoterpene a of emission by Lima bean plants: transcription synthase gene and volatile 65, 4821-4831. emission. Journal of Experimental Botany an amino-terminus cysteine-rich domain. Plant Molecular Biology 17, 243-253. an amino-terminus cysteine-rich domain. as biopesticides. Insect Biochemistry and Molecular Biology 27, 887-900. Biochemistry and Molecular Biology as biopesticides. Insect caused by inter-plant communications. PLOS ONE 6. cell content-feeding herbivores is regulated by the octadecanoid-signaling pathway. Plant cell content-feeding herbivores is regulated the octadecanoid-signaling by pathway. Ozawa R, Arimura GI, Takabayashi J, Shimoda T, Nishioka T. Nishioka T. J, Shimoda T, Arimura GI, Takabayashi Ozawa R, Synergism in the effect of prior jasmonic acid application prior herbivore-induced of on volatile Synergism in the effect 2011. 2011. The composite effect of transgenic plant volatiles for acquired immunity to herbivory attenuata 435. 11280. Chapter 4

Ramadan A, Muroi A, Arimura GI. 2011. Herbivore-induced maize volatiles serve as priming cues for resistance against post-attack by the specialist armyworm Mythimna separata. Journal of Plant Interactions 6, 155-158.

Roda A, Halitschke R, Steppuhn A, Baldwin IT. 2004. Individual variability in herbivore- specific elicitors from the plant’s perspective. Molecular Ecology 13, 2421-2433.

Rodriguez-Saona CR, Rodriguez-Saona LE, Frost CJ. 2009. Herbivore-induced volatiles in the perennial shrub, Vaccinium corymbosum, and their role in inter-branch signaling. Journal of Chemical Ecology 35, 163-175.

Schlumbaum A, Mauch F, Vögeli U, Boller T. 1986. Plant chitinases are potent inhibitors of fungal growth. Nature 324, 365-367.

Schmelz EA, LeClere S, Carroll MJ, Alborn HT, Teal PEA. 2007. Cowpea chloroplastic ATP synthase is the source of multiple plant defense elicitors during insect herbivory. Plant Physiology 144, 793-805.

Schoonhoven LM, Van Loon JJA, Dicke M. 2005. Insect-Plant Biology. Oxford: Oxford University Press.

Smits PH, Van De Vrie M, Vlak JM. 1986. Oviposition of beet armyworm (Lepidoptera: Noctuidae) on greenhouse crops. Environmental Entomology 15, 1189-1191.

Stam JM, Kroes A, Li Y, Gols R, Van Loon JJA, Poelman EH, Dicke M. 2014. Plant interactions with multiple insect herbivores: From community to genes. Annual Review of Plant Biology 65, 689–713.

Strauss SY, Rudgers JA, Lau JA, Irwin RE. 2002. Direct and ecological costs of resistance to herbivory. Trends in Ecology and Evolution 17, 278-285.

Turlings TCJ, McCall PJ, Alborn HT, Tumlinson JH. 1993. An elicitor in caterpillar oral secretions that induces corn seedlings to emit chemical signals attractive to parasitic wasps. Journal of Chemical Ecology 19, 411-425.

Turlings TCJ, Tumlinson JH, Lewis WJ. 1990. Exploitation of herbivore-induced plant odors by host-seeking parasitic wasps. Science 250, 1251-1253.

Vadassery J, Reichelt M, Mithöfer A. 2012. Direct proof of ingested food regurgitation by Spodoptera littoralis caterpillars during feeding on Arabidopsis. Journal of Chemical Ecology 38, 865-872.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3.

Yoshinaga N, Alborn HT, Nakanishi T, Suckling DM, Nishida R, Tumlinson JH, Mori N. 2010. Fatty acid-amino acid conjugates diversification in lepidopteran caterpillars. Journal of Chemical Ecology 36, 319-325.

Chapter 4 69 2009. Effect of feeding and oral secretions of caterpillars secretions oral and of feeding Effect Zhang PJ, Zheng SJ, Van Loon JJA, Boland W, David A, Mumm R, Dicke M. Dicke R, Mumm A, David W, Boland JJA, Loon Van SJ, Zheng PJ, Zhang Whiteflies interfere with indirect plant defense against spider mites in Lima bean. Proceedings bean. Lima in mites spider against defense plant indirect with interfere Whiteflies of the National Academy of Sciences of the United States of America 106, 21202-21207. America States of Sciences of the United Academy of of the National 70 Chapter 5 Effect of sequential induction by Mamestra brassicae L. and Tetranychus urticae Koch on Lima bean plant indirect defense Tila R. Menzel, Tze-Yi Huang, Berhane T. Weldegergis, Rieta Gols, Joop J.A. van Loon, Marcel Dicke

In press in Journal of Chemical Ecology.

71 Chapter 5

Abstract Attack by multiple herbivores often leads to modification of induced plant defenses compared to single herbivory, yet little is known about the effects on induced indirect plant defense. Here, we investigated the effect of sequential induction of plant defense by Mamestra brassicae caterpillar oral secretion and an infestation by Tetranychus urticae spider mites on the expression of indirect plant defense in Lima bean plants. The effect on indirect defense was assessed using behavior assays with the specialist predatory mite Phytoseiulus persimilis in an olfactometer, headspace analysis of 11 major herbivore-induced plant volatiles (HIPVs) including (E)-β-ocimene and transcript levels of the corresponding gene Phaseolus lunatus β-ocimene synthase (PlOS). Predatory mites were found to distinguish between plants induced by spider mites and caterpillar oral secretion but not between plants with single spider mite infestation and plants induced by caterpillar oral secretion prior to spider mite infestation. Indeed, the volatile blends emitted by plants induced by spider mites only and the sequential induction treatment of caterpillar oral secretion followed by spider mite infestation, were similar. Our results suggest that plant indirect defense is not affected by previous treatment with oral secretion of M. brassicae caterpillars.

72 Chapter 5 , , 73 et al. et al. et , 2013). , , 2008), , , 2010). , , 2008). -induced Boisduval et al. et et al. et al. et et al. Hübner, and Hübner, -infested Lima (Erb S. littoralis S. 2008; Zhang , 2014). However, most 2014). However, , T. urticae T. , 2000; Van Poecke and 2000; Van , et al. , 2009; Rodriguez-Saona , 2009; Zhang , 1999; Kessler and Baldwin 1999; Kessler and et al. et S. littoralis S. et al. et et al. Spodoptera littoralis , 2011). Multiple herbivory can , 2011). et al. et Spodoptera exigua , 2008). However, simultaneous , 2008). However, et al. et al. , 2010). et al. et al. , 2007; De Boer Kirshbaum and et al. Sequential induction by caterpillar and mites and by caterpillar induction Sequential , 2013). The few studies on indirect plant defense , 2013). Koch, results in a synergistic increase in volatile Koch, , 2004; Erb et al. 2010; Schwartzberg et al. et al. , 2003; Delphia Athias-Henriot (De Boer , 2009) show that volatile blends of multiple herbivore-attacked , 2009). The underlying mechanism for the differences in the effect effect in the differences the The underlying mechanism for 2009). , , 2004; Koornneef and Pieterse, 2008). Depending 2004; Koornneef and Pieterse, , on the signaling et al. Euscelidius variegatus Tetranychus urticae Tetranychus , 2011; Mathur , 2011; et al. et al. et et al. , 2013; Erb et al. et al. , 2013), allowing natural enemiesto distinguish between plants infested by prey et al. Phytoseiulus persimilis , 2010; Erb thereby differentially modulate defense responses. Multiple defense pathways and alter plant thereby differentially bean plants (Zhang herbivory is subject to an increasing (Stam studies herbivory is subject to number of known to play modulating roles via pathway cross-talk (Ozawa play modulating roles known to known as indirect plant defense. Phytohormones, such as jasmonic acid (JA), salicylic acid jasmonic acid (JA), Phytohormones, such as indirect plant defense. known as known as herbivore-induced plant volatiles Natural enemies (HIPVs). of herbivorous influence indirect plant defense in a positive, neutral, or negative manner. In Lima bean plants, bean Lima In manner. negative or neutral, positive, a in defense plant indirect influence feeding of the cicadellid for example, simultaneous feeding of non-prey caterpillars, Phytohormone crosstalk may also be involved in interactions between herbivores that attack attack that Phytohormone crosstalk may also be involved in interactions between herbivores Dicke, 2002; Ament Plants that are under attack by herbivores produce and release complex mixtures of volatiles, volatiles, complex mixtures of and release herbivores produce by are under attack Plants that whereby prior feeding by one herbivore can result in a kind of “vaccination” which can responses to herbivory. The phytohormone JA and its volatile derivative methyl jasmonate volatile its and phytohormone JA The herbivory. responses to mite prey, the spider mite the prey, mechanisms (Dicke plants can differ quantitatively or qualitatively compared to single induction (Shiojiri pathways induced by an herbivore, the composition of HIPV blends can differ significantly play an important role in indirect plant defense (e.g. Dicke Whitefly infestation has a negative effect on predator attraction to of multiple herbivory is likely phytohormone cross-talk (Zhang of of these studies focus on the effect on direct plant defenses and plant-mediated on direct plant defenses interactions effect on the these studies focus of In nature, prey and non-prey herbivores can simultaneously feed on the same plant and nature, In a plant in temporally spaced (Kessler events and Baldwin, 2004; Poelman emission and an increased attraction of a natural enemy of the spider mite, i.e. the predatory amongst the herbivores (Kaplan and Denno, 2007; Brunissen and non-prey herbivores (De Boer and Dicke (2010). The phenomenon release is enemies via HIPV recruitment of natural of (2010).and Dicke arthropods arthropods to locate can use these HIPVs their herbivorous (reviewed prey or host by Mumm caterpillars on maize plants did not alter volatile emission differently from differently alter volatile emission caterpillars on maize plants did not Sequential herbivory can have long-lasting effects on plant defenses (Poelman volatile emission and did not affect behavior of a parasitoid of behavior of volatile emission not affect and did 2001; Rodriguez-Saona 2009; Zhang 2002). However, other phytohormones, such as salicylic acid (SA) and ethylene (ET) are (ET) and ethylene such as salicylic acid (SA) other phytohormones, 2002). However, (Zhang (MeJA) are the main signaling molecules for induction of plant defense against herbivores and plant defense signaling (MeJA) are the main of induction molecules for (SA) and ethylene (ET) are involved in generating are involved (ET) (SA) and ethylene modulatingand defense induced plant et al. Introduction Chapter 5 affect direct and indirect plant defenses against a later-arriving second herbivore (Kessler and Baldwin, 2004). Voelckel and Baldwin (2004) suggest that the order/identity of arrival is crucial because some herbivore-induced stress effects on plant defense seem to be more stable than others. This is likely also dependent on the intensity and timing of subsequent defense inductions. In Zea mays seedlings, for example, a low dose of exogenously applied SA increases endogenous JA levels and volatile production upon a second induction by an insect-derived elicitor (Engelberth et al., 2011). However, higher doses resulted in reduced JA responses due to negative cross-talk. Moreover, shorter incubation times than 15 h with the phytohormone did not result in accumulation of JA or enhanced volatile production.

Volatile induction by leaf-chewing lepidopterans, and certain cell-content feeding herbivores, such as the spider mite Tetranychus urticae, is primarily regulated by the jasmonic acid pathway, and therefore antagonistic effects on plant defense are not expected between the two (Ozawa et al., 2000). Here, we investigated the effect of sequential induction of plant defense by M. brassicae caterpillar oral secretion and an infestation by T. urticae spider mites, which were temporally separated by a period of 48 h. We hypothesized that sequential induction of JA-induced plant defenses would result in increased attraction of P. persimilis through changes in the HIPV blend. Moreover, we investigated whether transcription levels of the JA-responsive gene Phaseolus lunatus β-ocimene synthase (PlOS), coding for a rate- limiting step in the biosynthesis of the spider-mite inducible plant volatile (E)-β-ocimene, would increase accordingly (Ament et al., 2004). The HIPV (E)- β-ocimene is known to be highly attractive to P. persimilis (Dicke et al., 1990).

Materials and Methods

Plants Lima bean plants (Phaseolus lunatus L., cv. Wonderbush, De Bruyn Seed Company, Michigan, USA) were cultivated in a greenhouse compartment at 23 ± 2 °C , 60 ± 10 % relative humidity (RH) and a 16 : 8 h light : dark (L : D) photoregime. Plants were grown in 5 × 5 cm plastic pots for gene transcription experiments or 11 × 11 cm for headspace volatile collection and behavioral experiments, respectively. After 12 - 14 days plants with two expanded primary leaves were transferred to a climate chamber and incubated at 25 ± 1 °C, 60 ± 10 % R.H. and 16 : 8 h L : D. In the climate chamber, plants of different treatments were kept separate in plastic cages (90 × 90 × 60 cm) that were connected to house vacuum to prevent volatile transfer between plants of different treatments.

Herbivores and predatory mites A colony of two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae), was maintained on Lima bean plants in a greenhouse compartment at 25 ± 5 °C, 50 – 70 % R.H., 16 : 8 h L : D. Adult female spider mites for experiments were selected randomly from the colony.

74 Chapter 5 75 colony. Only gravid female predatory colony. T. urticae T. Sequential induction by caterpillar and mites and by caterpillar induction Sequential received per 20 mites plant, followed by another Athias-Henriot (Acari: Phytoseiidae), were reared in L. (Lepidoptera: L. (Lepidoptera: Noctuidae) caterpillars on reared were T. urticae T. var. gemmifera cv. Cyrus, Syngenta seeds BV, Enkhuizen, The Enkhuizen, seeds BV, Cyrus, Syngenta gemmifera cv. var. caterpillars using a glass Pasteur pipette. The stock was diluted caterpillars pipette. using a glass Pasteur Phytoseiulus persimilis B. oleracea B. Mamestra brassicae Mamestra M. brassicae under same conditionsthe as the leaves were treated with 10 µl per leaf of diluted caterpillar oral secretion using a fine paint treatment or single infestation by the application with oral secretion, plants were incubated for 48 h. Control plants and plants brush for application. Oral secretion consisted of a pooled stock, freshly collected from 18 - freshly collected from brush for application.a pooled stock, Oral secretion consisted of kept in a climate chamber and incubated separately according to treatment in cages. Samples treatment climate chamber and incubated separately according to in a kept incubation). Plants treated with caterpillar oral secretion were first artificially damaged with a in the experimental room prior to the experiment in order to acclimate. room prior to the experiment in order to in the experimental for transcriptional analysis were taken 6, 20, 26, or 46 h following treatment. followed with 20 mites (48 h incubation), by infestation v) caterpillar oral secretion (96 h Netherlands) Netherlands) the same photoregime 50 - 70 % R.H., under at 22 ± 1 °C and as for plants and Plant treatments included i) control, ii) infestation with 20 spider mites, and iii) caterpillar oral control, ii) infestation with 20 spider mites, included i) Plant treatments Plants were kept in a climate chamber and incubated in groups separated by treatment in the treatment separated by climate chamber and incubated in groups in a Plants were kept Plant treatments were: i) control, ii) infestation with 20 spider mites (48 h incubation), iii) were: i) control, ii) infestation Plant treatments Predatory mites, which were heavily infested with 9 cm) on detached Lima bean leaves Petri dishes (diameter were sampled for transcriptional, volatile or behavioral analysis. Plants with dual induction receiving single infestation by spider mites did not receive mechanical damage or oral secretion. randomly selected from the colony. randomly pattern wheel by drawing six lines of ca. 7 cm length on each primary leaf. Then the damaged on each primary leaf. ca. 7 cm length pattern wheel by drawing six lines of piece of moist cotton wool to avoid dehydration. piece of moist cotton h and left Predatory mites were starved for 24 mites were used for behavioral experiments, ca. 1 - 2 days after their final molt. For behavioral For molt. final their after days 2 - 1 ca. experiments, behavioral for used were mites mites. Oral secretions were collected from 18 - 20 caterpillars in the 5th instar, which were were collectedOral secretions mites. the 5th instar, - 20 caterpillars in from 18 secretion. Plant treatments were executed the same as in the previous section. Plants were experiments the females were individually confined in 0.5 ml Eppendorf tubes containing a cages described above. After 48 h, plants to be analyzed 48 h, plants After for single induction by oral secretion cages described above. caterpillar oral secretion (48 h incubation), iv) caterpillar oral secretion (48 h incubation) oral secretion (48 h caterpillar incubation), iv) caterpillar oral secretion (48 h cabbage plants ( cabbage plants Plant treatments 20 5th instar Cabbage Cabbage moth, Sequential induction experiments induction Sequential 48 h of incubation before sample collection. Time series experiment series Time 1:1 with tap water and on ice prior kept to use to avoid degradation of compounds. Upon T. urticae T. Chapter 5

Y-tube olfactometer Responses of predatory mites were tested in a Y-tube olfactometer (Takabayashi & Dicke 1992). A Y-shaped metal wire was located in the center of a glass Y-tube, each arm was connected to a 5-L glass jar. Glass jars containing plants were connected to air inlets providing a 2 L/min charcoal-filtered air influx to carry volatiles into the two arms of the Y-tube. For behavior experiments, plant pots and loose soil were gently removed and roots with soil were carefully wrapped in aluminum foil. Three plants of a treatment were placed in a glass jar as odor source and the system was purged for 30 - 60 min without closing the vessels. Afterwards glass jars were sealed with viton-lined glass lids and the whole Y-tube olfactometer setup was flushed with air for 7 - 10 min before commencing the behavior experiment. Individual predatory mites were placed downwind on the Y-shaped wire and their choice for either odor source recorded when they passed a line located halfway up one of the two olfactometer arms or no-choice was recorded when they had not passed the line within 5 min. Sides of treatments were alternated after every five predatory mites to avoid positional bias. Plants were replaced after every 20 predatory mites or approximately 90 min after the first mite was tested, whichever came first. Each comparison was tested on two different days with 40 - 60 predatory mites per day.

Dynamic headspace collection of plant volatiles Plants were prepared for volatile collection by gently removing pots and loose soil, and wrapping roots with soil in aluminum foil. Two plants of each treatment were transferred to a 5-L glass jar. Glass jars were sealed with viton-lined glass lids equipped with an air inlet and outlet. The setup was flushed with 100 ml/min synthetic air (Linde Gas Benelux B.V., The Netherlands) filtered by passing through charcoal before entering the glass jar. Glass jars with plant samples were flushed with air for 30 - 45 min. A stainless steel tube filled with 200 mg Tenax TA (20/35 mesh; CAMSCO, Houston, TX, USA) was connected to the outlet of each glass jar and volatile collection was done by sucking air out of the jars at 100 ml/min for 2 h. A total of eight replicates of each treatment were sampled over two days. Fresh weight of above-ground plants tissue was determined immediately after volatile collection using an analytical balance (NewClassic ML, Mettler Toledo, Greifensee, Switzerland).

Chemical analysis of plant volatiles A Trace Ultra gas chromatograph (GC) coupled with Trace DSQ quadrupole mass spectrometer (MS) both from Thermo (Thermo Fisher Scientific, Waltham, USA) were used for separation and identification of plant volatiles as described previously (Menzel et al. 2014).

Standards of (Z)-3-hexen-1-ol, (Z)-3-hexen-1-ol, acetate, (Z)-3-hexen-1-ol, butanoate, (Z)- 3-hexen-1-ol, isovalerate, linalool, methyl salicylate, indole, (E)-β-ocimene, as well as alloocimene were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Additional standards (E)-4,8-dimethylnona-1,3,7-triene [(E)-DMNT] and (E,E)-4,8,12-trimethyltrideca-1,3,7,11- tetraene [(E,E)-TMTT] were kindly provided by Prof. W. Boland (Max Planck Institute for Chemical Ecology, Jena, Germany). For quantification, calibration lines were constructed for

76 Chapter 5

77 PlOS PlPR-4 PlACT1 PlNMP1 P. lunatus P. ; GenBank accession primers were F- PlOS acidic pathogenesis-related PlACT1 ( ; GenBank accession AF289260.1). GenBank accession ; (2014). primers were F- 5’-AGCCAGATCAAGACGAAGGA-3’, 5’-AGCCAGATCAAGACGAAGGA-3’, 5’-TGCTGCTTCCCCTCTCTCTA-3’, 5’-TGCTGCTTCCCCTCTCTCTA-3’, et al. PlNMP1 ( PlOS P. lunatus Actin1 P. PlACT1 PlACT1 Sequential induction by caterpillar and mites and by caterpillar induction Sequential 5’-CCGGAATGGAGTGTTGACGAGCA-3’ and R- 5’-CCGGAATGGAGTGTTGACGAGCA-3’ 5’-ACGCTTTCCTCAGTGCTCTC-3’ and R- 5’-ACGCTTTCCTCAGTGCTCTC-3’ ; GenBank accession EU194553),; PlNMP1 PlPR-4 PlOS ( ), and the two reference genes P. lunatus Nuclear matrix protein 1 P. primers F- PlPR-4 ( were F- PlNMP1 ocimene synthase - the statistical software SPSS version 19 (SPSS Inc., Chicago, IL, USA). Data that violated Data that USA). Chicago, IL, Inc., version 19 (SPSS the statistical software SPSS thawing. Total thawing. RNA Total was isolated and purified using the Qiagen(Hilden, Germany) RNeasy treatments using paint brush. Plant material was obtained a soft discs out by cutting four leaf for cDNA synthesis. cDNA was generated from total RNA by using the Bio-Rad iScript cDNA Bio-Rad iScript cDNA by using the total RNA was generated from synthesis. cDNA cDNA for Kruskal-Wallis followed tests by Mann-Whitney U tests applying the Bonferroni correction for Kruskal-Wallis Log transformation was applied to data from gene transcription experiments and volatiles in DQ159907) and Plant Mini kit with integrated DNAse treatment, according to manufacturer’s instructions. RNA manufacturer’s to according integrated DNAse treatment, with Plant Mini kit RNA instructions. using one-way ANOVA or generalized linear model (GLM) followed by Tukey’s Honestly Tukey’s followed by or generalized linear model (GLM) ANOVA using one-way using a NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, DE, Wilmington, Technologies, using a NanoDrop® spectrophotometer (NanoDrop ND-1000 multiple comparisons. primary leaf of three plants were pooled to yield one biological replicate. All samples were All yield one biological pooled to three plants were replicate. primary leaf of leaf material was homogenized processing. Frozen avoiding using mortar and pestle while 5’-CCAGCTCAGAAACATCTGGCAATGG-3’. 5’-CCAAGGCTAACCGTGAAAAG-3’ and R- 5’-CCAAGGCTAACCGTGAAAAG-3’ 5’-TCCTCGTCGTCGCAGTAATCCTT-3’, 5’-TCCTCGTCGTCGCAGTAATCCTT-3’, 5’-TGCATGGGTCTCAGTCTCTG-3’ and R- 5’-TGCATGGGTCTCAGTCTCTG-3’ Statistical analysis Statistical synthesis kit (Bio-Rad, Hercules, CA, USA) according to manufacturer’ssynthesis kit (Bio-Rad, Hercules, CA, USA) instructions. shock-frozen in liquid nitrogen immediately after collection and then stored at - 80 °C until further until °C 80 - at stored then and collection after immediately nitrogen liquid in shock-frozen USA). Only RNA samples with 260 / 280 wavelength ratio > 2 and a RIN value > 7 were used 7 value > a RIN 2 and 280 wavelength ratio > samples with 260 / Only RNA USA). order to meet assumptions of normality and homogeneity meet assumptions of of variances. Data were analyzed order to of one primary leaf per plant using a cork borer (diameter 2 cm). Leaf discs obtained plant using a cork borer (diameter 2 of one primary leaf per from the data point. data point. quality was assessed Agilent using 2100 Bioanalyzer with the 6000 RNA Nano Labchip® kit assumptions on normality and equal variance after log transformation were analyzed by and a 72 - well rotor; for a detailed description see Menzel each compound using seven data points at different concentrations and two replicates of each of each two replicates and concentrations at different data points using seven compound each β RNA extraction and cDNA synthesis cDNA and extraction RNA Quantitative RT-PCR was performed in a Rotor-Gene 6000 machine (Corbett Research) with Quantitative RT-PCR Significant Difference (HSD) post-hoc tests for pairwise comparisons between treatments in Spider mites, eggs, feces and webbing were gently removed from plants of the respective respective the plants of removed from webbing were gently and feces eggs, Spider mites, protein 4 PlPR-4 (all from Agilent Santa Technologies, Clara, CA, USA) and RNA quantifications were done Quantitative RT-PCR Quantitative A A real-time quantitative RT-PCR was used to quantify gene transcript levels of Chapter 5

Predator choices in the Y-tube olfactometer experiments were analyzed using a binomial test to examine whether the choice distribution significantly differed from 50 : 50.

Volatile profiles of plants exposed to different treatments were analyzed using multivariate data analysis. Data were expressed per unit of plant fresh weight, log-transformed, and univariate- scaled. Then an Orthogonal Projection to Latent Structures-Discriminant Analysis (OPLS-DA) was performed using the software SIMCA P+ version 12 (Umetrics, Umeå, Sweden). Pairwise comparisons for individual volatiles among treatments were executed using Mann-Whitney U tests.

Results

Response of predatory mites to single or multiple herbivore infestation The attraction of predatory mites to Lima bean plants exposed to different treatments was tested in a two-choice behavioral assay (Fig. 1). Feeding by T. urticae, with and without prior treatment with M. brassicae oral secretion, were preferred over control plants (binomial test, P < 0.001 in both comparisons). However, predators did not distinguish between control plants and plants treated with M. brassicae oral secretion alone (binomial test, P > 0.05). Moreover, predatory mites showed a preference for odors from plants infested by their prey compared to odors from plants induced by caterpillar oral secretion (binomial test, P < 0.001). The volatile blend of plants induced by the combination of non-prey oral secretion and prey herbivores was more attractive than the volatile blend of plants induced by oral secretion of M. brassicae (binomial test, P <0.001). Predators did not display a significant preference when they were offered plants infested by prey herbivores versus volatiles from plants induced by the combination of non-prey oral secretion and prey herbivores (binomial test, P > 0.05). Fig. 1. Responses of P. persimilis in a Y-tube olfactometer to volatiles emitted by Lima bean plants induced by mechanical damage and oral secretion of non-prey (M. brassicae; M.b.), prey (T. urticae; T.u.) infestation, or a combination of the two (M.b.+ T.u.). Volatile sources consisted of three Lima bean plants per treatment, i) control plants, or ii) plants infested with 20 T. urticae for 48 h, iii) induction by mechanical damage and M. brassicae oral secretion incubated for 48 h, or iv) induction by mechanical damage and M. brassicae oral secretion incubated for 48 h followed by infestation by 20 T. urticae for 48 h. Bars represent the overall percentages of predators choosing either odor source. Numbers in bars correspond to the number of predators choosing either odor source. For each comparison the number of mites that did not make a choice within 5 min ranged between zero and three. As- terisks indicate significance of predatory mite choices (binomial test; n.s. = not significant, *** P < 0.001).

78 Chapter 5 79

. . -3-hexen- T.u

T.u .

(Z)

, which were 7 0.5 M.b T.u

Ctrl M.b.+ M.b.+

T.u.

7 0.4 4 9 was most similar to

11 1 T. urticae T.

-ocimene, oral secretion followed 5 0.3 β

- 3 6

(E) 4 0.2

T. urticae T. 8

0.1 -TMTT, -TMTT, M. brassicae M.

2 ; eight replicates for for replicates ; eight 48 h (M.b.+T.u.) for 0.0 (E,E) infestation alone. Together with the third with the Together infestation alone.

plants with i) no treatment (Ctrl), or treated treated or (Ctrl), withi) no treatment plants

. ) and (2) ) 0.1 - PLS1 PLS1 %) (58.87 M.b

T.u. 0

Sequential induction by caterpillar and mites and by caterpillar induction Sequential ( versus treatments without T. urticae T. 0.2 - ), did not yield significant principal components and components principal significant yield not did ),

1 -

PLS1 PLS1 %) (58.87 T.u. 0.3 + -

2 T. urticae T. - T. urticae T.

oral secretion (Suppl. Information, Fig. S1 and Fig. S2). Fig. S1 and (Suppl. Information, oral secretion M.b. 0.4 - (

3 - 0.5 -

4 - T. urticae T. 0.6 -

5 M. brassicae , iv) M., iii) M. brassicae brassicae oral oral secretion(M.b.) 48 h 48 h (T.u.) for -

0.7 -

Ctrl 6 - 0.8

0.4 0.2 0.6 0.8

4.0 3.0 2.0 1.0 0.0 0.2 0.4 0.6

1.0 3.0 2.0 0.0

------

PLS2 (16.08 %) (16.08 PLS2

B A thus were not significantly different. than 1, thus contributing to discrimination most between Pairwise treatments. comparison the volatile blend from plants treated with plants the volatile blend from treatments: OPLS-DA resulted in a model with three significant principal components (Fig. to treatment with to treatment by infestation with 20 between the two treatments (1) 20 (1) treatments two between the including exposure to feeding by response to the treatments showed that volatile blends were significantly different between model showed that four volatile compounds, namely model showed that four principal component 89 % of the total variability of the data could be explained. Moreover, the principal component total variability 89 % of the could be explained. of the data Moreover, separated by the first principal component. The second component separated treatments with treatments separated component second The component. principal first the by separated of the combination treatment of caterpillar oral secretion and caterpillar oral secretion from treatments that did not include oral secretion. The volatile The blend caterpillar oral secretion from treatments that did not include oral secretion. Spider mite feeding Spider mite resulted in higher emission rates of several compounds compared 2). Figure 2B shows that the first distinction between treatments was made for treatments Comparison Comparison of volatile profiles consisting of the 11 major compounds HIPV emitted in each treatment. The first two principal components are depicted (panel A) with the percentage of are depicted A)with the(panel percentage principalcomponents two The first each treatment. 1) (Z)-3-hexen- in the loading (panel B) represent plot Numbers explained in parentheses. variation 3) (E)-β-ocimene 4) linalool, 5) (E)-4,8-dimethylnona-1,3,7-triene acetate, 2) (Z)-3-hexen-1-ol, 1-ol, salicylate, 8) methyl 6) alloocimene 9) (Z)-3-hexen-1- [(E)-DMNT], , 7) (Z)-3-hexen-1-ol,butanoate 10) indole, 11) (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene [(E,E)-TMTT].ol,isovalerate, T. urticae with ii) 20 T. urticae with 20 T. secretion 48 h followed infestation by Fig. 2. Multivariate data analysis by orthogonal PLS-DA (OPLS-DA) (panel A) and corresponding corresponding (panel A) and (OPLS-DA) PLS-DA orthogonal by analysis data 2. Multivariate Fig. lunatus P. of blends volatile loadingof (panel B) plot Volatile analysis Volatile 1-ol acetate, and alloocimene, 1-ol acetate, and alloocimene, variable had importance in the projection (VIP) values higher Chapter 5

Relative gene transcription Transcript levels for PlOS, the gene encoding for the enzyme mediating the rate-limiting step in the biosynthesis of (E)-β-ocimene, which is a principal attractant for P. persimilis (Dicke et al., 1990; Zhang et al., 2009), were compared between treatments. Treatments significantly affected PlOS transcript levels (GLM, P < 0.001; Fig. 3). Plants infested by T. urticae, with or without prior treatment with M. brassicae oral secretion, showed increased levels of PlOS compared to control plants and plants treated with M. brassicae oral secretion alone (post hoc Tukey’s HSD, P < 0.01). Oral secretion from M. brassicae did not increase PlOS transcript levels different from control (post hoc Tukey’s HSD, P > 0.05). Transcript levels of PlPR-4 were also affected by the treatments (ANOVA, F4,15 = 16.86 P < 0.001). Infestation by T. urticae and treatment with M. brassicae oral secretion both resulted in significantly increased PlPR-4 transcript levels compared to control plants (post hoc Tukey’s HSD, P < 0.001). Sequential treatment induced an increase in transcript levels compared to control plants, and transcript levels were even higher than in response to single treatments (post hoc Tukey’s HSD, P < 0.001).

Fig. 3. Relative gene transcript levels of P. lunatus β-ocimene synthase (PlOS) and P. lunatus pathogenesis-related protein 4 (PlPR-4) of plants i) without treatment (control), or treated with ii) 20 T. urticae for 48 h (T.u.), iii) M. brassicae oral secretion for 48 h [M.b. (48 h)], iv) M. brassicae oral secretion for 48 h followed by infestation with 20 T. urticae for 48 h (M.b.+T.u.), v) M. brassicae oral secretion 96 h [M.b.(96 h)]. Values are the mean (± SE) of 10 to 12 biological replicates, pooled from three replications of the same treatment. Different letters above bars indicate significant differences in transcript levels between treatments (Tukey’s HSD tests, P < 0.05). Gene transcript levels were normalized to the normalization factor obtained from geometrically averaging the Ct values of the two reference genes P. lunatus Actin1 (PlACT1) and P. lunatus nuclear matrix protein 1 (PlNMP1) for each sample.

Time series for relative PlOS gene transcription in response to single treatments To investigate PlOS gene transcript levels for plants infested with T. urticae and plants with caterpillar oral secretion treatment, a time series experiment was conducted. The time series of PlOS gene transcription for the single treatment with 20 T. urticae or M. brassicae oral secretion showed clear differences in the gene transcription patterns between the two treatments (Fig. 4). Infestation by 20 T. urticae led to 14 times higher transcript levels than in control plants already after 6 h post treatment (hpt). At 20 hpt expression levels of both the plants treated with M. brassicae oral secretion and plants exposed to T. urticae feeding

80 Chapter 5 81

a M.b. T.u. M.b. < 0.05), whereas whereas < 0.05), 46 hpt

a Ctrl . .

a T.u

a M.b. M.b. 26 hpt

a was lower. Treatment with caterpillar oral with Treatment lower. was Ctrl

PlOS b Sequential induction by caterpillar and mites and by caterpillar induction Sequential < 0.05). Before and after this time point transcript time point transcript and after this < 0.05). Before transcript levels compared to control levels but only but control levels to levels compared transcript

P b hpt M.b. T.u. T.u. M.b. PlOS for each sample. 1 (PlNMP1)for protein matrix nuclear lunatus P. 20

a Ctrl . .

b T.u < 0.05) within each point. Gene normalized levels time transcript were to

P a hpt ). Values are the mean (± SE) of four biological four of replicates. the mean (± SE) are ). Values oral secretion (M.b. M.b. M.b. 6 , urticae(T.u.) ii) 20 T. Ctrl), (control; with i) no treatment treated were plants

a Ctrl

8 6 4 2 0

P. lunatus P. 14 12 10 16 18 and and (PlACT1) Actin1 lunatus P. Relative gene transcription of of transcription gene Relative PlOS levels were not different from control levels. levels were not different found although the degree of upregulation of upregulation the degree of found although were significantly higher than control levels (ANOVA, Tukey’s HSDTukey’s tests, were significantly higher than control levels (ANOVA, secretion also led to an increase of to also led secretion at 20 hpt (ANOVA, Tukey’s HSD tests, HSD Tukey’s (ANOVA, at 20 hpt at 26 hpt these differences had disappeared. At 46 hpt the same pattern as at 6 hpt was pattern as at At 46 hpt the same had disappeared. these differences at 26 hpt over four time points separated by hours post treatment post separated treatment hours points by time four levels PlOS transcript over gene of Fig. 4. Relative (hpt). M. iii) brassicaeor Different letters above bars indicate significant differences in transcript levels between treatments transcript in levelssignificant treatments between above indicate differences bars letters Different tests, HSD (Tukey’s geometrically reference the two values from of obtained the Ct factor averaging the normalization genes Chapter 5

Discussion Plants are frequently attacked by multiple herbivores, which may arrive at different moments in time. The resulting sequential herbivory has been shown to have long-lasting effects on plant resistance against the subsequent herbivores (Viswanathan et al., 2007; Poelman et al., 2008; Mathur et al., 2011; Stam et al., 2014). However, little is known about the effect of sequential herbivory on plant indirect defense (but see e.g. Zhang et al., 2009; Erb et al., 2010; Zhang et al., 2013). Here, we investigated the effect of the sequential attack by different herbivore species on plant indirect defense against T. urticae spider mites through the attraction of the specialist predator P. persimilis. Previous studies suggest that plants can form memories after stressful events such as herbivory, which enables them to adjust their defense accordingly in order to respond in an enhanced manner to a second stress (Frost et al., 2008; Conrath, 2009). Our results show that prior treatment of plants with oral secretions of the generalist caterpillar M. brassicae, as a mimic of caterpillar feeding, does not affect the attraction of P. persimilis to plants infested with its prey T. urticae. The modulating effects caused by interactions with two herbivore species may thus depend on several factors such as severity of initial damage or infestation, timing between attacks, identity of the herbivore, and the associated defense pathway induced (Voelckel and Baldwin, 2004; Viswanathan et al., 2007; Dicke et al., 2009; Zhang et al., 2009). The defense pathway commonly induced by leaf-chewing insects such as M. brassicae, is the octadecanoid pathway with JA as signaling molecule (McConn et al., 1997; Kessler and Baldwin, 2002). The same defense pathway is also induced by feeding by cell-content feeders such as T. urticae (Dicke et al., 1999; Ozawa et al., 2000; Li et al., 2002). Multiple herbivory that occurs simultaneously by herbivores that induce the same pathway may lead to changes in volatile emission that results in increased attraction of predators (De Boer et al., 2008). In our study with sequential herbivory, predators did not distinguish between volatile blends from plants fed upon by T. urticae only and plants pre-treated with caterpillar oral secretions and subsequently exposed to feeding by T. urticae. Headspace analysis demonstrated that the volatile profiles of plants that had been exposed to these two treatments largely overlapped. It is thus possible that the initial defense induction was not strong enough to induce a memory effect or that the memory had decayed at the onset of the second attack by T. urticae. However, the volatile profile from plants with caterpillar oral secretion treatment was notably different compared to plants treated with T. urticae and predators were significantly more attracted to plants infested with their prey. This is in accordance with data of De Boer and co-workers (2008) who found that the specialist predator P. persimilis can distinguish between volatiles induced by prey and non-prey herbivores. Nevertheless, De Boer et al. (2008) found an increased attraction of P. persimilis to dual-infested Lima bean plants, which were fed upon by T. urticae and Spodoptera exigua caterpillars. In their study the dual infestation resulted in the emission of increased amounts of a subset of the plant volatiles. In the study by De Boer et al. (2008), the plants were exposed to simultaneous infestation with spider mites and S. exigua caterpillars. Whether the differentiation by the predators in their study and not in ours was due to the different caterpillar species, simultaneous compared to sequential treatments, or to caterpillar feeding instead of the use of oral secretion, remains to be elucidated.

82 Chapter 5

- - , , , β - is 83 (E) et al. (E,E) PlOS PlOS et al. et , also were , 2009). resulted T. urticae T. PlPR-4 transcript transcript PlOS et al. PlPR-4 induced PlOS PlOS transcript levels transcript levels. T. urticae T. oral secretion, and transcription peaked , 1991; Arimura PlOS PlOS S. littoralis S. , 1990; Zhang et al. PlOS -treated plants, the latter being -treated plants, the et al. , showed M. brassicae infestation and showed different infestation and showed different (2008) found that Lima bean plants (2008) found that T. urticae T. et al. et T. urticae T. -treated plants, spider mites do both emit large amounts of spider mites do both emit large amounts P. persimilis P. -3-hexen-1-ol acetate, and alloocimene, -3-hexen-1-ol were acetate, oral secretion on transcription of (1999) found that the emission that the emission (1999) found of the two rates (Z) Sequential induction by caterpillar and mites and by caterpillar induction Sequential et al. oral secretion. T. urticae T. T. urticae T. oral secretion resulted in increased is attracted to five HIPVs, two ofwhich, namely , 2014). To investigate the temporal effect of caterpillar caterpillar of investigate the temporal effect To 2014). , -DMNT, as well as the phenolic were involved ester MeSA, -DMNT, -ocimene, M. brassicae β - (E) , 1986; Kramer and Muthukrishnan, 1997). Consequently, other Consequently, 1997). Muthukrishnan, and Kramer 1986; , et al. et . The two other compounds, namely the terpene alcohol linalool two other compounds, namely the terpene The . (E) -ocimene are also known to play important roles in the attraction important play to -ocimene are also known M. brassicae -infested Lima bean plants (Dicke -infested Lima bean β et al. - feeding as well as application of M. brassicae P. persimilis P. transcript levels a time series experiment conducted. Results was transcript caterpillars or (E) and -TMTT, -TMTT, after 6 h. Continuous and thus feeding by infestation -TMTT and -TMTT transcript levels in three out of four time points. Moreover, transcript levels of four time points. Moreover, in three out PlOS P. persimilis P. T. urticae T. -ocimene, -ocimene, were indeed found to play a significant role in separating the (E,E) transcript levels of plants treated with caterpillartranscript levelsof oral secretion peaked at T. urticae T. β PlOS (E,E) - to PlOS S. exigua S. (2008) found a close relationship between JA levels and transcription of levels relationship (2008) found a close JA between T. urticae T. (E) PlOS et al. (2008) who found that feeding by the generalist (2008) who found that feeding by the caterpillar -ocimene. Moreover, it is likely that a time lag exists between gene transcription and -ocimene. Moreover, β - P. persimilis P. levels of plants treated with caterpillar oral secretion declined levels of plants treated with caterpillar oral to control levels within 6 h after levels in Lima bean plants, but not different from each other. Treatment with caterpillar with caterpillar oral Treatment from each other. levels in Lima bean plants, but not different treatment of the peak. Apart from an effect of Apart from an effect the peak. levels of Transcription another gene investigated was affected. transcription of transcript levels only after 24 hpt but not at 6 hpt, whereas JA treatment and wounding did transcript levels hpt but not at 6 hpt, whereas only after 24 JA transcriptional patterns. Compared to herbivory can cause different patterns of gene transcription patterns of gene transcription when compared to individual herbivory can cause different homoterpenes homoterpenes in elevation of infested with in the differentiation between JA-treated plants and between JA-treated in the differentiation feeding by the two different herbivores. In our study, treatments with the prey with the treatments our study, herbivores. In different two feeding by the Moreover, Moreover, upregulated by which the enzyme that leadsis the production to principalof the predator attractant metabolite production (Stam more attractive to show that treatment with caterpillar oral secretion does increase show that treatment with caterpillar oral secretion, which in attraction of did not result oral secretion on ocimene in and Lima Baldwin bean. (2004) Voelckel found that simultaneous and sequential of In our study, predatory mite behavior was well reflected in volatile blend analysis. Four It has been shown that shown has been It defense mechanisms apart from indirect defense could in fact be affected by the combination defense mechanisms apart from indirect in fact be affected defense could quite late for plants treated with caterpillar oral secretion. This is in Arimura accordance with arthropods (Schlumbaum an acidic chitinase, which is MeSA-responsive (Margis-Pinheiro already induce a different time point than in plants infested with a different and the prey plus with and the monoterpene and the monoterpene compounds, i.e. combined application than either single treatment. led to higher transcript levels comparable to control levels. However, De Boer control levels. However, comparable to volatile profiles of Lima bean plants that received had different treatments (Dicke 2000). Chitinases are commonly involved in plant direct defense against plant pathogens and et al. Arimura (E) TMTT and and TMTT 1990; De Boer and Dicke, 2004). Dicke Boer and Dicke, 2004). 1990; De Chapter 5 found to have an important function in discrimination between treatments. The involvement of (E,E)-TMTT and (E)-β-ocimene in the attraction of P. persimilis to prey-infested Lima bean plants have been described previously (Dicke et al., 1990). Nevertheless, there are other well-known compounds that play an important role in predator attraction (Dicke et al., 1990; De Boer et al., 2004). In fact, volatile blends convey more information than a single compound e.g. on herbivore identity and herbivore developmental stage (Takabayashi et al., 1995; De Moraes et al., 1998; Mumm and Dicke, 2010).

Conclusion In our study, sequential herbivore treatment involving herbivores that induce the same plant defense pathway did not enhance or interfere with indirect defense against T. urticae. Yet, differences in volatile blends perceived by the predator play an important role in distinguishing between prey and non-prey infested plants. Volatiles attractive to natural enemies are perceived in the context of other volatiles in order to extract specific information about the presence of prey. Furthermore, gene transcription is differently induced in terms of timing and magnitude by different herbivore species and a considerable time lag exists between gene transcription of genes relevant in indirect defense and metabolite emission.

84 Chapter 5 85 1990. 2000. Herbivory- 1998. Herbivore-infested 2009. Host-plant mediated 2009. Host-plant mediated 2004. Jasmonic acid is a key 2004. Jasmonic on Lima bean leaves: IV. Diurnal Diurnal and leaves: IV. on Lima bean 2008. Prey and non-prey arthropods 1999. Jasmonic acid herbivory and 2011. Low concentrations of salicylic acid 2011. seedlings. Journal of Chemical 37, Ecology 2011. Sequence 2011. of arrival determines plant- 2007. Induction of plant volatiles by herbivores 2007. Induction of Sequential induction by caterpillar and mites and by caterpillar induction Sequential 2004. Identification of volatiles that are used in . Journal of Chemical Ecology 30, 255-271. . Journal of Chemical Zea mays Spodoptera Spodoptera littoralis 2009. Chemical complexity of volatiles from plants induced volatiles from 2009. Chemical complexity of 2010. A tritrophic signal that attracts parasitoids to host- parasitoids to attracts tritrophic signal that A 2010. 2004. The role of methyl salicylate in prey searching The role of methyl salicylate of the behavior 2004. Phytoseiulus persimilis 2009. Priming of induced plant defense responses. Advances in Botanical 2009. Priming of induced plant defense responses. 2008. Effects of feeding 2008. Effects thrips. Journal of Chemical Ecology 33, 997-1012. thrips. Journal of Chemical Ecology 33, by multiple attack. Nature Chemical Biology 5, 317-324. by multiple attack. Nature Chemical Biology induced volatiles elicit defence genes in Lima bean leaves. Nature 406, 512-515. defence genes in Lima bean leaves. induced volatiles elicit Applicata 132, 30-38. et species. Entomologia Experimentalis aphid two interactions between Dicke M, Van Beek TA, Posthumus MA, Ben Dom N, Van Bokhoven H, De Groot A. Bokhoven H, De Groot N, Van Posthumus MA, Ben Dom Beek TA, Dicke M, Van Dicke M, Gols R, Ludeking D, Posthumus MA. Dicke D, Posthumus M, Gols R, Ludeking Delphia CM, Mescher MC, De Moraes CM. Mescher MC, CM, Delphia De Moraes CM, Lewis WJ, Paré PW, Alborn HT, Tumlinson JH. Tumlinson HT, Alborn Paré PW, WJ, Lewis De Moraes CM, De Boer JG, Posthumus MA, Dicke M. MA, Posthumus De Boer JG, De Boer JG, Dicke M. De Boer JG, MA, Dicke De Boer JG, Hordijk CA, Posthumus M. Erb M, Robert CA, Hibbard BE, Turlings TC. Turlings BE, Hibbard CA, Robert M, Erb Erb M, Foresti N, Turlings TCJ. Turlings N, Foresti M, Erb Engelberth J, Viswanathan S, Engelberth MJ. Engelberth J, Viswanathan Dicke M, Van Loon JJA, Soler R. JJA, Soler Loon Van Dicke M, Brunissen L, Cherqui A, Pelletier Y, Vincent C, Giordanengo P. C, Giordanengo Vincent Y, A, Pelletier Cherqui Brunissen L, Boland W. Boland Plant Physiology 135, 2025-2037. Plant Physiology Research 51, 361-395. with different feeding habits and the effects of induced defenses on host-plant selection by feeding habits and the effects with different regulator of spider mite-induced volatile terpenoid and methyl salicylate emission in tomato. salicylate emission and methyl volatile terpenoid spider mite-induced regulator of plants selectively attract parasitoids. Nature 393, 570-573. plants selectively attract parasitoids. Nature predatory mite nocturnal damage differentially initiate plant volatile emission. Plant Physiology 146, 965-973. Plant initiate plant volatile emission. nocturnal damage differentially mediated interactions between herbivores. Journal of Ecology 99, 7-15. sharing a host plant: Effects on induced volatile emission and predator attraction. Journal of on induced volatile emission and predator attraction. sharing a host plant: Effects stimulate insect elicitor responses in Involvement of host plant in its production. Journal of Chemical Ecology 16, 381-396. Involvement of host plant in its production. Isolation and identification of volatile kairomone that affects acarine predator-prey interactions predator-prey acarine affects that kairomone volatile of identification and Isolation differentially induce carnivore-attractingbean plants. Journal of plant volatiles in Lima differentially discrimination between plants infested with prey or nonprey herbivores by a predatory mite. damaged plants withstands disruption by non-host herbivores. BMC Plant Biology 10, art. no. art. non-host herbivores. BMC Plant Biology 10, damaged plants withstands disruption by 247. 263-266. Conrath U. Chemical Ecology 25, 1907-1922. Chemical Ecology 34, 281-290. Arimura GI, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J. Takabayashi W, Boland Nishioka T, T, Ozawa R, Shimoda Arimura GI, Arimura GI, Köpke S, Kunert M, Volpe V, David A, Brand P, Dabrowska P, Maffei ME, P, Dabrowska P, A, Brand David V, S, Kunert M, Volpe Köpke Arimura GI, Ament K, Kant MR, Sabelis MW, Haring MA, Schuurink RC. Schuurink Haring MA, Sabelis MW, Kant MR, Ament K, References Journal of Chemical Ecology 30, 2215-2230. Chapter 5

Frost CJ, Mescher MC, Carlson JE, De Moraes CM. 2008. Plant defense priming against herbivores: Getting ready for a different battle. Plant Physiology 146, 818-824.

Kaplan I, Denno RF. 2007. Interspecific interactions in phytophagous insects revisited: a quantitative assessment of competition theory. Ecology Letters 10, 977-994.

Kessler A, Baldwin IT. 2002. Plant responses to insect herbivory: The emerging molecular analysis. Annual Review of Plant Biology 53, 299-328.

Kessler A, Baldwin IT. 2004. Herbivore-induced plant vaccination. Part I. The orchestration of plant defenses in nature and their fitness consequences in the wild tobacco Nicotiana attenuata. Plant Journal 38, 639-649.

Koornneef A, Pieterse CMJ. 2008. Cross talk in defense signaling. Plant Physiology 146, 839-844.

Kramer KJ, Muthukrishnan S. 1997. Insect chitinases: Molecular biology and potential use as biopesticides. Insect Biochemistry and Molecular Biology 27, 887-900.

Li C, Williams MM, Loh YT, Gyu IL, Howe GA. 2002. Resistance of cultivated tomato to cell content-feeding herbivores is regulated by the octadecanoid-signaling pathway. Plant Physiology 130, 494-503.

Margis-Pinheiro M, Metz-Boutigue MH, Awade A, de Tapia M, le Ret M, Burkard G. 1991. Isolation of a complementary DNA encoding the bean PR4 chitinase: an acidic enzyme with an amino-terminus cysteine-rich domain. Plant Molecular Biology 17, 243-253.

Mathur V, Ganta S, Raaijmakers CE, Reddy AS, Vet LEM, Van Dam NM. 2011. Temporal dynamics of herbivore-induced responses in Brassica juncea and their effect on generalist and specialist herbivores. Entomologia Experimentalis et Applicata 139, 215-225.

Mathur V, Tytgat TOG, de Graaf RM, Kalia V, Sankara Reddy A, Vet LEM, van Dam NM. 2013. Dealing with double trouble: Consequences of single and double herbivory in Brassica juncea. Chemoecology 23, 71-82.

Menzel TR, Weldegergis BT, David A, Boland W, Gols, R, Van Loon, JJA, Dicke M. 2014. Synergism in the effect of prior jasmonic acid application on herbivore-induced volatile emission by Lima bean plants: transcription of a monoterpene synthase gene and volatile emission. Journal of Experimental Botany 65, 4821-4831.

Mumm R, Dicke M. 2010. Variation in natural plant products and the attraction of bodyguards involved in indirect plant defense. Canadian Journal of Zoology 88, 628-667.

86 Chapter 5

, : 87 2009. 2014. Plant 2010. Molecular, 2010. Molecular, Arabidopsis thaliana Acyrthosiphon pisum 2008. Early season herbivore herbivore Early season 2008. 1995. Developmental stage of . Journal of Chemical . Journal of Chemical Ecology 37, 2003. Volatile emissions triggered by 2003. Volatile 2011. Pea Pea aphids, 2011. 2001. Infochemically 2001. Infochemically mediated tritrophic Vicia faba Vicia 1986. Plant chitinases are potent inhibitors 1986. Plant chitinases of 2007. Consequences of sequential attack for Sequential induction by caterpillar and mites and by caterpillar induction Sequential . New Phytologist 197, 1291-1299. affects production of herbivore-induced synomone by corn affects 2002. Induced parasitoid attraction by 1992. Response of predatory mites with different rearing histories 1992.Response mites with different of predatory 2004. Herbivore-induced plant vaccination. Part II. Array-studies 2004. Herbivore-induced plant vaccination. Part II. Arabidopsis thaliana Pseudaletia separata to volatiles of uninfested plants. Entomologia Experimentalis et Applicata 64: 187-193. Applicata 64: Experimentalis et to volatiles of uninfested plants. Entomologia herbivore biochemical, and organismal analyses of tomato plants simultaneously herbivores tomato by attacked analyses of biochemical, and organismal interactions with multiple insect herbivores: From community to genes. Annual Review of interactions with multiple insect herbivores: From community to genes. interaction webs on cabbage plants. Population Ecology 43, 23-29. cabbage plants. Population Ecology 43, interaction webs on fungal growth. Nature 324, 365-367. fungal growth. Nature from two feeding guilds. Journal of Chemical Ecology 36, 1043-1057. from two feeding guilds. Poelman EH, Broekgaarden C, Van Loon JJA, Dicke M. JJA, Dicke Loon Van C, Broekgaarden EH, Poelman Botany 53, 1793-1799. Rodriguez-Saona CR, Musser RO, Vogel H, Hum-Musser H, Hum-Musser SM, Thaler JS. Musser RO, Vogel Rodriguez-Saona CR, Rodriguez-Saona C, Crafts-BrandnerRodriguez-Saona LA. SJ, Cañas Plant Biology 65, 689-713. reveal the transience of herbivore-specific transcriptional imprints and a distinct imprint from resistance to herbivores when plants have specific induced responses. Oikos 116, 1389-1399. 116, Oikos responses. induced specific have plants when herbivores to resistance plant defense in plants. Journal of Chemical Ecology 21, 273-287. plants. Journal of Chemical Ecology 21, multiple herbivore damage: Beet armyworm and whitefly feedingon cotton plants. Journal of Zhang PJ, Broekgaarden C, Zheng SJ, Snoeren TAL, Van Loon JJA, Gols R, Dicke M. Van Zhang PJ, Broekgaarden C, Zheng SJ, Snoeren TAL, Zhang PJ, Zheng SJ, Van Loon JJA, Boland W, David A, Mumm R, Dicke M. R, Mumm A, David W, JJA, Boland Loon Van SJ, Zheng PJ, Zhang Whiteflies interfere with indirect plant defense against spider mites in Lima bean. Proceedings bean. Lima in mites spider against defense plant indirect with interfere Whiteflies stress combinations. Plant Journal 38, 650-663. suppress induced plant volatiles in broad bean, of the National Academy of Sciences of the United States of America 106, 21202-21207. of the United States of Academy of Sciences of the National Involvement of the octadecanoid and the salicylic acid pathway. Journal of Experimental Involvement of the octadecanoid and the salicylic acid pathway. differentially affects plant defence affects responses differentially to subsequently colonizing herbivores and their Stam JM, Kroes A, Li, Y., Gols, R., Van Loon, JJA, Poelman, EH, Dicke, M. Loon, Gols, R., Van Y., A, Li, Stam JM, Kroes Shiojiri K, Takabayashi J, Yano S, Takafuji A. S, Takafuji Yano J, K, Takabayashi Shiojiri Schwartzberg EG, Böröczky K, Tumlinson JH. Böröczky Schwartzberg EG, K, Tumlinson Schlumbaum A, Mauch F, Vögeli U, Boller T. Vögeli A, Mauch F, Schlumbaum abundance in the field. Molecular Ecology 17, 3352-3365. in the field. Molecular abundance 2013. Jasmonate and ethylene signaling mediate whitefly-induced interferencewith indirect Voelckel C, Baldwin IT. Voelckel Viswanathan DV, Lifchits OA, Thaler JS. Thaler OA, Lifchits DV, Viswanathan Van Poecke RMP, Dicke M. Poecke RMP, Van Chemical Ecology 29, 2539-2550. Chemical

Takabayashi J & Dicke M. Takabayashi MA. Posthumus Dicke M, S, Takahashi J, Takabayashi 1055-1062. 88 Chapter 6 Effect of sequential herbivory by conspecific herbivorous spider mites on gene transcription and volatile emission in Lima bean plants Tila R. Menzel, Berhane Weldegergis, Joop J.A. van Loon & Marcel Dicke

89 Chapter 6

Abstract Plants possess a manifold of defense mechanisms that protect them against herbivorous arthropods in nature. However, due to their sessile nature plants may frequently encounter attacks of the same species of herbivores. It can be expected that plants have evolved defense mechanisms which help them in recognizing and defending themselves more efficiently against re-occurring attacks. Here, we studied the effect of herbivory bytwo- spotted spider mites (Tetranychus urticae) on two components of Lima bean induced indirect defense against subsequent herbivory by conspecifics. We studied the emission of (E)-β- ocimene and 12 other plant volatiles commonly associated with herbivory, as well as the transcription of two genes involved in Lima bean defense, namely P. lunatus β-ocimene synthase (PlOS) and P. lunatus pathogenesis-related protein 4 (PlPR-4). Volatile profiles and gene transcript level of plants attacked by herbivores differed significantly from that of control plants. Emission of volatiles did not differ between plants that experienced two bouts of herbivore attack by conspecific spider mites compared to plants that experienced only one bout of spider mite attack. Moreover, transcript levels of PlOS and PlPR-4 did not differ for these treatments. Our results suggest that Lima bean plants do no increase defense in response to sequential herbivory by two-spotted spider mites under the exposure regime tested.

90 Chapter 6

, et 91 et al. , 1998; , 2010). de novo et al. et al. , 2012; Stam , et al. et , 2009). Some of the , 2009; Erb , 2003). et al. et al. et al. Sequential induction by conspecifics induction Sequential 2008). Other studies suggest no impact or a 2008). Other , 2005). Moreover, a distinction is made between , 2005). Moreover, , 2004). Consequently, plants do not only provide et al. et et al. et al. , 1995), and herbivore density (Gols , 2009). However, only only few studies have investigated the modifying , 2009). However, 2007; De Boer et al. et al. et al. et , 1998), but also more detailed information such as on herbivore developmental , 2014). et al. et al. , 2014). their herbivorous (Mumm and Dicke, 2010). Plants release prey a number of volatile organic the effects may persist for hours to weeks, thereby affecting defense against other herbivores defense weeks, thereby affecting hours to persist for may effects the herbivore (Paré and Tumlinson, 1997; Dicke and Baldwin, 2010). Extensive research has Tumlinson, herbivore (Paré and been carried out to study simple tritrophic interactions between a plant, an herbivore, and its herbivore or microbial challenge. This memory This memory formation enables plants to respond in a faster herbivore or microbial challenge. induction and antagonistic effects among phytohormones, such as jasmonic acid (JA) and as jasmonic acid (JA) among phytohormones, such induction and antagonistic effects interactions (Dicke information species on the of the attacking herbivore to carnivores (De Moraes in the blend composition (Dudareva Moreover, initial initial herbivore-induced changes in plant chemistry may occur within hours, and Moreover, Nevertheless, plants are freely accessible Nevertheless, plants are freely accessible to herbivores to in nature, and plants are likely Plants are sessile organisms without chance of evading attacks by herbivorous arthropods. herbivorous arthropods. by evading attacks chance of without sessile organisms Plants are utilization of an arsenal of sophisticated defense mechanisms. These defense mechanisms utilization of an arsenal of sophisticated defense mechanisms. with multiple herbivore species over volatiles from single by either of the herbivore infestation negative impact of multiple herbivory on plant defense (Zhang natural enemy. HIPV blends are complex mixtures consisting of 20 to 200 compounds, which 200 compounds, 20 to blends are complex mixtures consisting of HIPV natural enemy. salicylic acid (SA), that are crucial in plant defense induction (Pieterse that salicylic acid (SA), species (Moayeri stage (Takabayashi stage (Takabayashi or are incrementally released and play a crucial role in attracting natural enemies of a specific a of enemies natural attracting in role crucial a play and released incrementally are or It has been suggested that plants are able to form some sort of ‘memory’ in response to a in response of ‘memory’ to form some sort that plants are able been suggested has It effects of multiple of herbivores on tritrophic interactions (Dicke effects available studies suggest that natural enemies tend to prefer volatiles from plants infested experience events of multiple herbivory, which can directly or indirectly influence tritrophic and enable plants to respond in a highly specific manner to an attacker (Agrawal, 2001). and indirect defense mechanisms,and indirect defense attraction and employment of natural which involve the enemies of herbivores (Schoonhoven and stronger manner to a subsequent attack, which is also known as “priming” (Frost can provide a manifold of information depending on quantitative and qualitative differences compounds, known as herbivore-induced plant volatiles (HIPVs), which are produced constitutive and induced defense mechanisms. The latter, induced defenses, are highly plastic, latter, The constitutive and induced defense mechanisms. can be roughly divided into direct defense mechanisms, such as thorns and toxic compounds, 2008; Conrath, 2009). In fact, plants defend themselves against herbivorous defend themselves against herbivorous plants In fact, 2008; Conrath, 2009). arthropods by Carnivorous enemies of herbivores use a number of chemical cues to locate and identify (Stam al. Introduction These differences in the effect on defense manifestation are likely caused by differential on defense manifestation are likely caused by differential in the effect These differences Turlings Turlings Chapter 6

Depending on herbivore species and feeding mode, the JA defense signaling pathway or the SA signaling pathway can be induced. Each of them activates a set of genes which are involved in generating a distinct plant defense response (Pieterse et al., 2012). Herbivory by arthropods with the same feeding mode or induction of the same defense signaling pathway are therefore expected to cause no interference with defense responses within the plant. In fact, De Boer et al. (2008) showed that simultaneous feeding of the beet armyworm Spodoptera exigua Hübner potentiated indirect defense against the spider mite Tetranychus urticae Koch. Defense against biting-chewing herbivores and certain cell-content feeding mites involves for example JA-related defenses (Ozawa et al., 2000; Pieterse et al., 2012). Nevertheless, De Vos et al. (2005) found that herbivores with different feeding modes induce distinctive transcriptional patterns in Arabidopsis. In fact, induced defense responses against these two groups of herbivores vary enough to allow predators a distinction between HIPV induced by a prey and a non-prey herbivore (De Boer et al., 2008).

Generally, feeding by conspecifics can increase plant resistance and HIPVs from plants fed upon by herbivores may even repel conspecific herbivores and render neighboring plants more resistant to attack by the same herbivore (Dicke, 1986; Karban, 1990; Bruin et al., 1992; De Moraes et al., 2001; Horiuchi et al., 2003). Moreover, Cui et al. (2012) found that feeding by conspecifics significantly reduced whitefly fitness. However, also effects induced by conspecifics may not always be of positive nature for plants. For instance, Underwoodet al. (2012) found that single damage by the generalist caterpillar Spodoptera exigua induced plant defense, but repeated damage by the caterpillar interfered with plant defense.

Here we investigated the effect of a low-density infestation by the spider mite T. urticae, on the induction of defenses by conspecifics at a later time point; we focused on the induction of HIPV and genes involved in induced defense. We hypothesized that a defense induction by conspecific herbivores would result in an enhanced induced defense response against the later-arriving conspecific herbivores.

Methods and materials

Plants and mites Lima bean plants (Phaseolus lunatus L., cv. Wonder Bush) were maintained in a greenhouse at 23 ± 2 °C with 60 ± 10 % R.H., and a photophase of 16 h. Plants were used for experiments 9 - 10 days after sowing. Two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae), were reared on Lima bean plants in a greenhouse compartment at 25 ± 5 °C, R.H. 50 – 70%, 16L : 8D. Only adult females were used for the infestation of the plants in experiments.

Plant treatments Plant treatments consisted of i) control, ii) four T. urticae, iii) four T. urticae followed by no treatment, iv) four T. urticae followed by no treatment and then 10 T. urticae, and v) 10 T. urticae (see also Suppl. Fig. S1 - 2). Control plants [i)] did not receive any treatment. The

92 Chapter 6

P. 93 PlOS PlPR-4 (2014). PlACT1 PlNMP1 T. urticae T. ), and the ), ; GenBank et al. per leaf. After per leaf. PlOS ( females per leaf per leaf females PlPR-4 ( primers were F- T. urticae T. T. urticae T. PlOS females per primary females per primary leaf for 48 h. (2014). ocimene synthase - β primers were F-

5’-AGCCAGATCAAGACGAAGGA-3’, 5’-AGCCAGATCAAGACGAAGGA-3’, 5’-TGCTGCTTCCCCTCTCTCTA-3’, 5’-TGCTGCTTCCCCTCTCTCTA-3’, et al. Sequential induction by conspecifics induction Sequential T. urticae T. ; GenBank accession DQ159907) and ; PlOS PlACT1 PlACT1 P. lunatus P. PlACT1 ; GenBank accession AF289260.1) were quantified. were AF289260.1) accession GenBank ; ( acidic pathogenesis-related protein 4

5’-CCGGAATGGAGTGTTGACGAGCA-3’ and R- 5’-CCGGAATGGAGTGTTGACGAGCA-3’ PlNMP1 ( 5’-ACGCTTTCCTCAGTGCTCTC-3’ and R- 5’-ACGCTTTCCTCAGTGCTCTC-3’ PlNMP1 P. lunatus P. PlPR-4 P. lunatus Actin1 P. primers F- were F- PlNMP1 two reference genes two two primary leaves leaves two primary ii) received treatment plants with of adulttwo that were transferred from a plant in the spider-mite culture by using a fine After paint brush. incubated incubated for another 48 h after removal of mites and their residues. Primary leaves of plants PCR was used. Relative transcript levels of Plant material was collected and processed as previously described in Menzel plants was pooled Plant material from primary leaves of three to give one biological replicate uncovered. Then four plants of each treatment were transferred to a 30-L glass jar. Glass jars Glass glass jar. Then four plants of each treatment were transferred to a 30-L uncovered. were sealed with viton-lined glass lids equipped with an air inlet and outlet. The setup was with treatment iv) were also with treatment iv) were infested with two adult replication. per primary leaf and were incubated per primary leaf and sampling. Each treatment was applied for 48 h before to For volatile collection, treatments i) ,and iii)-v) (see Suppl. Fig. samples were taken from plant For treatment v) at 96 h after start of experiments plants were infested with five adult five with infested were plants experiments of start after h 96 at v) treatment For 6 - 15 plants, except the control treatment for which the number of plants was 48 - 60. the control treatment for which the number 6 - 15 plants, except 5’-CCAAGGCTAACCGTGAAAAG-3’ and R- 5’-CCAAGGCTAACCGTGAAAAG-3’ 5’-CCAGCTCAGAAACATCTGGCAATGG-3’. 5’-TGCATGGGTCTCAGTCTCTG-3’ and R- 5’-TGCATGGGTCTCAGTCTCTG-3’ 5’-TCCTCGTCGTCGCAGTAATCCTT-3’, lunatus Nuclear matrix protein 1 soil and roots with aluminum foil, thereby leaving only the above-ground part of the plants sampled. of treatment iii) Plants received were ii), except that they treatment as for the same In order to assess relative transcript levels of the genes of interest, a real time quantitative RT- quantitative time real a interest, of genes the of levels transcript relative assess to order In and a 72-well rotor; for a detailed description see Menzel accession EU194553), and three to five biological replicates were collected for each treatment per experimental another 48 h, the mites and their residues were removed plant leaf tissue was sampled. and RNA extraction and cDNA synthesis cDNA and extraction RNA Quantitative RT-PCR was performed in a Rotor-Gene 6000 machine (Corbett Research) with Quantitative RT-PCR flushed with 200 ml/min The synthetic Netherlands), air which (Linde was Gas Benelux B.V., Glass jars with plants were filtered by passing through charcoal before entering the glass jar. S2). Plants were prepared for volatile collection carefully wrapping their pots containing by Dynamic headspace collection volatiles plant of PlPR-4 Quantitative RT-PCR Quantitative 48 h. the Subsequently, primary leaves were infested with five adult 48 h mites and their residues were removed by using a fine paint brush and leaf tissue was After 48 h, the mites and their residues were removed, and plants were incubated for another and plants were incubated for mites and their residues were removed, the 48 h, After Chapter 6 flushed with air for 45-60 min before sampling. For sampling a stainless steel tube filled with 200 mg Tenax TA (20/35 mesh; CAMSCO, Houston, TX, USA) was connected to the outlet of each glass jar and volatiles were collected by sucking air out of the jars at 200 ml/min for 2 h. A total of 10 replicates of each treatment were sampled over 10 days. Immediately after volatile collection, the fresh weight of above-ground tissue of sampled plants was determined using an analytical balance (NewClassic ML, Mettler Toledo, Greifensee, Switzerland).

Chemical analysis of plant volatiles Separation and identification of plant volatiles was done using a Trace Ultra gas chromatograph (GC) coupled with Trace DSQ quadrupole mass spectrometer (MS) (Thermo Fisher Scientific, Waltham, USA). For a detailed description we refer to Menzel et al. (2014).

Standards of (E)-2-hexenal, (Z)-3-hexen-1-ol, (Z)-3-hexen-1-ol, acetate, (Z)-3-hexen-1-ol, butanoate, (Z)-3-hexen-1-ol, isovalerate, linalool, methyl salicylate, indole, β-caryophyllene, (E)-β-ocimene, and alloocimene were acquired from Sigma-Aldrich (Saint Louis, MO, USA). Additional standards (E)-4,8-dimethylnona-1,3,7-triene [(E)-DMNT] and (E,E)-4,8,12- trimethyltrideca-1,3,7,11-tetraene [(E,E)-TMTT] were kindly provided by Prof. W. Boland (Max Planck Institute for Chemical Ecology, Jena, Germany). For quantification, calibration lines were constructed for each compound using seven data points at different concentrations and two replicates for each data point.

Statistical analysis Normally distributed 2−ΔΔCt values for gene transcript levels of PlOS and PlPR-4 with homogeneity of variances were analyzed by Student’s t-test for pairwise comparisons or one-way ANOVA followed by Tukey’s Honestly Significant Difference (HSD) for post hoc pairwise comparisons. Gene transcription data of PlPR-4 had to be log-transformed prior to analysis by one-way ANOVA in order to meet the assumptions. Gene transcription data that violated assumptions of normality and homogeneity of variances were analyzed with Mann-Whitney tests for pairwise comparisons. Statistical analyses were performed in SPSS version 19 (SPSS Inc., Chicago, IL, USA). Volatile profiles of plants exposed to different treatments were analyzed using multivariate data analysis. Data were expressed as amount emitted per hour per unit of plant fresh weight, log-transformed, and univariate-scaled. Then a Latent Structures-Discriminant Analysis (PLS-DA) was performed using the software SIMCA P+ version 12 (Umetrics, Umeå, Sweden).

Results

Volatile emission Emission profiles of plants with different treatments were compared for 13 well-known HIPVs emitted by spider-mite infested Lima bean plants (Fig. 1). PLS-DA with all four treatments resulted in a model with one significant principal component that explained 50 % of the total variability of the data. Volatile profiles of control plants were clearly different from plants with

94 Chapter 6 95 ), iv) Tu +NT+10 for 48 h followed for had been removed, -ocimene, were compared β - T. urticae T. for 48 h (4 Tu for (E) ). The first two principal components are principalcomponents two ). The first T. urticae T. +NT), iii) four Sequential induction by conspecifics induction Sequential were significantly increased in plants that plants in increased significantly were -caryophyllene, -caryophyllene, and indole had variable β PlOS PlPR-4 and and for 48 h (10 Tu for = 0.001). However, after = 0.001). However, PlOS P , the gene which codes enzyme for the ocimene synthase, plants with i) no treatment (Ctrl), or treatment with ii) four T. with ii) four treatment or (Ctrl), with i) no treatment plants for 48 h compared to transcript levels in control plants without PlOS -3-hexen-1-ol, -3-hexen-1-ol, acetate, (Z) T. urticae T. with and without previous infestation by conspecifics overlapped largely and for 48 h followed by no treatment for 48 h (4 Tu 48 h (4 for no treatment 48 h followed by for transcript levels returned to control levels and were not significantly different from control from different significantly not were and levels control to returned levels transcript T. urticae T. the different treatments (see also pairwise comparisons Suppl. Fig. S3 - 5). Three volatile volatile Three pairwise (see also treatments comparisons S3 - 5). Suppl. Fig. the different infestation (Mann-Whitney U test, importance importance the projection in values (VIP) higher model, meaning than 1 in the those that DA DA did not result in a significant model. For the results of single volatile analyses between Relative transcript levels of were infested by four which is involved volatile in the synthesis of the compound were closely located to each other (Fig. 1 A & B). Moreover, pairwise comparisons by OPLS- pairwise comparisons Moreover, B). & A 1 (Fig. each other to were closely located different treatments refer to supplemental data Table S1. Table supplemental data treatments refer to different among treatments (Fig. 2). Transcript levels of Transcript 2). among treatments (Fig. Relative gene transcript levels transcript gene of Relative compounds, compounds, namely volatiles contributed most to discrimination between treatments. Volatile profiles of plants with plants of profiles Volatile treatments. between discrimination to most contributed volatiles PlOS T. urticae 10 T. 96 h and for no treatment in the loading Numbers explained parentheses. in variation of A) with (panel given the percentage (E)- 4) acetate, 3) (Z)-3-hexen-1-ol, 2) (Z)-3-hexen-1-ol, 1) (E)-2-hexenal, (panel B) represent plot 7) alloocimene,-DMNT], [(E) β-ocimene, 5) linalool, 8) (Z)- 6) (E)-4,8-dimethylnona-1,3,7-triene 11) indole, 12) salicylate, 9) methyl isovalerate, 10) (Z)-3-hexen-1-ol, butanoate, 3-hexen-1-ol, β-caryophyllene, 13) (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene and [(E,E)-TMTT]. Figure 1. Multivariate data analysis by PLS-DA (panel A) and corresponding loading (panel plot corresponding A) and (panel PLS-DA by analysis data 1. Multivariate Figure lunatus P. of blends volatile B) of urticae T. urticae 10 T. infestation subsequent 48 h and for no treatment by 10 Chapter 6 levels after 48 h (Student’s t-test, P = 0.43). When plants were infested with 10 T. urticae,

PlOS transcript levels were significantly affected (ANOVA F2,17 = 4.40, P < 0.05). Nevertheless, PlOS transcript levels in plants that had previously experienced an infestation by conspecifics were not significantly different from transcript levels of plants that had only experienced a 48 h period of spider-mite infestation (Tukey’s post hoc test, P > 0.05). Moreover, PlOS transcript levels of plants with infestation experience by conspecifics were not different from control levels (Tukey’s post hoc test, P > 0.05).

Figure 2. Relative gene transcript levels of PlOS in Lima bean plants. Plant were i) control, or treated with ii) four T. urticae for 48 h, iii) four T. urticae 48 h followed by no treatment for 48 h, iv) four T. urticae for 48 h followed by no treatment for 48 h and then 10 T. urticae for 48 h, v) 10 T. urticae for 48 h. Values are the mean (± SE) of five to nine biological replicates, pooled from two replications of the same treatment. Different letters above bars indicate significant differences in transcript levels between treatments (Mann-Whitney U test, Student’s t-test, and Tukey’s HSD tests respectively, P < 0.05). Gene transcript levels were normalized to the normalization factor obtained from geometrically averaging the Ct values of the two reference genes P. lunatus Actin1 (PlACT1) and P. lunatus nuclear matrix protein 1 (PlNMP1) for each sample.

Relative gene transcript levels of PlPR-4, a chitinase which may be involved in plant direct defense were compared among treatments (Ward et al., 1991; Arimura et al., 2000). Gene transcript levels of PlPR-4 were significantly increased compared to control levels when plants were infested with four T. urticae (Fig. 2; Mann-Whitney U test, P = 0.001). After four T. urticae had been removed and plants had recovered for 48 h, PlPR-4 transcript levels were not different from control levels (Student’s t-test, P = 0.20). When plants were infested with 10 T. urticae, treatment significantly affected PlPR-4 transcript levels compared to control plants

(ANOVA F2,17 = 14.28, P < 0.001). Plants that were infested with 10 T. urticae and plants that had also previously experienced an infestation by conspecifics showed increasedPlPR-4 levels compared to control plants (Tukey’s post hoc both, P < 0.01), while they were not different from each other (Tukey’s post hoc both, P < 0.05).

96 Chapter 6

97 for 48 h followed by no treatment for 48 for 48 h followed no treatment by for Sequential induction by conspecifics induction Sequential in Lima bean plants. Plants were i) control, or or control, i) Limain were bean Plants plants. PlPR-4 T. urticae T. 48 h, iii) four for for 48 h v) 10 urticae for 10 T. then 48 h and for no treatment 48 h followed by for 0.05). Gene transcript levels were normalized to the normalization factor obtained obtained factor P < 0.05). Gene normalized levels the normalization transcript were to

for 48 h. Values are the mean (± SE) of five to nine biological nine to five replicates, of pooled two meanthe (± SE) are from h. Values 48 for igure 3. Relative gene transcript levels transcript gene of Relative 3. igure T. urticae T. with ii) four treated F T. urticae T. h, iv) four (PlACT1) Actin1 lunatus P. genes geometrically reference two the valuesfrom of the Ct averaging each. sample 1 (PlNMP1) for protein matrix nuclear lunatus P. and T. urticae T. in significant above indicate differences bars letters Different treatment. the same of replications tests HSD Tukey’s and t-test, Student’s U test, (Mann-Whitney levelstranscript between treatments respectively, Chapter 6

Discussion Induced defense responses of plants involve a time-lag between recognition of attack by herbivores and activation of defenses. In order to reduce this time lag between attacker recognition and defense induction, it might be advantageous for plants to retain information by memory formation about a previous herbivore attack. In this study, we investigated whether a low density infestation by the spider mite T. urticae could affect the emission of 13 relevant HIPVs and two genes involved in induced plant defense, when plants experienced a second bout of defense induction by conspecifics at a later time point. Our results show that volatile profiles and transcript levels of genes which function in Lima bean defense,PlOS and PlPR-4, did not differ for plants infested with T. urticae, irrespective of their previous encounter with the same herbivore.

Indirect plant defense by natural enemies of herbivores relies on the emission of HIPVs as reliable host location cues. The tritrophic interaction of Lima bean plants with the specialist predator mite Phytoseiulus persimilis Athias-Henriot, an important natural enemy of spider mites, has been extensively studied and the principal volatile compounds that mediate the tritrophic interaction have been identified (Dicke et al., 1990; De Boer and Dicke, 2004). We have previously found that indirect defense against T. urticae mediated by P. persimilis can be enhanced when plants are treated with exogenous JA for up to 7 days after defense induction (Gols et al., 2003; Menzel et al., 2014). Nevertheless, in the present study we found no effect on indirect defense for sequential infestation by conspecifics in terms of volatile emission. The acyclic monoterpene hydrocarbon compound (E)-β-ocimene constitutes one of the most common volatile chemicals released from plants in response to herbivory and is a principle attractant for the specialist predatory mite P. persimilis (Dicke et al., 1990; Paré and Tumlinson, 1999; Pichersky and Gershenzon, 2002; Arimura et al., 2009). The corresponding gene, β-ocimene synthase is also known as PlOS in Lima bean and codes for the enzyme which is involved the last step of the synthesis of this compound (Ament et al., 2004; Arimura et al., 2004). Arimura et al. (2008) showed that PlOS is a JA-regulated gene that responds to increases in JA levels with increased transcript accumulation. Infestation by T. urticae mites has previously been shown to induce JA and de novo synthesis of HIPVs, such as (E)-β- ocimene (Dicke et al., 1990; Ozawa et al., 2000). Accordingly, we found that an infestation by four T. urticae, and later 10 T. urticae, increased transcript levels of PlOS compared to control transcript levels. However, after removal of T. urticae gene transcript levels returned to control levels within 48 h. In fact, plant induced defenses, such as the activation of defense genes and synthesis of HIPVs, are considered as costly in the absence of herbivores due to a metabolic trade-off between plant growth and reproduction with these defenses (Heil and Baldwin, 2002; Kessler and Baldwin, 2002). Consequently, induced defenses are usually quickly down-regulated in the absence of attackers. However, “priming” of defenses against subsequent herbivores is thought to outweigh the costs for this type of defense (Van Hulten et al., 2006). Nevertheless, in accordance with volatile results we did not find an effect on PlOS gene transcription. Moreover, direct defense mediated by chitinases such as PR-4 seemed to follow the same transcriptional pattern as for PlOS, not indicating a memory effect. Previously, Underwood (1998) showed that soybean plants became more resistant

98 Chapter 6

et 99 , 2004). The is a cell–content is a cell–content et al. , such as the four Solanum dulcamara , it has been shown that , it has been shown T. urticae T. T. urticae T. , 2000; Ament T. urticae T. et al. infestation. Sequential induction by conspecifics induction Sequential does not enhance in indirect plant defense T. urticae T. T. urticae T. , 2003). The two spotted-spider The two spotted-spider mite 2003). , et al. (2007) found season-long effects of sequential flea beetle feeding in terms of a memory effect for volatile emissions or defense gene transcription. Absence for volatile emissions gene transcription. or defense terms of a memory effect to other herbivores. Consequently, initial infestation might have been too low or lasted too to Mexican bean beetles by 3 days after attack by conspecifics. Moreover, Viswanathan between inductions can have a significant impact on induced plant defense responses (e.g. in the formation of defense memory against in the formation of defense feeder, which causes small lesions causes small lesions which numbers plant tissues. Large in of these herbivorous mites feeder, L. However, factors such as infestation levels such as infestation factors interval level, and time and severity of damage L. However, Moreover, while JA is a key regulator defense against of while JA Moreover, used for the first infestation in our experiments, cause little plant tissue damage compared pathways or low herbivore density. short in order to generate a defense response potent enough to induce memory formation. induce memory response potent enough to generate a defense short in order to Underwood, Underwood, Gols 1998; of a priming effect might be caused be antagonistic of phytohormone cross-talk signaling of a priming effect defense against these mites also induces SA (Ozawa mites also induces defense against these SA antagonistic cross-talk between the JA and SA signaling pathway might thus pose an obstacle and SA antagonistic cross-talk between the JA are known to overexploit host plants, but low numbers their of Conspecific herbivore infestation by al. Conclusion Chapter 6

References Agrawal AA. 2001. Phenotypic plasticity in the interaction and evolution of species. Science 294, 321-326.

Arimura GI, Matsui K, Takabayashi J. 2009. Chemical and molecular ecology of herbivore- induced plant volatiles: Proximate factors and their ultimate functions. Plant and Cell Physiology 50, 911-923.

Arimura GI, Ozawa R, Kugimiya S, Takabayashi J, Bohlmann J. 2004. Herbivore-induced defense response in a model legume. Two-spotted spider mites induce emission of (E)-β- ocimene and transcript accumulation of (E)-β-ocimene synthase in Lotus japonicus. Plant Physiology 135, 1976-1983.

Arimura GI, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J. 2000. Herbivory- induced volatiles elicit defence genes in Lima bean leaves. Nature 406, 512-515.

Bruin J, Dicke M, Sabelis MW. 1992. Plants are better protected against spider-mites after exposure to volatiles from infested conspecifics. Experientia 48, 525-529.

Conrath U. 2009. Priming of induced plant defense responses. Plant Innate Immunity 51, 361-395.

Cui H, Sun Y, Su J, Li C, Ge F. 2012. Reduction in the fitness ofBemisia tabaci fed on three previously infested tomato genotypes differing in the jasmonic acid pathway. Environmental Entomology 41, 1443-1453.

De Boer JG, Dicke M. 2004. The role of methyl salicylate in prey searching behavior of the predatory mite Phytoseiulus persimilis. Journal of Chemical Ecology 30, 255-271.

De Moraes CM, Lewis WJ, Paré PW, Alborn HT, Tumlinson JH. 1998. Herbivore-infested plants selectively attract parasitoids. Nature 393, 570-573.

De Moraes CM, Mescher MC, Tumlinson JH. 2001. Caterpillar-induced nocturnal plant volatiles repel conspecific females. Nature 410, 577-579.

Dicke M. 1986. Volatile spider-mite pheromone and host-plant kairomone, involved in spaced- out gregariousness in the spider mite Tetranychus urticae. Physiological Entomology 11, 251- 262.

Dicke M, Baldwin IT. 2010. The evolutionary context for herbivore-induced plant volatiles: beyond the ‘cry for help’. Trends in Plant Science 15, 167-175.

100 Chapter 6 101 1990. 1990. 2014. 2003. A 2003. A Phytoseiulus 2000. Involvement of (Lepidoptera: Noctuidae). Applied (Lepidoptera: Noctuidae). 2008. Plant defense priming against 2008. Plant defense Sequential induction by conspecifics induction Sequential (Acari: Tetranychidae) and Tetranychidae) (Acari: 2003. Induction of direct and indirect plant direct and indirect 2003. Induction of 2004. Biochemistry of plant volatiles. Plant 2004. Biochemistry biosynthesis of volatiles induced by insect herbivory volatiles induced by biosynthesis of Spodoptera exigua or 2009. Chemical complexity of volatiles from plants induced plants induced volatiles from complexity of 2009. Chemical 2010. A tritrophic signal that attracts parasitoids to host- parasitoids to attracts tritrophic signal that A 2010. De novo Tetranychus urticae Tetranychus 2002. The formation and function of plant volatiles: Perfumes formation and function of The 2002. T. urticae T. 1999. Plant volatiles as a defense against insect herbivores. Plant 1997. 2002. Plant responses to insect herbivory: The emerging molecular 2002. Plant responses to insect herbivory: 2002. Fitness costs of inducedresistance: Emerging experimentalof costs 2002. Fitness 2010. Variation in natural plant products and the attraction of bodyguards in natural plant products and 2010. Variation 1990. Herbivore outbreaks on only young trees: testing hypothesestesting 1990. Herbivore trees: about aging outbreaks on only young (Acari: Phytoseiidae) to volatiles emitted from Lima bean leaves with different (Acari: Phytoseiidae) volatilesto emitted from Lima bean leaves with different levels of damagemade by levels of treatment and spider mite infestation. Journal of Chemical Ecology 29, 2651-2666. mite infestation. Journal of Chemical treatment and spider jasmonate- and salicylate-related signaling pathways for the production of specific herbivore- herbivores: Getting ready for a different battle. Plant Physiology 146, 818-824. battle. Plant Physiology ready for a different herbivores: Getting by multiple attack. Nature Chemical Biology 5, 317-324. attack. Nature Chemical by multiple in cotton plants. Plant Physiology 114, 1161-1167. in cotton plants. Plant Physiology 114, induced volatiles in plants. Plant and Cell Physiology 41, 391-398. induced volatiles in plants. Plant and Cell involved in indirect plant defense. Canadian Journal of Zoology 88, 628-667. involved in indirect plant defense. Canadian for pollinator attraction and defense. Current Opinion in Plant Biology 5, 237-243. Mumm R, Dicke M. Menzel TR, Weldegergis BT, David A, Boland W, Gols R, Van Loon JJA, Dicke Loon M. Gols R, Van A, Boland W, David BT, Menzel TR, Weldegergis Pichersky E, Gershenzon J. Gershenzon Pichersky E, Paré PW, Tumlinson JH. Tumlinson Paré PW, Paré PW, Tumlinson JH. Tumlinson Paré PW, Erb M, Foresti N, Turlings TCJ. Turlings N, Foresti M, Erb Dudareva N, Pichersky E, Gershenzon J. Gershenzon E, Dudareva N, Pichersky Dicke M, Van Loon JJA, Soler R. JJA, Soler Loon Van Dicke M, Dicke M, Van Beek TA, Posthumus MA, Ben Dom N, Van Bokhoven H, De Groot A. Groot H, De Bokhoven N, Van Ben Dom MA, Posthumus TA, Beek M, Van Dicke Horiuchi JI, Arimura GI, Ozawa R, Shimoda T, Takabayashi J, Nishioka T. Takabayashi Ozawa R, Shimoda T, Arimura GI, Horiuchi JI, Heil M, Baldwin IT. Baldwin M, Heil Frost CJ, Mescher MC, Carlson JE, De Moraes CM. JE, De Mescher MC, Carlson Frost CJ, Karban R. A, Baldwin IT. Kessler Physiology 121, 325-331. Physiology 135, 1893-1902. responses by jasmonic acid, low spider mite densities, or a combination acid, low spider mite densities, or responses by jasmonic acid of jasmonic Entomology and Zoology 38, 109-116. support for a slippery concept. Trends in Plant Science 7, 61-67. Trends support for a slippery concept. Involvement of host plant in its production. Journal of Chemical Ecology 16, 381-396. of Chemical Ecology production. Journal of host plant in its Involvement Isolation and identification of volatile kairomone that affects acarine predator-prey interactions predator-prey acarine affects that kairomone volatile of identification and Isolation damaged plants withstands disruption by non-host herbivores. BMC Plant Biology 10, art. no. no. art. Plant Biology 10, non-host herbivores. BMC disruption by damaged plants withstands emission. Journal of Experimental Botany 65, 4821-4831. emission. Journal of Experimental Botany analysis. Annual Review of Plant Biology 53, 299-328. Annual Review of Plant Biology analysis. monoterpene a of emission by Lima bean plants: transcription synthase gene and volatile and induced resistance. Oikos 59, 27-32. comparison responses of the of Ozawa R, Arimura GI, Takabayashi J, Shimoda T, Nishioka T. Nishioka T. J, Shimoda T, Arimura GI, Takabayashi Ozawa R, Synergism in the effect of prior jasmonic acid application prior herbivore-induced of on volatile Synergism in the effect 247. Gols R, Roosjen M, Dijkman H, Dicke M. Dicke H, Dijkman M, Roosjen R, Gols persimilis Chapter 6

Pieterse CMJ, Van Der Does D, Zamioudis C, Leon-Reyes A, Van Wees SCM. 2012. Hormonal modulation of plant immunity. Annual Review of Cell and Developmental Biology 28, 489-521.

Schoonhoven LM, Van Loon JJA, Dicke M. 2005. Insect-Plant Biology. Oxford: Oxford University Press.

Stam JM, Kroes A, Li Y, Gols R, Van Loon JJA, Poelman EH, Dicke M. 2014. Plant interactions with multiple insect herbivores: From community to genes. Annual Review of Plant Biology 65, 689–713.

Turlings TCJ, Bernasconi M, Bertossa R, Bigler F, Caloz G, Dorn S. 1998. The induction of volatile emissions in maize by three herbivore species with different feeding habits: Possible consequences for their natural enemies. Biological Control 11, 122-129.

Underwood N. 2012. When herbivores come back: Effects of repeated damage on induced resistance. Functional Ecology 26, 1441-1449.

Underwood NC. 1998. The timing of induced resistance and induced susceptibility in the soybean-Mexican bean beetle system. Oecologia 114, 376-381.

Van Hulten M, Pelser M, Van Loon LC, Pieterse CMJ, Ton J. 2006. Costs and benefits of priming for defense in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 103, 5602-5607.

Viswanathan DV, Lifchits OA, Thaler JS. 2007. Consequences of sequential attack for resistance to herbivores when plants have specific induced responses. Oikos 116, 1389-1399.

Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, Alexander DC, Ahl-Goy P, Métraux JP, Ryals JA. 1991. Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3, 1085-1094.

102 Chapter 6 103 Sequential induction by conspecifics induction Sequential 104 Chapter 7 General discussion Tila R. Menzel

105 Chapter 7

Introduction Plants live in complex environments which require them to interact with a manifold of arthropods, including insects, of which ca. 50% are herbivores. However, interactions with arthropods can also benefit plants, in particular interactions with natural enemies of herbivores or even be vital for plant reproduction through interactions with pollinators. Chemical cues, such as plant volatiles, play an important role in the interactions of plants with their surrounding community (Dicke and Van Loon, 2000). Volatiles can be used by herbivores to locate their respective host plants or repel herbivores from a potential host plant (Bolter et al., 1997; Arimura et al., 2000; Kalberer et al., 2001; Conrath, 2009). Moreover, herbivore-induced plant volatiles (HIPVs) emitted from plants that are attacked by a herbivore can prime the defense of other plants for enhanced defense induction upon herbivory or they can attract natural enemies of herbivores that can act as bodyguards of the plants by ridding them of the herbivores (e.g. Dicke et al., 1990; Bruin et al., 1992; Karban et al., 2003). The latter is also known as plant indirect defense, as it affects the herbivores indirectly by attracting predators or parasitoids that attack the attackers of the plants. Indirect plant defense has been subject to a manifold of studies. However, only by using behavioral studies in combination with molecular and chemical methods the underlying mechanisms that regulate and modulate plant defense are starting to be unraveled. Already a rapidly growing body of knowledge exists on the expression of plant defense to a single herbivore in terms of changes in plant gene transcription, metabolite biosynthesis, and arthropod behavior (e.g. Dicke and Van Loon, 2000; Kessler and Baldwin, 2002; Heidel and Baldwin, 2004; De Vos et al., 2005; Thompson and Goggin, 2006; Howe and Jander, 2008; Mithöfer and Boland, 2012). However, plants are subjected to a multitude of herbivores which can attack in spatially and temporally separated events. Induced herbivore- specific fluctuations in defense-related phytohormones and cross-talk between phytohormone signaling pathways, may thus significantly alter the plant phenotype and significantly change defense expression compared to single herbivory (Dicke et al., 2009; Stam et al., 2014). Such changes in plant phenotype can affect all members of the community associated with a plant through direct or indirect interactions (e.g. Denno et al., 1995; Soler et al., 2005; Soler et al., 2007; De Boer et al., 2008). Establishing a solid basis in the understanding of molecular and chemical plant responses to multiple herbivores can help us to comprehend how plants deal with the complexity of interactions that they are exposed to in their natural environment.

The aim of the thesis was to use a multidisciplinary approach, with focus on molecular and chemical methods, combined with behavioral investigations, to elucidate the mechanisms of plant responses to multiple herbivory that affect tritrophic interactions through phenotypic changes in plants. I addressed the following research questions:

Question I: Can plant genes and metabolites that are involved in indirect defense be primed with low doses of phytohormones?

Question II: Does minor herbivory change or prime plant genes and metabolites for enhanced induction of indirect defense by subsequent herbivory?

Question III: Do herbivores that differ in feeding mode differentially influence plant indirect

106 Chapter 7

), 107 PlOS ations PlOS , 2011; , 2011; ( T. urticae T. has been et al. in situ , namely the , , 2008). According T. urticae T. , 1999; Heidel and General discussion General transcript levels were et al. et , 2004). However, , 2004). However, P. persimilis P. et al. ocimene synthase - PlOS et al. β

, 2009; Engelberth and one of its natural enemies, et al. infestation in a dose-response and . The principal volatile compounds that volatile compounds principal The . , 1999; Ament , 2008). Depending on the type of herbivore T. urticae T. et al. , 1999; Heil, 2004). In Chapter 2, I have reported I Chapter 2, 1999; Heil, 2004). In , Phaseolus lunatus et al. Tetranychus urticae Tetranychus et al. et Phytoseiulus persimilis Phytoseiulus , 2014). Moreover, it has been proposed it has been proposed that in certa , 2014). Moreover, , 2005; Ozawa ene, makes it possible to study the effects of herbivory from gene to gene from herbivory of the effects study possible to ene, makes it et al. , 2005). It has been suggested that when multiple herbivores attack a et al. ocim - are known and some defense gene sequences are available. Particularly, β - et al. (E) (2009) there are different mechanisms by which this memory formation might (2009) there are different , 2011; Wei Wei , 2011; et al. P. persimilis P. et al. transcript levels were decreased in a dose-dependent manner in response to SA application. transcript levels were decreased in a dose-dependent manner in response to SA the sequence of arrival, defense pathways induced, magnitude of defense induction, and time and induction, defense of magnitude induced, pathways defense arrival, of sequence the to Gális these phytohormonal pathways are induced and lead to activation of distinct sets of defense defense of distinct sets activation of and lead to these phytohormonal pathways are induced the synthesis of defensive metabolites. Application of these phytohormones has been shown metabolites. the synthesis of defensive induce plant direct and indirect defense responses in terms of gene transcription, to effectively the availability of the gene sequence of of attractant principal a biosynthesis of leads to that enzyme the the specialist predatory mite specialist predatory the interval between infestations (Underwood, 1998; Zhang increased in response to JA application and increased in response to JA Lima bean plants respond to exogenous application of the phytohormone JA with the Lima bean plants respond to exogenous application of the phytohormone JA Baldwin, 2004; Lou Defense signaling the the jasmonic acid (JA) pathway and major ones being pathways, the Erb plant, plant defense can become affected in different manners depending manners on factors such as in different plant, plant defense can become affected plants are able to form a sort of memory in response to a biotic stress, such as herbivory, which herbivory, as such biotic stress, a memory in response to of sort a form plants are able to metabolite resulting synthesis, and arthropod behavior (Dicke monoterpene behavior. metabolite to resulting plants, the generalistplants, the herbivorous mite shown to require JA-related signaling (Dicke signaling JA-related require to shown salicylic acid (SA) pathway, with JA and SA as hormonal signals, regulating are involved in and SA JA with pathway, salicylic acid (SA) genes (De Vos genes (De Vos on transcript levels of several relevant defense genes. Indeed, on the effect of different doses of JA, SA, and infestation levels of the herbivore of different on the effect of phytohormones. due In fact, to their importance in plant defense signaling, phytohormones occur, that is via changes the response in time, increased signal amplitude, or baseline levels occur, density-dependent manner. In fact, induction In fact, indirect defense against of density-dependent manner. defense in terms of gene transcription and metabolites? transcription of gene in terms defense activation of induced indirect defense mechanisms comprised of excretion and volatile emission (Dicke extrafloral nectar (EFN) are likely to play a pivotal role in priming. enhances the plant defense response to subsequent stresses (Frost affect affect herbivore context Activation of plant defense genes results from the induction of the signaling plant defense genes results from the induction of and pathways Activation of Phytohormones priming and in defense plant The tritrophic system which was used to study these research questions consists of Lima bean bean Lima of consists questions research these study to used was which system tritrophic The A role of phytohormones and priming in plant indirect defense mechanisms in a multiple a multiple in mechanisms defense indirect plant in priming and phytohormones of A role Chapter 7 can be affected by antagonistic cross-talk (Chapter 2; Zhang et al., 2009; Wei et al., 2014). Consequently, antagonistic cross-talk between signaling pathways provides a regulatory mechanism at the transcriptional level to fine-tune plant defense responses (Pieterse et al., 2012; Wei et al., 2014).

Interestingly, exogenous application of low doses of phytohormones that do not induce plant defenses directly, can lead to enhanced induction of defense mechanisms when followed by herbivory (Gols et al., 2003; Engelberth et al., 2011). In Chapter 3, I investigated the underlying mechanisms of this enhanced indirect defense mediated by P. persimilis. Application of a low dose of JA resulted in an increase in endogenous JA levels, which could induce synergistic effects on gene transcription and metabolites when followed by herbivory by T. urticae. Moreover, continuous feeding by herbivores would be expected to lead to increased phytohormone levels; however, feeding by a low density of only four T. urticae per plant did not affect phytohormone levels. Nevertheless, herbivory by T. urticae might induce JA accumulation at earlier time points which is supported by observed increases in the transcript level of the JA-responsive gene PlOS (Chapter 3; Zhang et al., 2009). Moreover, whereas 45 T. urticae induced transcription of enzyme genes involved in the biosynthesis of monoterpenes and diterpenes in tomato within 24 h, volatile emissions and predator attraction is only increased at day four (Kant et al., 2004). Nevertheless in Lima bean, we reported priming of the monoterpene synthase PlOS and differences in volatile profiles already after 48 h. The emission of the predator-attracting volatile (E)-β-ocimene showed evidence of priming during the afternoon, but not during the morning. Consequently, temporal dynamics as well as the diurnal cycle in biosynthesis affect plant defenses significantly (Loughrin et al., 1994; Underwood, 2012). In fact a study by Underwood et al. (2012) suggested that plant resistance against a subsequent herbivore can even change from enhanced resistance due to priming to enhanced susceptibility over time.

Gális et al. (2009) suggested that plant memory formation, or priming, is expressed by an increase in baseline levels of phytohormones, shorter response time to subsequent attack, or increased response amplitude after attack. However, the priming effect in Chapter 3 appeared not to be caused by an increase in baseline levels of endogenous phytohormone levels or increased response amplitude for phytohormone levels in plants with multiple bouts of defense induction compared to control plants. Because we did not further investigate the temporal dynamics of the phytohormone levels, we cannot exclude other mechanisms. However, priming can occur at different levels of biological organization as shown in Chapter 3. In fact, in Arabidopsis accumulation of mitogen-activated protein kinases (MAPKs) or inactive forms of transcription factors is related to priming (Beckers et al., 2009).

A role of previous herbivory in induced (indirect) plant defense mechanisms in a multiple herbivore context Plant defense responses can become primed in response to biotic stress caused by e.g. herbivory, oviposition, or HIPVs from neighboring plants (Arimura et al., 2000; Kessler and Baldwin, 2004; Kim et al., 2012; Pashalidou et al., 2013). Phytohormonal signaling underlies

108 Chapter 7

, et et did 109 et al. et might , 2008). 2008). , T. urticae T. et al. et . Caterpillars . T. urticae T. T. urticae T. , 2012; Zhang , 1997; Li , General discussion General et al. T. urticae T. et al. et , 2003). However, phytohormone phytohormone , 2003). However, , 2011; Soler , 2011; transcript levels compared to control transcript levels compared et al. et al. , 2008). Consequently, PlOS (2006) showed that caterpillar feeding can (2006) showedthat et al. et et al. et (2000) showed that an infestation by 100-150 (2000) showed that an infestation by induction, but also able to quickly downregulate induced et al. PlOS gene in response to tissue damage by either herbivore tissue damage by either gene in response to . De Vos De Vos . , 2009; Schwartzberg T. urticae T. PlOS et al. T. urticae T. , 2005). In fact, plants increase , 2005). In fact, in proportion resistance to the extent , 2000). Memory formation might thus be more complicated against 2000). , , are known to adapt to and overcome constitutive and induced are known to adapt to and overcome constitutive and , defenses et al. , 2012). In contrast, piercing-sucking herbivores are categorized as SA- et al. et et al. T. urticae T. are expected to induce the JA signaling pathway and accordingly up- we found are expected to induce the JA , 2002; Zhang results in increased levels of a gene potentially involved in plant direct defense, for 24 h induced defenses under control and of both, the JA- SA-related signaling mites can cause a priming effect in indirect defense against in indirect priming effect mites can cause a et al. , 2003; Mithöfer T. urticae T. T. urticae T. , 2013). In the arms race between plants and herbivores, , 2013). In the arms race between plants it is hypothesized that herbivores , 1999). In chapters 4, 5, and 6 I investigated investigated by caterpillars 6 I whether minor herbivory 1999).In chapters 4, 5, and , and the antagonistic cross-talk between signaling pathways. After all, spider mites induce both the antagonistic between cross-talk signaling pathways. throughout the chapters. However, previous previous defense induction by caterpillars or throughout the chapters. However, the induction of plant defense and it has been shown that priming priming shown that has been and it plant defense of the induction is feasible defense of indirect by i.e. the pathogenesis-related protein PR 4. in Chapter 4 the previous application of caterpillar oral secretions followed by an infestation induce JA induce responses, JA as reflected by within few generations (Agrawal, 2000; Kant within few generations (Agrawal,2000; response to mite treatment. Generally, repeated feeding by herbivoresrepeated feeding in results in increases Generally, response to mite treatment. regulation of the JA-inducible regulation of mites are categorized as JA-signaling-pathway categorized as are inducers (McConn mites mediated interaction with pests such as pathway. SA-inducing herbivores have been shown to interfere with JA-related plant defenses pathway. plant defense according to the herbivore density and damage level (Underwood, to the herbivore density and damage plant defense according Gols 2000; plants in all chapters, we did not record an increase in endogenouswe did not levels in phytohormone all chapters, plants in not result in enhanced transcription or volatile gene not result level of or primed indirect defense on the plants compared to blends plants emitted from that experienced actual herbivory (Dicke Underwood, 2000). However, Ozawa Underwood, 2000). However, of feeding. Biting-chewing herbivores, as caterpillars, and certain cell-content feeding such oral secretions did not enhance or sensitize indirect defense mechanisms against of damage until approximately 90% of leaf area has been lost (Baldwin and Schmelz, 1994; develop mechanisms to avoid detection and suppress plant defenses. Highly polyphagous enhance PR- gene transcription in Arabidopsis when followed by pathogen infestation. In fact, enhance transcription PR- gene in either. Investigating time points other and mechanisms be useful may in studying the plant- either. emission. Moreover, although emission. Moreover, and application application by herbivory (Ozawa the defense induction not fully resemble does certain herbivores than others. Interestingly, caterpillar feeding and application caterpillar caterpillar of Interestingly, certain herbivoresthan others. Specialist predators are able to distinguish HIPV blends emitted from phytohormone-induced via application of exogenous JA (Chapter 3; Gols (Chapter via application of exogenous JA 2002; Pieterse (Moran et al. al. al. Effect of herbivore feeding mode on plant defenseplant modeon induction feeding herbivore of Effect JA accumulation. This might be achieved by using the plant’s own regulatory mechanisms, e.g. This might be achieved by using the plant’s accumulation. JA (Ozawa and SA JA Traditionally, plant herbivores are characterized as JA or SA inducers based on their mode or SA plant herbivores are characterized as JA Traditionally, T. urticae T. T. urticae T. Chapter 7 signaling-pathway inducers (Kempema et al., 2007). Exogenous application of the respective phytohormones has been used to induce defense responses that mainly resemble the ones induced by the corresponding herbivore feeding category (Dicke et al., 1999; Zhang et al., 2009). In fact, herbivorous arthropods induce phytohormonal signal signatures, whereby insects of the same or different feeding guilds induce the activation of specific sets of defense-related genes and transcriptomic changes (De Vos et al., 2005; Bidart-Bouzat and Kliebenstein, 2011). Other members of the community such as natural enemies of herbivores, rely on host-location cues provided by plants, when searching for their arthropod food source. Particularly specialist carnivores are expected to be able to distinguish between volatile blends induced by specific herbivores in order to locate their respective prey or host. Consequently, especially specialist natural enemies are expected to rely on differences in volatile blends emitted from plants attacked by different herbivores and herbivore combinations. Stam et al. (2014) suggested that when herbivores attack a plant sequentially different effects on interactions can occur. The effects include priority effects, overriding effects, and canalization. Hereby factors such as sequence of arrival, defenses induced, amplitude of defense induction and time lag between multiple herbivores might play an important role. Attack by herbivores with different feeding modes can have beneficial or adverse effects on plant indirect defense depending on the defense signaling pathways induced (De Boer et al., 2008; Zhang et al., 2009).

A role of feeding mode in induced (indirect) plant defense mechanisms in a multiple herbivore context Using molecular tools and chemical analysis there is a rapidly growing body of evidence that plants themselves are able to distinguish different herbivores and even show distinct gene activation in response to herbivores which induce the same signaling pathway(s) (De Vos et al., 2005). In Chapters 4-6 we investigated whether herbivores with different feeding modes, namely caterpillars and mites, that are known to induce the same defense signaling pathway, i.e. the JA pathway, differentially affect plant indirect defense mechanisms against T. urticae. Zhang et al. (2009) showed that T. urticae-induced defense can be reduced by an infestation by herbivores with a different feeding mode, i.e. whiteflies, through antagonistic cross-talk of the defense signaling pathways (Zhang et al., 2009). Moreover, this cross-talk induced by piercing-sucking whiteflies, also caused suppression of plant defenses against caterpillars in Arabidopsis (Zhang et al., 2013).

We found that PlOS gene transcript levels did not significantly differ between plants that were exposed to multiple bouts of herbivory by herbivores with the same or different feeding mode compared to plants that experienced only one bout of herbivory. However, temporal gene transcription patterns of PlOS differ in response to herbivory by arthropods with different feeding mode (Chapter 5). Predatory mites, such as P. persimilis are able to distinguish between volatile blends of plants with prey and non-prey herbivores (Chapter 5; De Boer et al., 2008) and, accordingly, we found quantitative differences in the emission rates of three principal attractants of P. persimilis and a green leaf volatile between plants infested by caterpillars and T. urticae. Moreover, we found that defense gene induction differed in

110 Chapter 7 111 (2011) showed (2011) et al. et General discussion General herbivory was compared to plants herbivory compared to plants with only plants to herbivory compared T. urticae T. , depending , depending on how damage the caterpillar by T. urticae T. feeding, has not been investigated. feeding, has not been , 2011). In fact, Bidart-Bouzart fact, In 2011). , T. urticae T. et al. et T. urticae T. , 1991). The gene’s transcript levels did not show the same levels did not show transcript The gene’s , 1991). feeding followed by et al. . herbivory. Whether this difference in gene induction between feeding and in Whether this difference herbivory. oral secretion followed by oral secretion followed M. brassicae , 2005; Bonaventure , had been applied had been codes for of PR-4, which Gene transcript levels (Chapter 4). et al. et T. urticae T. herbivory (Ward herbivory (Ward M. brassicae M. that arthropods with the same feeding mode induce different transcriptomic changes in that arthropods the same feeding with mode induce different by feeding or mechanical damage with oral secretions by the same herbivore. Moreover, by feeding or mechanical by the same herbivore. Moreover, damage with oral secretions increase when feeding rhythm to identify herbivore identity and adjust their defense responses accordingly identify herbivore identity and adjust their to feeding rhythm while herbivores same signaling may induce the and even cause induction pathway of the with only with response to a subsequent exposure to exposure subsequent a to response However, this suggests that mechanisms of plant indirect defense are indeed affected are indeed affected plant indirect defense mechanisms of suggests that this However, same genes, shifts in temporal patterns and amplitude same genes, shifts plants of transcript levels show that specifically bydifferent herbivores and that plants differentiate between damage caused oral secretions also caused a shift in the temporal pattern of induction caused a shift in the temporal pattern oral secretions also like for of this gene, do distinguish between those herbivores. Plants are likely to use more herbivore-specific a chitinasein plants treated were increased plant direct defense, be involved in and might cues besides feeding mode, such as for example herbivore-associatedexample cues besides feeding mode, such as for and elicitors (HAE), caterpillar oral secretions versus caterpillar oral secretions M. brassicae (Mithöfer Arabidopsis thaliana T. urticae T. Chapter 7

Conclusions and future perspectives In this thesis we explored the effects of multiple herbivory on the mechanisms that guide plant indirect defense against the pest T. urticae. The work gives important new insights into understanding plant defense mechanisms (see Main conclusions below) and provides helpful leads for future research concerning how plants deal with multiple stresses.

Topics for future research are to investigate whether application of low doses of other phytohormones, that do not interfere with JA-related defense signaling (e.g. ethylene; see Horiuchi et al., 2001) have a potential for priming of Lima bean indirect defenses and to test the applicability under field conditions. Also, the temporal dynamics of plant defense mechanisms and the effect of the diurnal cycle on priming should receive further attention. In this context it would be interesting to use a larger transcriptomic approach to see differences in priming by phytohormones versus actual herbivory. To achieve this the genome sequence of Lima bean needs to become available. In contrast to our attempt to induce priming by actual herbivory for indirect defense mechanisms, this phenomenon has been successfully observed for plant direct defense. This also means that it needs to be investigated whether the finding of the research in this thesis can be applied to other plant species and tritrophic systems. Investigating whether low-dose phytohormone application results in priming of defense mechanisms in other plant species can provide more solid knowledge about this plant defense mechanism and on what level of biological organization plant memory to stressful events is retained in different plant species. It could also give information whether plant species vary in their capability of forming memories. It would be interesting to study whether certain herbivores, such as T. urticae or aphids, that induce both JA and SA signaling pathways (Ozawa et al., 2000; Moran and Thompson, 2001; Moran et al., 2002) are able to manipulate plant resistance to avoid memory formation and thus do not lead to increased resistance against subsequent herbivores. Therefore, induction of direct defense mechanisms might also be included. Because application of caterpillar oral secretions is frequently used to mimic herbivory to induce plant defenses in a dose- and damage-controlled manner, another issue that needs to be investigated is the observed difference in defense induction between caterpillar feeding and oral secretions. For this, it would be useful to study the temporal dynamics of phytohormones and the volatile profiles of plants treated with feeding or oral secretions. This would give more information on whether our results represent temporal shifts in defense or different mechanisms being induced by these treatments.

Finally, it should be recognized that deepening our knowledge of the mechanistic aspects underlying and guiding tritrophic interactions is not only important to gain a principal understanding of complex ecological interactions. Despite the use of pesticides and other artificially introduced plant protection measures, agricultural losses due to herbivorous insects and plant pathogens, some of which can use insects as vectors, is estimated to amount to 25 % for the USA (Pimentel and Andow, 1997). At the same time, the human population keeps growing, thus increasing the demand for plant-based food and material, while the agricultural area keeps diminishing. Consequently, it remains of great importance to keep investigating the underlying mechanisms of plant defense against herbivorous arthropods to expand

112 Chapter 7 113 General discussion General Feeding by arthropods or the combination of artificial wounding and elicitor application elicitor and wounding artificial of combination the or arthropods by Feeding Different herbivores induce differential temporal dynamics in induced indirect plant herbivores induce differential Different Priming of plant indirect defense mechanisms is not necessarily based on defense mechanisms is not necessarily Priming of plant indirect Plant indirect defense mechanisms are stable to multiple herbivory by conspecific Plant indirect defense mechanisms can be primed. Plant indirect defense iv) ii) iii) i) v) in feeding mode. increased levels of phytohormones. defense mechanisms. shifting temporal patterns. on defense gene transcription by their effects in differ and heterospecific herbivores that induce the same defense pathway even if they differ fundamental fundamental knowledge be used that can understandto not only and conserve but nature, underlie Lima bean indirect defense against multiple herbivores. The main conclusions the for underlie herbivores. Lima bean indirect defense against multiple systems studied in this project are: systems studied in this I thank Marcel Dicke and Joop J.A. van Loon I thank Marcel Dicke and Joop J.A. van for helpful an and constructive comments on earlier version of this chapter. also develop tools to secure human food supply in a sustainable way. food supply in tools to secure human also develop Main ConclusionsMain The work described The work has increased in this thesis the understanding in the mechanisms that Acknowledgments Chapter 7

References Agrawal AA. 2000. Host-range evolution: Adaptation and trade-offs in fitness of mites on alternative hosts. Ecology 81, 500-508.

Arimura GI, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J. 2000. Herbivory- induced volatiles elicit defence genes in Lima bean leaves. Nature 406, 512-515.

Baldwin IT, Schmelz EA. 1994. Constraints on an induced defense: The role of leaf area. Oecologia 97, 424-430.

Bonaventure G, VanDoorn A, Baldwin IT. 2011. Herbivore-associated elicitors: FAC signaling and metabolism. Trends in Plant Science 16, 294-299.

Bruin J, Dicke M, Sabelis MW. 1992. Plants are better protected against spider-mites after exposure to volatiles from infested conspecifics. Experientia 48, 525-529.

Conrath U. 2009. Priming of induced plant defense responses. Plant Innate Immunity 51, 361-395.

De Vos M, Van Zaanen W, Koornneef A, Korzelius JP, Dicke M, Van Loon LC, Pieterse CMJ. 2006. Herbivore-induced resistance against microbial pathogens in Arabidopsis. Plant Physiology 142, 352-363.

Denno RF, McClure MS, Ott JR. 1995. Interspecific interactions in phytophagous insects: Competition reexamined and resurrected. Annual Review of Entomology 40, 297-331.

Dicke M, Gols R, Ludeking D, Posthumus MA. 1999. Jasmonic acid and herbivory differentially induce carnivore-attracting plant volatiles in Lima bean plants. Journal of Chemical Ecology 25, 1907-1922.

114 Chapter 7 , ) to 115 2001. Exogenous Phaseolus lunatus Oreina cacaliae General discussion General 2004. Differential timing of 2004. Differential 2003. Herbivore damage to 2008. Plant defense priming against 2008. Plant defense 2008. Intraspecific variation in a generalist a in variation Intraspecific 2008. 2011. Low concentrations Low concentrations of salicylic acid 2011. 2007. Arabidopsis transcriptome changes 2007. seedlings. Journal of Chemical Ecology 37, 2011. Sequence 2011. of arrival determines plant- 2003. Induction of direct and indirect plant direct and indirect 2003. Induction of 2009. Molecular mechanisms underlying 2009. Molecular mechanisms plant 2001. Attraction of a leaf beetle ( 2001. Zea mays to attack by insects from multiple feeding guilds. Plant, to attack by insects from multiple feeding 2009. Chemical complexity of volatiles from plants induced plants induced volatiles from complexity of 2009. Chemical 2000. Multitrophic effects of herbivore-induced of plant volatiles in an Multitrophic 2000. effects 2002. Plant responses to insect herbivory: The emerging molecular 2002. Plant responses to insect herbivory: 2008. Plant immunity to insect herbivores. Annual Review of Plant 2008. Plant immunity to insect herbivores. 2004. Microarray analysis of salicylic acid- and jasmonic acid-signaling Nicotiana attenuata 2004. Induction of two indirect defences benefits Lima bean ( treatment and spider mite infestation. Journal of Chemical Ecology 29, 2651-2666. treatment and spider mite infestation. Journal herbivore accounts for differential induction and impact of host plant defences. Proceedings herbivore for differential accounts herbivores: Getting ready for a different battle. Plant Physiology 146, 818-824. battle. Plant Physiology ready for a different herbivores: Getting by multiple attack. Nature Chemical Biology 5, 317-324. attack. Nature Chemical by multiple in response to phloem-feeding silverleaf whitefly nymphs. Similarities and distinctions in in responses of Erb M, Robert CA, Hibbard BE, Turlings TC. Turlings BE, Hibbard CA, Robert M, Erb Engelberth J, Viswanathan S, Engelberth MJ. S, Engelberth J, Viswanathan Engelberth Dicke M, Van Loon JJA, Soler R. JJA, Soler Loon Van Dicke M, Dicke M, Van Loon JJA. M, Van Dicke Howe GA, Jander G. Biology 59, 41-66. Horiuchi JI, Arimura GI, Ozawa R, Shimoda T, Takabayashi J, Nishioka T. J, Nishioka Takabayashi T, R, Shimoda Ozawa Arimura GI, JI, Horiuchi Letters 509, 332-336. Heil M. Heidel AJ, Baldwin IT. Heidel Frost CJ, Mescher MC, Carlson JE, De Moraes CM. JE, De Mescher MC, Carlson Frost CJ, Kessler A, Baldwin IT. IT. A, Baldwin Kessler Kempema LA, Cui X, Holzer FM, Walling LL. Walling Holzer FM, Cui X, Kempema LA, Karban R, Maron J, Felton GW, Ervin G, Eichenseer H. GW, Karban R, Maron J, Felton Kant MR, Sabelis MW, Haring MA, Schuurink RC. Kant MR, Sabelis MW, Kant MR, Ament K, Sabelis MW, Haring MA, Schuurink RC. Haring MA, Schuurink Ament K, Sabelis MW, Kant MR, Kalberer NM, Turlings TCJ, Rahier M. Kalberer NM, Turlings responses to aphids. Plant Physiology 143, 849-865. responses by jasmonic acid, low spider mite densities, or a combination responses by jasmonic acid, low spider of jasmonic acid memory in JA-mediated defence responses. Plant, Cell and Environment 32, 617-627. defence responses. Plant, Cell and memory in JA-mediated mediated interactions between herbivores. Journal of Ecology 99, 7-15. between herbivores. Journal of Ecology mediated interactions Fabaceae) in nature. Journal of Ecology 92, 527-536. Fabaceae) in nature. Journal of Ecology sagebrush induces resistance in wild tobacco: Evidence for eavesdropping between plants. spider mite-induced and indirect defenses in tomato plants. Plant Physiology direct 483- 135, stimulate insect elicitor responsesstimulate insect elicitor in of the Royal Society B: Biological Sciences 275, 443-452. of the Royal Society B: Biological Sciences damaged host plants. Journal of Chemical Ecology 27, 647-661. damaged host plants. Journal of Chemical analysis. Annual Review of Plant Biology 53, 299-328. analysis. evolutionary context. Entomologia Experimentalis et Applicata 97, 237-249. et Experimentalis context. Entomologia evolutionary 263-266. Gols R, Roosjen M, Dijkman H, Dicke M. Dicke H, Dijkman M, Roosjen R, Gols Gális I, Gaquerel E, Pandey SP, Baldwin IT. Baldwin Pandey SP, E, Gaquerel I, Gális Oikos 100, 325-332. Cell and Environment 27, 1362-1373. 495. ACC enhances volatiles production mediated by jasmonic acid in Lima bean leaves. FEBS Chapter 7

Kim J, Tooker JF, Luthe DS, De Moraes CM, Felton GW. 2012. Insect eggs can enhance wound response in plants: a study system of tomato Solanum lycopersicum L. and Helicoverpa zea Boddie. PLOS ONE 7.

Li C, Williams MM, Loh YT, Gyu IL, Howe GA. 2002. Resistance of cultivated tomato to cell content-feeding herbivores is regulated by the octadecanoid-signaling pathway. Plant Physiology 130, 494-503.

Loughrin JH, Manukian A, Heath RR, Turlings TCJ, Tumlinson JH. 1994. Diurnal cycle of emission of induced volatile terpenoids by herbivore- injured cotton plants. Proceedings of the National Academy of Sciences of the United States of America 91, 11836-11840.

Mithöfer A, Boland W. 2012. Plant defense against herbivores: Chemical aspects. Annual Review of Plant Biology 63, 431-450.

Moran PJ, Cheng Y, Cassell JL, Thompson GA. 2002. Gene expression profiling of Arabidopsis thaliana in compatible plant-aphid interactions. Archives of Insect Biochemistry and Physiology 51, 182-203.

Moran PJ, Thompson GA. 2001. Molecular responses to aphid feeding in Arabidopsis in relation to plant defense pathways. Plant Physiology 125, 1074-1085.

Ozawa R, Shiojiri K, Sabelis MW, Takabayashi J. 2008. Maize plants sprayed with either jasmonic acid or its precursor, methyl linolenate, attract armyworm parasitoids, but the composition of attractants differs. Entomologia Experimentalis et Applicata 129, 189-199.

Pashalidou FG, Lucas-Barbosa D, Van Loon JJA, Dicke M, Fatouros NE. 2013. Phenotypic plasticity of plant response to herbivore eggs: Effects on resistance to caterpillars and plant development. Ecology 94, 702-713.

Pieterse CMJ, Van Der Does D, Zamioudis C, Leon-Reyes A, Van Wees SCM. 2012. Hormonal modulation of plant immunity. Annual Review of Cell and Developmental Biology 28, 489-521.

Pimentel D, Andow DA. 1997. Techniques for reducing pesticides: Environmental and economic benefits. Chichester, UK: Wiley.

116 Chapter 7

, 117 2009. 2014. Plant 2014. Reciprocal General discussion General Acyrthosiphon Acyrthosiphon pisum 2005. Root herbivore 2005. Root herbivore . Journal of Chemical . Journal of Ecology 37, 2011. Pea aphids, 2011. Vicia faba Vicia 2007. Root herbivores influence the behaviour of an . New Phytologist 197, 1291-1299. 2006. Transcriptomics and functional genomics of plant defence Transcriptomics 2006. 1991. Coordinate gene activity in response to agents that induce agents that 1991. Coordinate gene activity in response to 1998. The timing of induced resistance and induced susceptibility in the 1998. Arabidopsis thaliana 2012. When herbivores come back: Effects of repeated damage on induced 2012. When herbivores come back: Effects 2000. Density dependence in induced plant resistance to herbivore damage: brassicaceous brassicaceous insect performance plant: From to gene Functional transcription. Ecology 26, induction by phloem-feeding insects. Journal of Experimental Botany 57, 755-766. induction by phloem-feeding insects. Journal interactions with multiple insect herbivores: From community to genes. Annual Review of interactions with multiple herbivores: insect to genes. From community in plant quality. Journal of Animal Ecology 74, 1121-1130. Animal Ecology 74, Journal of in plant quality. P, Métraux JP, Ryals JA. JP, Métraux P, Plant Biology 65, 689–713. resistance. Functional Ecology 26, 1441-1449. Wei J, Van Loon JJA, Gols R, Menzel TR, Li N, Kang L, Dicke M. R, Menzel JJA, Gols Loon J, Van Wei Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, Alexander DC, Ahl-Goy Alexander DC, DL, Wiederhold SS, Dincher SC, Williams SJ, Uknes ER, Ward plant defense in Underwood NC. Underwood N. Underwood Underwood N. Underwood Zhang PJ, Zheng SJ, Van Loon JJA, Boland W, David A, Mumm R, Dicke M. R, Mumm A, David W, JJA, Boland Loon Van SJ, Zheng PJ, Zhang Zhang PJ, Broekgaarden C, Zheng SJ, Snoeren TAL, Van Loon JJA, Gols R, Dicke M. Van Zhang PJ, Broekgaarden C, Zheng SJ, Snoeren TAL, Whiteflies interfere with indirect plant defense against spider mites in Lima bean. Proceedings bean. Lima in mites spider against defense plant indirect with interfere Whiteflies systemic acquired resistance. Plant Cell 3, 1085-1094. systemic acquired resistance. Plant Cell soybean-Mexican bean beetle system. Oecologia 114, 376-381. Oecologia 114, soybean-Mexican bean beetle system. suppress inducedsuppress plant volatiles bean, in broad of the National Academy of Sciences of the United States of America 106, 21202-21207. Academy of Sciences of the United States of of the National emission and herbivore behavior. Journal of Experimental Botany 65, 3289-3298. emission and herbivore behavior. antagonism between two defense-signaling antagonism between two pathways in Lima bean modulates volatile aboveground parasitoid through changes in plant-volatile signals. Oikos 116, 367-376. Oikos 116, through changes in plant-volatile signals. aboveground parasitoid JJA,Poelman EH, Dicke M. Loon Gols R, Van Y, A, Li Stam JM, Kroes Soler R, Harvey JA, Kamp AFD, Vet LEM, Van Der Putten WH, Van Dam NM, Stuefer Dam NM, Van WH, Der Putten Van LEM, Vet AFD, HarveyJA, Kamp R, Soler Soler R, Bezemer TM, Van Der Putten WH, Vet LEM, Harvey JA. Der Putten WH, Vet Soler R, Bezemer TM, Van on above-ground herbivore, parasitoid and hyperparasitoid changes performance via effects Soler R, Badenes-Pérez FR, Broekgaarden C, Zheng SJ, David A, Boland W, Dicke Dicke M. W, A, Boland Soler R, Badenes-Pérez FR, Broekgaarden C, Zheng SJ, David Schwartzberg EG, Böröczky K, Tumlinson JH. EG, Böröczky Schwartzberg K, Tumlinson 2013. Jasmonate and ethylene signaling mediate whitefly-induced interference with indirect 2012. Plant-mediated facilitation between leaf-feeding a and a phloem-feeding insect in a

Thompson GA, Goggin FL. Thompson GA, Goggin JF, Gols R, Hordijk CA, Bezemer Hordijk Gols R, TM. JF, Threshold, strength and genetic variation. Oikos 89, 295-300. Threshold, strength and genetic variation. 156-166. 1055-1062. 118 Summary

Summary

Plants live in complex environments and are under constant threat of being attacked by herbivores, such as insects and mites. Next to a remarkable ability to regenerate, plants possess sophisticated defense mechanisms. Two types of defenses are generally distinguished: constitutive and induced defenses. Constitutive defense includes for example thorns and trichomes (hairs). Induced defenses include chemical compounds such as toxins and herbivore-induced plant volatiles or extrafloral nectar. Induced defense can be further divided into direct and indirect mechanisms. The first directly act against the attacker by affecting its behavior or reducing its growth rate, whereas indirect defenses involve the attraction of natural enemies of herbivores that can act as a kind of bodyguards to plants. Natural enemies of herbivores use odors emitted by damaged plants that serve as a “cry for help” to find their prey or host herbivore. The odors emitted by damaged plants can consist of up to 200 compounds, of which a fraction is used by a natural enemy to find its food source, the herbivore. Nevertheless, based on the odor blend natural enemies can gain information about the identity of the plant, identity of the herbivore, developmental stage of the herbivore and so on.

Many studies have investigated the herbivore-induced changes that occur at different levels of biological organization such as plant hormone levels (phytohormones), gene level, metabolite level, and the behavior of natural enemies and herbivores in response to such changes. However, in their natural environment these interactions are much more complicated than investigated in these studies. For instance, plants are frequently attacked by more than one herbivore. This attack by multiple herbivores can occur simultaneously or in events spaced over time, on the same organ or on different organs, which can have different effects on how plant defenses are expressed. This, however, has not been investigated for many systems and different scenarios.

Recently, it has been suggested that plants even possess a sort of memory, which allows them to respond to a second attack more quickly and more strongly than when the first attack had not taken place. This phenomenon, sometimes referred to as “priming”, is often compared to mammalian vaccination, however, plants do not possess an immune system comparable to that of mammals. Nevertheless, plants do possess a complex signaling network, which allows them to sense and recognize a herbivorous attack and initiate adequate defense responses. Many studies have investigated these signaling networks and their interactions. Yet, studies addressing how multiple herbivore attacks shape defense responses and the underlying signaling networks have only been initiated relatively recently.

The aim of this thesis was therefore to use a multidisciplinary approach, with focus on molecular and chemical methods, combined with behavioral investigations, to elucidate the mechanisms of plant responses to multiple herbivory that affect a tritrophic system consisting of a plant, an herbivore and a natural enemy. In Lima bean plants the five principal components that mediate

119 the attraction of the predatory mite Phytoseiulus persimilis to plants infested with its prey, the herbivorous mite Tetranychus urticae, are known to consist of (E)-4,8-dimethylnona-1,3,7- triene [(E)-DMNT] and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene [(E,E)-TMTT], linalool, methyl salicylate (MeSA), and (E)-β-ocimene. Moreover, despite the fact that the Lima bean genome has not been sequenced yet, sequences of a limited number of genes potentially involved in Lima bean defense have been identified. Since the predatory mite P. persimilis is a specialist predator that preys only on mites in the genus Tetranychus, this predator heavily relies on accurate information conveyed by plant odors.

In Chapter 1, relevant literature is reviewed about plant defense and tritrophic interactions and the study system is introduced in more detail.

In Chapter 2, I present studies on the response of three defense-related genes, Phaseolus lunatus lipoxygenase (PlLOX), Phaseolus lunatus β-ocimene synthase (PlOS) and Phaseolus lunatus pathogenesis-related protein 4 (PlPR-4) to different doses of phytohormones and different densities of the herbivore Tetranychus urticae. Exogenous application of phytohormones was used to simulate herbivory and to induce defense gene transcription. The jasmonic acid (JA)-responsive gene PlOS responded to exogenous JA and salicylic acid (SA), reflecting the antagonistic interaction between the JA and SA phytohormone signaling pathways. Furthermore, PlOS transcript levels positively correlated to density of JA-inducing T. urticae mites. Transcript levels of another JA-responsive gene, PlLOX, did not show a correlation to phytohormone doses or herbivore densities at the time point investigated here. The previously reported methyl salicylate-responsive gene, PlPR-4, did not respond to treatment with SA.

In Chapter 3, I report on experiments to investigate how application of a low dose of JA followed by minor herbivory by T. urticae spider mites affects gene transcript levels of PlOS, emission of (E)-β-ocimene and nine other plant volatiles commonly associated with herbivory. Furthermore, I investigated the plants’ phytohormonal response. Application of a low dose of JA increased PlOS transcript levels in a synergistic manner when followed by minor herbivory for both simultaneous and sequential infestation. Emission of (E)-β-ocimene was also increased, and only JA, but not SA, levels were affected by treatments. Analysis of other volatiles showed overlap between volatile blends of plants exposed to different treatments. Thus, a low-dose JA application results in a synergistic effect on gene transcription and an increased emission of a volatile compound involved in indirect defense after herbivore infestation. This connects well to earlier experiments that had shown that the application of a low JA dose enhanced the attraction of P. persimilis when the plants were subsequently infested with spider mites.

In Chapter 4, I used feeding damage or the combination of mechanical damage and oral secretions of the caterpillar species Mamestra brassicae and Spodoptera exigua to study the damage- and species-specific effects on transcript levels of three defense-related genes in Lima bean, i.e. PlLOX, PlOS, and PlPR-4. Since induction of defense can affect subsequent herbivores, I also investigated how the induction of defense genes by feeding damage or mechanical damage plus oral secretion affected the defense response to subsequent

120 Summary herbivory by T. urticae spider mites. Whereas patterns of gene transcription were mostly identical in response to the two caterpillar species, feeding damage or mechanical damage plus caterpillar oral secretion caused differential induction of the transcription of defense genes. Nevertheless, compared to plants with single herbivory, plants with dual herbivory only showed differential gene induction for PlPR-4. Plants responded differently to caterpillar feeding than to mechanical damage plus caterpillar oral secretion, which resulted in different effects on plant direct and indirect defense against subsequent herbivores as well.

In Chapter 5, I investigated the effect of sequential induction of plant defense by M. brassicae caterpillar oral secretion and an infestation by T. urticae spider mites on the expression of indirect plant defense in Lima bean plants. The effect on indirect defense was assessed using behavioral assays with the specialist predatory mite P. persimilis in an olfactometer, headspace analysis of 11 major herbivore-induced plant volatiles including (E)-β-ocimene and transcript levels of the corresponding gene PlOS. Predatory mites were found to distinguish between plants induced by spider mites and plants induced by the combination of artificial mechanical damage and caterpillar oral secretion but not between plants with single spider mite infestation and plants induced by caterpillar oral secretion prior to spider mite infestation. Indeed, the volatile blends emitted by plants induced by spider mites only and the sequential induction treatment of caterpillar oral secretion followed by spider mite infestation, were similar. The data presented in this thesis suggest that the induction of plant indirect defense is not affected by previous treatment with oral secretion of M. brassicae caterpillars.

In Chapter 6, I studied the effect of herbivory by T. urticae mites on two components of induced indirect defense against subsequent herbivory by conspecific mites on Lima bean. We studied the emission of (E)-β-ocimene and 12 other plant volatiles commonly associated with herbivory, as well as the transcription of two genes involved in Lima bean defense, namely PlOS and PlPR-4. Volatile profiles and gene transcript level of plants attacked by herbivores differed significantly from that of control plants. Emission of volatiles did not differ between plants that experienced two bouts of herbivore attack by conspecific spider mites compared to plants that experienced only one bout of spider mite attack. Moreover, transcript levels of PlOS and PlPR-4 did not differ for these treatments. The results suggest that Lima bean plants do no increase defense in response to sequential herbivory by two-spotted spider mites under the exposure regime tested.

The results are discussed in the context of recent literature in Chapter 7. In conclusion, the work compiled in this thesis presents new insights in the mechanisms of induction of indirect plant defense and tritrophic interactions in a multiple herbivore context and provides helpful leads for future research concerning how plants deal with multiple stresses.

121 122 Samenvatting

Samenvatting

Planten leven in complexe omgevingen en staan onder de constante dreiging aangevallen te worden door plantenetende organismen zoals insecten en mijten. Naast een opmerkelijk vermogen om te regenereren, bezitten planten verfijnde verdedigingsmechanismen. In het algemeen worden twee soorten verdediging onderscheiden: constitutieve en induceerbare verdediging. Constitutieve verdediging omvat bijvoorbeeld doorns en trichomen (haren). Induceerbare verdediging is met name gebaseerd op chemische verbindingen, zoals toxines, afwerende vluchtige stoffen of extraflorale nectar. Geïnduceerde verdediging kan verder onderverdeeld worden in directe en indirecte geïnduceerde verdediging. De eerste beïnvloedt de aanvaller direct via bijvoorbeeld het veranderen van het gedrag of het verminderen van de groei, terwijl indirecte verdediging werkt via natuurlijke vijanden van planteneters. Deze natuurlijke vijanden van planteneters gebruiken geuren die door beschadigde planten verspreid worden. Het geurmengsel dat verspreid wordt door beschadigde planten kan uit enkele tientallen tot wel 200 verbindingen bestaan. Een aantal daarvan wordt gebruikt door een natuurlijke vijand van planteneters om zijn voedselbron te vinden. Niettemin kan het geurmengsel informatie bevatten over de identiteit van de plant, de identiteit van de planteneter, het ontwikkelingsstadium van de planteneter, enz.

Veranderingen die gepaard gaan met geïnduceerde verdediging zijn onderzocht op verschillende niveaus van biologische organisatie, zoals gen-niveau, metaboliet-niveau, en individu-niveau. Op het individu-niveau wordt bijvoorbeeld het gedrag van natuurlijke vijanden en planteneters bestudeerd in reactie op veranderingen in de emissie van metabolieten. In hun natuurlijke omgeving zijn deze interacties veel ingewikkelder dan onderzocht in zulke studies. Zo worden planten vaak aangevallen door meer dan één planteneter. Deze aanval door meerdere planteneters kan gelijktijdig of verspreid in de tijd plaatsvinden, op hetzelfde orgaan of op verschillende organen, wat verschillende effecten op verdedigingsmechanismen van planten kan hebben. Deze effecten op verdedigingsmechanismen zijn slechts voor enkele plantensoorten onderzocht.

Recentelijk is gesuggereerd dat planten zelfs een soort geheugen bezitten, dat hen, na een eerste aanval, in staat stelt om sneller en sterker te reageren op een tweede aanval. Dit verschijnsel, ook wel “priming” genoemd, wordt vaak vergeleken met inenten, echter planten hebben geen immuunsysteem zoals zoogdieren. Planten, daarentegen, bezitten een complex signaleringsnetwerk, waardoor ze planteneters kunnen herkennen, en hun verdediging kunnen opstarten. Veel studies hebben deze signaleringsnetwerken en hun interacties onderzocht, maar onderzoek naar verdedigingsreacties tegen meerdere planteneters en de onderliggende signalerende netwerken is schaars.

Het doel van dit proefschrift was dan ook om een multidisciplinaire benadering te volgen door gebruik te maken van moleculair-genetische en chemisch-analytische methoden, gecombineerd met gedragsonderzoek, om de invloed van meerdere planteneters op

123 de mechanismen van verdediging in een tritroof systeem te onderzoeken, dat bestaat uit een plant, een planteneter en een natuurlijke vijand. In Limaboon zijn de vijf belangrijkste componenten van het geurmengsel die een rol spelen in de aantrekking van de roofmijt Phytoseiulus persimilis nadat de planten geïnfecteerd zijn door plantenetende mijten (Tetranychus urticae), bekend. Deze componenten zijn (E)-4,8-dimethylnona-1,3,7-trieen en (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraeen, linalool, methylsalicylaat en (E)-β-ocimeen. Bovendien zijn de sequenties van een beperkt aantal genen, die mogelijk betrokken zijn bij verdediging, geïdentificeerd. De roofmijt P. persimilis is een specialistische roofvijand die alleen mijten in het geslacht Tetranychus eet. Daarom is deze roofmijt sterk afhankelijk van nauwkeurige informatie die beschikbaar is in de vorm van plantengeuren.

In Hoofdstuk 1 wordt een overzicht gegeven van de relevante literatuur over verdediging van planten en tritrofe interacties en het studiesysteem wordt in detail geïntroduceerd.

In Hoofdstuk 2 presenteer ik het effect van behandeling van planten met verschillende doses van enkele fytohormonen en verschillende dichtheden van de planteneter Tetranychus urticae op drie genen die een rol spelen in de verdediging van Limaboon, Phaseolus lunatus lipoxygenase (PlLOX), Phaseolus lunatus β-ocimene synthase (PlOS) en Phaseolus lunatus pathogenesis-related protein 4 (PlPR-4). Uitwendige toediening van plantenhormonen wordt vaak gebruikt voor het simuleren van herbivorie en het induceren van de transcriptie van genen die betrokken zijn bij plantenverdediging. De transcriptie van het gen PlOS, dat door behandeling met het fytohormoon jasmonzuur (JA) wordt aangeschakeld, wijst op een antagonistische interactie tussen de JA- en SA-signaalwegen. Bovendien was de transcriptie van PlOS positief gecorreleerd met herbivorie door T. urticae mijten. De vraat van deze mijten induceert JA in planten. De transcriptie van het andere gen dat op JA reageert, PlLOX, liet geen correlatie zien met T. urticae-dichtheden of plantenhormonen op het tijdstip dat hier onderzocht is. Het salicylzuur(SA)-responsieve gen PlPR-4 reageerde niet op de behandeling met SA.

In Hoofdstuk 3 beschrijf ik hoe de toediening van een lage dosis van JA de transcript-niveaus van het gen PlOS en de emissie van (E)-β-ocimeen en negen andere vluchtige plantenstoffen beïnvloedt als het gevolgd wordt door een infectie met een klein aantal T. urticae spintmijten. Verder heb ik de inductie van fytohormonen in de planten onderzocht. Toepassing van een lage dosis van JA verhoogde de transcript-niveaus van PlOS op een synergistische manier, wanneer dit gevolgd werd door infectie met een klein aantal T. urticae. Dit was zowel het geval bij simultane als bij sequentiële behandeling met JA en spintmijten. Emissie van (E)-β- ocimeen werd ook verhoogd, en het JA niveau, maar niet het SA niveau, werd beïnvloed door de behandelingen. Uit de analyse van andere vluchtige stoffen blijkt dat de samenstelling van de geurmengsels van planten met verschillende behandelingen overlapt. Zo heeft een lage dosis JA een synergistisch effect op gen-transcriptie en een verhoogde emissie van een vluchtige verbinding die betrokken is bij indirecte verdediging na besmetting door spintmijten. Dit sluit goed aan op eerdere experimenten, die hadden aangetoond dat de toepassing van een lage dosis JA in combinatie met een aantasting door spintmijten de aantrekking van P. persimilis versterkt.

124 Samenvatting

In Hoofdstuk 4 gebruikte ik rupsenvraat of de combinatie van mechanische schade en rupsenspuug van twee rupsensoorten, Mamestra brassicae en Spodoptera exigua, om de effecten op transcript-niveaus van drie verdedigings-genen, PlLOX, PLOS, PlPR-4, te bestuderen. Inductie van verdediging tegen een eerste aanvaller kan de verdediging tegen latere planteneters beïnvloeden. Daarom heb ik ook onderzocht hoe de inductie van genen door vraatschade of door de combinatie van mechanische schade plus rupsenspuug de verdediging in reactie op een secundaire infectie met T. urticae spintmijten beïnvloedt. Terwijl de patronen van gentranscriptie meestal identiek waren in reactie op vraat door de twee soorten rupsen, veroorzaakten mechanische beschadiging in combinatie met rupsenspuug differentiële inductie van de transcriptie van verdedigings-genen. In vergelijking met planten met enkelvoudige herbivorie, vertoonden planten met dubbele herbivorie alleen differentiële inductie van het gen PlPR-4. Planten reageerden dus verschillend op rupsenvraat en op mechanische beschadiging in combinatie met rupsenspuug, wat ook resulteert in verschillende effecten op directe en indirecte verdediging van de planten tegen latere planteneters.

In Hoofdstuk 5 beschrijf ik onderzoek naar het effect van opeenvolgende inductie van verdediging door M. brassicae spuug gevolgd door vraat door T. urticae spintmijten op de expressie van indirecte verdediging in Limaboonplanten. Het effect op de indirecte verdediging werd beoordeeld met behulp van gedragsobservaties van de specialistische roofmijt P. persimilis in een olfactometer, headspace analyse van 11 vluchtige plantenstoffen zoals (E)-β-ocimeen en de transcriptie van het PlOS gen. Roofmijten bleken onderscheid te maken tussen de geuren van planten geïnduceerd door spintmijten en de geuren van planten geïnduceerd door mechanische beschadiging in combinatie met rupsenspuug, maar niet tussen de geuren van planten met enkelvoudige spintmijt-infectie en planten geïnduceerd door rupsenspuug gevolgd door spintmijtvraat. Chemische analyse liet zien dat de geurmengsels afkomstig van planten met spintmijtvraat en van planten behandeld met rupsenspuug gevolgd door spintmijtvraat een vergelijkbare samenstelling hadden. De inductie van verdediging in Limaboonplanten werd niet beïnvloed door eerdere behandeling met spuug van M. brassicae rupsen.

Hoofdstuk 6 beschrijft de studie van het effect van spintmijtvraat op de expressie van twee componenten van indirecte verdediging en op een latere aanval door soortgenoten. Ik bestudeerde de emissie van (E)-β-ocimeen en 12 andere vluchtige stoffen geassocieerd met spintvraat, alsmede de transcriptie van twee genen, namelijk PlOS en PlPR-4. Geuremissie en gentranscriptie van planten met spintvraat verschilde significant van die van controle planten. Geuremissie verschilde niet tussen planten die twee aanvallen van mijten hadden ervaren in vergelijking met planten die slechts één aanval van mijten hadden ervaren. Bovendien waren transcript-niveaus van PlOS en PlPR-4 niet verschillend voor deze behandelingen. De resultaten suggereren dat verdediging in Limaboon niet toeneemt in reactie op sequentiële aanvallen door spintmijten.

Tenslotte worden de resultaten besproken in de context van recente literatuur in Hoofdstuk 7. Samenvattend, het onderzoek gebundeld in dit proefschrift presenteert nieuwe inzichten in de mechanismen van inductie van indirecte verdediging van planten en tritrofe interacties in

125 de context van meer dan één planteneter en biedt perspectieven voor toekomstig onderzoek naar de manier waarop planten omgaan met combinaties van planteneters.

126 Samenvatting

127 128 Acknowledgements

Acknowledgements

Receiving a PhD degree is so much more than a logical consequence of a series of correct choices and actions. It is not simply the doing or work of one person alone. Being able to do a PhD is mainly a privilege that is made possible by hard work in combination with support from a strong support system consisting of many people. The further I progressed with my PhD project and with increasing complexity of my project, the more I came to realize that I wouldn’t be where I am today without some major support from the wonderful people around me. Some of them I would like to acknowledge here. Unfortunately, certain people are not able to read this because they have recently passed away or are too young to read, but nevertheless they need to be mentioned.

To start with, I would like to thank my closer family: Mona and Decio, Jascha and Jule (and the three kids), Jürgen and Pia. Without your support, encouragement, and trust in my abilities, I would not even be close to where I am today. You are indeed the pillars that hold me up and keep me going. You all live in my heart where I hold you close no matter how far we might be apart. I know I can always come home to you when the world knocks me down, just so I can head back out swinging.

The three musketeers in my life: Karin for always being a friend who I can literally steal horses with and who remains an inspiration in my life. Josip for filling my heart (and ears) with music and making me smile while teaching me not to take life and science too serious. For reminding me of my roots, friendship, and honesty. Uli for his unfailing friendship, loyalty, and reading drafts and who is just always “there”.

Dennis Oonincx, my favorite brainiac, who sees everything in life as a (solvable) experiment. Thank you for being a pleasant and clever seat neighbor, and not only a colleague with a great scientific mind, but a true friend who brings laughter in my life when I cannot see the sun behind the (Dutch) clouds. Who is always there in an emergency and brutally honest when I need it.

Fr. Manteuffel for believing in me and her kindness and generosity to practically a stranger during the last years.

Dr. Nawaporn Onkokesung with her sharp mind (and tongue) for good food and deep lunch conversations as well as company in the molecular lab.

Nurmi whose endless kindness, empathy, optimism, and ‘Friday hugs’ I would not want to have missed during long lab days and lonely lab weekends.

My “Doktorväter” Prof. Marcel Dicke and Prof. Joop J. van Loon without whose great scientific minds this PhD project would not have been possible and who steered me back on the path when I was drifting off or getting lost in details. Marcel, thank you also particularly for the race

129 in the end of writing my thesis, which I greatly enjoyed. Joop, thank you especially for your understanding and kindness during rough times.

The whole Ento team for taking me in so kindly and enriching my working days. It pains me that I got to spend so little time with all of you since the birth of my daughter. Nevertheless, every single person in the Ento group has always been helpful and great to talk to when time allows. Particularly thanks also to the rearing team Léon, André, Frans, Joop, and the technicians Patrick, Rieta, and Jeroen. Without your advice, effort, flexibility and great help, this thesis would not have worked out! Also special thanks to our Ento-secretary Angelique without who all the paper work and administrative stuff would be a mess.

My old colleagues from Bioscience (and Plant Physiology), who I might not have seen that often anymore but who I still enjoy to talk to and also always have an open ear: Wei, Aldana, Ting & Qing, Lemeng, Katarina, Jimmy, Benyamin, Geert, Maarten, Natalia, Liping, Katja, and Roland.

Dr. Jenny de Jonge for being a neighbor, a colleague, cat sitter, and so much more, but especially a loyal friend since our MSc studies in Wageningen. You are dearly missed here.

Other friends along the way like the ‘twins” Andres and Skye. My girls Danica, Bianca, Elli, Krissy, Susann, and of course the remaining German clique Jacqui, Lea, and Jule. You all made my life in the Netherlands enjoyable and exciting. Two amazing women and mothers: Neli and Liset for making me the mother I am and want to be, and helping out when I need someone to discuss life, work, or just need to laugh.

The old crew of the EPS PhD student council, Jenny, Padraic, Jule, Martine, Anitha, Wilma, Pierre, Vid. I will always remember the good times we had planning and organizing PhD days, symposia, and other social events.

Shane for being a good partner for 10 years who has tolerated many ups and downs during my studies.

Last but not least my daughter Nynke L. Imhausen, my Minime, who drives me crazy and makes me whole again when I struggle with (PhD-)life. You are the love of my life. Your smile and your little kisses are the reason why I want to keep waking up every morning.

This is just to mention the few people that helped me during the last few years to grow as a person and researcher. Since life is a journey this can be no more but a little snapshot of who I should all thank for being here and succeeding at what I do.

130 Acknowledgements

131 132 Curriculum vitae

Curriculum vitae

Tila Romina Menzel was born on April 24th, 1984 in Berlin, Germany. In 2001 she obtained her American highschool diploma during her 1-year stay in the state of Oregon as a foreign exchange student. Afterwards she went back to Germany to complete her education there and obtain the German highschool diploma in 2003. During her highschool time she found her passion for travelling and biology.

She went on to study in Italy in order to pursue an International first level degree in Job Creation Oriented Biotechnology, which has been established by 12 European universities with the objective to create a common mentality in students coming from universities of different countries. During her studies she travelled to different countries and gained practical experience by working in different laboratories across the world. She did an internship at the Centre d’Estudis Avançats de Blanes (Planes, Spain) where she worked with algae and nutrient-diffusing substrata, another internship at the Institut für Getreideverarbeitung (IGV) (Potsdam, Germany) where she investigated disruption methods for different algae cells to facilitate protease extraction. Finally she did a bachelor thesis project at Oregon State University (Corvallis, OR, USA) studying errantivirus sequences in insect cell lines. During an inspiring course of Plant Biotechnology taught by Prof. Dr F. Veronesi she finally decided to focus her studies on plants.

In 2010 she obtained her MSc degree at Wageningen University (Wageningen, The Netherlands) in Plant Biotechnology with emphasis on Molecular Plant Breeding and Pathology. Also during her MSc studies she participated in applied research within the Department of Plant Breeding and Bioscience, the latter in collaboration with the Department of Entomology. She worked on her PhD project from 2010-2014 under supervision of Prof. Dr M. Dicke and Prof. Dr J.J.A van Loon to study the induction of indirect plant defense in the context of multiple herbivory. During the first two years of her PhD traject, she was a member of the PhD council of the Experimental Plant Sciences (EPS) graduate school, where she was particularly involved in organizing events such as the ExPectationS career day and other social events for PhD students.

133 134 List of publications

List of publications

Menzel TR, Huang T-Y, Weldegergis BT, Gols R, Van Loon JJA, Dicke M. 2014. Effect of sequential induction by Mamestra brassicae L. and Tetranychus urticae Koch on Lima bean plant indirect defense. Journal of Chemical Ecology (in press), DOI 10.1007/s10886 - 014 - 0499 - 9.

Ramirez AM, Stoopen G, Menzel TR, Gols R, Bouwmeester HJ, Dicke M, Jongsma MA. 2012) Bidirectional secretions from glandular trichomes of pyrethrum enable immunization of seedlings. Plant Cell 24, 4252 – 4265.

Wang Y, van Kronenburg B, Menzel T, Maliepaard C, Shen X, Krens F. 2012. Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars. Plant Cell, Tissue and Organ Culture 111, 113 – 122.

Menzel T, Rohrmann GF. 2008. Diversity of errantivirus (retrovirus) sequences in two cell lines used for baculovirus expression, Spodoptera frugiperda and Trichoplusia ni. Virus Genes 36., 583 – 586.

135 Education Statement of the Graduate School Experimental Plant Sciences

Issued to: Tila Romina Menzel

Date: 19 November 2014

Group: Entomology

University: Wageningen University & Research Centre

1) Start-up phase date

► First presentation of your project

Priming in indirect defenses of Lima bean plants against herbivorous arthropods Feb 19, 2010

► Writing or rewriting a project proposal

► Writing a review or book chapter

► MSc courses

Insect-Plant Interactions (ENT-50806) Apr 26- May 27, 2010

► Laboratory use of isotopes Subtotal Start-up Phase 7.5 credits*

2) Scientific Exposure date

► EPS PhD student days

PhD student day, Utrecht University Jun 01, 2010

PhD student day, Wageningen University May 20, 2011

► EPS theme symposia

EPS symposium Theme 2, 'Interactions between plants and biotic agents', Leiden University Jan 15, 2010

EPS symposium Theme 2, 'Interactions between plants and biotic agents', Wageningen University Feb 03, 2011

EPS symposium Theme 2, 'Interactions between plants and biotic agents', Utrecht University Feb 10, 2012

► NWO Lunteren days and other National Platforms

NWO-ALW meeting 'Experimental Plant Sciences', Lunteren, NL Apr 19-20, 2010

NWO-ALW meeting 'Experimental Plant Sciences', Lunteren, NL Apr 04-05, 2011

NWO-ALW meeting 'Experimental Plant Sciences', Lunteren, NL Apr 14-15, 2014

Entomologendag (NEV) Dec 16, 2010

Entomologendag (NEV) Dec 13, 2013

► Seminars (series), workshops and symposia

Invited seminars at Entomology 2010-2014

Invited seminars at department PSG 2010-2014

5th Plant-Insect Interactions workshop, Wageningen University Nov 11, 2010

ExPectationS (EPS career day) Nov 19, 2010

ExPectationS (EPS career day) Nov 18, 2011

8th Plant-Insect Interactions workshop, Wageningen University Oct 24, 2013

CONTINUED ON NEXT PAGE 136 ► Seminar plus

Invited lecturer Georg Jander, Boyce Thompson Institute for Plant Research, USA Jan 10, 2011

International symposia and congresses (highly recommended)

14th International Symposium on Insect-Plant interactions (SIP-14), Wageningen, NL Aug 13-18, 2011

► Presentations

Poster: Transcriptional response of Lima bean plants to herbivory after low dose phytohormone application (SIP-14; Poster) Aug 13-18, 2011

Presentation: Transcriptional response of Lima bean plants to herbivory after low dose phytohormone application (Summerschool Environmental Signaling) Aug 22-24, 2011

Poster: Effect of heterospecific herbivores on biological pest control (EuroVOL Summer School) Sep 09, 2013

Presentation: The effects of sequential infestation by herbivores on the response of the predator Phytoseiulus perimilis (Entomologendag 2013) Dec 13, 2013

► IAB interview

Meeting with a member of the International Advisory Board of EPS Nov 15, 2012

► Excursions

Keygene excursion Jan 26, 2012 Subtotal Scientific Exposure 14.0 credits*

3) In-Depth Studies date

► EPS courses or other PhD courses

Postgraduate course 'Generalized linear models' Jun 28-29, 2010

Utrecht Summer School of Environmental Signaling Aug 22-24, 2011

Postgraduate course 'Advanced Statistics: Design of Experiments' Oct 10-12, 2012

EuroVOL Summer School Sep 09-12, 2013

► Journal club

Entomology (IPI) journal club 2010-2014

Entomology PhD lunches 2010-2014

Priming group 2010-2014

► Individual research training Subtotal In-Depth Studies 7.5 credits*

4) Personal development date

► Skill training courses

Scientific writing/Techniques for writing and presenting a scientific paper Feb 14-17, 2012

Project and Time Management Nov-Dec 2011

PhD competence assessment May-Jun.2010

► Organisation of PhD students day, course or conference

ExPectationS (EPS career day) Nov 19. 2010

EPS social event: PhD movie Dec 14, 2011

► Membership of Board, Committee or PhD council

member of the EPS PhD student council Jan. 2010-2012 Subtotal Personal Development 7.4 credits*

TOTAL NUMBER OF CREDIT POINTS* 36.4 Herewith the Graduate School declares that the PhD candidate has complied with the educational requirements set by the Educational Committee of EPS which comprises of a minimum total of 30 ECTS credits

* A credit represents a normative study load of 28 hours of study.

137 Supplemental data Supplemental data Chapter 3

25 c

20 PlOS

b b 5 a Relative gene transcription 0 water water+4Tu 0.1 mM JA 0.1 mM JA+4Tu (control)

Supplemental Figure 1. Relative gene transcript levels of PlOS of three independent experiments spaced in time, quantified in P. lunatus plants treated with i) water (control), ii) 0.1 mM JA, iii) four T. urticae (water + 4Tu), or iv) 0.1 mM JA with four T. urticae mites (0.1 mM JA + 4Tu). Simultaneous application of four T. urticae on plants for 48 h. Values are the mean (± SE) of ten to twelve biological replicates, different letters above bars indicate significant differences in transcript levels between treatments (Fisher’s LSD tests, α = 0.05). PlOS transcript levels were normalized to the normalization factor obtained from geometrically averaging the Ct values of the two reference genes PlACT1 and PlNMP1 for each sample. Baseline represents transcript level in control plants.

45 c 40 PlOS 35 30 25 20 b 15 10 5 Relative gene transcription a a 0 water 0.1 mM JA water+4Tu 0.1 mM JA+4Tu (control) (7 days) (7 days) (9 days) (9 days)

Supplemental Figure 2. Relative gene transcript levels of PlOS of two experiments spaced in time, quantified in P. lunatus plants treated with i) water (control), ii) 0.1 mM JA, iii) four T. urticae (water + 4Tu), or iv) 0.1 mM JA with four T. urticae mites (0.1 mM JA + 4Tu). Sequential application of four T. urticae placed on plants for 48 h after prior application with water or 0.1 mM JA seven days before. Values are the mean (± SE) of six to eight biological replicates, different letters above bars indicate significant differences in transcript levels between treatments (Fisher’s LSD tests, α = 0.05). PlOS transcript levels were normalized to the normalization factor obtained from geometrically averaging the Ct values of the two reference genes PlACT1 and PlNMP1 for each sample. Baseline represents transcript level in control plants.

138 Supplemental data

Supplemental Figure 3. SA levels in ng SA/g FW in P. lunatus plants treated with i) water (control), ii) 0.1 mM JA, iii) four T. urticae (water + 4Tu), or iv) 0.1 mM JA with four T. urticae mites (0.1 mM JA + 4Tu). (A) Inoculation of four adult female T. urticae on plants was done immediately following JA-treatment and mites had since been feeding for 48 h, and (B) inoculation of four adult female T. urticae for 48 h was done seven days after incubation with water or 0.1 mM JA started and mites had since been feeding for 48 h. Values are the mean (± SE) of four biological replicates, and were analyzed by ANOVA (A) or Kruskal-Wallis test (B) respectively (α = 0.05).

139 Effect of sequential induction by Mamestra brassicae L. and Tetranychus urticae Koch on Lima bean plant indirect defense

Supplementary Information Supplemental data Chapter 5

(E)-DMNT

M.b. T.u.

(Z)-3-Hexen-1-ol-butanoate MeSA

M.b. M.b. T.u. T.u.

(Z)-3-Hexen-1-ol isovalerate (E,E)-TMTT

M.b. M.b. T.u. T.u.

Fig.Supplemental S1. Pairwise Figure comparisons S1. Pairwise between comparisons average emission between rates average of volatiles emission show ratesing of highvolatiles VIP valueshowings high VIP values in plants treated with 20 T. urticae (T.u.) for 48 h or caterpillar oral secretion in( M.b.plants) for treated 48 h. with Values 20 T.are urticae the mean (T.u. )(± for SE) 48 hof or eight caterpillar biological oral secretionreplicates. (M Diff.b.) erentfor 48 letters h. Values above arebars the indicate mean ( signifi± SE) of cant eight diff biological erences replicatesin emission. Different rates between letters treatmentsabove bars indicate(Mann significantWhitney U test, α = 0.05). differences in emission rates between treatments (Mann Whitney U test, α = 0.05).

140 Supplemental data

300 b 250

200

150 ab ng/g ng/g FW/h 100 ab

50 a

Supplemental Figure S2. Average emission rates of (E)-β-ocimene in clean P. lunatus plants or/and treated with 20 T. urticae for 48 h (T.u.) or caterpillar oral secretion [M.b. (48 h)] for 48 h or the combination of M. brassicae oral secretion for 48 h followed by infestation with 20 T. urticae for 48 h (M.b.+T.u.). Values are the mean (± SE) of eight biological replicates. Different letters above bars indicate significant differences in emission rates between treatments (ANOVA, Tukey’s HSD tests, α= 0.05).

141 Supplemental data Chapter 6

Gene transcription sampling

Treatment i

4 T. urticae Treatment ii

4 T. urticae No treatment Treatment iii

4 T. urticae No treatment 10 T. urticae Treatment iv

4 T. urticae No treatment 10 T. urticae Treatment v

48 h 96 h 144 h

Time

Sampling

Suppl. Fig. 1. Treatments and sampling for gene transcription analysis. Treatments consisted of i) control, ii) four T. urticae, iii) four T. urticae + no treatment, iv) four T. urticae followed by no treatment and then 10 T. urticae, and v) 10 T. urticae.

Volatile sampling

Treatment i

4 T. urticae No treatment Treatment iii

4 T. urticae No treatment 10 T. urticae Treatment iv

10 T. urticae Treatment v

48 h 96 h 144 h

Time

Sampling

Suppl. Fig. 2. Treatments and sampling of samples for volatile sampling. Treatments consisted of i) control, iii) four T. urticae followed by no treatment, iv) four T. urticae followed by no treatment and then 10 T. urticae, and v) 10 T. urticae.

142 Supplemental data 1.9 0.9 0.9 0.59 0.46 1.41 1.78 0.48 0.03 0.92 0.44 0.71 0.48 VIP value value VIP c -value < 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 < 0.001 ≤ 0.001 P 9 (n= 10) 0.08±0.00 0.06±0.01 1.54±0.32 0.66±0.15 0.28±0.03 3.57±1.38 0.02±0.00 0.05±0.01 0.14±0.02 0.21±0.03 40.09±5.58 42.04±6.69 0.04±0.01 10 T.urticae T.urticae 10 9 (n= 10) 0.05±0.01 1.74±0.40 0.69±0.12 0.25±0.05 3.93±1.61 0.05±0.01 0.02±0.00 0.11±0.01 0.04±0.01 0.25±0.06 30.96±3.08 39.68±6.00 0.08±0.00 10 T. urticae urticae T. 10 4 T.urticae + Nothing+ T.urticae 4 8 + Nothing + Nothing 1±0.19 (n= 10) 0.2±0.02 0.04±0.01 0.33±0.08 0.29±0.03 2.35±0.69 0.01±0.00 0.04±0.00 0.09±0.01 0.05±0.01 28.1±2.83 0.09±0.01 36.28±12.52 T.u. 4 9 9 Treatment (n= 10) Control 0.03±0.00 1.61±0.54 0.05±0.01 0.23±0.04 0.22±0.05 4.11±2.32 0.08±0.01 0.03±0.01 0.19±0.03 27.14±5.43 33.28±6.93 0.07±0.00 0.01±0.00 )-TMTT )-DMNT indole linalool E )-2-hexenal by Lima bean plants of the four different treatments with one-way ANOVA results of single volatile analysis and VIP values. analysis single of volatile results ANOVA one-way with treatments different Lima by the four bean of plants )-β-ocimene E-E ( a ( )-3-hexen-1-ol alloocimene E E ( ( Z β-caryophyllene ( methyl salicylate Volatile compound )-3-hexen-1-ol, acetate )-3-hexen-1-ol, )-3-hexen-1-olisovalerate )-3-hexen-1-ol, butanoate )-3-hexen-1-ol, Z ( Z Z ( ( b 1 2 3 4 5 6 7 8 9 10 11 12 13 ID ID corresponds with the numbers presented in Fig. 1 Fig. in presented numbers the with corresponds ID Underlined P-values indicate significant differences in emission among treatments of a compound (one-way ANOVA) (one-way a compound of treatments among emission in differences significant indicate P-values Underlined Volatile emissions are given as mean peak area ± SE/g fresh weightof foliage with the number of samples between brackets !Numbers in superscript number the emission quantities offollowing samples give in whichcompound the was it if not wasfound detected, samples the in all of thattreatment a b c Table 1 Volatile emissions 1 Volatile Table

143 A 6 Ctrl

4 4 Tu+ NT

(50.36%) 0

-2 PLSo

-4

-6

-4.0 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 PLS1 (17.39%)

B 6 0.40 10 2 8 12 3

4 13 0.30 5 9 (50.36%) 0.20 1

PLSo 7 0.10

11 0.00 -0.3 -0.2 -0.1 -0.0 0.1 0.2 0.3 0.4 0.5 0.6 PLS1 (17.39%) Supplemental Figure 3. Multivariate data analysis by orthogonal PLS-DA (OPLS-DA; panel A) and corresponding loading plot (panel B) of volatile blends of P. lunatus plants with no treatment (Ctrl), or treatment with 4 T. urticae for 48 h + Nothing for 48 h (4 Tu+NT). The first two principal components are given (panel A) with the percentage of variation explained in parentheses. Numbers in the loading plot (panel B) represent 1) (E)-2-hexenal, 2) (Z)-3-hexen-1-ol, 3) (Z)-3-hexen-1-ol, acetate, 4) (E)-β-ocimene, 5) linalool, 6) (E)-4,8-dimethylnona-1,3,7-triene [(E)-DMNT], 7) alloocimene, 8) (Z)-3-hexen-1-ol, butanoate, 9) methyl salicylate, 10) (Z)-3-hexen-1-ol, isovalerate, 11) indole, 12) β-caryophyllene, and 13) (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene [(E,E)-TMTT].

A Ctrl 4 10 Tu

(44.87%) 0

-2 PLSo

-4

-5 -4 -3 -2 -1 0 1 2 3 4 5 PLS1 (27.98%)

B 0.50 10 6 8 13 9 0.40 2

4 5 0.30 3 7 12 (44.87%)

0.20

PLSo 0.10 1

0.00 11

-0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 PLS1 (27.98%)

Supplemental Figure 4. Multivariate data analysis by orthogonal PLS-DA (OPLS-DA; panel A) and corresponding loading plot (panel B) of volatile blends of P. lunatus plants with no treatment (Ctrl), or treatment with 10 T. urticae for 48 h (10 Tu). The first two principal components are given (panel A) with the percentage of variation explained in parentheses. Numbers in the loading plot (panel B) represent 1) (E)-2-hexenal, 2) (Z)-3-hexen-1-ol, 3) (Z)-3-hexen-1-ol, acetate, 4) (E)-β-ocimene, 5) linalool, 6) (E)-4,8-dimethylnona-1,3,7-triene [(E)-DMNT], 7) alloocimene, 8) (Z)-3-hexen-1-ol, butanoate, 9) methyl salicylate, 10) (Z)-3-hexen-1-ol, isovalerate, 11) indole, 12) β-caryophyllene, and 13) (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene [(E,E)-TMTT]. 144 Supplemental data

A Ctrl 4

4 Tu+NT+10 Tu

(42.78%) 0

PLSo -2

-4

-5 -4 -3 -2 -1 0 1 2 3 4 5 PLS1 (22.73%)

B 6 10 0.50 13 8 2

9 0.40 5 4 12

0.30 1 7

(42.78%) 3

0.20 PLSo

0.10

0.00 11

-0.10 -0.05 -0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 PLS1 (22.73%)

Supplemental Figure 5. Multivariate data analysis by orthogonal PLS-DA (OPLS-DA; panel A) and corresponding loading plot (panel B) of volatile blends of P. lunatus plants with i) no treatment (Ctrl), or treatment with four T. urticae for 48 h followed by no treatment for 48 h and then 10 T. urticae for 48 h (4 Tu+ NT +10 Tu). The first two principal components are given (panel A) with the percentage of variation explained in parentheses. Numbers in the loading plot (panel B) represent 1) (E)-2-hexenal, 2) (Z)-3-hexen-1-ol, 3) (Z)-3-hexen-1-ol, acetate, 4) (E)-β-ocimene, 5) linalool, 6) (E)-4,8-dimethylnona-1,3,7-triene [(E)-DMNT], 7) alloocimene, 8) (Z)-3-hexen-1-ol, butanoate, 9) methyl salicylate, 10) (Z)-3-hexen-1-ol, isovalerate, 11) indole, 12) β-caryophyllene, and 13) (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene [(E,E)-TMTT].

145 This work was performed at the Laboratory of Entomology, Wageningen University. Layout by the author. Cover by the author with photo courtesy of Neli Prota and Hans Smid.

Printed by: Gildeprint Drukkerijn -The Netherlands.

146