Chapter 13 ETHYLENE: MULTI-TASKER IN PLANT–ATTACKER ... · doi: 10.1002/9781118223086.ch13 Chapter 13 ETHYLENE: MULTI-TASKER IN PLANT–ATTACKER INTERACTIONS Sjoerd Van der Ent

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Annual Plant Reviews (2012) 44, 343–377 http://onlinelibrary.wiley.comdoi: 10.1002/9781118223086.ch13

Chapter 13

ETHYLENE: MULTI-TASKERIN PLANT–ATTACKERINTERACTIONSSjoerd Van der Ent and Corne M.J. PietersePlant-Microbe Interactions, Department of Biology, Faculty of Science,Utrecht University, PO Box 800.56, 3508 TB Utrecht, The Netherlands; Centre forBioSystems Genomics, PO Box 98, 6700 AB Wageningen, The Netherlands

Abstract: In the past decades, the role of ethylene in the regulation of plant re-sponses to biotic stress has been intensively studied. Analyses of plant genotypesthat are impaired in ethylene biosynthesis, perception or signalling revealed an im-portant role for ethylene in the primary response to pathogen attack. In addition,ethylene has been demonstrated to fulfil a key function in the control of systemicimmune responses that are induced by beneficial micro-organisms. Although theimportance of ethylene in the regulation of plant immune responses is evident, itsrole in stimulating disease resistance or susceptibility appears to depend greatlyon the plant–attacker combination. Whereas in many studies ethylene was demon-strated to facilitate disease resistance or tolerance, in other studies ethylene wasshown to support pathogen infection. Recent advances in defence-signalling re-search have revealed that ethylene plays an important role in modulating inter-actions between defence-signalling pathways that are regulated by either salicylicacid (SA) or jasmonic acid (JA). By functioning as a modulator of these importantdefence-regulatory pathways, ethylene may play a decisive positive or negativerole in the final outcome of the immune response of a plant.

Keywords: hormonal crosstalk; plant immune signalling; induced systemic re-sistance; jasmonic acid; salicylic acid; disease resistance

Annual Plant Reviews Volume 44: The Plant Hormone Ethylene, First Edition. Edited by Michael T. McManus.C© 2012 Blackwell Publishing Ltd. Published 2012 by Blackwell Publishing Ltd.

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344 � The Plant Hormone Ethylene

13.1 Introduction

As photosynthetically competent, plants are able to convert carbon dioxideand water into energy-rich sugars at the expense of solar energy. These sugarsare subsequently distributed over the plant tissues and provide the energyrequired for growth and development. As most other organisms lack the abil-ity to photosynthesize, they are directly or indirectly dependent on carbonassimilates of plants. As a result, many different types of organisms can befound in the vicinity of plants. While many thrive solely on organic litter,others directly interact with living plant tissues. Many micro-organisms areinvolved in plant relationships that are mutually beneficial (e.g. mycorrhizalfungi, nitrogen-fixing Rhizobium bacteria and other beneficial rhizospherebacteria and fungi), while others are harmful as they obtain energy-rich or-ganic compounds. In nature, plants are continuously threatened by a widerange of harmful pathogens and pests, including viruses, bacteria, fungi,oomycetes, nematodes and insect herbivores. Each of these attackers exploitshighly specialized features to establish a parasitic relationship with the hostplant. According to their lifestyles, plant pathogens are generally divided intobiotrophs, necrotrophs or hemi-biotrophs (Figure 13.1) (Glazebrook, 2005).Necrotrophs first destroy host cells, often through the production of phy-totoxins, after which they feed on the contents. Biotrophs derive nutrients

Figure 13.1 Disease symptoms on Arabidopsis thaliana leaves caused by the biotrophicoomycete Hyaloperonospora arabidopsidis, the necrotrophic fungus Botrytis cinerea andthe hemi-biotrophic bacterium Pseudomonas syringae. (Photos: Hansvan Pelt.) (For a colour version of this figure, please see Plate 13.1.)


Ethylene: Multi-Tasker in Plant–Attacker Interactions � 345

from living host tissues, commonly through specialized feeding structures(haustoria) that invaginate the host cell without disrupting it. Many plantpathogens display both lifestyles, depending on the stage of their life cycle,and are called hemi-biotrophs.

While pre-existing thorns, needles and trichomes are designed to harm ordeter herbivores, the cell wall poses a constitutively present physical barrierto many micro-organisms. In addition, plants constitutively produce a rangeof secondary metabolites that inhibit the growth of microbes or render thetissue less attractive for herbivores, such as caffeine, nicotine and opiates(Osbourn, 1996; Tierens et al., 2001). When these pre-existing physical andchemical barriers are broken, a second line of defence, consisting of a widespectrum of inducible plant defence responses, becomes activated. Theseprevent the attacker from causing further damage by blocking colonization,or by directly targeting the attacker’s physiology.

To minimize attacker-inflicted damage, plants have evolved a wide ar-ray of inducible defensive mechanisms. Active defence against biotrophicpathogens, which require a living host to fulfil their life cycle, is often largelydependent on programmed cell death, whereas defence against necrotrophicpathogens, which first kill host cells and live on the contents, require a dif-ferent defence strategy that often involves the production of anti-microbialcompounds (Glazebrook, 2005). Defence against insect herbivores is basedon two strategies: (1) direct defence and (2) indirect defence. Upon herbivoreattack, plants activate defence responses that target the attacker directly, suchas the accumulation of toxic and repellent compounds, or the decrease inthe nutritional value of the leaf tissue, which affects the feeding behaviourof the insect (Howe & Jander, 2008). Direct defences often act systemicallythroughout the plant, thereby protecting the plant against future attack. As asecond, indirect, line of defence, plants signal for help by emitting a volatileodour blend that attracts carnivorous enemies of the herbivores that feed onthe plant (Dicke et al., 2009).

Most inducible defence mechanisms are tightly controlled throughhormone-regulated signalling pathways (Pieterse et al., 2009). For decades,the plant hormone ethylene is known to function as an important modula-tor of plant defence (Boller, 1991). Perception of pathogen attack leads to arapid increase in ethylene production. This enhanced production in the earlystages of the infection process is associated with the induction of defence re-actions (Boller, 1991). While it is generally assumed that ethylene productioncontributes to disease resistance, several plant pathogens have been demon-strated to produce ethylene as a virulence factor, which promotes the ability ofthe pathogen to colonize host tissue (Arshad & Frankenberger, 1992; Chagueet al., 2002, 2006). This chapter highlights the role of ethylene in the regulationof the plant immune response, with special emphasis on its modulating role ininduced disease resistance and crosstalk among different hormone-regulateddefence-signalling pathways.



13.2 Hormones in plant defence signalling

13.2.1 Hormones as defence regulators

Many studies with plant mutants or transformants that are impaired inhormone signalling have demonstrated that these signalling molecules arecrucial for the activation of plant defence responses (Figure 13.2). The im-portance of salicylic acid (SA), jasmonic acid (JA) and ethylene as primarysignals in the regulation of these hormone-regulated immune responses iswell established (Van Loon et al., 2006a; Loake & Grant, 2007; Von Dahl &Baldwin, 2007; Howe & Jander, 2008; Pieterse et al., 2009; Vlot et al., 2009).In addition, abscisic acid (ABA) (Mauch-Mani & Mauch, 2005; Asselberghet al., 2008; Ton et al., 2009), auxins (Navarro et al., 2006; Wang et al., 2007),gibberellins (Navarro et al., 2008), cytokinins (Siemens et al., 2006; Walters& McRoberts, 2006) and brassinosteroids (Nakashita et al., 2003; Shan et al.,2008) have also emerged as players, but their role(s) in plant disease resistanceis less well characterized (Pieterse et al., 2009). It is commonly accepted thatSA-inducible defences are generally effective against biotrophic pathogens,whereas JA/ethylene-dependent defences are predominantly active againstnecrotrophic pathogens and insect herbivores (Glazebrook, 2005; Grant &Lamb, 2006; Pieterse & Dicke, 2007; Howe & Jander, 2008). However, it isbecoming increasingly clear that there are many exceptions to this notion(Thaler et al., 2004; Pieterse et al., 2009). In fact, plants react with a surpris-ing specificity to pathogens and insects with very different modes of attack(Reymond & Farmer, 1998; Rojo et al., 2003; De Vos et al., 2005). Upon pathogenor insect attack, plants produce a specific combination of hormones, which

Figure 13.2 Plant hormones implicated in the regulation of plant defence.



varies greatly in quantity, composition and timing, and results in the activa-tion of differential sets of defence-related genes that eventually determine thenature of the defence response against the attacker encountered (Reymond &Farmer, 1998; Rojo et al., 2003; De Vos et al., 2005). In the upcoming sections,we give an overview of the roles of SA, JA and ethylene in basal resistanceagainst pathogen and insect attack.

13.2.2 Salicylic acid

A central role for SA became apparent with the use of NahG transformants.NahG plants constitutively express the bacterial NahG gene, encoding salicy-late hydroxylase, which converts SA into inactive catechol, and tobacco andArabidopsis thaliana NahG plants show enhanced disease susceptibility to abroad range of oomycete, fungal, bacterial and viral pathogens (Delaney et al.,1994; Kachroo et al., 2000). Genetic screens in Arabidopsis with the aim of un-ravelling plant defence pathways have identified recessive mutants affectedin SA signalling that also show enhanced susceptibility to pathogen infec-tion (Vlot et al., 2009). For instance, the salicylic acid induction deficient1 (sid1),salicylic acid induction deficient2 (sid2) and phytoalexin deficient4 (pad4) mutantsare defective in SA accumulation in response to pathogen infection. As a re-sult, these mutants display enhanced susceptibility to the bacterial pathogenPseudomonas syringae pv. tomato and the oomycete pathogen Hyaloperonosporaarabidopsidis (Zhou et al., 1998; Nawrath & Metraux, 1999; Wildermuth et al.,2001), confirming the importance of SA in basal resistance against these dif-ferent types of pathogens.

13.2.3 Jasmonic acid

JA and its oxylipin derivatives (collectively called jasmonates (JAs)) weresimilarly demonstrated to play a role in basal disease resistance. For ex-ample, both the Arabidopsis jasmonic acid resistant1 (jar1) mutant, with re-duced sensitivity to methyl jasmonate (MeJA), and the fatty acid desaturase(fad3fad7fad8) triple mutant, which is defective in JA biosynthesis, exhibitsusceptibility to normally non-pathogenic soil-borne oomycetes of the genusPythium (Staswick et al., 1998; Vijayan et al., 1998). In another study, mu-tant fad3fad7fad8 showed extremely high mortality from attack by larvae ofthe common saprophagous fungal gnat, Bradysia impatiens (McConn et al.,1997), demonstrating an important role for JA in primary defence againstherbivorous insects. Increased susceptibility of jar1 to Fusarium oxysporum(Berrocal-Lobo & Molina, 2004) and impairment of induced resistance againstCucumber mosaic virus in fad3fad7fad8 mutants have been reported (Ryuet al., 2004). The JA-insensitive mutant coronatine insensitive1 (coi1) shows en-hanced susceptibility not only to the bacterial leaf pathogen Erwinia carotovora(Norman-Setterblad et al., 2000), the necrotrophic fungi Alternaria brassici-cola and Botrytis cinerea (Thomma et al., 1998), but also to insect herbivores



such as Pieris rapae, Spodoptera exigua and Spodoptera littoralis (Cui et al., 2005;Bodenhausen & Reymond, 2007; Van Oosten et al., 2008). Conversely, over-expression of a JA carboxyl methyl transferase increased endogenous levelsof MeJA and resulted in higher resistance to B. cinerea (Seo et al., 2001). More-over, constitutive activation of the JA-signalling pathway in the Arabidopsismutant constitutive expressor of VSP1 (cev1) resulted in enhanced resistance toP. syringae and the mildew fungi Erysiphe cichoracearum, Erysiphe orontii andOidium lycopersicum (Ellis et al., 2002a). All these examples clearly point toa role of JAs in resistance against a diverse range of pathogens, challengingthe general notion that JA-dependent defence responses are predominantlyeffective against necrotrophic pathogens and insect herbivores.

13.2.4 Ethylene

The role of ethylene in plant defence against microbial attackers is ratherambiguous (Broekaert et al., 2006; Van Loon et al., 2006a; Adie et al., 2007).On the one hand, stress-induced production of ethylene is clearly relatedto adaptation to the stress condition, while on the other hand pathogen-produced ethylene can act as a virulence factor and, as such, favour diseasedevelopment. In the past, a wealth of information on the role of ethylenein plant–microbe interactions has accumulated. In the upcoming sections,an array of aspects of the role of ethylene in plant–microbe interactions isdiscussed. Since the role of ethylene in plant–microbe interactions clearlydiverges among plant species, the most important findings for a number ofimportant model plant species in ethylene research are summarized first.

13.3 Implications of ethylene in basal defenceand disease susceptibility

13.3.1 Studies with Arabidopsis thaliana

In 1993, Chang and co-workers reported on the dominant nature of mutationsin the Arabidopsis ethylene receptor gene ETHYLENE RESPONSE1 (ETR1)(Chang et al., 1993). They subsequently demonstrated that transformationwith the mutant etr1 gene rendered Arabidopsis plants unable to perceive ethy-lene (see Chapter 4). The generation of Arabidopsis mutants and transgeniclines with a reduced ability to produce or perceive ethylene greatly facili-tated studies on the role of this hormone in plant–microbe interactions. Forseveral plant–attacker combinations, ethylene signalling has been positivelycorrelated with basal disease resistance. In these cases, mutants disruptedin ethylene signalling have been proven to be more susceptible to pathogenattack than the respective wild-type plants. Arabidopsis mutants insensitiveto ethylene as a result of a disruption in, for instance the ethylene receptor



ETR1 or in the central regulatory protein ETHYLENE INSENSITIVE2 (EIN2),are more susceptible to different strains of the oomycete Pythium (Ger-aats et al., 2002) as well as to fungal pathogens B. cinerea (Thomma et al.,1999; Berrocal-Lobo et al., 2002), Chalara elegans (Geraats et al., 2003), Plec-tosphaerella cucumerina (Berrocal-Lobo et al., 2002) and different subspecies ofF. oxysporum, namely conglutinans (Berrocal-Lobo & Molina, 2004), lycopersici(Berrocal-Lobo & Molina, 2004) and matthiolae (Geraats et al., 2003). Strikingly,the same ein2 and etr1 mutants are more resistant to F. oxysporum f.sp. raphani(Geraats et al., 2003), which seems to be a contradictory result. However, thelatter isolate is partly biotrophic, which is in contrast to that of the other threenecrotrophic F. oxysporum strains. Therefore, these results are in line withthe notion that ethylene-mediated defences are primarily effective againstnecrotrophs and not against biotrophic pathogens. Similarly, ein2 mutantsare more susceptible to the necrotrophic bacterium E. carotovora, but moreresistant to P. syringae that is partly biotrophic (Bent et al., 1992).

Exceptions to the SA or JA/ethylene categorization exist as well. Despitethe fact that the sugar beet cyst nematode Heterodera schachtii is biotrophic,both ein2 and etr1 and other mutations that negatively affect ethylenesignalling show less disease symptoms than wild-type Arabidopsis plants(Wubben et al., 2001). In line with these results, the same study shows that H.schachtii leads to increased disease severity in several ethylene overproducer (eto)mutants, which produce increased levels of ethylene due to a disruption ofa negative regulator of the rate-limiting step in ethylene biosynthesis (Wanget al., 2004). Furthermore, the necrotizing bacterium Ralstonia solanacearumcauses less severe disease development on ein2 mutants, when comparedwith wild type (Hirsch et al., 2002). Likewise, infection of Arabidopsis bythe necrotrophic fungus Verticillium dahliae is hampered by a mutation inETR1 (Veronese et al., 2003). Another predominantly necrotrophic fungusthat causes more disease symptoms and produces more conidia on ethylene-insensitive mutants when compared with wild-type Arabidopsis plants isFusarium graminearum. In a bioassay set-up that was especially designedto allow this pathogen of wheat and barley to infect Arabidopsis plants (Xuet al., 2006), the ethylene-signalling mutants etr1, ein2 and ein3 showed lit-tle F. graminearum-inflicted disease development, while in contrast, eto1, eto2and constitutive triple response1 (ctr1), a mutant with constitutive ethylenesignalling, were more susceptible to the fungus (Chen et al., 2009). These ob-servations were confirmed by data from experiments with chemical amend-ment. Reduction of ethylene perception by the application of silver ions orapplication of ethylene resulted in a reduction and enhancement of diseaseseverity, respectively. Together, these examples underpin the complex role ofethylene in plant–pathogen interactions and this role is highly dependent onthe type of attacker encountered.

For certain interactions with pathogens, it remains elusive due to contra-dicting results whether ethylene signalling contributes to the resistance ofArabidopsis or, inadvertently, aids the attacker in the infection process, for



example by modulating the outcome of other hormone-regulated defencepathways (Pieterse et al., 2009). For example, ein2 mutants were demonstratedto have an increased level of resistance against Xanthomonas campestris pv.campestris (Bent et al., 1992), whereas etr1 was more susceptible to this bac-terial pathogen (O’Donnell et al., 2003). A similar contrast applies to theinfection by P. syringae pv. tomato. When vacuum infiltrated into the leaves,the bacteria did not cause any characteristic water-soaked lesions on ein2mutants (Bent et al., 1992). However, these symptoms did arise on ein2 aswell as on etr1 upon dipping the above-ground plant tissues into a P. syringaepv. tomato suspension (Pieterse et al., 1998). These results demonstrate thatboth the degree of limitation of ethylene sensitivity as well as the methodof inoculation can affect the outcome of the biotic interaction. Furthermore,the assessment of symptom development on ethylene mutants is thwartedby the fact that ethylene itself is involved in the amplification of senescence.As a result, wild-type plants respond to infection-induced ethylene by devel-oping chlorosis near infection sites, while in ethylene-signalling mutants thisresponse is absent solely due to their inability to perceive ethylene. Becauseof this lack of chlorosis, the ethylene-signalling mutants can easily be misclas-sified as being more resistant, while in fact they are only more tolerant to thepathogen. Indeed, in the study of Bent and co-workers (1992), ein2 mutantsdeveloped less P. syringae pv. tomato-inflicted disease symptoms, but werecolonized to the same extent as wild-type plants.

13.3.2 Studies with tobacco

When heterologously overexpressed, the mutant etr1 gene from Arabidopsisconfers ethylene insensitivity in tomato and Petunia (Wilkinson et al., 1997),potato (Haines et al., 2003) and tobacco (Knoester et al., 1998). The transgenictobacco line, designated as Tetr (Tobacco etr), has been used, especially forthe study of ethylene-signalling plants other than Arabidopsis. In line withthe notion that ethylene-mediated defences are primarily effective againstnecrotrophs, Tetr tobacco plants are more severely diseased, when comparedwith wild-type tobacco, by E. carotovora pv. carotovora and marginally more byR. solanacearum – two necrotrophic bacterial species (Geraats et al., 2003). Cor-respondingly, Tetr tobacco displays increased levels of susceptibility to the(partially) necrotizing fungal pathogens B. cinerea, Cercospora nicotianae andColletotricum destructivum, but not to that inflicted by the biotrophic fungusOidium neolycopersici (Chen et al., 2003; Geraats et al., 2003). In addition, Tetrplants were more vulnerable to infection when compared to wild type by thegenerally non-pathogenic, soil-borne fungi C. elegans, Fusarium solani, F. oxys-porum and Rhizopus stolonifer, which are all necrotrophs (Geraats et al., 2002,2003). A similar classification can be made for disease-inflicting oomycetes.Ethylene insensitivity renders tobacco more susceptible to Pythium sylvaticumand other necrotizing Pythium spp., but not to infection by the biotrophPeronospora tabacina (Knoester et al., 1998; Geraats et al., 2002, 2003).



13.3.3 Studies with tomato

Tomato has also frequently been used to study the role of ethylene inhost–microbe interactions. Due to a spontaneous single base substitutionin Le-ETR3, the tomato Never ripe (Nr) mutants show suppressed or blockedresponses to ethylene (Lanahan et al., 1994; Wilkinson et al., 1995; see Chap-ter 11). Unexpectedly, Nr mutants show lower levels of disease severityupon infection by the necrotizing fungus F. oxysporum f.sp. lycopersici or byX. campestris pv. vesicatoria and P. syringae pv. tomato, two partially necrop-trophic bacterial species (Lund et al., 1998; Ciardi et al., 2000). These resultsimply that in tomato, in contrast to Arabidopsis and tobacco, ethylene sig-nalling contributes to disease development in wild-type plants. Results fromother tomato mutants and transformants with disrupted ethylene signallingare in line with this observation. Constitutive expression of the mutant alleleof Arabidopsis ETHYLENE RESPONSE FACTOR1 (ERF1) rendered tomatoplants more resistant against X. campestris pv. vesicatoria (Lund et al., 1998).Furthermore, a transgenic tomato line overexpressing bacterial 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase with the result of ham-pered ethylene synthesis, showed reduced levels of X. campestris pv. vesicato-ria-inflicted necrotic symptoms (Lund et al., 1998) and less V. dahliae-causedwilting (Robison et al., 2001). Likewise, infection by the post-harvest fungalpathogen Colletotrichum gloeosporioides progressed more slowly on transgenictomato fruit in which ethylene biosynthesis had been inhibited by an ACCoxidase (ACO) antisense transgene, compared with fruits of wild-type plants(Cooper et al., 1998). Recently, Lin et al. (2008) demonstrated that ethylene con-tributes to disease development in tomato. In this study, the authors used atransgenic line that constitutively produces the N-terminal region of LeCTR2.This protein kinase shares similarity with Arabidopsis CTR1 and is a negativeregulator of ethylene signalling. In common with Arabidopsis (Huang et al.,2003), overexpression of solely the N-terminal region of the LeCTR2 increasesethylene responsiveness of the transformants through competition with non-truncated LeCTR2 for binding to ethylene receptors (Lin et al., 2008). Theleaves and fruits of these transgenic tomato lines displayed enhanced sus-ceptibility to infection by B. cinerea, implying that ethylene has a negativeeffect on resistance against this fungus in wild-type plants (Lin et al., 2008).However, the results are contradictory to those published earlier by Dıaz et al.(2002). In the latter study, transgenic tomato plants that were hampered inethylene production through the continuous expression of ACC deaminasewere more susceptible, rather than more resistant, to B. cinerea. Additionally,the mutant epi (epinastic; Fujino et al., 1988), which is constitutively activatedin a subset of ethylene responses (Barry et al., 2001), showed a significant re-duction in the amount of B. cinerea-imposed symptoms, when compared withwild-type tomato plants (Diaz et al., 2002). Chemical modulation of ethylenelevels altered B. cinerea pathogenicity in a comparable way. Plants that weretreated with ethylene prior to challenge inoculation were less susceptible. In



contrast, inhibitors of ethylene perception (1-methylcyclopropene (1-MCP) or2,5-norbornadiene (NBD)) increased disease development (Diaz et al., 2002).In some cases, ethylene has been implicated in systemic acquired tolerance,which consists of a reduction in tissue damage in response to a secondarychallenge with a virulent pathogen with no effect on pathogen growth (Blocket al., 2005). Together these data demonstrate that in tomato also the effect ofethylene on defence signalling is rather ambiguous.

13.3.4 Studies with soybean

Hoffman and colleagues examined whether insensitivity to ethylene con-tributed to disease resistance in the important field crop, soybean. To thisend, they screened plants from mutagenized seeds (soybean var Hobbit 87)for absence of the ethylene triple response (short hypocotyl, exaggerated curlof the hypocotyl hook and radial bulging of hypocotyl) and a number ofstrong and weak ethylene-insensitive mutants were subsequently tested forresistance against pathogens with different lifestyles and infection strategies(Hoffman et al., 1999). Tests conducted using the fungus Septoria glycines,which is the causal agent of the foliar disease Septoria brown spot, revealedslightly worse symptoms in the ethylene-insensitive mutants relative to thewild-type controls. Similarly, the mutants were slightly, though not statisti-cally different, significantly more susceptible to the root rot causing fungusRhizoctonia solani. Contrastingly, ethylene insensitivity modestly improvedthe plant reaction to virulent isolates of the bacterial pathogen P. syringae pv.glycinea and of the oomycete Phytophthora sojae.

A subsequent study, performed by Bent and colleagues (2006), used thesame mutants to study the effect of ethylene insensitivity in the field.Under these conditions, the enhancement of S. glycines-inflicted brownspot symptoms in ethylene-signalling mutants observed in the laboratory(Hoffman et al., 1999) could not be detected (Bent et al., 2006). However,the mutants exhibited a clear increase in susceptibility to another fungalpathogen, white mould-causing Sclerotinia sclerotiorum. In contrast, disrup-tion of ethylene signalling reduced colonization by the soybean cyst nema-tode Heterodera glycines, which is the most damaging pathogen of soybeanin the United States (Wrather & Koenning, 2006). These latter observationsare consistent with the earlier results that ethylene contributes to disease sus-ceptibility in the interaction between H. schachtii and Arabidopsis (Wubbenet al., 2001), possibly by modulating the outcome of other effective hormone-regulated defence pathways (Pieterse et al., 2009).

13.3.5 Other plant species

The role of ethylene in resistance has been studied in other plant speciesas well although not as extensively as in the species described previously.Recently, Penmetsa and colleagues characterized multiple mutant alleles of



MtSkl1 (sickle), the Medicago trunculata orthologue of Arabidopsis EIN2, andtested one of these, skl1–1, for its interaction with R. solani and the oomycetouspathogen Phytophthora medicaginis (Penmetsa et al., 2008). The sickle mutantswere less able to control infection by both necrotizing pathogens when com-pared with wild-type M. trunculata, indicating that in the latter, ethylenepositively contributes to the resistance against both necrotrophs.

In fruits of Citrus, a positive correlation has been observed to exist betweenethylene synthesis and pathogen susceptibility (Ortuno et al., 2008). Ortunoand co-workers demonstrated that relatively high ethylene-producing For-tune (Citrus clementina × Citrus reticulata) fruits were more susceptible tothe necrotrophic fungus Alternaria alternate pv. citri than lower ethylene-producing Citrus paradisi and Citrus limon fruits. Furthermore, disease devel-opment increased when fruits were pre-treated with the ethylene-precursorACC or with the ethylene-releasing compound ethephon. In contrast to theresults obtained with Medicago (Penmetsa et al., 2008), and to the general no-tion that ethylene contributes to the resistance against necrotrophic pathogens(Thomma et al., 2001; Ton et al., 2002b; Glazebrook, 2005), these findings sug-gest that ethylene produced during infection of Citrus fruits promotes diseaserather than alleviating it (Ortuno et al., 2008).

Recently, the role of ethylene in disease signalling has been studied inmonocot species. In line with the results gained with Arabidopsis, attenua-tion of EIN2-expression through RNA interference-induced gene silencingreduced F. graminearum-caused Fusarium head blight symptoms in wheat(Triticum aestivum cv. Hobbit ‘sib’) (Chen et al., 2009). Furthermore, amend-ment with silver ions, which blocks ethylene perception, reduced F. gramin-earum disease development in both wheat and barley (Hordeum vulgare cv.‘Golden Promise’), while application of ethylene-supported disease develop-ment (Chen et al., 2009). Thus, in these monocot species ethylene contributesto susceptibility to infection by F. graminearum.

In conclusion, the often contradictory results described previously indi-cate that the role of ethylene in disease signalling is highly complex. In somecases ethylene promotes disease, whereas in other cases ethylene seems tocontribute to basal resistance. This highlights the multi-faceted role of ethy-lene in the regulation of basal resistance responses and demonstrates that therole of ethylene in plant defence cannot easily be generalized.

13.4 Implications of ethylene in systemicimmune responses

13.4.1 Systemic induced immunity

Plants possess sophisticated systems to perceive attackers and to subse-quently translate this perception into an effective defence response (Chisholm



et al., 2006; Jones & Dangl, 2006). Besides activation of effective defences atthe site of pathogen attack, plants also systemically induce defence responsesthat protect the non-infected tissues against future pathogen attack (Van Loon,2000). The development of a broad-spectrum, systemic acquired resistance(SAR) after primary infection with a necrotizing pathogen is well knownand the signal transduction pathway has been extensively studied (Durrant& Dong, 2004; Hammerschmidt, 2009). SA is the dominant signal involvedin the regulation of SAR (Gaffney et al., 1993; Delaney et al., 1994; Loake &Grant, 2007; Vlot et al., 2008; Attaran et al., 2009; Vlot et al., 2009). In Arabidop-sis, studies with mutants impaired in ethylene signalling showed that SARfunctions independently of ethylene signalling (Lawton et al., 1994; Lawtonet al., 1995; Pieterse et al., 1998; Knoester et al., 1999). However, in tobacco,grafting experiments with wild-type and transgenic ethylene-insensitive Tetrplants revealed that ethylene plays an important role in the synthesis, releaseor transport of the mobile SAR signal as wild-type scions did not developSAR when grafted on Tetr rootstocks after applying the SAR inducer to therootstock (Verberne et al., 2003).

Plants of which the roots have been colonized by specific strains of non-pathogenic fluorescent Pseudomonas spp. develop a phenotypically similarform of systemic protection that is called rhizobacteria-induced systemicresistance (ISR) (Van Loon et al., 1998; Van Wees et al., 2008; Van der Ent et al.,2009a). In contrast to pathogen-induced SAR, which is regulated by SA,rhizobacteria-ISR is controlled by a signalling pathway in which ethylene andJA play key roles (Pieterse et al., 1998). Recent advances in research on plantimmune responses to beneficial micro-organisms revealed that ethylene andJAs play a dominant role in the regulation of immune responses that aretriggered by beneficial soil-borne microbes, such as mycorrhizal fungi, andplant growth-promoting fungi (PGPF) and plant growth-promoting bacteria(PGPR) (reviewed in Van Wees et al., 2008). In the past 15 years, Arabidopsishas been used to study the molecular basis of rhizobacteria-ISR (Pieterseet al., 2002; Van der Ent et al., 2009a). In the upcoming sections, a historicalperspective and a review is provided of our current knowledge of the signaltransduction steps involved in the JA/ethylene-dependent ISR pathwaythat leads from recognition of the rhizobacteria in the roots to systemicexpression of broad-spectrum disease resistance in above-ground foliartissues.

13.4.2 Rhizobacteria-mediated ISR

Plants produce exudates and lysates at their root surface, where rhizobacteriaare attracted in large numbers (Walker et al., 2003). Selected strains of non-pathogenic rhizobacteria appear to be plant growth promoting, because theypossess the capability to stimulate plant growth (Kloepper et al., 1980). Al-though rhizobacteria can stimulate plant growth directly, in the field growth



promotion results mainly from the suppression of soil-borne pathogens andother deleterious micro-organisms (Schippers et al., 1987). Fluorescent Pseu-domonas spp. are among the most effective plant growth-promoting rhizobac-teria and have been shown to be responsible for the reduction of soil-bornediseases in naturally disease-suppressive soils (Raaijmakers & Weller, 1998;Weller et al., 2002; Duff et al., 2003). This type of natural biological controlcan result from the competition for nutrients, siderophore-mediated compe-tition for iron, antibiosis or the production of lytic enzymes (Van Loon &Bakker, 2003). Apart from such direct antagonistic effects on soil-bornepathogens, some rhizobacterial strains are also capable of reducing diseaseincidence in above-ground plant parts by triggering an effective immuneresponse in the below-ground plant parts (Van Peer et al., 1991; Wei et al.,1991). This rhizobacteria-ISR has been demonstrated in many plant speciesand has a broad-spectrum of effectiveness (Van Loon et al., 1998; Kloepperet al., 2004). Among the ISR-inducing PGPR documented to date are manynon-pathogenic Pseudomonas spp. and Bacillus spp. (Kloepper et al., 2004; VanLoon & Bakker, 2006).

To study the signal transduction pathway of ISR, an Arabidopsis-basedmodel system was developed. In this model system, the non-pathogenic rhi-zobacterial strain Pseudomonas fluorescens WCS417r was used as the inducingagent (Pieterse et al., 1996). Colonization of Arabidopsis roots by ISR-inducingWCS417r bacteria protects the plants against different types of pathogens,including the bacterial pathogens P. syringae pv. tomato, X. campestris pv.armoraciae and E. carotovora pv. carotovora, the fungal root pathogen F. oxys-porum f.sp. raphani, the fungal leaf pathogens A. brassicicola, B. cinerea and P.cucumerina, the oomycete pathogen H. arabidopsidis (Pieterse et al., 1996; VanWees et al., 1997; Ton et al., 2002b; Van der Ent et al., 2008; Segarra et al., 2009;Van der Ent et al., 2009b) and even insect herbivores such as the generalistS. exigua (Van Oosten et al., 2008) (Figure 13.3). Research on the molecularmechanism of ISR was focused initially on the role of pathogenesis-related(PR)-proteins, as the accumulation of these proteins was considered to bestrictly correlated with induced disease resistance (Van Loon et al., 2006b).However, Arabidopsis plants expressing WCS417r-ISR showed enhancedresistance against F. oxysporum and P. syringae pv. tomato, but this did notcoincide with the activation of the SAR marker genes PR-1, PR-2 and PR-5(Pieterse et al., 1996; Van Wees et al., 1997). Determination of SA levels inISR-expressing Arabidopsis plants revealed that ISR is not associated withincreased accumulation of SA (Pieterse et al., 2000). Moreover, WCS417r-ISRwas expressed normally in SA-non-accumulating Arabidopsis NahG plants(Pieterse et al., 1996), and in the SA biosynthesis mutants enhanced diseasesusceptibility5 (eds5)/sid1 and sid2 (Pieterse et al., 2002). This led to the conclu-sion that WCS417r-ISR is an SA-independent resistance mechanism, and thatWCS417r-ISR and pathogen-induced SAR are regulated by distinct signallingpathways.



WCS417r-ISR

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Figure 13.3 Spectrum of effectiveness of Pseudomonas fluorescens WCS417r-mediatedinduced systematic resistance (ISR) in Arabidopsis. Colonization of the roots of Arabidopsisplants leads to enhanced resistance against the bacterial pathogens Pseudomonas syringaeand Xanthomonas campestris, the fungal pathogens Fusarium oxysporum, Alternariabrassicicola and Botrytis cinerea, the oomycete Hyaloperonospora arabidopsidis and theinsect herbivore Spodoptera exigua. WCS417r-ISR is not effective against Turnip crinklevirus and the insects Pieris rapae and Myzus persicae (Pieterse et al., 1996; Van Wees et al.,1997; Ton et al., 2002b; De Vos et al., 2007; Van der Ent et al., 2008; Van Oosten et al.,2008). (Photos: Hans van Pelt.) (For a colour version of this figure, please see Plate 13.2.)

13.4.3 Genetic dissection of the ISR pathway in Arabidopsis

Since WCS417r-ISR appeared to function independently of SA, the Arabidop-sis JA-response mutants jar1, jasmonic acid insensitive1 (jin1), coi1 and eds8, andthe ethylene-response mutant etr1 were tested for their ability to express ISR.None of these mutants displayed resistance against P. syringae pv. tomato aftercolonization of the roots by WCS417r (Pieterse et al., 1998; Ton et al., 2002c;Pozo et al., 2008), indicating that ISR requires responsiveness to both ethyleneand JA. To further elucidate the role of ethylene in the ISR-signalling pathway,a large set of well-characterized ethylene-signalling mutants was analyzed.None of these mutants showed an ISR response against P. syringae pv. tomatoafter colonization of the roots by WCS417r (Knoester et al., 1999). These resultsconfirmed that an intact ethylene-signalling pathway is required for the es-tablishment of ISR. Of particular interest was the analysis of the eir1 (ethyleneinsensitive in the roots1) mutant, which is ethylene insensitive in the roots, butnot in the shoot. This eir1 mutant was incapable of showing ISR after root col-onization by WCS417r, while in contrast, after leaf infiltration with WCS417rit did show ISR, indicating that responsiveness to ethylene is required at thesite of rhizobacterial induction (Knoester et al., 1999). The observation thatthe ethylene-responsive Arabidopsis AtTLP1 gene, encoding a thaumatin-likeprotein, is activated in the roots upon colonization by WCS417r confirmsthat ethylene signalling is initiated in the roots by ISR-inducing rhizobacteria(Leon-Kloosterziel et al., 2005). Further evidence for the involvement of the



ethylene-response pathway came from the identification of the ArabidopsisISR1 locus (Ton et al., 1999). Genetic analysis of the progeny of a cross be-tween the WCS417r-responsive accession Col-0 and the ISR-impaired acces-sion RLD1 revealed a single locus, designated ISR1, to be important in theexpression of ISR against several different pathogens (Ton et al., 2002a). Ac-cessions with the recessive isr1 allele have reduced sensitivity to ethylene andenhanced susceptibility to P. syringae pv. tomato (Ton et al., 2001). These resultsstrongly indicate that the Arabidopsis ISR1 locus encodes a novel componentin the ethylene-signal transduction pathway that is important for both basalresistance and ISR in Arabidopsis.

In the past decade, research on the defence-signalling pathways that areactivated by beneficial micro-organisms revealed that ethylene and JA areoften central players in the regulation of ISR. As well as WCS417r, the roleof ethylene and JAs in the regulation of the ISR response of Arabidopsis hasbeen established for other PGPR (Iavicoli et al., 2003; Ryu et al., 2004; Ahnet al., 2007). Likewise, ISR triggered by the PGPF Penicillium sp. GP16–2, Tri-choderma harzianum T39 and Pirimorfospora indica was shown to be blocked inJA- and/or ethylene-signalling mutants of Arabidopsis (Hossain et al., 2008;Korolev et al., 2008; Stein et al., 2008). In other plant species too, evidenceis accumulating for a role for ethylene and JAs in the regulation of ISR.For instance, in tomato the JA-insensitive mutant def1 (defenceless 1) and theethylene-insensitive mutant Never ripe (Nr) were not capable of mountingISR against the oomycete pathogen Phytophthora infestans upon colonizationof the roots by the PGPR Bacillus pumilus SE34 or P. fluorescens 89B61 (Yan et al.,2002). Similarly, colonization of tomato roots by the non-pathogenic oomycetePythium oligandrum resulted in a decrease in R. solanacearum-inflicted diseasesymptoms, whereas the ISR response was blocked in the JA-insensitive mu-tant jasmonic acid insensitive1 (jai1) plants (Hase et al., 2008). In addition, usingnahG-expressing rice, an ethylene-insensitive OsEIN2 antisense rice line, andthe JA-deficient rice mutant hebiba, De Vleesschauwer et al. (2008) demon-strated that the ability of P. fluorescens WCS374r to trigger ISR against the riceleaf blast pathogen Magnaporthe oryzae is regulated by an SA-independent butJA/ethylene-modulated signalling pathway. In cucumber, application of thechemical inhibitors silver thiosulphate and diethyldithiocarbamate, whichblock the action of ethylene and the synthesis of JA, respectively, reducedTrichoderma asperellum T203-mediated ISR against P. syringae pv. lachrymans,indicating a role for JA/ethylene-dependent signalling in ISR in this species(Shoresh et al., 2005). Hence, the picture is emerging that JAs and ethyleneare the dominant hormonal players in the regulation of the SA-independentplant immune response that is triggered by beneficial micro-organisms.

To investigate a possible involvement of the SAR regulatory proteinNON-EXPRESSER OF PR GENES1 (NPR1) in ISR signalling, the Arabidopsisnpr1 mutant was tested in the ISR bioassay. Surprisingly, the npr1 mutantwas incapable of developing WCS417r-ISR (Pieterse et al., 1998; Van Weeset al., 2000). This result clearly showed that WCS417r-ISR, in common with



SA-dependent SAR, is an NPR1-dependent defence response. Several studieswith different beneficial ISR-inducing microbes confirmed the important roleof NPR1 in ISR signalling (reviewed in Van Wees et al., 2008; Van der Entet al., 2009a), and further analysis of the ISR signal-transduction pathway re-vealed that NPR1 acts downstream of the JA- and ethylene-dependent steps(Pieterse et al., 1998). Because SAR is associated with NPR1-dependent PR-gene expression, and ISR is not, the action of NPR1 in ISR differs from thatin SAR. These different activities are not mutually exclusive because simulta-neous activation of ISR and SAR can lead to an enhanced defensive activitycompared to that observed with either type of induced resistance alone (VanWees et al., 2000). This suggests that NPR1 is important in regulating andconnecting different hormone-dependent induced defence pathways (Dong,2004; Pieterse & Van Loon, 2004). While the role of NPR1 in SA-signallingis clearly connected to a function of this regulatory protein in the nucleus(Dong, 2004), evidence is accumulating that the role of NPR1 in JA/ethylenesignalling is connected to a cytosolic function of NPR1 (Glazebrook et al.,2003; Johansson et al., 2006; Yuan et al., 2007; Stein et al., 2008; Leon-Reyeset al., 2009; Pieterse et al., 2009). However, the exact molecular mechanismsby which NPR1 exerts its role in these JA/ethylene-dependent ISR remain tobe elucidated.

Recently, the transcription factor MYB72 has been identified as an impor-tant regulator of ISR in Arabidopsis (Van der Ent et al., 2008). In a microarray-based search for root-specific, WCS417r-responsive genes, MYB72 appearedto be specifically up-regulated in the roots upon colonization by WCS417r(Verhagen et al., 2004). Analysis of myb72 knockout mutants revealedthat MYB72 is required for the onset of WCS417r-ISR against a set of(hemi)biotrophic and necrotrophic pathogens (Van der Ent et al., 2008). Inter-estingly, MYB72 was also shown to be required for the development of ISRtriggered by the PGPF T. asperellum T34 (Segarra et al., 2009), indicating thatthe ISR pathways triggered by these very different beneficial microbes mustconverge.

13.4.4 Priming for enhanced JA/ethylene-dependent defences

In Arabidopsis, both ethylene and JA activate specific sets of defence-relatedgenes (Schenk et al., 2000) but, when applied exogenously, each can induce re-sistance (Pieterse et al., 1998; Van Wees et al., 1999). To investigate how far ISRis associated with these changes in JA/ethylene-responsive gene expression,Van Wees et al. (1999) monitored the expression of a set of well-characterizedethylene- and/or JA-responsive, defence-related genes in Arabidopsis plantsexpressing WCS417r-ISR. None of these genes were up-regulated in inducedplants, neither locally in the roots nor systemically in the leaves. This sug-gested that the resistance attained was not associated with major increase inthe levels of either ethylene or JA. Indeed, analysis of ethylene and JA levelsin leaves of ISR-expressing plants revealed no changes in the production of



these signal molecules (Pieterse et al., 2000; Hase et al., 2003). Therefore, it wasconcluded that the ethylene and JA dependency of ISR must be based on anenhanced sensitivity to these hormones, rather than on an increase in theirproduction.

To identify ISR-related genes, the transcriptional response of over 8,000Arabidopsis genes was monitored during WCS417r-ISR (Verhagen et al., 2004).However, systemically in the leaves, none of the ∼8,000 genes tested showeda consistent change in expression in response to effective colonization ofthe roots by WCS417r, indicating that the onset of ISR in the leaves is notassociated with detectable changes in gene expression. However, after chal-lenge inoculation of WCS417r-induced plants with P. syringae pv. tomato,81 genes showed an augmented expression pattern in ISR-expressing leavescompared to inoculated control leaves, suggesting that ISR-expressing plantsare primed to respond faster and/or more strongly upon pathogen attack.The majority of the primed genes was predicted to be regulated by JA and/orethylene signalling, confirming earlier findings that colonization of the rootsby WCS417r primed Arabidopsis plants for augmented expression of the JA-and/or ethylene-responsive genes VSP2, PDF1.2 and HEL (Van Wees et al.,1999; Hase et al., 2003). Interestingly, Arabidopsis leaves expressing WCS417r-ISR also displayed potentiated expression of PDF1.2 and HEL upon feedingby the generalist insect herbivore S. exigua, but not when the leaves weredamaged by the specialist herbivore P. rapae (Van Oosten et al., 2008). Ac-cordingly, colonization of Arabidopsis roots by WCS417r reduced growth anddevelopment of S. exigua but not that of P. rapae, indicating that primingfor enhanced defence-related gene expression is associated with enhancedresistance.

This PGPR-mediated sensitization of the tissue for enhanced defence ex-pression is termed ‘priming’ and is characterized by a faster and/or strongeractivation of cellular defences upon pathogen or insect attack resulting inenhanced resistance to the invader encountered (Conrath et al., 2006; Frostet al., 2008). Priming for enhanced JA/ethylene-dependent defences duringISR has been demonstrated in many plant–microbe interactions, including theinteraction of Arabidopsis with the PGPR P. putida LSW17S (Ahn et al., 2007)and the Bradyrhizobium sp. strain ORS278 (Cartieaux et al., 2008), and withthe PGPF T. asperellum T34 (Segarra et al., 2009) and the Penicillium sp. strainGP16–2 (Hossain et al., 2008). Although priming for enhanced JA/ethylene-dependent defences is well documented, it should be noted that primingfor JA/ethylene-independent defences by PGPR and PGPF has also beenreported (Tjamos et al., 2005; Waller et al., 2005; Pozo and Azcon-Aguilar,2007; Conn et al., 2008). Also, priming for enhanced formation of callose-containing papillae at the site of attempted pathogen entry has been reportedfor rhizobacteria-mediated ISR (Van der Ent et al., 2008). However, it remainsto be investigated to what extent this latter priming phenomenon is regu-lated by plant hormones. A model of rhizobacteria-mediated ISR is shown asFigure 13.4.



MYB72

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Figure 13.4 Recognition of beneficial rhizosphere-colonizing micro-organisms, such asPseudomonas fluorescens WCS417, leads to a local activation of the transcription factorgene MYB72 in the roots. Downstream of, or in parallel with MYB72, a so far unidentifiedethylene-signalling component is required in the roots for the onset of inducedsystematic resistance (ISR) in the leaves. The ISR signal transduction cascade requiresNPR1, likely in the systemic tissue. Systemically, induction of ISR is associated withpriming for enhanced expression of a set of jasmonic acid (JA)- and/orethylene-responsive genes and increased formation of callose-containing papillae at thesite of attempted pathogen entry. Attack by pathogens or insects, as depicted on theright side of the figure, activates defence responses in the plant (yellow arrows), which isaccelerated in ISR-primed plants (combined blue and yellow arrows). (For a colourversion of this figure, please see Plate 13.3.)

13.4.5 Molecular mechanisms of priming for enhanced defence

Priming provides the plant with an enhanced capacity for rapid and effectiveactivation of cellular defence responses to effectively combat pathogen or in-sect attack. However, the molecular mechanisms underlying priming are stillpoorly understood. Hypothetically, the primed state is based on the accumu-lation, or post-translational modification of one or more signalling proteinsthat, after being expressed and/or modified, still remain inactive. Upon per-ception of a pathogen- or insect-derived stress signal, this enhanced defence-signalling capacity would enable a faster and stronger defence reaction. Sincepriming is clearly expressed at the transcriptional level, transcription factorproteins are likely candidates in this two-step regulatory mechanism.



To identify transcription factors involved in the regulation of priming,Pozo et al. (2008) followed a whole-genome transcript profiling approach toidentify the set of JA-responsive genes that are primed upon induction ofWCS417r-ISR. To this end, uninduced and WCS417r-ISR-expressing plantswere treated with MeJA after which ISR-primed, JA-responsive genes suchas LOX2 and PYK10 (encoding a �-glucosidase) were selected. Interestingly,the set of ISR-primed genes was enriched for JA-responsive genes that werepreviously identified as being responsive to the JA-inducing pathogens andinsects including P. syringae, A. brassicicola, P. rapae and Frankliniella occidentalis(Western flower thrips) (De Vos et al., 2005). This suggests that JA-responsivegenes that are activated by JA-inducing attackers are selectively primed dur-ing ISR (Van der Ent et al., 2009a). Analysis in silico of the promoters of 442ISR-primed, JA-responsive genes revealed that the primed genes were signifi-cantly enriched for a cis-acting G-box-like motif in comparison to non-primed,JA-responsive genes. This promoter element can serve as a binding site forthe basic helix-loop-helix leucine zipper transcription factor MYC2 (originallycalled JIN1 for JASMONATE INSENSITIVE1), which plays a central role inJA- and ABA-regulated signalling (Lorenzo & Solano, 2005). MYC2-impairedjin1 mutants were unable to mount WCS417r-ISR against P. syringae andH. arabidopsidis (Pozo et al., 2008) or P. indica-mediated ISR against Golovino-myces orontii (Stein et al., 2008), pinpointing MYC2 as an important regulatorin priming during ISR.

In another approach to identify transcription factors involved in prim-ing, Van der Ent et al. (2009b) analyzed the expression profile of over2,000 potential Arabidopsis transcription factor genes upon induction of theprimed state by WCS417r using a robotized real-time reverse transcrip-tion (RT)-PCR-based resource for quantitative measurement of transcripts(Czechowski et al., 2004). In the absence of a pathogen, colonization of theroots by WCS417r caused a consistent change in the expression in the leavesof more than 100 transcription factor genes, including MYC2 (Van der Entet al., 2009b). Different types of transcription factor genes were induced, butthe APETALA2/ETHYLENE-RESPONSIVE FACTORS (AP2/ERF) family oftranscription factors was notably over-represented. Several members of theAP2/ERF family have been implicated in the regulation of JA- and ethylene-dependent defences (Lorenzo et al., 2003; Pre et al., 2008). However, theirexact role in the regulation of the priming response during ISR remains to beelucidated.

Although WCS417r directly induced the expression of several transcrip-tion factor genes, such as MYC2, no significant downstream activation ofdefence-related gene was observed in the absence of a pathogen (Verhagenet al., 2004). This suggests that the transcription factors remain inactive un-til the perception of a secondary pathogen- or insect-derived signal. Hence,regulatory mechanisms that act post-translationally are likely to be involvedin priming as well. Recently, the inactive forms of the mitogen-activated pro-tein kinases (MAPKs) – MPK3 and MPK6 – were found to accumulate upon



priming induced by the SA-analogue benzothiadiazole (BTH) (Beckers et al.,2009). These signalling components only became activated upon treatmentwith a secondary stress, suggesting a role for MAPKs in priming. Epigeneticregulation of gene expression has also been suggested to play a role in priming(Bruce et al., 2007). However, future research is required to fully understandthe molecular mechanisms underlying the priming phenomenon.

13.4.6 Costs and benefits of priming for enhanced defence

Priming for enhanced defence is a common feature of induced resistance andcan explain the broad-spectrum effectiveness that is typical for many inducedresistance phenomena (Conrath et al., 2006; Frost et al., 2008). Since plant de-fences are costly and involve diversion of resources away from plant growthand development (Heil & Baldwin, 2002; Walters & Heil, 2007), priming forenhanced defence is generally considered to be a cost-effective defence mech-anism (Walters & Boyle, 2005; Pieterse & Dicke, 2007). Through the study ofthe costs and benefits of priming in Arabidopsis, it was shown that the fit-ness costs of priming are indeed lower than those of constitutively activateddefences (Van Hulten et al., 2006). Moreover, the fitness benefit of primingwas shown to outweigh its costs under pathogen pressure. Recent findingson priming for enhanced defence in barley are in good agreement with theseresults (Walters, 2009).

13.5 Ethylene modulates crosstalk amongdefence-signalling pathways

13.5.1 Crosstalk in defence signalling

Ethylene, JA and SA play pivotal roles in the regulation of basal and induceddefence responses, and the accumulation of these hormones triggers the ac-tivation of a cascade of defence-signalling pathways (Dong, 1998; Grant &Lamb, 2006; Van Loon et al., 2006a; Loake & Grant, 2007; Von Dahl & Baldwin,2007; Howe & Jander, 2008; Van Wees et al., 2008; Vlot et al., 2008; Pieterseet al., 2009; Van der Ent et al., 2009a). However, the final outcome of the de-fence response is greatly influenced by the timing and composition of thehormones produced (De Vos et al., 2005; Mur et al., 2006; Koornneef et al.,2008b; Leon-Reyes et al., 2009; Pieterse et al., 2009). In nature, plants oftendeal with simultaneous or subsequent invasion by multiple pathogens andinsects, which can influence the primary induced defence response of thehost plant (Van der Putten et al., 2001; Bezemer & Van Dam, 2005; Stout et al.,2006; Poelman et al., 2008; Verhage et al., 2010). Since activation of plant de-fence mechanisms is associated with ecological fitness costs (Walters & Heil,



2007), plants need regulatory mechanisms to effectively adapt to changes intheir environment. Recent advances in defence-signalling research revealedthat SA, JA and ethylene function in a complex network of interconnectingsignalling pathways (Pieterse et al., 2009). Interactions among these pathwaysprovide the plant with a powerful regulatory potential that may allow theplant to tailor its defence response to the invaders encountered (Reymond &Farmer, 1998; Kunkel & Brooks, 2002; Bostock, 2005; Pieterse & Dicke, 2007;Verhage et al., 2010).

13.5.2 Interplay among SA, JA and ethylene signalling

One of the best-studied examples of signal crosstalk is the antagonistic inter-action between SA and JA signalling. Many studies have demonstrated thatendogenously accumulating SA antagonizes JA-dependent defences, therebyprioritizing SA-dependent resistance over JA-dependent defence (Bostock,1999; Van Wees et al., 1999; Felton & Korth, 2000; Kunkel & Brooks, 2002;Thaler et al., 2002; Glazebrook et al., 2003; Beckers & Spoel, 2006; Koornneef &Pieterse, 2008; Spoel & Dong, 2008; Verhage et al., 2010). For example, in-duction of the SA pathway in Arabidopsis by exogenous application of SAor infection by the SA-inducing pathogen P. syringae suppressed JA sig-nalling and rendered infected leaves more susceptible to the necrotrophicfungus A. brassicicola (Spoel et al., 2007; Leon-Reyes et al., 2009). Similarly,the biotrophic oomycete pathogen H. arabidopsidis strongly suppressed JA-mediated defences that were activated upon feeding by caterpillars of P. rapae(Koornneef et al., 2008b). Pharmacological experiments with Arabidopsis re-vealed that JA-responsive marker genes, such as PDF1.2 and VSP2, are highlysensitive to suppression by exogenous application of SA (Spoel et al., 2003;Koornneef et al., 2008a, 2008b). SA-mediated suppression of JA-responsivegene expression was observed in a large number of Arabidopsis accessionscollected from very different geographic origins, highlighting the potentialsignificance of this phenomenon in the regulation of induced plant defencesin nature (Koornneef et al., 2008b).

Although many reports describe an antagonistic interaction between SA-and JA-dependent signalling, synergistic interactions have been describedas well (Schenk et al., 2000; Van Wees et al., 2000; Mur et al., 2006). For ex-ample, in Arabidopsis, treatment with low concentrations of MeJA and SAresulted in a synergistic effect on the JA- and SA-responsive genes PDF1.2and PR-1, respectively. However, at higher concentrations the effects wereantagonistic, demonstrating that the outcome of the SA–JA interaction is de-pendent on the relative concentration of each hormone (Mur et al., 2006).Koornneef et al. (2008b) and Leon-Reyes et al. (2010) demonstrated that thetiming and sequence of initiation of SA and JA signalling are also importantfor the outcome of the SA–JA signal interaction. Hence, the kinetics of phy-tohormone biosynthesis and signalling during the interaction of a plant with



Figure 13.5 Eggs of the cabbage white butterfly (Pieris rapae) activate the salicylic acid(SA)-responsive PR1::GUS reporter gene in Arabidopsis at the site of oviposition. Activationof the SA pathway suppresses jasmonic acid (JA)-dependent herbivore resistance,providing an advantage for newly hatched larvae of generalist herbivores that feed fromthis de-immunized tissue. (Photo: Hans van Pelt.) (For a colour version of this figure,please see Plate 13.4.)

its attacker(s) could be highly decisive in the final outcome of the defenceresponse to the attacker encountered. Interplay among defence pathwaysmay provide the plant with a powerful regulatory potential, but it is also apossible target for plant attackers to manipulate the plant defence-signallingnetwork for their own benefit (Pieterse & Dicke, 2007; Robert-Seilaniantzet al., 2007; Walling, 2008; Pieterse et al., 2009; Verhage et al., 2010). A niceexample is the observation of Bruessow et al. (2010) that elicitors from insecteggs activate the SA pathway in Arabidopsis at the site of oviposition. As aresult, JA-dependent defences are suppressed resulting in an advantage forthe newly hatched offspring that fed from the undefended tissue (Bruessowet al., 2010; Figure 13.5).

Several proteins with an important regulatory role in SA–JA crosstalk havebeen identified in Arabidopsis. Mutation or ectopic expression of the corre-sponding genes was shown to have contrasting effects on SA and JA sig-nalling and on resistance against biotrophs and necrotrophs (Koornneef &Pieterse, 2008; Spoel & Dong, 2008; Pieterse et al., 2009). The defence-regulatory protein NPR1 was identified as a key signalling node in the inter-action between the SA and JA pathways, as mutant npr1 plants were blockedin SA-mediated suppression of JA-responsive genes (Spoel et al., 2003). Re-cently, it was demonstrated that ethylene bypasses the need of NPR1 in SA–JAcrosstalk, while it enhances NPR1-dependent, SA-responsive PR-1 expression(De Vos et al., 2006; Leon-Reyes et al., 2009). These findings indicate that thefinal outcome of the SA–JA signal interaction during the complex interactionof plants with their attackers can be shaped by ethylene.



13.5.3 Ethylene: an important modulatorof defence-signalling pathways

In many cases, ethylene has been shown to act as an important modulator ofplant responses to SA and JAs (Adie et al., 2007; Kazan & Manners, 2008). Theinteraction between ethylene and JAs is often synergistic. A classic example isthe regulation of PDF1.2, which requires concomitant activation of the JA- andthe ethylene-response pathways (Penninckx et al., 1998). Two members of thelarge plant-specific AP2/ERF superfamily of transcription factors, ERF1 andOCTADECANOID-RESPONSIVE ARABIDOPSIS59 (ORA59), have emergedas principal integrators of the JA- and ethylene-signalling pathways (Lorenzoet al., 2003; Pre et al., 2008). The expression of ERF1 and ORA59 is inducedby JA or ethylene and is synergistically activated by both hormones. Over-expression of ORA59 or ERF1 in the JA-insensitive mutant coi1–1, and over-expression of ERF1 in mutant ein2 constitutively activated the PDF1.2 gene,indicating that these transcription factors are important nodes of conver-gence of the JA- and ethylene-signalling pathways (Lorenzo et al., 2003; Preet al., 2008). Ectopic expression of ERF1 and ORA59 was shown to enhanceresistance of Arabidopsis to necrotrophic pathogens such as B. cinerea andP. cucumerina, and F. oxysporum (Berrocal-Lobo et al., 2002; Berrocal-Lobo &Molina, 2004; Pre et al., 2008). Moreover, silencing of ORA59 was shown toenhance susceptibility to B. cinerea (Pre et al., 2008), indicating that theseAP2/ERF-type transcription factors play an important role in the regulationof JA/ethylene-dependent defences. Another point of convergence betweenJA and ethylene signalling is CEV1 (Ellis & Turner, 2001), which is also knownas cellulose synthase CeSA3 (Ellis et al., 2002b). Arabidopsis cev1 mutants con-stitutively express ethylene- and JA-dependent responses, as evidenced byhigh PDF1.2 and VSP2 transcript levels and enhanced pathogen resistance(Ellis & Turner, 2001; Ellis et al., 2002a). Recently, Leon-Reyes et al. (2010)provided evidence that the synergistic effect of ethylene on JA signalling isresponsible for counteracting the antagonistic effect of SA on JA signalling,and that the AP2/ERF transcription factor ORA59 is an important player inthe regulation of this process. This points to a model in which simultaneousinduction of the ethylene and JA pathway renders the plant insensitive tofuture SA-mediated suppression of JA-dependent defences, which may pri-oritize the JA/ethylene pathway over the SA pathway during multi-attackerinteractions. A simplified model of the interplay among SA, JA and ethyleneis depicted in Figure 13.6.

13.6 Concluding remarks

Plant diseases and pests are responsible for large crop losses in agriculture.Knowledge of defence-signalling pathways has proven to be instrumentalfor the development of new strategies for broad-spectrum disease resistance.



Figure 13.6 Model illustrating the interplay among salicylic acid (SA), jasmonic acid(JA) and ethylene in the SA-mediated down regulation of JA/ethylene-responsive genessuch as PDF1.2. (For a colour version of this figure, please see Plate 13.5.)

Ethylene has been proven to play an important modulating role in manyaspects of plant defence. While in some plant–pathogen interactions thehormone contributes to enhanced resistance, in other systems ethylenepromotes symptom development or confers tolerance to specific pathogeninfections. At the level of defence signal transduction, ethylene seems tobe a crucial modulator in crosstalk among other defence-related hormonalsignals. As such, ethylene functions as a spider in the web of induceddefence-signalling pathways and plays a crucial role in the final outcomeof the resistance reaction. Because of its pluriform function in plant defence,ethylene remains a difficult, yet intriguing hormone of study in plant diseaseresistance research. Ultimately, unravelling the complexity of plant defencesignalling will not only provide fundamental insights into how plantscope with different enemies, it will also be instrumental in developingstrategies for biologically based, environmentally friendly and durable cropprotection.

Acknowledgements

Our laboratory has been financially supported by grants 865.04.002,813.06.002 and 863.04.019 of the Earth and Life Sciences Foundation (ALW),which is subsidized by the Netherlands Organization for Scientific Research(NWO), and ERC Advanced Grant no. 269072 of the European ResearchCouncil.



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Chapter 13 ETHYLENE: MULTI-TASKER IN PLANT–ATTACKER ... · doi: 10.1002/9781118223086.ch13 Chapter 13 ETHYLENE: MULTI-TASKER IN PLANT–ATTACKER INTERACTIONS Sjoerd Van der Ent