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Annemart Koornneef
CROSS-TALK IN PLANT DEFENSE SIGNALING ANTAGONISM BETWEEN SALICYLATE AND JASMONATE PATHWAYS IN ARABIDOPSIS
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Cross-talk in plant defense signaling
A n t a g o n i s m b e t w e e n s a l i c y l a t e a n d j a s m o n a t e p a t h w a y s i n A r a b i d o p s i s
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Cross-talk in plant defense signaling
A n t a g o n i s m b e t w e e n s a l i c y l a t e a n d j a s m o n a t e p a t h w a y s i n A r a b i d o p s i s
Interact ies tussen s ignaal - t ransduct ieroutes
t i jdens de immuunrespons van Arabidops is
A n t a g o n i s m e t u s s e n v a n s a l i c y l z u u r e n v a n
j a s m o n z u u r a f h a n k e l i j k e a f w e e r
(met een samenvatting in het Nederlands)
P ro e f s c h r i f t
ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. J.C. Stoof, ingevolge het besluit van het college voor promoties
in het openbaar te verdedigen opmaandag 21 april 2008 des middags te 14.30 uur
door
A n n e m a r t K o o r n n e e f
geboren op 4 september 1980 te Wageningen
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v e r s i e 3 • d d 4 - 3 - 2 0 0 8
P r o m o t o r e n : Prof. dr. ir. C.M.J. Pieterse
Prof. dr. ir. L.C. van Loon
I S B N : 978-90-393-4783-6C o v e r a n d l a y - o u t : Marjolein Kortbeek-Smithuis; Communications & Design, Faculty of Science, Universiteit Utrecht, The NetherlandsC o v e r p h o t o g r a p h : Hans van Pelt & Adriaan VerhageP r i n t e d b y : Grafisch Bedrijf Ponsen & Looijen, Wageningen, The Netherlands
The research described in this thesis was performed at the Graduate School Experimental Plant Sciences, Plant-Microbe Interactions, Institute of Environmental Biology, Faculty of Science, Utrecht University (Padualaan 8, 3584 CH Utrecht, The Netherlands) and financed by grants 813.06.002 and 865.04.002 of the Earth and Life Sciences Foundation (ALW), which is subsidized by The Netherlands Organization of Scientific Research (NWO).Printing of this thesis was financially supported by the J.E. Jurriaanse Stichting.
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C O N T E N T S
C h a p t e r 1 9
General introduction
C h a p t e r 2 2 5
NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol
C h a p t e r 3 4 3
Kinetics of cross-talk in plant defense signaling
C h a p t e r 4 6 1
Towards the identification of novel key players of cross-talk in plant defense
C h a p t e r 5 8 5
Development of a reporter system to identify regulators of cross-talk between salicylate and jasmonate signaling pathways in Arabidopsis
C h a p t e r 6 9 9
General discussion
R e f e r e n c e s 1 0 7
S u m m a r y 1 2 3
S a m e n v a t t i n g 1 2 5
D a n k w o o r d 1 2 7
C u r r i c u l u m v i t a e 1 2 9
L i s t o f p u b l i c a t i o n s 1 3 1
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C H A P T E R 1
General introduction
Adapted from:
Koornneef, A. and Pieterse, C.M.J. (2008) Cross talk in defense signalingPlant Physiology 146, 839-844.
Van der Ent, S.*, Koornneef, A.*, Ton, J., and Pieterse, C.M.J. (2008)Induced resistance - orchestrating defence mechanisms through cross-talk and priming In: Annual Plant Reviews - Molecular Aspects of Plant Disease Resistance (J.E. Parker, ed), Blackwell, in press.* Equal contribution.
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Chapter 1 • 1 1
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General Introduction
P L A N T S E L F D E F E N S E
Plants are equipped with an array of defense mechanisms to protect themselves against herbivorous insects and microbial pathogens. During their lifetime, they encounter numerous potential invaders with diverse modes of attack. In order to survive, plants have to perceive attack by these deleterious organisms and respond adequately. Some defense mechanisms are pre-existing while others are only activated upon insect attack or pathogen invasion. However, induced defense responses entail fitness costs (Heil, 2002; Heil and Baldwin, 2002). Therefore, plants possess elaborate regulatory mechanisms that efficiently coordinate activation of attacker-specific defenses. In this way, optimal resistance is attained while fitness costs are minimized (Pieterse and Dicke, 2007). A major focus in plant defense signaling research is to uncover key mechanisms by which plants tailor their responses to different attackers, and to investigate how plants cope with simultaneous interactions with multiple aggressors.
Plants are immune to most potential pathogens (non-host resistance), and can restrict disease caused by virulent pathogens (basal resistance). Recognition of common features of micro-organisms, such as flagellin, chitin, lipopolysaccharides, and ergosterol is a common initial step for resistance induction (Bittel and Robatzek, 2007). These pathogen-associated molecular patterns (PAMPs) activate signaling events that lead to PAMP-triggered immunity (PTI). However, successful pathogens can secrete effector proteins that interfere with PTI and overcome or suppress basal resistance responses. Recognition of these effector proteins mediated through production of specific disease resistance (R) genes elicits an immune response called effector-triggered immunity (ETI). ETI is often associated with a hypersensitive response (HR) that arrests further pathogen ingress through tissue necrotization (Jones and Dangl, 2006).
In addition to the attacker-specific primary immune response, plants can activate another line of defense that is referred to as ‘induced resistance’. This type of resistance often acts systemically throughout the plant and is typically effective against a broad spectrum of
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attackers (Walters et al., 2007). Plants are able to activate different types of induced resistance, depending on the organism that interacts with the plant. Well-studied examples of induced resistance are systemic acquired resistance (SAR), which is triggered by pathogens causing limited infection such as hypersensitive necrosis (Durrant and Dong, 2004), rhizobacteria-induced systemic resistance (ISR), which is activated upon colonization of roots by selected strains of non-pathogenic rhizobacteria (Van Loon et al., 1998), and wound-induced resistance (WIR), which is typically elicited upon tissue damage, such as that caused by insect feeding (Kessler and Baldwin, 2002; Howe, 2004). The role of phytohormones in the regulation of these induced defenses is well established. Salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are recognized as key players in the regulation of the signaling pathways involved (Howe, 2004; Pozo et al., 2004; Lorenzo and Solano, 2005; Grant and Lamb, 2006; Van Loon et al., 2006; Von Dahl and Baldwin, 2007). Other plant hormones, including abscisic acid (ABA) (Mauch-Mani and Mauch, 2005), brassinosteroids (Nakashita et al., 2003), and auxin (Navarro et al., 2006; Wang et al., 2007) have also been implicated in plant defense, but their significance is less clear.
The alarm signals SA, JA, and ET are produced in response to pathogen or insect attack in a specific blend, which varies greatly in quantity, composition, and timing. It is thought that this so-called ‘signal signature’ contributes to the specificity of the plant’s primary defense response (Reymond and Farmer, 1998; De Vos et al., 2005). The signaling pathways that are activated upon endogenous accumulation of these signals regulate different defense mechanisms that are effective against partially distinct classes of attackers. Although there are exceptions (Thaler et al., 2004), generally it can be stated that pathogens with a biotrophic lifestyle are more sensitive to SA-mediated induced defenses, whereas necrotrophic pathogens and herbivorous insects are resisted more through JA/ET-mediated defenses (Thomma et al., 2001; Kessler and Baldwin, 2002; Glazebrook, 2005). In nature, however, plants often have to deal with simultaneous or subsequent invasion by multiple aggressors, which can influence the primary induced defense response (Van der Putten et al., 2001; Bezemer and Van Dam, 2005; Stout et al., 2006). Hence, plants need regulatory mechanisms to effectively adapt to changes in their hostile environment. Cross-talk between induced defense signaling pathways is thought to provide the plant with such a powerful regulatory potential. Signaling interactions can be either (mutually) antagonistic or synergistic, resulting in negative or positive functional outcomes. Hence, cross-talk can be interpreted as an inclusive term for the interaction between signaling pathways (Bostock, 2005). Cross-talk helps the plant to minimize energy costs and create a flexible signaling network that allows it to finely tune its defense response to the invaders encountered (Reymond and Farmer, 1998; Pieterse et al., 2001; Bostock, 2005). Yet, it appears that insect herbivores and pathogens have also evolved to manipulate plants for their own benefit by suppressing induced defenses through modulation of the plant defense signaling network (Pieterse and Dicke, 2007). In this way, they can suppress or evade resistance mechanisms and retain their virulence on the host plant.
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Chapter 1 • 1 3
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S I G N A L I N T E R A C T I O N S T O F I N E - T U N E D E F E N S E
Molecular and genomic tools are now being used to unravel the complexity of the induced defense signaling networks that have evolved during the arms race between plants and their attackers (Pieterse and Dicke, 2007). Global expression profiling studies provided ample evidence that SA, JA and ET pathways interact, either positively or negatively (Schenk et al., 2000; Glazebrook et al., 2003; De Vos et al., 2005). One of the best characterized examples of defense-related signal cross-talk is the interaction between the SA and JA response pathways (Rojo et al., 2003; Bostock, 2005; Beckers and Spoel, 2006). Although most reports indicate a mutually antagonistic interaction between SA- and JA-dependent signaling, synergistic interactions have been described as well (Schenk et al., 2000; Van Wees et al., 2000; Mur et al., 2006). As a result of negative cross-talk between SA and JA, activation of the SA response renders a plant more susceptible to attackers that are resisted through JA-dependent defenses and vice versa. Indeed, many examples of trade-offs between SA-dependent resistance against biotrophic pathogens and JA-dependent defense against insect herbivores and necrotrophic pathogens have been reported (Pieterse et al., 2001; Bostock, 2005; Stout et al., 2006). However, comparative analysis of a large number of plant-microbe-insect interactions has revealed a more complex reality, which can be partially explained by differences in experimental conditions. These complexities make predictions about the outcome of such tripartite interactions difficult (Stout et al., 2006).
A n t a g o n i s t i c i n t e r a c t i o n s
In Arabidopsis thaliana, Spoel et al. (2007) recently showed that SA-mediated defenses that are triggered upon infection by a virulent strain of the biotrophic pathogen Pseudomonas syringae rendered infected tissues more susceptible to infection by the necrotrophic pathogen Alternaria brassicicola by suppressing the JA signaling pathway. Earlier, Moran (1998) demonstrated that pathogen-induced SAR in cucumber was associated with reduced resistance against feeding by spotted cucumber beetles (Diabrotica undecimpunctata howardi) and enhanced reproduction of melon aphids (Aphis gossypii). Similarly, induction of SAR by tobacco mosaic virus (TMV) resulted in increased feeding by the tobacco hornworm Manduca sexta compared to control tobacco plants (Preston et al., 1999). Analysis of transgenic tobacco plants overexpressing the phenylalanine ammonia-lyase (PAL) gene demonstrated increased SAR to TMV while resistance to the tobacco budworm (Heliothis virescens) was severely reduced. Conversely, silencing of PAL reduced SA accumulation and SAR, and enhanced herbivore-induced resistance against H. virescens (Felton et al., 1999). Application of the SA analog benzothiadiazole S-methyl ester (BTH) has been shown to reduce resistance to the corn earworm Helicoverpa zea (Stout et al., 1999) and to the beet armyworm Spodoptera exigua (Thaler et al., 1999) on tomato. SA treatment similarly inhibited JA-induced resistance against S. exigua in Arabidopsis (Cipollini et al., 2004). Conversely, S. exigua larvae gained significantly less weight on transgenic SA-nonaccumulating NahG plants and on the SA signaling mutant npr1 (nonexpressor of PR genes 1) (Cipollini et al., 2004;
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Mewis et al., 2005; Van Oosten, 2007). Likewise, the SA mutants npr1 and sid2 (salicylic acid induction-deficient 2) exhibited increased resistance to the Egyptian cotton worm Spodoptera littoralis (Stotz et al., 2002; Bodenhausen and Reymond, 2007) and the cabbage looper Trichoplusia ni (Cui et al., 2002).
These examples demonstrate the negative effect of SA-mediated responses on JA-dependent defenses. Negative cross-talk of JA on SA-mediated signaling has been less well documented for tripartite plant-microbe-insect interactions, but does occur (Bostock, 2005). For instance, infestation with the alfalfa hopper Spissistilus festinus rendered alfalfa more susceptible to the fungus Fusarium oxysporum (Moellenbeck et al., 1992). In addition, herbivore feeding on willow and creeping thistle rendered these plants more susceptible to rust fungi (Kluth et al., 2001; Simon and Hilker, 2003). The antagonistic effect of JA on SA signaling has been suggested to involve also indirect defenses. Upon herbivore feeding, plants emit volatiles as an indirect defense mechanism that attracts natural enemies of the herbivore (Van Poecke and Dicke, 2004). Methyl salicylate (MeSA) comprises one component of the complex volatile blend that is emitted by wounded plants (Van Poecke and Dicke, 2004). SA is converted by SA-methyltransferase (SAMT) to MeSA. JA was found to induce SAMT transcription and increase MeSA emission in several plant species (Martin et al., 2003; Ament et al., 2004; Filella et al., 2006; Koo et al., 2007). Overexpression of the rice SAMT gene in Arabidopsis generated a MeSA-overproducing, but SA-depleted transgenic plant, as evidenced by decreased SA-responsive PR-1 expression and impaired resistance to the biotrophs P. syringae and Golovinomyces orontii. Consequently, it was suggested that JA-induced SAMT may contribute to the antagonistic effect on SA signaling by depleting the pool of SA in plants (Koo et al., 2007).
S y n e r g i s t i c i n t e r a c t i o n s
Next to antagonistic effects, synergistic interplay between SA and JA routes has been described as well. Microarray analysis performed by Schenk et al. (2000) revealed a number of genes that were induced or repressed by both SA and methyl jasmonate (MeJA), indicating coordinated regulation of SA- and JA-dependent defense responses. Pharmacological experiments with SA and JA likewise revealed transient synergistic interactions when the chemicals were applied at low doses (Mur et al., 2006). Furthermore, in Arabidopsis, SA-dependent SAR and JA/ET-dependent ISR have an additive effect on induced resistance against virulent P. syringae (Van Wees et al., 2000). Recently, a role for jasmonates (JAs) in SAR signaling was suggested (Truman et al., 2007). However, other lines of evidence demonstrate that mutants disrupted in JA signaling are still able to mount wild-type levels of SAR (Lawton et al., 1996; Pieterse et al., 1998; Ton et al., 2002b). Hence, the precise role of JAs in SAR needs to be explored further.
Cross-resistance between pathogens and insects has been described as well. Infestation of tomato by H. zea caterpillars enhanced resistance against P. syringae, and vice versa, demonstrating reciprocal induced resistance for these attackers (Stout et al., 1999). Moreover, insect-induced resistance triggered by feeding of caterpillars of the small cabbage white
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Chapter 1 • 1 5
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(Pieris rapae) was locally effective against two bacterial pathogens, Xanthomonas campestris and P. syringae, and locally and systemically effective against turnip crinkle virus (De Vos et al., 2006). These variable outcomes, depending on plant-attacker combinations, highlight the complexity of the induced defense response.
D e c o y o f p l a n t d e f e n s e
Cross-talk between defense signaling pathways is thought to help the plant ‘decide’ which defensive strategy to follow, depending on the type of attacker it is encountering. Yet, it seems that attackers have also evolved to manipulate plants for their own benefit by suppressing induced defenses or modulating the defense signaling network (Pieterse and Dicke, 2007). For instance, herbivorous nymphs of the silverleaf whitefly (Bemisia tabaci) may activate the SA signaling pathway as a decoy strategy to effectively suppress JA-dependent defenses, thus enhancing insect performance (Zarate et al., 2007). Similarly, egg-derived elicitors from P. rapae and the large cabbage white, Pieris brassicae, have been suggested to suppress JA-dependent defenses through SA/JA cross-talk to benefit hatching larvae (Little et al., 2007). Microbial pathogens have acquired the ability to manipulate the plant signaling infrastructure by producing phytohormones or their functional mimics to ‘trick’ the plant into activating inappropriate defenses (Robert-Seilaniantz et al., 2007). For instance, virulent P. syringae bacteria produce the toxin coronatine that functions as a potent mimic of jasmonates (Nomura et al., 2005). It is assumed that coronatine triggers induction of JA signaling responses, which results in suppression of SA-dependent defenses through pathway cross-talk, and thereby promotes P. syringae pathogenesis (Zhao et al., 2003; Brooks et al., 2005; Cui et al., 2005; Laurie-Berry et al., 2006). Recently, coronatine was also demonstrated to prevent PAMP-induced stomatal closure, thereby facilitating bacterial entry into the leaf (Melotto et al., 2006).
S I G N A L T R A N S D U C T I O N PAT H WAY S
SA- and JA-dependent defense signaling pathways have been studied extensively over the last decades in the model plant Arabidopsis thaliana. In order to elucidate the molecular mechanism underlying SA/JA cross-talk, detailed knowledge of the signaling events leading to SA- or JA-dependent defense reactions is mandatory. Below, an overview of SA- and JA-dependent signaling pathways is provided.
S A - d e p e n d e n t s i g n a l i n g
The importance of SA in the regulation of plant defense became evident through experiments with transgenic NahG plants that convert SA into catechol through the activity of an introduced salicylate hydroxylase (NahG) gene. Expression of this enzyme renders tobacco and Arabidopsis plants incapable of accumulating SA and developing SAR (Gaffney et al., 1993), and increases their susceptibility to many different pathogens,
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including bacteria, viruses, fungi, and oomycetes (Delaney et al., 1994; Kachroo et al., 2000). Similarly, Arabidopsis mutants that are not able to enhance the production of SA upon pathogen infection, such as eds1 (enhanced disease susceptibility 1) (Rogers and Ausubel, 1997), sid1 (=eds5), sid2 (=eds16) (Nawrath and Métraux, 1999), and pad4 (phytoalexin-deficient 4) (Zhou et al., 1998), display a higher level of susceptibility to different pathogens, indicating that SA also plays an important role in basal defense. Transduction of the SA signal to activate pathogenesis-related (PR) gene expression requires the function of the regulatory protein NPR1, also known as NIM1 (NON-IMMUNITY 1) and SAI1 (SALICYLIC ACID-INSENSITIVE 1) (Cao et al., 1994; Delaney et al., 1995; Shah et al., 1997). Mutations in the NPR1 gene render the plant largely unresponsive to pathogen-induced SA production, thereby blocking the induction of SA-dependent PR genes and SAR (Cao et al., 1994; Delaney et al., 1995; Shah et al., 1997).
NPR1 is expressed throughout the plant at low levels and shows only a modest induction upon pathogen infection or SA treatment in wild-type Arabidopsis (Cao et al., 1997; Ryals et al., 1997). Overexpression of NPR1 does not result in a massive induction of the PR-1 gene, indicating that NPR1 requires post-translational activation in order to transduce the SA signal (Cao et al., 1998; Friedrich et al., 2001). Indeed, SA-induced redox changes have been shown to reduce intermolecular disulfide bonds that hold NPR1 together as an inactive oligomer. This reduction converts the inactive oligomeric complex into an active monomeric form that is translocated into the nucleus to activate PR gene expression (Mou et al., 2003). Although NPR1 acts as a modulator of PR gene expression, it does not posses a DNA-binding domain. However, it does contain an ankyrin-repeat and a BTB/POZ (Broad-complex, Tramtrack, and Bric-a-brac/Pox virus and Zinc finger) domain, which are involved in protein-protein interactions (Cao et al., 1997; Ryals et al., 1997). NPR1 has been shown to interact with a negative modulator of PR gene expression, NIMIN1 (NIM-interacting 1), which is thought to provide a fine-tuning mechanism for the activation of SAR (Weigel et al., 2005). Furthermore, NPR1 interacts with seven out of ten members of the TGA subclass of the basic leucine zipper (bZIP) family of transcription factors (Zhang et al., 1999; Després et al., 2000; Zhou et al., 2000; Subramaniam et al., 2001; Fan and Dong, 2002; Jakoby et al., 2002; Kim and Delaney, 2002; Després et al., 2003; Johnson et al., 2003). TGA transcription factors can bind to both positive and negative cis-elements in the PR-1 promoter (Lebel et al., 1998) and act as either positive or negative regulators of PR-1 gene expression (Pontier et al., 2001; Zhang et al., 2003; Rochon et al., 2006; Kesarwani et al., 2007), indicating that PR gene expression during SAR must be under tight regulatory control. Moreover, NPR1 is required for removal of the suppressor protein SNI1 (SUPPRESSOR OF npr1-1, INDUCIBLE 1), which negatively affects PR expression, possibly through chromatin modifications (Li et al., 1999; Mosher et al., 2006).
Recently, a genomics-directed approach demonstrated that upon induction of SAR, a select group of WRKY transcription factor genes is induced after nuclear translocation of NPR1 monomers (Wang et al., 2006). Like the TGAs, WRKY transcription factors have both positive and negative effects on the expression of PR genes, thus contributing further to the
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complexity of the SA- and NPR1-dependent signaling network involved in SA-dependent SAR (Wang et al., 2006). Besides regulating PR genes, NPR1 was also shown to target the transcription of genes that are involved in the protein secretory pathway. Expression of these proteins ensures proper processing of PR transcripts and secretion of PR proteins required for SA-based resistance (Wang et al., 2005). Thus, multiple positive and negative integrators play a role in fine-tuning SA-dependent defense signaling (Figure 1.1).
J A - d e p e n d e n t s i g n a l i n g
In the past twenty years, JA and its functionally active derivatives (e.g. jasmonoyl-isoleucine (JA-Ile) and MeJA) emerged as important regulators of induced plant defense. JAs are produced by the octadecanoid pathway from linolenic acid that is released from chloroplast membranes upon wounding caused by insect herbivory or pathogen attack (Howe and Schilmiller, 2002; Wasternack, 2007). Downstream target genes include defense-related genes, such as the defensin PDF1.2 (PLANT DEFENSIN 1.2) and thionin Thi2.1 (THIONIN 2.1), but also genes that are required for the biosynthesis of JA itself. A central role for JA in plant resistance was demonstrated in several mutants that are defective in different steps of the JA pathway, such as biosynthesis, perception, and/or signaling (Creelman and Mullet, 1997; Devoto and Turner, 2005; Lorenzo and Solano, 2005). For example, the fad3 fad7 fad8 (fatty acid desaturation 3, 7, 8) JA biosynthesis mutant showed increased susceptibility to normally non-pathogenic Pythium spp. (Staswick et al., 1998; Vijayan et al., 1998) and cucumber mosaic virus (Ryu et al., 2004), and suffered high mortality from attack by
Figure 1.1. SA signal transduction pathway.Accumulation of SA triggered by pathogen attack leads to a change in the redox state of the cell. After an initial oxidative burst, the cell attains a more reducing environment that changes the inactive NPR1 oligomeric complex into active monomers. Monomeric NPR1 is translocated to the nucleus, where it interacts with the negative regulator NIMIN1. NPR1 is required for removal of the repressor SNI1, interacts with TGA transcription factors, and directly targets several WRKY transcription factors that can have positive and negative effects on PR gene expression. Furthermore, NPR1 upregulates genes involved in the protein secretory pathway, which ensures proper processing and secretion of PR-proteins (Scheme adapted from Durrant and Dong, 2004).
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larvae of the fungus gnat, Bradysia impatiens (McConn et al., 1997). In addition, mutant coi1 (coronatine insensitive 1) plants are unresponsive to JA and show enhanced susceptibility to the bacterial soft rot pathogen Erwinia carotovora (Norman-Setterblad et al., 2000) and the necrotrophic fungi Alternaria brassicicola and Botrytis cinerea (Thomma et al., 1998).
Virtually all JA responses are dependent on the presence of the COI1 protein (Feys et al., 1994; Xie et al., 1998). COI1 encodes an F-box protein (Xie et al., 1998), which is part of an SCF (Skp/Cullin/F-box) E3 ubiquitin ligase complex involved in proteasome-mediated protein degradation (Devoto et al., 2002; Xu et al., 2002). The F-box protein confers specificity to the E3 ligase complex by interacting with proteins that are targeted for ubiquitination and subsequent degradation. Therefore, COI1 is thought to mediate the removal of repressors that keep JA responses inactive (Devoto et al., 2003). Recently, JAZ (jasmonate ZIM-domain) proteins have been identified as likely candidates for COI1-targeted transcriptional repressors of JA-responsive genes (Chini et al., 2007; Thines et al., 2007). JAZ proteins repress JA-responsive gene expression by actively suppressing transcriptional activators of JA-responsive genes, such as MYC2 (Chini et al., 2007). MYC2 regulates the expression of wound-inducible JA-responsive genes, such as VSP2 (VEGETATIVE STORAGE PROTEIN 2) and LIPOXYGENASE (LOX2), whereas the JA/ET-responsive transcription factor ERF1 (ETHYLENE RESPONSE FACTOR 1) regulates the expression of pathogen-responsive genes, such as PDF1.2 and HEL (HEVEIN-LIKE) (Lorenzo et al., 2004; Lorenzo and Solano, 2005). It is currently not known whether JAZ repressors also interact with the ERF1 transcription factor. Upon stimulation of the JA response, the physical interaction of JA-Ile with JAZ proteins allows COI1 to target JAZ proteins for degradation by the proteasome (Thines et al., 2007). As a result, repression by the JAZ proteins is lifted, causing enhanced transcription of JA-responsive genes (Figure 1.2). Notably, JAZ biosynthesis genes are induced by JA itself, indicating a negative feedback loop that allows for a pulsed response to the JA-inducing stimulus (Chini et al., 2007; Thines et al., 2007).
M O L E C U L A R P L AY E R S I N C R O S S - TA L K B E T W E E N S A A N D J A PAT H WAY S
Mutations that affect the induction of either SA- or JA-dependent signaling are often associated with a shift in the balance between SA- and JA-dependent defenses. Overexpressors of SA signaling show decreased JA responses and vice versa, indicating that under wild-type conditions SA- and JA-dependent pathways are mutually antagonistic. For example, SA-deficient mutants, such as NahG, sid2, eds4, eds5, and pad4, produced increased levels of JA in response to infection with P. syringae, or showed higher PDF1.2 induction in response to exogenous MeJA or infection by A. brassicicola (Penninckx et al., 1996; Gupta et al., 2000; Heck et al., 2003; Spoel et al., 2003). The opposite phenotype was observed in mutants that accumulate high levels of SA and/or show enhanced PR gene expression. For
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example, the bos3 (botrytis susceptible 3) mutant showed enhanced susceptibility to B. cinerea and A. brassicicola, but was more resistant to virulent Hyaloperonospora parasitica and P. syringae. Defense gene expression was similarly altered, showing an increase in SA-dependent PR-1 expression and a decrease in JA-dependent PDF1.2 induction (Veronese et al., 2004). Thus, the balance between SA and JA responses is altered in this mutant. A similar phenotype was observed after inactivation of BIK1 (BOTRYTIS-INDUCED KINASE 1). The bik1 mutant accumulated SA and showed enhanced resistance to virulent P. syringae, while PDF1.2 expression and resistance to A. brassicicola and B. cinerea was reduced. Transgenic bik1/NahG plants demonstrated that these responses were dependent on SA (Veronese et al., 2006). Additionally, a WRKY triple mutant, wrky18 wrky40 wrky60 was more resistant to P. syringae than wild-type plants, which correlated with enhanced PR-1 expression. Conversely, the triple mutant was more susceptible to B. cinerea than wild-type plants, which correlated with reduced expression of PDF1.2 (Xu et al., 2006).
Mutations in the JA signaling route equally have an effect on SA-dependent defenses. For example, overexpression of the transcription factor ERF1 resulted in increased resistance to the necrotrophic pathogens B. cinerea and Plectosphaerella cucumerina, but enhanced susceptibility to P. syringae (Berrocal-Lobo et al., 2002). Furthermore, SA-induced PR-1 expression was reduced in the cev1 (constitutive expression of VSP1) mutant, which constitutively expresses JA and ET responses compared to wild-type plants (Ellis and Turner, 2001; Ellis et al., 2002). Conversely, increased PR-1 expression was reported in JA-defective tomato mutants jai1-1 (jasmonic acid insensitive 1-1) and def-1 (defenseless-1) (Zhao et al., 2003; Ament et al., 2004), and Arabidopsis coi1 (Kloek et al., 2001; Ellis et al., 2002; Devoto et al., 2005), demonstrating a reciprocal suppression by JA on the SA pathway.
Figure 1.2. JA signal transduction pathway.JA is synthesized upon wounding by insect herbivory or pathogen attack. In the presence of JA-Ile, JAZ repressor proteins are recruited to COI1, which targets these repressors for degradation by the proteasome. Removal of JAZ de-represses transcription factors, such as MYC2, which induces JA-responsive genes (Scheme adapted from Farmer, 2007).
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Elucidating the molecular mechanism underlying the antagonistic interactions between SA- and JA-dependent defense signaling pathways provides an excellent model to start unravelling the multifaceted signal interactions that shape the plant immune response. Over the past years, various regulatory components with a role in SA/JA cross-talk have been identified. Below, the most prominent molecular players in SA/JA cross-talk are discussed in more detail.
N P R 1
Several key regulatory proteins involved in pathway cross-talk have been identified. In Arabidopsis, SA-mediated suppression of JA-inducible gene expression is blocked in mutant npr1 plants, demonstrating a crucial role for NPR1 in the cross-talk between SA and JA signaling (Spoel et al., 2003). Using npr1 plants expressing recombinant NPR1 protein with a glucocorticoid receptor hormone-binding domain to control the nucleocytoplasmic localization of the NPR1 protein, Spoel et al. (2003) showed that nuclear localization of NPR1 is not required for SA-mediated suppression of JA-responsive genes. This result indicates that the SA-induced suppression of the JA response is controlled by NPR1 functioning in the cytosol. Recently, a similar function of NPR1 in cross-talk was reported in rice (Oryza sativa) (Yuan et al., 2007). Overexpression of cytosolic OsNPR1 suppressed JA-responsive transcription and enhanced the level of susceptibility to insect herbivory. Moreover, NPR1-dependent suppression of the JA response was no longer present in plants when the OsNPR1 protein was constitutively targeted to the nucleus.
However, a recent report on NPR1-silenced wild tobacco plants (Nicotiana attenuata) demonstrated that these transgenic plants accumulated increased levels of SA upon insect herbivory and were highly susceptible to herbivore attack (Rayapuram and Baldwin, 2007). It was proposed that in wild-type plants NPR1 is required to negatively regulate SA production during herbivore attack and thus suppress SA/JA cross-talk to allow induction of JA-mediated defenses against herbivores. These results indicate a diverse regulatory role of NPR1 in cross-talk. Yet, it also demonstrates that molecular mechanisms of induced defense as identified in model systems should be tested in an ecological context, in which the plant species under study co-evolved with its natural enemies, to fully understand its biological function.
W R K Y t r a n s c r i p t i o n f a c t o r s
In Arabidopsis, the family of WRKY transcription contains 74 members of which many have been implicated as either positive or negative regulators of defense responses (Maleck et al., 2000; Wang et al., 2006; Eulgem and Somssich, 2007). Induction of SA-dependent defenses altered the expression of 49 WRKY genes (Dong et al., 2003), and eight WRKYs were identified as direct targets of NPR1 (Wang et al., 2006). In addition, some WRKY factors have been implicated in SA/JA cross-talk. Arabidopsis WRKY70 was identified as a node of convergence between SA and JA signaling when Li et al. (2004) showed that overexpression of WRKY70 caused enhanced expression of SA-responsive PR genes and
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concomitantly suppressed MeJA-induced expression of the marker gene PDF1.2. Hence, WRKY70 has been suggested to act as a positive regulator of SA-mediated defenses, while repressing the JA response.
WRKY11 and WRKY17 have been shown to act as cross-regulators within the WRKY family of transcription factors by influencing the expression of other WRKYs (Journot-Catalino et al., 2006). In the double mutant wrky11 wrky17 transcripts of SA-responsive genes accumulated to higher levels, whereas those of JA-responsive genes were notably lower. The expression of WRKY70 was up-regulated in this double mutant, suggesting that WRKY11 and WRKY17 function as negative regulators of WRKY70 (Journot-Catalino et al., 2006). Recently, WRKY62 was added to the list of WRKY transcription factors with a putative role in SA/JA cross-talk. Mao et al. (2007) reported that the expression of WRKY62 was induced by SA and boosted by MeJA in wild-type Col-0 plants, but not in mutant npr1. Furthermore, transposon-tagged wrky62 plants showed enhanced MeJA-induced transcription of the JA-responsive genes LOX2 and VSP2, whereas overexpression of WRKY62 resulted in suppression of these genes. These findings suggest an NPR1-mediated repressive effect of WRKY62 on the JA response.
G l u t a r e d o x i n G R X 4 8 0
SA-induced redox changes activate NPR1 to induce PR gene expression (Mou et al., 2003), and NPR1 is required for the SA/JA antagonism (Spoel et al., 2003). Glutaredoxins have been implicated in redox-dependent regulation of protein activities and catalyze thiol disulfide reductions (Lemaire, 2004). Thus, glutaredoxins may function as transducers of the SA-induced redox change by catalyzing reduction of disulfides or glutathione mixed disulfides. Recently, Ndamukong et al. (2007) identified glutaredoxin GRX480 (GLUTAREDOXIN 480) as a putative regulator of SA/JA cross-talk. This glutaredoxin was found in a two-hybrid screen for interactors with TGA transcription factors. Expression of GRX480 was found to be inducible by SA and dependent on NPR1. Overexpression of GRX480 completely abolished MeJA-induced PDF1.2 expression, but hardly affected the induction of the JA-responsive genes LOX2 and VSP2. This suggests that GRX480 affects only a subset of the JA-responsive genes that are sensitive to SA-mediated suppression. The suppressive effect of GRX480 on PDF1.2 induction was abolished in the tga2-1 tga5-1 tga6-1 triple mutant, indicating that the interaction between GRX480 and TGA transcription factors is essential for the GRX480-dependent cross-talk (Ndamukong et al., 2007). These results suggest a model in which SA-activated NPR1 induces GRX480, which in turn interacts with TGA transcription factors to suppress JA-responsive gene induction.
M P K 4
Mitogen-activated protein kinases (MAPKs) transfer information from sensors to cellular responses in all eukaryotes. Therefore, it is not surprising that several MAP kinases have been implicated in plant defense signaling (Menke et al., 2004; Nakagami et al., 2005). Petersen
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et al. (2000) identified MAP KINASE 4 (MPK4) as a negative regulator of SA signaling and a positive regulator of JA signaling in Arabidopsis. Inactivation of MPK4 in mutant mpk4 plants resulted in elevated SA levels and constitutive expression of SA-responsive PR genes, suppression of JA-responsive genes and enhanced susceptibility to the necrotroph A. brassicicola (Petersen et al., 2000; Brodersen et al., 2006). The mpk4 mutation blocked JA-responsive gene expression independently of SA accumulation, as SA-nonaccumulating mpk4/NahG transgenics still exhibited increased susceptibility to A. brassicicola and suppression of MeJA-induced PDF1.2 expression (Petersen et al., 2000; Brodersen et al., 2006). EDS1 and PAD4 were identified as downstream effectors of MPK4 function, acting oppositely to MPK4 by behaving as activators of SA signaling and repressors of JA signaling (Brodersen et al., 2006).
MAP KINASE 4 SUBSTRATE 1 (MKS1) has been identified as a substrate of MPK4. Phosphorylation of MKS1 by MPK4 is thought to repress SA signaling, since MKS1-RNAi could partially rescue the PR-1-overexpressing phenotype of mutant mpk4. However, over- or under-expression of MKS1 did not affect PDF1.2 gene expression, indicating that other downstream targets of MPK4 must be involved in JA signaling. MKS1 was demonstrated to interact with two WRKY transcription factors, WRKY25 and WRKY33, both of which can be phosphorylated by MPK4 (Andreasson et al., 2005). These WRKYs may be downstream targets of MPK4 that contribute to the repression of SA responses, because overexpression of both WRKY25 and WRKY33 resulted in decreased pathogen-induced PR-1 expression and enhanced susceptibility to P. syringae (Zheng et al., 2006; Zheng et al., 2007). Conversely, wrky33 mutant plants showed increased susceptibility to the necrotrophs B. cinerea and A. brassicicola and reduced PDF1.2 expression (Zheng et al., 2006), consistent with a role of WRKY transcription factors in SA/JA cross-talk.
S S I 2
Mutant ssi2 (suppressor of SA insensitivity 2) is defective in stearoyl ACP desaturase, resulting in altered fatty acid (FA) composition. This mutant shows NPR1-independent constitutive PR-1 expression and enhanced resistance to H. parasitica, but is impaired in PDF1.2 transcription and resistance to B. cinerea. Inhibition of PDF1.2 expression is not dependent on elevated SA levels, since the ssi2/NahG plants were still unable to express JA-induced PDF1.2 (Kachroo et al., 2001). Mutations that restored the lowered 18:1 FA levels rescued the ssi2 mutant phenotype, suggesting involvement of fatty acid signaling in SA/JA cross-talk (Kachroo et al., 2003; Kachroo et al., 2004).
O U T L I N E O F T H E T H E S I S
In the past years, significant progress has been made in elucidating the molecular mechanism underlying the interplay between hormone-regulated defense signaling pathways. Several molecular components in pathway cross-talk have been identified. However, translation of
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molecular mechanisms into predictability of trade-offs between herbivore and pathogen resistance requires additional research. So far, studies on trade-offs between induced insect and pathogen resistance are often performed with single plant-attacker interactions under a limited set of abiotic conditions. This type of research is highly valuable, because only under controlled conditions the highly flexible induced defense signaling network functions reproducibly, such that novel mechanisms of regulation can be elucidated. However, because plant defense mechanisms evolved during the co-evolutionary arms race between plants and their natural enemies and entail costs in addition to benefits, insights into their biological significance should ideally come from ecological studies. Therefore, to understand the functioning of the complex defense signaling network in nature, molecular biologists and ecologists should join forces to place molecular mechanisms of induced plant defenses in an ecological perspective.
De Vos (2006) and Van Oosten (2007) employed a combinatorial phytopathological and entomological approach to study pathogen and insect resistance in Arabidopsis. Transcriptomics and mutant analysis were used to evaluate how plants defend themselves against different types of attackers. Based on the complexity of the responses seen, they concluded that elucidation of the underlying molecular mechanisms is of utmost importance to understand the regulatory potential of plant defenses. Therefore, the main goal of this study was to unravel the molecular mechanism underlying cross-communicating SA- and JA-dependent defense signaling pathways in Arabidopsis. In order to study this highly complex phenomenon, we employed a predominantly pharmacological approach to control experimental conditions.
In Chapter 2, treatment with SA or infection with virulent P. syringae was shown to suppress MeJA-responsive gene expression, indicating an antagonistic effect of SA on JA signaling. Mutation of the regulatory protein NPR1 abolished this suppressive effect of SA, demonstrating an essential role for NPR1 in SA/JA cross-talk. Furthermore, treatment of transgenic plants expressing recombinant NPR1 under nucleocytoplasmic control demonstrated that nuclear localization of NPR1 is not required for the suppression of MeJA-responsive genes. Hence, cytosolic NPR1 mediates cross-talk between SA and JA pathways.
The kinetics of SA/JA cross-talk were further investigated in Chapter 3. We used biological inducers, A. brassicicola, B. cinerea, F. occidentalis, H. parasitica, and P. rapae, to show that SA-mediated suppression of JA-responsive genes can be triggered biologically as well as chemically. Furthermore, the robustness of the phenomenon was shown by its conservation among 18 different Arabidopsis accessions. The longevity and sensitivity of SA/JA cross-talk was investigated by time course analyses, as well as dose-response assays. JA-responsive gene expression was readily suppressed by SA for several days, even when triggered by very low doses of SA. Time interval studies revealed that SA has a window of opportunity to suppress MeJA-responsive gene expression, and that this time interval correlates with the SA-induced redox change in the plant tissue. Thus, redox modulation is likely to play a central role in the regulatory mechanism underlying SA/JA cross-talk.
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In Chapter 4, we aimed at identifying additional signaling components involved in the regulation of SA/JA cross-talk. Mutant and transcriptome analysis revealed NPR1-dependent as well as NPR1-independent cross-talk, and confirmed that TGA transcription factors play a role in SA/JA cross-talk. Furthermore, we found no evidence for an involvement of histone modifications in the SA-mediated suppression of MeJA-responsive gene expression. Finally, analysis of overrepresented motifs in the promoters of co-regulated genes affected by the SA/JA cross-talk suggested that the GCC box in JA-responsive promoters may be a target for cross-talk regulation.
In Chapter 5 we designed a mutant screen to identify essential cross-talk regulators through an unbiased mutagenesis screen. The MeJA-responsive PDF1.2 promoter was fused to a herbicide resistance gene, to allow for MeJA-inducible herbicide tolerance. EMS mutagenesis of this transgenic line would allow for the identification of mutants that survive the SA/JA cross-talk and herbicide treatments. However, the SA/JA antagonism proved insufficient to fully suppress herbicide tolerance. A mutant screen that allows detection of quantitative differences in gene expression would be better suited for identification of cross-talk mutants.
Finally, the results presented in this thesis are discussed in Chapter 6 with respect to current knowledge of plant-attacker cross-talk and hormone-regulated defense signaling pathways in Arabidopsis.
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C H A P T E R 2
NPR1 modulates cross-talk between
sal icylate- and jasmonate-dependent
defense pathways through a
novel function in the cytosol
Steven H. Spoel 1,2, Annemart Koornneef 1, Susanne M.C. Claessens 1, Jerôme P. Korzelius 1, Johan A. Van Pelt 1, Martin J. Mueller 3, Antony J. Buchala 4, Jean-Pierre Métraux 4, Rebecca Brown 5, Kemal Kazan 5, L.C. Van Loon 1, Xinnian Dong 2, and Corné M.J. Pieterse 1
1 Graduate School Experimental Plant Sciences, Plant-Microbe Interactions,
Institute of Environmental Biology, Faculty of Science, Utrecht University,
P.O. Box 800.56, 3508 TB, The Netherlands2 Developmental, Cell, and Molecular Biology Group, Department of Biology,
Duke University, Durham, North Carolina 27708-1000, USA3 Pharmaceutical Biology, Julius-von-Sachs Institute of Biological Sciences,
University of Wuerzburg, D-97082 Wuerzburg, Germany4 Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland5 Cooperative Research Centre for Tropical Plant Pathology, John Hines Building,
The University of Queensland, St. Lucia, Queensland 4072, Australia
The Plant Cell 15, 760-770 (2003)
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NPR1 modulates cross-talk between
sal icylate- and jasmonate-dependent
defense pathways through a
novel function in the cytosol
A B S T R A C T
Plant defenses against pathogens and insects are differentially regulated by cross-communicating signal transduction pathways in which salicylic acid (SA) and jasmonic acid (JA) play key roles. In this study we investigated the molecular mechanism of the antagonistic effect of SA on JA signaling. Arabidopsis plants unable to accumulate SA produced 25-fold higher levels of JA, and showed enhanced expression of the JA-responsive genes LOX2, PDF1.2, and VSP in response to infection by Pseudomonas syringae pv tomato DC3000, indicating that in wild-type plants, pathogen-induced SA accumulation is associated with suppression of JA signaling. Analysis of the Arabidopsis mutant npr1, impaired in transducing the SA signal, revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for suppression of JA signaling, indicating that cross-talk between SA and JA is modulated through a novel function of NPR1 in the cytosol.
I N T R O D U C T I O N
To effectively combat invasion by microbial pathogens and herbivorous insects, plants are able to activate distinct defense responses that are effective specifically against the invader encountered (Van Loon, 2000). These induced defenses often are expressed not just locally but also in parts distant from the site of primary infection, thereby protecting the plant systemically against subsequent attack. Induced resistance is regulated by a network of interconnecting signal transduction pathways in which salicylic acid (SA) and jasmonic acid (JA) function as key signaling molecules (Reymond and Farmer, 1998; Pieterse and Van Loon, 1999; Glazebrook, 2001; Thomma et al., 2001). SA and JA accumulate in response to pathogen infection or herbivore damage, resulting in the activation of distinct sets of defense-related genes. Mutant and transgenic plants that are affected in SA accumulation
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often are more susceptible to pathogen infection than wild-type plants (Delaney et al., 1994; Nawrath and Métraux, 1999; Wildermuth et al., 2001). Blocking the response to JA generally renders plants more susceptible to herbivore damage (Howe et al., 1996; McConn et al., 1997), although enhanced susceptibility toward necrotrophic pathogens has been reported as well (Thomma et al., 2001). SA- and JA-dependent defense pathways have been shown to cross-communicate (Felton and Korth, 2000; Feys and Parker, 2000; Pieterse et al., 2001), providing the plant with a regulatory potential to fine-tune the defense reaction depending on the type of attacker encountered.
One of the most studied induced defense responses in plants is systemic acquired resistance (SAR). SAR is triggered after local infection with pathogens, causing hypersensitive necrosis, and is effective against a broad spectrum of plant pathogens (Ryals et al., 1996). The onset of SAR is accompanied by a local and systemic increase in the endogenous levels of SA (Malamy et al., 1990; Métraux et al., 1990) and the concomitant upregulation of a large set of genes (Ward et al., 1991), including genes encoding pathogenesis-related (PR) proteins (Van Loon and Van Strien, 1999). Several PR proteins possess antimicrobial activity and are thought to contribute to the state of resistance attained. Transduction of the SA signal requires the function of NPR1 (also known as NIM1), a regulatory protein that was identified in Arabidopsis through genetic screens for SAR-compromised mutants (Cao et al., 1994; Delaney et al., 1995; Shah et al., 1997). Mutant npr1 plants accumulate normal levels of SA after pathogen infection but are impaired in their ability to express PR genes and to mount a SAR response. The NPR1 gene encodes a protein with a BTB/POZ and an ankyrin-repeat domain (Cao et al., 1997; Ryals et al., 1997; Aravind and Koonin, 1999). Both domains are known to mediate protein-protein interactions and are present in proteins with diverse functions (Bork, 1993; Aravind and Koonin, 1999), including the transcriptional regulator IκB, which mediates animal innate immune responses (Baldwin, 1996). Upon induction of SAR, NPR1 is translocated to the nucleus (Kinkema et al., 2000), where it interacts with members of the TGA/OBF subclass of basic domain/Leu zipper (bZIP) transcription factors (Zhang et al., 1999; Després et al., 2000; Zhou et al., 2000; Subramaniam et al., 2001; Fan and Dong, 2002) that are involved in the SA-dependent activation of PR genes (Lebel et al., 1998; Niggeweg et al., 2000). Physical interaction between NPR1 and TGA transcription factors has been shown to be required for the binding activity of these factors to promoter elements that play a crucial role in the SA-mediated activation of PR genes (Després et al., 2000; Fan and Dong, 2002).
The activation of SAR has been shown to suppress JA signaling in plants, thereby prioritizing SA-dependent resistance to microbial pathogens over JA-dependent defense against insect herbivory (Felton and Korth, 2000; Pieterse et al., 2001). Moreover, pharmacological and genetic experiments have shown that SA is a potent suppressor of JA-inducible gene expression (Doherty et al., 1988; Peña-Cortés et al., 1993; Doares et al., 1995; Harms et al., 1998; Gupta et al., 2000). The antagonistic effect of SA on JA signaling shows a striking resemblance to the effect of the nonsteroidal anti-inflammatory drug acetylsalicylic acid (aspirin), a derivative of SA, on the formation of prostaglandins in
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animal cells. Prostaglandins are related structurally to JA and play a role in diverse biological processes, such as inflammation at sites of infection or tissue injury (Straus and Glass, 2001). JA and prostaglandins originate biosynthetically from linolenic acid and arachidonic acid, respectively, which are released from cell membranes upon phospholipid hydrolysis. Linolenic acid and arachidonic acid are metabolized rapidly via the oxylipin pathway, in which the enzymatic reactions leading to JA and prostaglandin formation are similar (Pan et al., 1998). In animal cells, aspirin inhibits the octadecanoid pathway by acetylating the key enzyme cyclooxygenase, ultimately leading to a decrease in prostaglandin formation (Van der Ouderaa et al., 1980).
In a similar process in plants, aspirin has been shown to inhibit the activity of the counterpart of cyclooxygenase, allene oxide synthase, which catalyzes the same step in the octadecanoid pathway in plants, thereby affecting the formation of JA and the subsequent activation of stress-related gene expression (Pan et al., 1998). Whereas aspirin is able to inhibit prostaglandin and JA biosynthetic enzymes by acetylating them, SA, which lacks the acetyl group, is ineffective in this respect. Indeed, in Arabidopsis and flax plants, no inhibitory effect of SA on allene oxide synthase activity was observed (Harms et al., 1998; Laudert and Weiler, 1998). Thus, given the fact that the acetylated form of SA does not occur naturally in plants (Pierpoint, 1997), it is unlikely that inhibition of the allene oxide synthase activity plays a major role in the cross-communication between SA and JA signaling in plants. Nevertheless, SA is a strong negative regulator of JA-dependent cellular defense responses in plants (Doherty et al., 1988; Doares et al., 1995; Harms et al., 1998; Gupta et al., 2000).
So how does SA negatively regulate JA-dependent cellular defense responses in plants? In animal cells, both aspirin and SA are able to reduce proinflammatory prostaglandin formation by inhibiting the activity of the transcription factor NF-κB (Kopp and Ghosh, 1994; Yin et al., 1998). NF-κB plays a key role in the transcriptional activation of many genes during the innate immune response (Baldwin, 1996; Hatada et al., 2000), including the gene that encodes CYCLOOXYGENASE2, which catalyzes a rate-limiting step in prostaglandin production (Newton et al., 1997). In resting cells, NF-κB is sequestered in the cytoplasm by association with its inhibitory protein IκB. In response to various cellular stress conditions, such as infection by microbial or viral pathogens, IκB kinase is activated and phosphorylates IκB. Subsequently, IκB is ubiquitinated and degraded by the proteasome, releasing NF-κB to migrate into the nucleus and activate gene expression (Baldwin, 1996; Hatada et al., 2000). Both aspirin and SA block the activation of NF-κB by inhibiting IκB kinase, preventing the degradation of IκB and retaining NF-κB in the cytosol (Kopp and Ghosh, 1994; Yin et al., 1998). Interestingly, IκB shares structural similarity with NPR1 in plants (Cao et al., 1997; Ryals et al., 1997). In addition to the ankyrin-repeat domain, the phosphorylated Ser residues important for IκB function also are conserved in the NPR1 protein (Ryals et al., 1997).
Because of the intriguing analogies between the actions of SA/aspirin, prostaglandin, and IκB in animals, and SA, JA, and NPR1 in plants, we investigated whether NPR1 plays a role in the SA-mediated negative regulation of JA signaling in Arabidopsis. In contrast
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to IκB in animal cells, which functions in the cytosol, NPR1 was reported previously to function in the nucleus when acting as a positive regulator of SA-dependent, defense-related gene expression (Kinkema et al., 2000; Subramaniam et al., 2001). Here, we report a novel function of NPR1 in the cytosol and provide evidence that cytosolic NPR1 plays a crucial role in cross-communication between SA- and JA-dependent plant defense responses.
R E S U LT S
A n t a g o n i s t i c e f f e c t o f p a t h o g e n - i n d u c e d S A o n J A s i g n a l i n g
Previously, pharmacological experiments have shown that SA and its derivative aspirin exert an antagonistic effect on JA biosynthesis and JA-responsive gene expression in plants (Doherty et al., 1988; Peña-Cortés et al., 1993). To investigate whether endogenously synthesized SA also functions as a negative regulator of JA signaling during pathogen infection, we analyzed the production of JA and the expression of JA-responsive genes in Arabidopsis wild-type Columbia (Col-0) and transgenic NahG plants after infection with the bacterial speck-inducing pathogen Pseudomonas syringae pv tomato DC3000. Infection of wild-type Col-0 plants with Pseudomonas DC3000 resulted in a strong increase in both free and conjugated SA levels, whereas SA levels in SA hydroxylase-expressing NahG plants remained unchanged (Figure 2.1A). The expression pattern of the SA-inducible PR-1 gene correlated with the SA levels in infected wild-type and NahG plants (Figure 2.1C). In Col-0 plants, JA levels increased slightly in response to pathogen infection. However, in NahG plants, JA accumulated to 25-fold higher levels (Figure 2.1B), suggesting that in wild-type plants JA formation was suppressed by endogenously accumulating SA.
To investigate the effect of pathogen-induced SA on JA-responsive gene expression, we analyzed the expression of three well-characterized Arabidopsis genes involved in various steps of the JA signaling pathway: LOX2 (LIPOXYGENASE2), which encoded LOX2, a key enzyme in the octadecanoid pathway leading to JA biosynthesis (Bell et al., 1995); VSP, which encodes a vegetative storage protein (Berger et al., 1995); and PDF1.2, which encodes a plant defensin with antimicrobial properties (Penninckx et al., 1996). In wild-type plants, LOX2, VSP, and PDF1.2 showed moderate increases in expression upon pathogen infection (Figure 2.1C). However, in NahG plants, mRNAs of the three JA-responsive genes accumulated to much higher levels. These results indicate that in wild-type plants, pathogen-induced SA accumulation is associated with the suppression of JA-responsive gene expression.
I n h i b i t i o n o f L O X 2 i s s u f f i c i e n t t o s u p p r e s s
p a t h o g e n - i n d u c e d J A p r o d u c t i o n
LOX2 is a key enzyme in the octadecanoid pathway leading to formation of JA. In transgenic Arabidopsis S-12 plants, which have severely reduced levels of LOX2 as a result
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of cosuppression of the LOX2 gene, the ability to accumulate JA in response to wounding is completely blocked (Bell et al., 1995). To investigate whether the SA-mediated suppression of LOX2, as observed during pathogen infection (Figure 2.1C), could explain the inhibitory effect of SA on JA formation (Figure 2.1B), JA production was analyzed in infected wild-type Col-0 and transgenic S-12 plants. As shown in Figure 2.1C, pressure infiltration of wild-type leaves with Pseudomonas DC3000 resulted in increased accumulation of LOX2 transcripts, whereas in S-12 plants, the activation of this gene was completely blocked (data not shown). Furthermore, JA levels increased significantly in wild-type plants after inoculation with virulent Pseudomonas DC3000, but in LOX2-deprived S-12 plants, pathogen-induced accumulation of JA was almost abolished (Figure 2.2). Note that leaves were inoculated by pressure infiltration instead of dip inoculation, leading to a synchronous infection of virtually all cells and greater JA accumulation in wild-type leaves than that observed in Figure 2.1B.
Compared with inoculation with virulent Pseudomonas DC3000, pressure infiltration of wild-type leaves with avirulent Pseudomonas DC3000/avrRpt2, carrying the avirulence gene avrRpt2 (Kunkel et al., 1993), led to a hypersensitive reaction and fourfold higher JA levels. However, similarly treated S-12 plants showed no significant increase in JA levels. These results demonstrate that LOX2 is required for the pathogen-induced production of JA and that there is a direct correlation between the level of LOX2 gene expression and JA production. During pathogen infection of wild-type plants, endogenously accumulating SA has an inhibitory effect on LOX2 gene expression and JA formation. Therefore, we postulate that the inhibitory effect of SA on JA biosynthesis during infection is regulated
Figure 2.1. Enhanced JA accumulation and JA-responsive gene expression in Pseudomonas DC3000-infected Arabidopsis NahG plants.(A) Endogenous levels of free and conjugated SA in wild-type Col-0 (closed squares) and SA-degrading NahG (open circles) plants after inoculation with the bacterial pathogen Pseudomonas DC3000. Error bars represent SE (n = 5).(B) JA levels in Pseudomonas DC3000-infected Col-0 (closed squares) and NahG (open circles) plants. The experiment was performed three times with similar results.(C) RNA gel blot analysis of SA-responsive (PR-1) and JA-responsive (LOX2, VSP, and PDF1.2) genes during pathogen infection. Plants were inoculated with virulent Pseudomonas DC3000 by dipping the leaves in a bacterial suspension containing 2.5 × 107 colony-forming units/mL. At different days after inoculation (dpi), leaves were harvested for SA, JA, and RNA extraction. To check for equal loading, RNA blots were stripped and hybridized with a gene-specific probe for β-TUBULIN (TUB). Transcript levels of the constitutively expressed TUB gene decreased during the course of the infection process as a result of progressing cell death. FW, fresh weight.
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at least partly at the transcriptional level, although post-translational effects of SA on JA formation cannot be excluded completely.
N P R 1 c o n t r o l s t h e s u p p r e s s i o n o f J A s i g n a l i n g
NPR1 is an important transducer of the SA signal in disease resistance. To investigate a possible role of NPR1 in the suppression of JA-responsive gene expression by SA, mutant Arabidopsis npr1-1 plants were tested. Like NahG plants, npr1-1 plants showed enhanced JA-responsive gene expression and increased levels of JA upon infection with Pseudomonas DC3000 (data not shown), suggesting that in wild-type plants NPR1 is involved in the SA-mediated suppression of JA signaling. To investigate the role of NPR1 in cross-talk in more detail, we followed a pharmacological approach. In wild-type Col-0 plants, exogenous application of SA activated PR-1, whereas treatment with methyl jasmonate (MeJA) resulted in the accumulation of LOX2, VSP, and PDF1.2 mRNA (Figure 2.3). Upon combined treatment with SA and MeJA, MeJA had no effect on SA-induced PR-1 transcript levels. By contrast, both background and MeJA-induced transcript levels of the JA-responsive genes were strongly suppressed by SA, confirming the negative effect of
Figure 2.2. Effect of cosuppression of LOX2 on pathogen-induced JA production in Arabidopsis S-12 plants.JA levels in wild-type Col-0 and LOX2-deprived S-12 plants at 2 days after inoculation with virulent Pseudomonas DC3000 or avirulent Pseudomonas DC3000/avrRpt2. Plants were inoculated by pressure-infiltrating the leaves with a bacterial suspension containing 107 colony-forming units/mL. FW, fresh weight; n.i., not inoculated.
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Figure 2.3. Effect of the npr1 mutation on the SA-mediated suppression of JA-responsive gene expression.RNA gel blot analysis of SA-responsive (PR-1) and JA-responsive (LOX2, VSP, and PDF1.2) genes after exogenous application of MeJA, SA, or a combination of both in Arabidopsis wild-type Col-0 and mutant npr1 plants. Five-week-old plants were induced by dipping the leaves in a 0.015% (v/v) Silwet L-77 solution containing 1 mM SA, 0.1 mM MeJA, or a combination of both. Two days later, leaves were harvested for RNA extraction. Equal loading of RNA samples was checked using a probe for the constitutively expressed β-TUBULIN (TUB) gene.
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SA on JA-responsive gene expression. Mutant npr1-1 plants, which are compromised in their ability to express PR-1 in response to SA, showed levels of MeJA-induced LOX2, VSP, and PDF1.2 gene expression that were similar to those observed in wild-type plants. However, in the combined treatment, SA had almost no inhibitory effect on background and MeJA-induced expression of the three JA-responsive genes (Figure 2.3). Two additional npr1 mutants, npr1-2 and npr1-3, each with a mutation in different domains in the NPR1 protein (Cao et al., 1997), showed similar expression patterns (data not shown), confirming that NPR1 is required for the SA-mediated suppression of JA-responsive gene expression.
T h e N P R 1 i n t e r a c t o r T G A 2 b i n d s t o t h e
T G A C G m o t i f i n t h e P D F 1 . 2 p r o m o t e r
NPR1 interacts with members of the TGA/OBF family of bZIP transcription factors, which have been shown to play both positive and negative regulatory roles in plant defense (Niggeweg et al., 2000; Pontier et al., 2001; Fan and Dong, 2002). TGA transcription factors specifically bind to the TGACG motif in target promoters, thereby regulating gene transcription (Zhang et al., 1999; Després et al., 2000; Zhou et al., 2000). The TGACG motif was shown by linker-scanning mutagenesis to be essential for the SA-induced expression of the Arabidopsis PR-1 gene (Lebel et al., 1998), but it has been implicated in JA-responsive gene expression as well (Xiang et al., 1996; Rouster et al., 1997). Interestingly, the JA-responsive genes LOX2, VSP, and PDF1.2 all contain one or more TGACG motifs in their promoters. Therefore, we investigated the possible role of this motif in cross-talk between SA and JA signaling.
The promoter of the PDF1.2 gene contains a single TGACG sequence at positions –445 to –441 relative to the predicted translational start of the PDF1.2 gene product (Manners et al., 1998). To determine whether TGA transcription factors can bind to this motif, we performed a mobility shift assay with partially purified TGA2 transcription factor protein (Zhang et al., 1999), and TGACG-containing oligonucleotide probes derived from the PR-1 and PDF1.2 promoters (Figure 2.4A). The as-1 element from the PR-1 promoter contains two inverted TGACG sequences (Lebel et al., 1998). As demonstrated previously (Zhang et al., 1999), TGA2 caused a mobility shift for the as-1 oligonucleotide probe (Figure 2.4B, lane 2). A similar mobility shift was observed for the PDF1.2 probe (Figure 2.4B, lane 4). To examine the specificity of the binding of TGA2 to the PDF1.2 probe, a competition experiment was performed using an excess of unlabeled PDF1.2 oligonucleotide probe. Increasing the amount of unlabeled oligonucleotides in the reaction reduced the binding of TGA2 to the labeled probe (Figure 2.4B, lanes 5 to 8). By contrast, the addition of an unlabeled oligonucleotide PDF1.2 probe with point mutations in the TGACG binding motif (mpdf; Figure 2.4A) barely affected the binding of TGA2 to the labeled PDF1.2 probe (Figure 2.4B, lanes 9 to 13), indicating that the binding of TGA2 to the TGACG motif in the PDF1.2 promoter is specific.
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T h e T G A C G m o t i f i s n o t r e q u i r e d f o r N P R 1 - d e p e n d e n t c r o s s - t a l k
To investigate the role of the TGACG motif in the PDF1.2 promoter in the negative regulation of JA-responsive gene expression by SA in planta, we analyzed transgenic Arabidopsis plants containing a series of PDF1.2 promoter deletions fused to the uidA reporter gene in the wild-type background (Brown et al., 2000). Seedlings of six transgenic lines with varying 5’ deletions were selected (Figure 2.5A) and treated with SA, MeJA, or a combination of SA and MeJA. Consistent with previous findings (Brown et al., 2000), lines P1 to P5 showed induced β-glucuronidase (GUS) activity after treatment with MeJA, whereas line P6 showed no GUS activity at all (Figure 2.5B). MeJA-induced GUS expression was inhibited strongly when SA was included in the medium as well as in lines P4 and P5, which lack the TGACG motif. SA and MeJA had no effect on GUS activity in the constitutive GUS expressor PG15, indicating that neither of these chemical agents affected the activity of the GUS enzyme. Together, these results demonstrate that the TGACG motif in the promoter of PDF1.2 is not essential for SA-mediated inhibition of JA-responsive gene expression.
Figure 2.4. In vitro binding of TGA2 to the TGACG motif in the promoter of the Arabidopsis PDF1.2 gene.(A) Oligonucleotides used in the gel mobility shift assay. The as-1 probe contains two inverted TGACG motifs and represents the SA-responsive as-1 element in the promoter of the Arabidopsis PR-1 gene. The pdf probe resembles the wild-type sequence surrounding the TGACG motif in the JA-responsive PDF1.2 gene. The mpdf probe is similar to the pdf probe but contains three point mutations in the TGACG motif.(B) Gel mobility shift assay to test the binding of TGA2 to the TGACG motif in the PDF1.2 promoter. Binding reactions contained 7 × 104 cpm of 32P-labeled probe incubated with 1 µg of either a control protein preparation (lanes 1, 3 and 14) or partly purified TGA2 (lanes 2, 4 to 13, and 15). The specificity of the binding of TGA2 to the TGACG motif in the PDF1.2 promoter was tested by the addition of 100×, 50×, 10×, 1×, or 0.5× molar excess amounts of unlabeled pdf (lanes 5 to 8) or mpdf (lanes 9 to 13) competitor probes. The double asterisks indicate specific binding of TGA2 to the oligonucleotide probe, and the single asterisk indicates specific TGA2 binding and dimerization as a result of the presence of two TGACG motifs in the oligonucleotide probe. FP, free probe.
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C r o s s - t a l k i s m o d u l a t e d b y c y t o s o l i c N P R 1
NPR1 is translocated to the nucleus in response to SA, a process that was shown to be essential for SA-induced PR-1 gene expression (Kinkema et al., 2000). To determine whether the nuclear localization of NPR1 is similarly required for the SA-mediated suppression of JA-responsive gene expression, we used mutant npr1 plants engineered to constitutively express a fusion protein of NPR1 and the hormone-binding domain (HBD) of the rat glucocorticoid receptor. The nucleocytoplasmic localization of this fusion protein
Figure 2.5. The TGACG motif is not required for SA-mediated suppression of the MeJA-induced activation of the Arabidopsis PDF1.2 promoter.(A) Scheme of the 5’ deletions of the PDF1.2 promoter fused to the uidA gene. Red bars represent the TGACG motif located at positions –445 to –441 upstream of the predicted translational start of the uidA reporter gene.(B) Histochemical staining of GUS activity in seedlings of transgenic Arabidopsis lines PG15, which constitutively expresses the uidA gene, and P1 to P6, which contain 5’ deletions of the Arabidopsis PDF1.2 promoter fused to the uidA reporter gene. Twelve-day-old seedlings grown on MS medium were transferred to fresh medium containing 0.5 mM SA, 0.02 mM MeJA, or a combination of both and stained for GUS activity 2 days later.
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can be controlled by the steroid hormone dexamethasone (DEX) (Aoyama and Chua, 1997; Kinkema et al., 2000). In the absence of DEX, the NPR1-HBD fusion protein was retained in the cytosol through an association with the heat-shock protein HSP90. In cells treated with DEX, HSP90 dissociated, allowing the NPR1-HBD fusion protein to translocate into the nucleus. As a control, we used mutant npr1 plants constitutively expressing the wild-type NPR1 gene under the control of the 35S promoter of Cauliflower mosaic virus (35S::NPR1).
Figure 2.6 shows that overexpression of NPR1 in mutant npr1 plants (35S::NPR1) restored the SA-induced activation of PR-1 and the SA-mediated suppression of MeJA-induced PDF1.2 gene expression, both of which were blocked in mutant npr1 plants. In the absence of SA, neither PR-1 nor MeJA-induced PDF1.2 gene expression was affected in NPR1-overexpressing plants, confirming the notion that NPR1 needs to be activated by SA (Cao et al., 1998). Treatment of 35S::NPR1 plants with DEX did not induce PR-1 or PDF1.2 gene expression, nor did it affect the expression of these genes in response to SA or MeJA, indicating that DEX did not affect the NPR1 protein or SA- and JA-responsive gene expression. In 35S::NPR1-HBD plants, the SA-induced expression of PR-1 was restored only when the plants were treated with DEX, confirming previous findings that SA-induced PR-1 expression requires the nuclear localization of NPR1 (Kinkema et al., 2000). Interestingly, treatment of 35S::NPR1-HBD plants with both SA and MeJA suppressed the MeJA-induced expression of PDF1.2, not only in the presence of DEX but also in its absence, when NPR1 was retained in the cytosol. This finding indicates that the nuclear localization of NPR1 is not required to suppress the MeJA-induced expression of PDF1.2 by SA.
D I S C U S S I O N
The defense response of plants under attack by microbial pathogens or herbivorous insects is regulated by a network of signal transduction pathways. Cross-communication between defense signaling pathways provides the plant with an elaborate regulatory potential that leads to the activation of the most suitable defense against the invader encountered. In some cases, different defense signal transduction pathways cooperate and enhance resistance against pathogen attack (Van Wees et al., 2000). In other cases, antagonism between pathways allows the defense response to be controlled in a focused manner. For instance, plants that are infected by SAR-inducing pathogens have been shown to suppress JA-dependent defenses against certain herbivorous insects or necrotrophic pathogens (Felton and Korth, 2000; Pieterse et al., 2001), thereby prioritizing SA-dependent defense responses over JA-dependent responses. We report evidence indicating that SA produced during pathogen infection plays an important role in the suppression of both JA biosynthesis and JA-responsive gene expression, which are involved in the reaction of plants to wounding and insect herbivory. Compared with wild-type plants, transgenic NahG plants showed enhanced expression of
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LOX2 and accordingly synthesized 25-fold higher levels of JA during pathogen infection (Figures 2.1B and 2.1C). Moreover, the expression of the JA-responsive genes VSP and PDF1.2 was enhanced strongly in NahG plants, suggesting that in wild-type plants JA signaling is inhibited by SA that accumulates during pathogen infection.
The LOX2 gene, which encodes LOX2 (which is involved in the octadecanoid pathway), is autoregulated by JA and thus controls a feed-forward loop in JA biosynthesis (Bell et al., 1995). Cosuppression of LOX2 gene expression in transgenic S-12 plants appeared to be effective in blocking JA biosynthesis during pathogen infection (Figure 2.2). Therefore, the inhibition of this JA biosynthetic gene by SA produced during pathogen infection may result in a strong inhibition of JA formation. Other genes involved in JA biosynthesis, such as AOS, which encodes ALLENE OXIDE SYNTHASE, have been shown to be regulated by JA as well (Laudert and Weiler, 1998). Thus, the SA-mediated inhibition of JA formation during pathogen attack might be the result of a coordinated suppression of JA-responsive genes that encode enzymes of the octadecanoid pathway.
NPR1 has been demonstrated to be an important transducer of the SA signal in the SA-mediated activation of PR gene expression and broad-spectrum resistance (Cao et al., 1994; Delaney et al., 1995; Shah et al., 1997). Our study revealed NPR1 as a key regulatory factor in the cross-communication between SA and JA signaling. SA-mediated suppression of the JA-responsive genes LOX2, VSP, and PDF1.2, which was observed in wild-type Col-0 plants, was abolished in mutant npr1 plants (Figures 2.3 and 2.6), indicating that NPR1 is essential for the inhibition of JA-responsive gene expression by SA. How does SA-activated NPR1 function as a negative regulator of JA-responsive genes? Previously, NPR1 was found to interact with members of the TGA subclass of the bZIP transcription factor family (Zhang et al., 1999; Després et al., 2000; Zhou et al., 2000). TGA factors specifically bind to TGACG motifs and have been shown to play both positive and negative regulatory roles in plant defense (Xiang et al., 1996; Rouster et al., 1997; Lebel et al., 1998; Niggeweg et al., 2000; Pontier et al., 2001; Fan and Dong, 2002). All JA-responsive genes tested in this study contain one or more TGACG motifs in their promoters. Therefore, we hypothesized that NPR1-TGA interactions might play a role in the SA-mediated suppression of JA-responsive
Figure 2.6. SA-mediated suppression of MeJA-induced PDF1.2 expression through a function of NPR1 in the cytosol.RNA gel blot analysis of the SA-responsive PR-1 gene and the MeJA-inducible PDF1.2 gene in wild-type Col-0, mutant npr1, and the NPR1-overexpressing transformants 35S::NPR1 (in npr1) and 35S::NPR1-HBD (in npr1). Seedlings were grown for 12 days on MS medium with or without DEX (5 µM). Subsequently, the seedlings were transferred to fresh medium with (+) or without (-) 0.5 mM SA and 0.02 mM MeJA. Two days after induction, the seedlings were harvested for RNA blot analysis. Equal loading of RNA samples was checked by staining rRNA bands with ethidium bromide. Mn; B-Koornneef; B 07-943; 02-06.ai
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gene expression. Experiments indicated that the strong NPR1 interactor TGA2 (Zhang et al., 1999) binds specifically to the TGACG motif in the JA-responsive PDF1.2 promoter in vitro (Figure 2.4). However, promoter-deletion analysis demonstrated that SA suppressed MeJA-induced expression in all JA-responsive transgenic plants, including those that lack the TGACG motif (P4 and P5; Figure 2.5). These results suggest that the TGACG motif is not essential for the SA-mediated inhibition of JA-responsive gene expression.
In animal cells, it has been demonstrated that SA exerts an inhibitory effect on IκB kinase, thereby preventing IκB from being phosphorylated and subsequently degraded by the proteasome (Kopp and Ghosh, 1994; Yin et al., 1998). As a result, IκB remains associated with the transcription factor NF-κB, retaining NF-κB in the cytosol and preventing it from activating target genes, such as the NF-κB-dependent, pro-inflammatory prostaglandin biosynthesis gene CYCLOOXYGENASE2 (Newton et al., 1997; Gallois et al., 1998). Thus, in animal cells, SA is able to suppress the formation of prostaglandin, the JA counterpart, by affecting the activity of IκB in the cytosol. Because of the structural similarity between IκB and NPR1 (Cao et al., 1997; Ryals et al., 1997), we investigated whether the NPR1-dependent inhibitory effect of SA on JA-responsive gene expression and, consequently, JA production, also functions through a role of NPR1 in the cytosol. Using a DEX-inducible system to control the nucleocytoplasmic localization of NPR1, we demonstrated that nuclear localization of NPR1 is not required for cross-talk between SA and JA signaling (Figure 2.6). By inference, SA-activated NPR1 must exert its negative effect on JA-responsive gene expression through an unknown function in the cytosol.
How does cytosolic NPR1 control the SA-mediated suppression of JA-responsive gene expression? In the absence of SA, cytosolic NPR1 might be involved in the control of JA-responsive gene expression, either by inhibiting negative regulators of JA-responsive gene expression or by facilitating the delivery of positive regulators of JA-responsive genes to the nucleus. However, in this scenario, npr1 null mutants and NPR1 overexpressors should show an altered JA-responsive phenotype in the absence of SA. This was clearly not the case (data not shown), which makes this possibility unlikely. A more plausible explanation for the cytosolic role of NPR1 is presented in the model shown in Figure 2.7. In this model, NPR1 is translocated to the nucleus upon activation by SA, where it facilitates the activation of SA-responsive PR genes. In the cytosol, the remaining SA-activated NPR1 pool is involved in suppression of JA-responsive gene expression, either by facilitating the delivery of negative regulators of JA-responsive genes to the nucleus or by inhibiting positive regulators of JA-responsive gene expression. However, alternative scenarios, such as an effect of SA-activated cytosolic NPR1 on the activity of JA-metabolizing enzymes, cannot be excluded. It is tempting to speculate that, analogous to the effect of SA and aspirin on IκB kinase, (de)phosphorylation of NPR1 plays a role in the process that leads to the SA-mediated activation of NPR1, because the phosphorylated Ser residues important in IκB function are conserved in NPR1 (Ryals et al., 1997).
In conclusion, our results clearly demonstrate the importance of NPR1 in cross-talk between the SA- and JA-dependent signaling pathways in plant defense and reveal a
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novel function of NPR1 in the cytosol. The striking parallels with processes involved in the animal innate immune response suggest that defense signaling pathways in plants and animals are at least partly conserved.
M AT E R I A L S A N D M E T H O D S
P l a n t g r o w t h a n d p a t h o g e n i n f e c t i o n
To grow plants for pathogen infection, seeds of wild-type Arabidopsis thaliana (accession Col-0), transgenic NahG plants harboring the bacterial nahG gene (Gaffney et al., 1993), mutant npr1-1 plants (Cao et al., 1994), and LOX2-cosuppressed S-12 plants harboring the LOX2 cDNA under the control of the 35S promoter of Cauliflower mosaic virus (Bell et al., 1995) were sown in quartz sand. Two-week-old seedlings were transferred to 60-mL pots containing a mixture of sand and potting soil that had been autoclaved twice for 20 min with a 24-h interval. Plants were cultivated in a growth chamber with an 8-h day (200 µE/m2/s at 24°C)/ 16-h night (20°C) cycle at 70% relative humidity.
The virulent bacterial leaf pathogen Pseudomonas syringae pv tomato DC3000, which causes bacterial speck disease, and the avirulent strain Pseudomonas DC3000/avrRpt2 with the plasmid pV288 carrying the avirulence gene avrRpt2 (Kunkel et al., 1993), were grown overnight at 28°C in liquid King’s medium B as described previously (Pieterse et al., 1998). Bacterial cells were collected by centrifugation and resuspended in 10 mM MgSO
4 to a
final density of 107 or 2.5 × 107 colony-forming units/mL. For leaf dip inoculation of
Figure 2.7. Proposed model for cytosolic NPR1 as a modulator of cross-talk between SA- and JA-dependent plant defense responses.In wild-type Col-0 plants, SA accumulates after pathogen infection, resulting in the activation of NPR1 (asterisk). Activated NPR1 then is localized to the nucleus, where it interacts with TGA transcription factors, ultimately leading to the activation of SA-responsive PR genes. In the cytosol, activated NPR1 negatively regulates JA-responsive gene expression, possibly by inhibiting positive regulators of JA-responsive genes or by facilitating the delivery of negative regulators of JA-responsive genes to the nucleus. The suppression of JA-responsive genes that encode enzymes from the octadecanoid pathway, such as LOX2, ultimately results in the inhibition of JA formation.
SA JA
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4 0 • NPR1 modulates c ros s - ta lk between SA and JA s igna l ing pathways
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plants, the surfactant Silwet L-77 (Van Meeuwen Chemicals BV, Weesp, The Netherlands) was added to a final concentration of 0.015% (v/v).
Infection of Col-0, NahG and npr1-1 plants with Pseudomonas DC3000 was carried out as described previously (Pieterse et al., 1998). One day before pathogen infection, the plants were placed at 100% relative humidity. Leaves of 5-week-old plants were dipped in a suspension of Pseudomonas DC3000 containing 2.5 × 107 colony-forming units/mL. At different times after inoculation, all of the rosette leaves of 25 plants for each genotype and time point were harvested for RNA extraction and to determine salicylic acid (SA) and jasmonic acid (JA) levels. For the analysis of pathogen-induced JA levels in LOX2-cosuppressed S-12 plants (Bell et al., 1995), a suspension of virulent Pseudomonas DC3000 or avirulent Pseudomonas DC3000/avrRpt2 at a density of 107 colony-forming units/mL was pressure-infiltrated into the leaves as described previously (Pieterse et al., 1998). As a control, Col-0 plants were inoculated similarly.
S A a n d J A d e t e r m i n a t i o n
Leaves were frozen in liquid nitrogen and pulverized with mortar and pestle. For each SA extraction, 0.5 g of ground leaf tissue was transferred to a 1.5-mL microfuge tube, and 100 µL of the internal standard ortho-anisic acid (1 µg/mL) and 0.5 mL of 70% ethanol were added. Subsequently, extraction and quantification of free and conjugated SA were performed as described previously (Meuwly and Métraux, 1993). For each JA extraction, a sample of 1 g was taken from the frozen leaf material, which consisted of at least 20 plants that received the same treatment. Subsequently, the sample was transferred to a 50-mL centrifuge tube. To the frozen samples were added 100 ng of the internal standard 9,10-dihydrojasmonic acid, 10 mL of saturated NaCl solution, 0.5 mL of 1 M citric acid, and 25 mL of diethylether containing 0.005% (w/v) butylated hydroxytoluene as antioxidant. Subsequently, extraction and gas chromatography-mass spectrometry quantification of JA were performed as described (Mueller and Brodschelm, 1994).
R N A e x t r a c t i o n a n d R N A g e l b l o t a n a l y s i s
Extraction and electrophoretic separation of RNA, preparation of RNA gel blots, and hybridization of the blots with gene-specific probes for PR-1, LOX2, VSP, PDF1.2, and β-TUBULIN (TUB) were performed as described previously (Cao et al., 1994; Pieterse et al., 1998). The AGI numbers for the genes studied are At2g14610 (PR-1), At3g45140 (LOX2), At5g24770 (VSP), At5g44420 (PDF1.2), and At5g44340 (TUB).
C h e m i c a l i n d u c t i o n
The effect of exogenously applied SA on methyl jasmonate (MeJA)-induced gene expression was studied in wild-type Col-0 plants, mutant npr1-1, npr1-2, and npr1-3 plants (Cao et al., 1994; Cao et al., 1997), and 35S::NPR1 and 35S::NPR1-HBD plants overexpressing NPR1 and NPR1-HBD, respectively, in the mutant npr1 background (Kinkema et al., 2000). Plants were grown in soil as described above or on plates containing Murashige
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and Skoog (1962) (MS) medium, pH 5.7, supplemented with 2% (w/v) sucrose and 0.8% (w/v) plant agar. Chemical induction of soil-grown plants was performed by dipping the leaves of 5-week-old plants in a solution containing 0.015% (v/v) Silwet L-77 and either 1 mM SA (Mallinckrodt Baker, Deventer, The Netherlands), 0.1 mM MeJA (Serva, Brunschwig Chemie, Amsterdam, The Netherlands), or a combination of these chemicals. Control plants were treated with 0.015% Silwet L-77 only. One day before application of the chemicals, the plants were placed at 100% relative humidity. Chemical induction of plants grown on MS medium was performed by transferring 12-day-old seedlings to fresh MS medium supplemented with 0.5 mM SA, 0.02 mM MeJA, or both chemicals. MeJA was added to the solutions from a 1000-fold concentrated stock in 96% ethanol. To the solutions without MeJA, a similar volume of 96% ethanol was added. To control the nucleocytoplasmic localization of NPR1 in 35S::NPR1-HBD plants, 5 µM dexamethasone (Sigma) was included in the growth medium (before and after induction with SA and/or MeJA). After induction treatment, plants were cultured for 2 days under climate chamber conditions as described above, after which leaf tissue was harvested for RNA extraction.
A n a l y s i s o f t h e T G A C G m o t i f i n t h e P D F 1 . 2 p r o m o t e r
The gel mobility shift assay was performed with partially purified TGA2 protein as described previously (Zhang et al., 1999). The wild-type and mutant oligonucleotide PDF1.2 probes used were designed according to the sequence surrounding the TGACG motif in the JA-responsive promoter of the Arabidopsis PDF1.2 gene (Manners et al., 1998).
The construction of a series of transgenic Arabidopsis lines (P1 to P6) containing translational fusions of 5’ deletions of the PDF1.2 promoter to the uidA reporter gene was described previously (Brown et al., 2000). Surface-sterilized seeds of homozygous progeny from five independent transformants per line as well as the constitutive uidA-expressing line PG15 were allowed to germinate on MS medium supplemented with 1% (w/v) sucrose and 0.6% (w/v) plant agar, pH 5.7. After 12 days, seedlings were transferred to fresh MS medium containing 0.5 mM SA, 0.02 mM MeJA, or a combination of both, as described above. After 2 days on induction medium, β-glucuronidase activity was assessed by transferring the seedlings to β-glucuronidase staining solution (1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, 100 mM NaPi buffer, pH 7.0, 10 mM EDTA, 0.1% [v/v] Triton X-100, 1 mM potassium ferrocyanide, and 1 mM potassium ferricyanide). After overnight incubation at 37°C, the seedlings were destained by repeated washes in 70% ethanol and evaluated for staining intensity.
Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes.
A C K N O W L E D G M E N T SWe thank Stefanie Parchmann and Ruth Imbusch for JA measurements, Weihua Fan and Meenu Kesarwani for technical assistance and valuable discussions, and Peter Bakker and
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4 2 • NPR1 modulates c ros s - ta lk between SA and JA s igna l ing pathways
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Bas Rutjens for critically reading the manuscript. This research was supported in part by grants from the Schuurman Schimmel-Van Outeren Foundation, the Dr. Hendrik Muller’s Vaderlandsch Fonds Foundation, and the Karel Frederik Foundation to S.H.S., by Swiss National Science Foundation Grant 55662.98 to J.-P.M., and by a U.S. Department of Agriculture grant to X.D.
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C H A P T E R 3
Kinetics of cross-talk in
plant defense signaling
Annemart Koornneef, Antonio Leon-Reyes, Tita Ritsema, Adriaan Verhage, Floor C. Den Otter, L.C. Van Loon, and Corné M.J. Pieterse
Graduate School Experimental Plant Sciences, Plant-Microbe Interactions,
Institute of Environmental Biology, Faculty of Science, Utrecht University,
P.O. Box 800.56, 3508 TB Utrecht, The Netherlands
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Kinetics of cross-talk in
plant defense signaling
A B S T R A C T
Cross-talk between salicylic acid (SA) and jasmonic acid (JA) signaling pathways plays an important role in the regulation and fine-tuning of induced defenses that are activated upon pathogen or insect attack. Pharmacological experiments revealed that transcription of JA-responsive marker genes, such as PDF1.2 and VSP2, is highly sensitive to suppression by SA. This antagonistic effect of SA on JA signaling was also observed when the JA pathway was biologically activated by the necrotrophic pathogens Alternaria brassicicola and Botrytis cinerea, or by the insect herbivores Frankliniella occidentalis and Pieris rapae, and when the SA pathway was triggered by the biotrophic pathogen Hyaloperonospora parasitica. Furthermore, all 18 Arabidopsis thaliana accessions tested displayed SA/JA cross-talk, highlighting the potential significance of this phenomenon in induced plant defenses in nature. During plant-attacker interactions, the kinetics of SA and JA signaling are highly dynamic. Mimicking this dynamic response by applying SA and methyl jasmonate (MeJA) at different concentrations and time intervals revealed that PDF1.2 transcription is readily suppressed when the SA response was activated at or after the onset of the JA response, and that this SA/JA antagonism is long-lasting. However, when SA was applied more than 30 h prior to the onset of the JA response, the suppressive effect of SA was completely absent. The window of opportunity of SA to suppress MeJA-induced PDF1.2 transcription coincided with a transient increase in glutathione levels. The glutathione biosynthesis inhibitor L-buthionine-sulfoximine (BSO) strongly reduced SA/JA cross-talk, suggesting that SA-mediated redox modulation plays an important role in cross-talk between SA and JA signaling pathways.
I N T R O D U C T I O N
In nature, plants interact with a wide range of microbial pathogens and herbivorous insects. During the evolutionary arms race between plants and their attackers, primary and secondary
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immune responses evolved to recognize common or highly specialized features of microbial pathogens (Chisholm et al., 2006; Jones and Dangl, 2006), resulting in sophisticated mechanisms of defense. Although the arms race between plants and herbivorous insects has been intensively debated (Musser et al., 2002; Schoonhoven et al., 2005), knowledge of the underlying mechanisms is relatively limited. In the past years, various genomics approaches exponentially expanded our understanding of the molecular mechanisms by which plants tailor their defense response to pathogen and insect attack (Glazebrook et al., 2003; Tao et al., 2003; Eulgem et al., 2004; Reymond et al., 2004; De Vos et al., 2005; Kempema et al., 2007). The plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) emerged as key players in the regulation of the signaling networks involved (Howe, 2004; Pozo et al., 2004; Grant and Lamb, 2006; Van Loon et al., 2006; Von Dahl and Baldwin, 2007). Other plant hormones, such as abscisic acid (Mauch-Mani and Mauch, 2005), brassinosteroids (Nakashita et al., 2003), and auxins (Navarro et al., 2006; Wang et al., 2007), have been reported to play a role in the plant immune response as well, but their significance is less well understood. SA-, JA-, and ET-dependent pathways regulate defense responses that are differentially effective against specific types of attackers. Pathogens with a biotrophic lifestyle are generally more sensitive to SA-dependent responses, whereas necrotrophic pathogens and herbivorous insects are commonly deterred by JA/ET-dependent defenses (Thomma et al., 2001; Kessler and Baldwin, 2002; Glazebrook, 2005).
There is ample evidence that SA and JA signaling pathways are mutually antagonistic (Pieterse et al., 2001; Kunkel and Brooks, 2002; Glazebrook et al., 2003; Rojo et al., 2003; Bostock, 2005; Beckers and Spoel, 2006). This pathway cross-talk is thought to provide the plant with a powerful regulatory potential that helps deciding which defensive strategy to follow, depending on the type of attacker encountered (Reymond and Farmer, 1998). Yet, it appears that attackers have also evolved ways to manipulate plants for their own benefit by suppressing induced defenses via modulation of the plant signaling network. A nice example is the response of Arabidopsis to silverleaf whitefly (Bemisia tabaci) nymphs. The nymphs of this phloem-feeding insect sabotage effectual JA-dependent host defenses by activating the antagonistic SA signaling pathway (Zarate et al., 2007). Pathogens suppress host defenses as well, by using virulence factors that antagonize the plant immune response (Nomura et al., 2005). One of these virulence factors is the Pseudomonas syringae phytotoxin coronatine, which functions as a jasmonate analog. During the interaction with susceptible Arabidopsis plants, coronatine suppresses SA-dependent defenses, thereby promoting susceptibility to this pathogen (Zhao et al., 2003; Brooks et al., 2005; Cui et al., 2005; Laurie-Berry et al., 2006).
Several key regulatory proteins involved in SA/JA cross-talk have been identified in Arabidopsis. For instance, the transcription factor WRKY70 was shown to act as an activator of SA-responsive genes and a repressor of JA-inducible genes, thereby functioning as a molecular switch between both pathways (Li et al., 2004). Previously, we demonstrated that the defense regulatory protein NPR1 is required for SA/JA cross-talk (Spoel et al., 2003; Chapter 2). Induction of the SA response, either by pathogen infection or by exogenous
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application of SA, strongly suppressed JA-responsive genes, such as PDF1.2, LOX2, and VSP2. However, in mutant npr1-1 plants, this SA-mediated suppression of JA-responsive gene expression was completely abolished. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression (Kinkema et al., 2000), was not required for the suppression of JA-responsive genes, indicating that SA/JA cross-talk is modulated through a function of NPR1 in the cytosol (Spoel et al., 2003; Chapter 2). Recently, overexpression of the SA-regulated glutaredoxin GRX480 was found to antagonize JA-responsive PDF1.2 transcription (Ndamukong et al., 2007), suggesting a role for redox regulation in SA/JA cross-talk.
While genetic approaches are ideal for identifying key players of pathway cross-talk, they do not provide full insight into the actual functioning of this regulatory mechanism in response to pathogen and insect attack. Previously, we monitored changes in the signal signature and transcriptome of Arabidopsis upon attack by various microbial pathogens and herbivorous insects (De Vos et al., 2005). Clearly, timing, magnitude, and composition of the blend of signals produced play a primary role in orchestrating the induced defense response (De Vos et al., 2005). However, additional layers of regulation, such as pathway cross-talk, are needed to fine-tune the final outcome of the resistance reaction (Thaler et al., 2002; De Vos et al., 2006; Mur et al., 2006). Here, we demonstrate that biological or chemical induction of the SA response strongly suppresses the expression of the JA-responsive genes PDF1.2 and VSP2, such as triggered upon treatment with MeJA or attack by the JA-inducing necrotrophs Alternaria brassicicola and Botrytis cinerea, or the herbivores Frankliniella occidentalis and Pieris rapae. Using a pharmacological approach to dissect the kinetics and mechanisms underlying SA/JA cross-talk, we demonstrate that the SA-mediated antagonistic effect on JA-responsive gene expression is conserved among Arabidopsis accessions and that the kinetics of SA and JA signaling play an important role in the outcome of the SA/JA interaction. Furthermore, we provide evidence that the antagonistic effect of SA on JA-responsive gene transcription is linked to SA-induced changes in glutathione levels, suggesting that SA/JA cross-talk is modulated by redox changes.
R E S U LT S
S A s u p p r e s s e s J A r e s p o n s e s t r i g g e r e d b y n e c r o t r o p h i c
p a t h o g e n s a n d h e r b i v o r o u s i n s e c t s
In Arabidopsis, pharmacological experiments revealed that SA can antagonize the expression of JA-responsive genes, such as PDF1.2 and VSP2 (Spoel et al., 2003; Chapter 2). To investigate the potential significance of this pathway cross-talk in the defense response of plants to multiple attackers, we tested the effect of SA on the JA response as triggered by necrotrophic pathogens and herbivorous insects. To this end, the JA response was biologically activated by inoculating wild-type Col-0 plants with the necrotrophic fungi Alternaria brassicicola or Botrytis cinerea, or by infesting Col-0 plants with cell-content-feeding Western
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flower thrips (Frankliniella occidentalis) or tissue-chewing caterpillars of the small cabbage white (Pieris rapae). Subsequently, non-induced and induced plants were treated with 1 mM SA and leaves were harvested 24 h later to analyze the expression levels of the SA-responsive marker gene PR-1 and the JA-responsive marker gene PDF1.2. Because P. rapae specifically suppresses the expression of PDF1.2 (De Vos, 2006), we used VSP2 as a JA-responsive marker in the Arabidopsis-P. rapae interaction. Figure 3.1A shows that the necrotrophic pathogens and the herbivorous insects activated the JA-responsive marker genes to similar levels as did the chemical agent MeJA. In combination with SA, the JA-responsive genes were consistently suppressed, indicating that exogenously applied SA is able to antagonize the JA response as induced by a broad range of attackers.
To investigate whether biological activation of the SA pathway would similarly antagonize JA signaling, Col-0 plants were inoculated with the SA-inducing biotrophic pathogen Hyaloperonospora parasitica. At 3 days after inoculation, PR-1 transcripts continuously accumulated to high levels (Figure 3.1B), confirming that the SA signaling pathway was activated. Subsequent treatment of H. parasitica-inoculated plants with 0.1 mM MeJA resulted in suppression of MeJA-induced PDF1.2 and VSP2 transcription (Figure 3.1B). When P. rapae larvae were allowed to feed on H. parasitica-infected Col-0 plants, the expression of VSP2 was strongly reduced in comparison to caterpillar-infested plants that were not inoculated with the pathogen (Figure 3.1C). Together, these results indicate that pathogen-induced SA negatively affects JA signaling and that during multitrophic interactions, the SA pathway can be prioritized over the JA pathway.
Figure 3.1. Biological induction of SA and JA signaling pathways results in SA/JA cross-talk.(A) Exogenous application of 1 mM SA suppresses the expression of the JA-responsive marker genes PDF1.2. and VSP2, triggered by MeJA, the necrotrophic pathogens A. brassicicola and B. cinerea, and the insect herbivores F. occidentalis and P. rapae. (B) Infection with the SA-inducing biotrophic pathogen H. parasitica antagonizes MeJA-induced expression of PDF1.2 and VSP2.(C) H. parasitica suppresses P. rapae-induced expression of the JA-responsive gene VSP2. For northern blot analysis, leaf tissue was harvested 24 h after the second treatment. Ribosomal RNA (rRNA) was used to check for equal loading of RNA samples.
Mn; B-Koornneef; B 07-943; 03-01.ai
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Chapter 3 • 4 9
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S A / J A c r o s s - t a l k i s c o n s e r v e d a m o n g A r a b i d o p s i s a c c e s s i o n s
Naturally occurring variation in Arabidopsis accessions can be exploited to study the biological relevance and genetics of specific plant traits, such as resistance to pathogens and pests (Koornneef et al., 2004). To investigate whether Arabidopsis displays natural variation for the SA/JA cross-talk phenomenon, we analyzed the antagonistic effect of SA on MeJA-induced PDF1.2 transcription in 18 Arabidopsis accessions collected from very different geographical origins. All accessions were treated with 1 mM SA, 0.1 mM MeJA, or a combination of both chemicals. One day later, the expression of SA-responsive PR-1 and JA-responsive PDF1.2 was assessed (Figure 3.2). The single treatments with SA or MeJA clearly activated their corresponding marker genes PR-1 and PDF1.2, although the basal PR-1 and PDF1.2 transcript levels varied among the accessions. In the SA/MeJA combination treatments, SA-induced PR-1 expression was not affected by MeJA in the majority of the accessions. Conversely, all accessions displayed a strong SA-mediated downregulation of both MeJA-induced and basal levels of PDF1.2 transcription. These results demonstrate that the SA-mediated antagonism on JA-responsive gene expression is conserved among Arabidopsis accessions, suggesting an important role of this phenomenon for plant survival.
Figure 3.2. SA/JA cross-talk is conserved among Arabidopsis accessions. Northern blot analysis of PR-1 and PDF1.2 gene expression in 18 Arabidopsis accessions after treatment with 1 mM SA, 0.1 mM MeJA, or a combination of both chemicals. Leaf tissue was harvested 24 h after chemical treatment. Equal loading of RNA samples was checked using a probe for 18S rRNA. Signal intensities were quantified using a Phosphor imager. PDF1.2 transcript levels in the single MeJA treatments were set to 100%. Mn; B-Koornneef; B 07-943; 03-02.ai
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S A t r i g g e r s a f a s t a n d l o n g - l a s t i n g a n t a g o n i s t i c
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In response to pathogen or insect attack, Arabidopsis reacts by producing an attacker-specific signal signature (De Vos et al., 2005). The kinetics of the defense signal production play an important role in shaping the final outcome of the induced defense response (Reymond and Farmer, 1998). To investigate the effectiveness of SA/JA cross-talk in view of the dynamic changes in defense signal production, we monitored the time frame during which SA is able to effectively suppress PDF1.2 transcription. Col-0 plants were treated with SA, MeJA, or a combination of both chemicals and the expression of PR-1 and PDF1.2 was assessed at several time points after induction. Figure 3.3 shows that in the single treatments PR-1 and PDF1.2 transcripts were detectable 3 h after chemical application. In the combination treatment, again no effect of MeJA on SA-induced PR-1 was observed. However, SA readily antagonized MeJA-induced transcription of PDF1.2. The suppression of PDF1.2 by SA was clearly visible up to 4 days after chemical treatment, even though by that time SA-induced PR-1 expression had decreased to almost undetectable levels. It can thus be concluded that the antagonistic effect of SA on JA-responsive gene expression is induced rapidly, and lasts up to several days after induction of the SA signal.
P D F 1 . 2 t r a n s c r i p t i o n i s a n t a g o n i z e d b y v e r y l o w d o s e s o f S A
The observation that MeJA-induced PDF1.2 expression is downregulated by SA under conditions where PR-1 transcripts were barely detectable (Figure 3.3) prompted us to investigate the dosage effect of SA on SA/JA cross-talk. SA was applied to Col-0 plants as a foliar drench in concentrations ranging from 1000 to 0.1 µM, either alone or in combination with 0.1 mM MeJA. After 1 day, leaf tissue was harvested and PR-1 and PDF1.2 expression was assessed. SA concentrations below 100 µM had no effect on PR-1
Figure 3.3. SA exerts a fast and long-lasting antagonistic effect on PDF1.2 transcription. Northern blot analysis of PR-1 and PDF1.2 transcript levels in Col-0 plants treated with 1 mM SA, 0.1 mM MeJA, or a combination of both chemicals. Leaf tissue was harvested 1, 3, 6, 12, 24, 72, and 96 h after chemical treatment. To check for equal RNA loading a probe for 18S rRNA was used. Signal intensities were quantified using a Phosphor imager. PDF1.2 transcript levels in the single MeJA treatments were set to 100%.
Mn; B-Koornneef; B 07-943; 03-03.ai
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transcription, but still antagonized MeJA-induced expression of PDF1.2 (Figure 3.4). In fact, MeJA-induced PDF1.2 transcription was suppressed by concentrations of SA as low as 0.1 µM, although the effect was less pronounced than the suppression observed by 1000 µM SA. A higher dose of MeJA (1 mM) could not overrule the suppressive effect of SA on PDF1.2 expression (data not shown). These results highlight the robustness and sensitivity of the antagonistic effect of SA on JA-responsive genes, such as PDF1.2.
L o n g e v i t y o f S A / J A c r o s s - t a l k
To investigate the longevity of the SA-mediated antagonistic effect on MeJA-induced PDF1.2 transcription, SA and MeJA were either applied simultaneously or with an interval of 3 days. Subsequently, leaf tissue was harvested 1 day after application of the last chemical for northern blot analysis of PR-1 and PDF1.2 expression. Simultaneous treatment with SA and MeJA resulted in a typical suppression of MeJA-induced PDF1.2 expression by SA (Figure 3.5A, left panel). When SA was applied 3 days after MeJA, a similar SA-mediated suppression of PDF1.2 was evident (Figure 3.5A, middle panel). Note that in the middle panel in Figure 3.5A MeJA-induced transcript levels of PDF1.2 are lower than in the other two panels, because RNA was isolated 4 days instead of 1 day after the MeJA treatment. However, when SA was applied 3 days prior to the MeJA treatment, the antagonistic effect on PDF1.2 expression could no longer be observed (Figure 3.5A, right panel). These results indicate that SA is capable of suppressing JA-responsive gene expression when it is produced simultaneously with, or after the onset of the JA response. However, when SA is produced prior to activation of the JA pathway, the antagonistic effect of SA on JA signaling is only effective within a certain time frame after induction of the SA signal.
To investigate the window of opportunity of SA to suppress MeJA-induced expression of PDF1.2, we applied SA at several time points before MeJA. In all cases, Col-0 leaf tissue
Figure 3.4. Very low doses of SA antagonize PDF1.2 transcription. Northern blot analysis of PR-1 and PDF1.2 gene expression in Col-0 plants treated with 1000, 100, 10, 1, or 0.1 µM SA, with or without 0.1 mM MeJA. Leaf tissue was harvested 24 h after chemical treatment. Equal loading of RNA samples was checked using a probe for 18S rRNA. Signal intensities were quantified using a Phosphor imager. PDF1.2 transcript levels in the single MeJA treatments were set to 100%.
Mn; B-Koornneef; B 07-943; 03-04.ai
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was harvested 1 day after the MeJA treatment for northern blot analysis of PDF1.2 gene expression. The antagonistic effect of SA on MeJA-induced PDF1.2 expression was evident when SA was applied simultaneously with MeJA, or up to 30 h before the MeJA treatment (Figure 3.5B). However, when the time interval between the SA and MeJA treatments was extended to 48 h, the SA-mediated suppression of MeJA-induced PDF1.2 was no longer observed. It can thus be concluded that the antagonistic effect of SA on JA signaling is transient and that the suppressive effect is lost between 30 and 48 h after induction of the SA signal.
If the antagonistic effect of SA on JA signaling is only apparent during a certain time frame after induction of the SA signal, then constant activation of the SA-dependent signaling pathway should result in continuous downregulation of JA-responsive genes, such as PDF1.2. We tested this hypothesis by comparing PDF1.2 expression in wild-type Col-0 and mutant cpr1-1 plants after application of 20 and 100 µM MeJA. The cpr1-1 mutant has elevated endogenous levels of SA and shows constitutive PR-1 expression (Bowling et al., 1994). Figure 3.5C shows that PDF1.2 expression was induced by both concentrations of MeJA in wild-type Col-0. However, in mutant cpr1-1, the effect of the MeJA treatment on the level of PDF1.2 expression was strongly reduced. These results indicate that a
Figure 3.5. Longevity of SA/JA cross-talk. Northern blot analysis of PDF1.2 gene expression in Col-0 plants treated with 1 mM SA, 0.1 mM MeJA, or a combination of both chemicals. In the combination treatments, SA and MeJA were applied in different orders and with different time intervals. Leaf tissue was harvested (indicated by an arrow) 24 h after application of the last chemical. To check for equal loading, a probe for 18S rRNA was used. (A) The effect of SA on MeJA-induced PDF1.2 transcription when SA was applied simultaneously with (left panel), 3 days after (middle panel), or 3 days before MeJA (right panel). (B) The effect of SA on MeJA-induced PDF1.2 transcription when SA was supplied 0, 7, 30, or 48 h before MeJA. (C) The effect of constitutive expression of the SA response on PDF1.2 transcription. PDF1.2 mRNA levels were determined in Col-0 and cpr1-1 plants 24 h after treatment with 20 or 100 μM MeJA.
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continuous activation of the SA response is associated with a constitutive suppression of JA-responsive gene expression.
S A / J A c r o s s - t a l k c o i n c i d e s w i t h a c e l l u l a r
i n c r e a s e i n g l u t a t h i o n e l e v e l s
Changes in the cellular redox state play a major role in SA signal transduction (Després et al., 2003; Mou et al., 2003). SA-mediated redox changes activate the regulatory protein NPR1 by monomerization of inactive NPR1 oligomers, which results in the induction of SA-responsive genes, such as PR-1 (Mou et al., 2003; Dong, 2004). SA-activated NPR1 is also essential in mediating the antagonism between SA- and JA-dependent signaling (Spoel et al., 2003; Chapter 2). Therefore, we hypothesized that the transient nature of the antagonistic effect of SA on JA signaling might be associated with changes in the cellular redox state. As a marker of the redox potential, we monitored the level of glutathione in Arabidopsis leaves upon application of SA (Figures 3.6A and 3.6B). Glutathione is a low-molecular weight antioxidant that functions as a major determinant of cellular redox homeostasis (Noctor and Foyer, 1998; Schafer and Buettner, 2001; Mullineaux and Rausch, 2005). Both the concentration of the total glutathione pool and the ratio between reduced (GSH) and oxidized (GSSG) glutathione can influence the redox potential of the cell (Schafer and Buettner, 2001). Basal glutathione levels fluctuated between 158 and 280 nmol/g fresh weight during the course of the experiment, which is in accordance to previously published data (Karpinski et al., 1997; Mou et al., 2003). In addition, glutathione levels were influenced diurnally, showing a general increase during daylight conditions, followed by a decrease during night-time (Figure 3.6A), as described previously (Bielawski and Joy, 1986; Koike and Patterson, 1988; Schupp and Rennenberg, 1988; Noctor et al., 1997). Pathogen attack and application of SA or one of its functional analogs have been shown to trigger an increase in total glutathione content (Fodor et al., 1997; Vanacker et al., 2000; Mou et al., 2003; Mateo et al., 2006). Similarly, SA treatment resulted in a transient increase in the level of glutathione that returned to baseline levels after 30 h (Figures 3.6A and 3.6B). Interestingly, the change in redox potential coincided with the window of opportunity in which SA was able to suppress MeJA-induced PDF1.2 transcription (Figure 3.6C). Hence, we postulate that the antagonism between SA and JA signaling pathways is redox modulated.
I n h i b i t i o n o f g l u t a t h i o n e b i o s y n t h e s i s s u p p r e s s e s S A / J A c r o s s - t a l k
In order to demonstrate a causal relationship between changes in glutathione levels and SA/JA cross-talk, we manipulated the glutathione content of the cell and monitored the effect on PDF1.2 suppression. To deplete glutathione levels, we grew Arabidopsis seedlings on Murashige and Skoog (1962) (MS) medium, supplemented with a non-toxic and highly specific inhibitor of the first enzyme of GSH synthesis, L-buthionine-sulfoximine (BSO) (Griffith and Meister, 1979; May and Leaver, 1993). Inclusion of BSO in the growth medium resulted in a strong reduction in SA-induced glutathione levels (data not shown).
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To assess the effect of BSO on SA/JA cross-talk, BSO was included in the medium either during the whole growth period (2 weeks) or only during the last 48 h prior to harvest. Twelve-day-old seedlings grown on MS or MS supplemented with 2.5 mM BSO were transferred to MS medium supplemented with 2.5 mM BSO and either 0.5 mM SA, 20 μM MeJA, or a combination of both chemicals. Leaf tissue was harvested 48 h after chemical induction and assessed for PDF1.2 marker gene expression. Figure 3.7 shows normal levels of SA/JA cross-talk when the seedlings were grown on MS medium without BSO (Ctrl). However, inclusion of BSO in the growth medium for 2 days clearly reduced
Figure 3.6. Suppression of PDF1.2 by SA coincides with increased glutathione levels. (A) Total glutathione levels (GSH+GSSG) in wild-type Col-0 plants, harvested 0 to 78 h after foliar drench with 1 mM SA (open triangles) or control (closed squares) solution. Error bars represent SE.(B) Glutathione levels after subtraction of control values from SA values. (C) Percentage of PDF1.2 suppression after SA/MeJA treatment, compared to MeJA treatment alone. Signal intensities were quantified using a Phosphor imager. Wild-type Col-0 plants were treated with control or 1 mM SA solutions at t=0 h. Subsequently, 0.1 mM MeJA was applied at t=6, 12, 24, 27, 30, 33, 36, and 48 h. Leaf tissue was harvested 24 h after application of MeJA.The shaded area represents the 30-h window of opportunity of SA to suppress PDF1.2 expression. The white horizontal bar indicates the light period, and the black horizontal bar indicates the dark period.
Mn; B-Koornneef; B 07-943; 03-06.ai
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the antagonistic effect of SA on MeJA-induced PDF1.2 expression (CtrlBSO). This effect was even more pronounced when BSO was present in the medium during the whole growth period (BSO). Hence, the glutathione biosynthesis inhibitor BSO affects SA/JA cross-talk, strengthening our hypothesis that cross-talk is redox modulated.
D I S C U S S I O N
Cross-talk between defense signaling pathways is thought to play an important role in the regulation of induced defenses in plants. The antagonism between SA and JA signaling emerged as one of the most prominent of all signal interactions studied to date (Dong, 2004; Pieterse and Van Loon, 2004; Bostock, 2005; Nomura et al., 2005). However, the underlying molecular mechanisms of SA/JA cross-talk are to a large extent unknown. In this paper, we demonstrate that biological or chemical induction of the SA pathway strongly antagonizes the expression of the JA-responsive marker genes PDF1.2 and VSP2 as triggered by necrotrophic pathogens or insect herbivores. Moreover, we show that all 18 Arabidopsis accessions tested display SA/JA cross-talk, suggesting that this trait is important in the ecology and evolution of plant defense. Furthermore, we provide insight into how the outcome of the SA/JA signal interaction is influenced by the kinetics of the individual signaling cascades. Activation of the SA pathway resulted in an antagonistic effect on the expression of JA-responsive genes. However, when SA was applied prior to the JA trigger, SA had only a limited time frame to exert its antagonistic effect on the JA pathway. This window of opportunity of SA to downregulate JA-responsive gene expression coincided with a transient SA-induced change in the level of the antioxidant glutathione. Moreover, inhibition of glutathione biosynthesis by BSO strongly affected SA-mediated suppression of MeJA-induced PDF1.2 expression, suggesting that redox modulation is involved in SA/JA cross-talk.
In this study, we predominantly observed an antagonistic effect of SA on JA-responsive gene expression, while MeJA had virtually no effect on the SA-responsive marker gene PR-1 (Figures 3.1 to 3.4). Early studies in tomato already revealed that SA and its acetylated form, aspirin, are potent suppressors of the JA-dependent wound response (Doherty et al.,
Figure 3.7. The glutathione biosynthesis inhibitor BSO affects SA/JA cross-talk.Northern blot analysis of PDF1.2 expression in 14-day-old Col-0 seedlings grown on MS medium with or without 2.5 mM BSO, 0.5 mM SA, 20 μM MeJA, or a combination of these chemicals. SA and MeJA treatments were performed by transferring 12-day-old seedlings to MS medium with the chemicals indicated. Two days later, seedlings were harvested for northern blot analysis. BSO was included in the medium either at day 12 (CtrlBSO), or during the whole growth period (BSO). Equal loading of RNA samples was checked using a probe for 18S rRNA.Mn; B-Koornneef; B 07-943; 03-07.ai
10-12-2007
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1988; Peña-Cortés et al., 1993; Doares et al., 1995). Thus, activation of the SA pathway, such as upon infection by a biotrophic pathogen, might result in suppression of JA-dependent defenses that are triggered by necrotrophic pathogens and insect herbivores. Indeed, we observed that inoculation with the biotrophic pathogen H. parasitica activated the SA pathway, resulting in downregulation of herbivore-induced expression of the JA-responsive gene VSP2 (Figure 3.1C), indicating that during multitrophic interactions, the SA pathway can be prioritized over the JA pathway. Trade-offs between SA-dependent pathogen resistance and JA-dependent defense against insect herbivory have been repeatedly reported (Thaler et al., 1999; Felton and Korth, 2000; Pieterse et al., 2001; Bostock, 2005). In Arabidopsis, the SA pathway has been shown to inhibit JA-dependent resistance against tissue-chewing herbivores, such as Spodoptera exigua (beet armyworm) (Cipollini et al., 2004; Bodenhausen and Reymond, 2007) and Trichoplusia ni (cabbage looper) (Cui et al., 2002; Cui et al., 2005), and necrotrophic pathogens, such as A. brassicicola (Kariola et al., 2005; Spoel et al., 2007). Intriguingly, some herbivores have been demonstrated to induce the SA pathway to actively suppress effectual JA-dependent defenses and thereby escape host defense (Zarate et al., 2007). Hence, depending on the plant-attacker combination, the antagonistic effect of SA on JA-dependent defense responses may either be beneficial or deleterious.
Our studies on the kinetics of SA and JA signaling in relation to the outcome of the SA/JA signal interaction revealed that very low doses of SA are already able to suppress JA-responsive PDF1.2 transcription, suggesting that this SA/JA cross-talk mechanism is highly sensitive (Figure 3.4). However, the antagonistic effect was only apparent when the SA pathway was activated after the onset of the JA response, or within a time frame of about 30 h prior to the activation of the JA response, indicating that the ability of SA to suppress JA-responsive gene expression is transient (Figures 3.5A and 3.5B). Although our results are to a large extent consistent with previous findings in tomato, tobacco, and Arabidopsis (Thaler et al., 2002; Mur et al., 2006), Mur et al. (2006) demonstrated that transient synergistic effects between SA and JA signaling may occur during early stages of the SA/JA signal interaction. So how does SA manipulate JA-dependent defenses? In this paper, we demonstrated that SA/JA signal antagonism coincides with a transient increase in the level of glutathione (Figure 3.6). Glutathione is a major cellular antioxidant and an important determinant of the redox state in eukaryotes (Schafer and Buettner, 2001). Previously, Mou et al. (2003) determined both total glutathione levels and the ratio of reduced (GSH) and oxidized (GSSG) glutathione in Arabidopsis upon application of the SA analog 2,6-dichloroisonicotinic acid (INA), and observed comparable changes in kinetics in both glutathione pool size and redox status. In addition, SA-accumulating mutants with constitutive PR-1 expression were shown to have an increased glutathione pool size (Mateo et al., 2006). Our data indicate that the SA-induced change in glutathione levels plays an important role in initiating the antagonistic effect on JA-responsive gene transcription. The involvement of redox modulation is supported by the observation that overexpression of the SA-regulated glutaredoxin GRX480 antagonizes JA-responsive transcription of PDF1.2 (Ndamukong et al., 2007). In addition, EDS1 and PAD4 have been implicated
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in transduction of redox signals in response to biotic and abiotic stresses (Wiermer et al., 2005), as well as in the regulation of cross-talk as activators and repressors of SA and JA defenses, respectively (Brodersen et al., 2006).
Previously, it was demonstrated that SA-activated NPR1 is required for the suppression of JA-responsive gene expression by SA (Spoel et al., 2003; Chapter 2) and that activation of NPR1 is redox regulated (Mou et al., 2003). In uninduced cells, NPR1 is present as an oligomer formed through intermolecular disulfide bonds. SA mediates a change in the cellular redox potential, resulting in the reduction of the NPR1 oligomer to its active monomeric form. Monomeric NPR1 is then translocated into the nucleus where it functions as a coactivator of SA-responsive genes, such as PR-1 (Dong, 2004). For the suppression of JA-responsive gene expression, translocation of SA-activated NPR1 into the nucleus is not required, as has been demonstrated in both Arabidopsis and rice (Spoel et al., 2003; Chapter 2; Yuan et al., 2007), suggesting an important role for cytosolic NPR1 in SA/JA cross-talk. Thus, although the role of NPR1 in SA/JA cross-talk and SA-induced PR-1 gene expression seems to be dissimilar, it is plausible that both defense responses are controlled by active NPR1 monomers that are produced upon changes in the redox state. However, for SA/JA cross-talk, additional SA-mediated signaling components are required, because Arabidopsis transgenic plants with constitutively monomerized NPR1 did not affect JA-responsive marker gene expression in the absence of SA (Beckers and Spoel, 2006). Uncovering these players in pathway cross-talk will be the focus of future research.
M AT E R I A L S A N D M E T H O D S
C u l t i v a t i o n o f p l a n t s
Seeds of Arabidopsis thaliana accessions Col-0 (N1092), An-1 (N944), Bur-0 (CS6643), C24 (N906), Cvi-0 (N8580), Di-0 (N1106), Eri-1 (CS22548), Fei-0 (CS22645), Kond (CS6175), Kyo-1 (W10372), Ler-0 (NW20), Ll-0 (N1338), Ren-0 (CS22535), RLD-1 (N913), Sha (CS929), Uk-4 (N1580), Wei-0 (N3110), Ws-2 (CS2360), and mutants npr1-1 and cpr1-1 (Col-0 background) were kindly provided by Maarten Koornneef (Wageningen University, Wageningen, The Netherlands) and Xinnian Dong (Duke University, Durham, USA). Seeds were sown in quartz sand. Two weeks later seedlings were transferred to 60-mL pots containing a sand-and-potting soil mixture (5:12 v/v) that was autoclaved twice for 20 min. Plants were cultivated in a growth chamber with an 8-h day (200 μE/m2/s at 24°C) and 16-h night (20°C) cycle at 70% relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland solution (Hoagland and Arnon, 1938) containing 10 μM Sequestreen (CIBA-Geigy, Basel, Switzerland) once a week.
C h e m i c a l i n d u c t i o n
Induction treatments were performed by dipping the leaves of 5-week-old plants in an aqueous solution containing 0.015% (v/v) Silwet L-77 (Van Meeuwen Chemicals BV,
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Weesp, The Netherlands), supplemented with 0.1, 1, 10, 100, or 1000 μM SA (Mallinckrodt Baker, Deventer, The Netherlands), or 20 or 100 μM MeJA (Serva, Brunschwig Chemie, Amsterdam, The Netherlands), or a combination of both chemicals. Control plants were treated with 0.015% Silwet L-77 only. MeJA was added to the medium from a 1000-fold stock solution in 96% ethanol. Solutions without MeJA were supplemented with equal amounts of ethanol. Plants were harvested between 1 and 96 h after induction treatment and immediately frozen in liquid nitrogen.
P a t h o g e n a n d i n s e c t b i o a s s a y s
Alternaria brassicicola strain MUCL 20297 and Botrytis cinerea strain B0510 were grown on potato dextrose agar (Difco Laboratories, Detroit, USA) plates for 2 weeks at 22°C. Subsequently, conidia were collected as described previously (Broekaert et al., 1990). Five-week-old Col-0 plants were inoculated by applying 5-μL drops of half-strength potato dextrose broth containing 5 x 105 spores/mL. Pieris rapae and Frankliniella occidentalis were reared as described previously (De Vos et al., 2005) and transferred to 5-week-old Col-0 plants. Infestation was carried out by transferring 5 first-instar larvae of P. rapae or 20 larvae of F. occidentalis to each plant using a fine paintbrush. SA (1mM) was applied as a foliar drench 24 h after pathogen inoculation or herbivore infestation and leaf tissue was harvested another 24 h later. Sporangia from Hyaloperonospora parasitica strain WACO9 were collected by rinsing sporulating Col-0 leaves in 10 mM MgSO
4 as described previously
(Ton et al., 2002a). Next, 5-week-old Col-0 plants were inoculated by spraying the leaves with the spore suspension containing 5 x 104 sporangia/mL. To ensure infection, plants were placed at 17°C and kept at 100% relative humidity for 24 hours. After this period, plants were kept at 70-80% relative humidity to facilitate growth of the pathogen. MeJA (0.1 mM) was applied as a foliar drench 3 days after H. parasitica inoculation. In case of two biological inducers, P. rapae larvae were applied 3 days after H. parasitica inoculation. Leaf material was harvested 24 h after MeJA or P. rapae treatment.
R N A e x t r a c t i o n a n d n o r t h e r n b l o t a n a l y s i s
Total RNA was extracted as described previously (De Vos et al., 2005). For northern blot analysis, 15 μg RNA was denatured using glyoxal and dimethyl sulfoxide (Sambrook et al., 1989), electrophoretically separated on a 1.5% agarose gel, and blotted onto Hybond-N+ membrane (Amersham, ‘s-Hertogenbosch, The Netherlands) by capillary transfer. The electrophoresis and blotting buffer consisted of 10 and 25 mM sodium phosphate (pH 7.0), respectively. Northern blots were hybridized with gene-specific probes for PR-1, PDF1.2, and VSP2 as described previously (Pieterse et al., 1998). After hybridization with α-32P-dCTP-labeled probes, blots were exposed for autoradiography and signals quantified using a BioRad Molecular Imager FX (BioRad, Veenendaal, The Netherlands) with Quantity One software (BioRad, Veenendaal, The Netherlands). To check for equal loading, the blots were stripped and hybridized with a probe for 18S rRNA. The AGI numbers for the genes studied are At2g14610 (PR-1), At5g44420 (PDF1.2), and At5g24770 (VSP2). The probe
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for 18S rRNA was derived from an Arabidopsis cDNA clone (Pruitt and Meyerowitz, 1986). All gene expression analyses have been repeated with similar results.
G l u t a t h i o n e a s s a y
Total levels of glutathione (GSH+GSSG) were measured using the Glutathione Assay Kit (Sigma, Schnelldorf, Germany) according to the manufacturer’s protocol. Leaf tissue was frozen in liquid nitrogen and ground to a fine powder. Subsequently, 500 µL of 5% 5-sulfosalicylic acid were added to 0.1 g of pulverized leaf tissue to deproteinize the sample. Glutathione was then determined in a kinetic assay in which the reduction of 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) to yellow TNB was spectrophotometrically measured at 415 nm. The amount of total glutathione was calculated using a standard curve of reduced glutathione. Five plants per treatment were harvested at each time point, and each sample was measured 6 times.
B S O a s s a y
Col-0 seedlings were grown for 12 days on Murashige and Skoog (1962) (MS) medium with or without 2.5 mM L-buthionine-sulfoximine (BSO; Sigma, Schnelldorf, Germany) and with 1% (w/v) sucrose and 0.6% (w/v) plant agar, pH 5.7. Seedlings were then transferred to MS plates containing 2.5 mM BSO, 0.5 mM SA, 20 μM MeJA, or a combination of these chemicals. Leaf tissue was harvested 48 h later. BSO was included in the MS medium either continuously or only during the last 48 h, together with the SA and MeJA treatments.
A C K N O W L E D G M E N T SThe authors thank Ruth Joosten for technical assistance, Marcel Dicke for providing the insect herbivores and Leo Koopman, Frans van Aggelen, André Gidding, and Dick Peeters for insect rearing. This research was supported by grants 813.06.002 and 865.04.002 of the Earth and Life Sciences Foundation (ALW), which is subsidized by The Netherlands Organization of Scientific Research (NWO).
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C H A P T E R 4
Towards the identif ication of novel
key players of cross-talk in plant
defense
Annemart Koornneef 1, Katja Rindermann 2, Christiane Gatz 2, L.C. Van Loon 1, and Corné M.J. Pieterse 1
1 Graduate School Experimental Plant Sciences, Plant-Microbe Interactions,
Institute of Environmental Biology, Faculty of Science, Utrecht University,
P.O. Box 800.56, 3508 TB Utrecht, The Netherlands2 Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Georg-August-Universität Göttingen,
Untere Karspüle 2, D-37073 Göttingen, Germany
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Towards the identif ication of novel
key players of cross-talk in plant
defense
A B S T R A C T
Cross-talk between salicylic acid (SA) and jasmonic acid (JA) defense signaling pathways allows a plant to finely tune its response to the attacker encountered. In Arabidopsis, pharmacological experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PDF1.2, indicating that the SA pathway can be prioritized over the JA pathway. Previously, NPR1 was identified as a key regulatory protein involved in this SA/JA cross-talk. Here, we attempted to identify novel key players in SA/JA cross-talk. Firstly, a large number of well-characterized mutants affected in SA-dependent systemic acquired resistance (SAR) signaling were tested for SA/JA cross-talk. In most SAR mutants the antagonistic effect of SA on methyl jasmonate (MeJA)-induced PDF1.2 expression was not affected, indicating that the signaling pathways leading to SAR and suppression of JA-responsive gene expression are partly divergent. The only newly identified SAR mutant that was also impaired in SA/JA cross-talk was the tga2-1 tga3-1 tga5-1 tga6-1 quadruple mutant, indicating that TGA transcription factors are essential for both SA-mediated phenomena. Secondly, we investigated the putative role of histone modifications in the regulation of SA/JA cross-talk. Chromatin immunoprecipitation analysis using an antibody directed against acetylated histone H3 revealed that SA does not affect the association of this histone modification at the PDF1.2 promoter, suggesting that chromatin remodeling does not play a major role in SA/JA cross-talk. Thirdly, we followed a whole-genome transcript profiling approach to identify Arabidopsis genes that are sensitive to SA/JA cross-talk and to search for common promoter elements. Of all 1538 MeJA-responsive genes, 258 (17%) appeared to be significantly affected by SA. Sixty percent of the MeJA-inducible genes that were suppressed by SA displayed this suppression in a NPR1-dependent manner, demonstrating that NPR1 is involved in the SA-mediated downregulation of a subset of MeJA-responsive genes. In silico promoter analysis revealed that the I box (GATAAG) and the GCC box (GCCGCC) motifs are significantly enriched in the 1-kb promoter regions of the SA-suppressed, MeJA-inducible genes. Disruption of the I box in the PDF1.2
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promoter did not affect the negative effect of SA on MeJA-induced expression of this gene, suggesting that this motif does not play a major role SA/JA cross-talk. Since the GCC box is also required for JA-responsive expression of PDF1.2, its role in cross-talk could not be readily investigated. Together, these results provide further insight into the complexity of cross-talk in plant defense signaling and identified TGA transcription factors and the GCC box as candidate elements in SA/JA cross-talk.
I N T R O D U C T I O N
Plants can activate specific defense responses in order to resist attack by deleterious organisms. Apart from pre-existing chemical or structural barriers, inducible defenses are triggered, which often act systemically throughout the plant and confer broad-spectrum resistance. They are able to activate different types of induced resistance, depending on the organism that interacts with the plant. Well-studied examples of induced resistance are systemic acquired resistance (SAR), which is triggered by pathogens causing limited infection, such as a hypersensitive necrosis (Durrant and Dong, 2004), rhizobacteria-induced systemic resistance (ISR), which is activated upon colonization of roots by selected strains of non-pathogenic rhizobacteria (Van Loon et al., 1998), and wound-induced resistance, which is typically elicited upon tissue damage, such as that caused by insect feeding (Kessler and Baldwin, 2002; Howe, 2004).
The plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play important roles in the regulation of these induced defenses (Howe, 2004; Pozo et al., 2004; Lorenzo and Solano, 2005; Grant and Lamb, 2006; Van Loon et al., 2006; Von Dahl and Baldwin, 2007). The corresponding signal transduction pathways cross-communicate, providing the plant with a highly flexible defense signaling network. Cross-talk between signaling pathways is thought to optimize the defense reaction to a particular attacker by enhancing the appropriate response, while suppressing suboptimal reactions. Indeed, trade-offs between defense signaling pathways have been demonstrated in several plant species (Pieterse et al., 2001; Bostock, 2005). For instance, transgenic tobacco plants with elevated levels of SA have been demonstrated to be more susceptible to insect herbivores, which are generally resisted through JA-dependent defenses, suggesting that the SA pathway is prioritized over the JA pathway (Felton et al., 1999). Similarly, Spoel et al. (2007) demonstrated that simultaneous infection of Arabidopsis with a biotrophic and a necrotrophic pathogen results in impaired resistance to the necrotrophic pathogen, again demonstrating that the SA pathway that was activated by the biotroph suppressed the level of JA-dependent resistance against the necrotroph. However, cross-talk in the reciprocal direction has been demonstrated as well. For instance, virulent Pseudomonas syringae bacteria produce the toxin coronatine that functions as a potent mimic of jasmonates (Nomura et al., 2005). It is assumed that coronatine triggers induction of JA signaling responses, which results in suppression of SA-dependent defenses through pathway cross-talk, and thereby
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promotes P. syringae pathogenesis (Zhao et al., 2003; Brooks et al., 2005; Cui et al., 2005; Laurie-Berry et al., 2006).
Elucidation of the molecular mechanism underlying this pathway cross-talk will provide insight into how a plant copes with multiple stress inputs and relays these to an appropriate defense reaction (Reymond and Farmer, 1998; Pieterse et al., 2001; Bostock, 2005; Koornneef and Pieterse, 2008). Several key regulatory proteins involved in SA/JA cross-talk have been identified in Arabidopsis. Previously, we demonstrated that the defense regulatory protein NPR1 is required for SA/JA cross-talk (Spoel et al., 2003; Chapter 2). In mutant npr1-1 plants, the SA-mediated suppression of JA-responsive gene expression was completely abolished. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression (Kinkema et al., 2000), was not required for the suppression of JA-responsive genes, indicating that SA/JA cross-talk is modulated through a function of NPR1 in the cytosol (Spoel et al., 2003; Chapter 2). The transcription factor WRKY70 was shown to act as an activator of SA-responsive genes and a repressor of JA-inducible genes, thereby functioning as a node of convergence between both pathways (Li et al., 2004; Li et al., 2006). Recently, overexpression of the SA-regulated glutaredoxin GRX480 was found to antagonize JA-responsive PDF1.2 transcription (Ndamukong et al., 2007). Furthermore, SA induced a transient change in the levels of the antioxidant glutathione, which correlated with the 30-h window of opportunity of SA to suppress methyl jasmonate (MeJA)-induced PDF1.2 expression. These observations point to a role for redox modulation in the SA/JA antagonism (Chapter 3).
To identify novel key players in SA/JA cross-talk we followed three approaches. Firstly, we investigated to what extent various components of the SA-dependent SAR pathway play a role in SA/JA cross-talk. SA/JA cross-talk signaling overlaps with SAR in terms of redox change (Chapter 3) and requirement of NPR1 (Spoel et al., 2003; Chapter 2). At the onset of SAR, endogenous SA increases both locally and systemically (Malamy et al., 1990; Métraux et al., 1990). Mutants that are defective in SA biosynthesis, such as sid2 (Nawrath and Métraux, 1999), or transgenic NahG plants that convert SA into catechol (Gaffney et al., 1993), are impaired in their ability to express pathogenesis-related (PR) genes and show enhanced susceptibility to different pathogens (Delaney et al., 1994; Kachroo et al., 2000). Transduction of the SA signal to activate PR gene expression requires the regulatory protein NPR1. Mutation of NPR1 renders the plant largely unresponsive to pathogen-induced SA production and abolishes PR gene expression and SAR (Cao et al., 1994; Delaney et al., 1995; Shah et al., 1997). SA induces a redox change that converts inactive NPR1 oligomers into active NPR1 monomers through reduction of intermolecular disulfide bonds. Monomeric NPR1 is translocated into the nucleus, where it interacts with several factors to activate PR gene expression (Mou et al., 2003; Dong, 2004). It interacts with a negative modulator of PR gene expression, NIMIN1, which is thought to provide a fine-tuning mechanism for the activation of SAR (Weigel et al., 2005), as well as seven out of ten members of the TGA subclass of the basic leucine zipper (bZIP) family of transcription factors (Zhang et al., 1999; Després et al., 2000; Zhou et al., 2000; Subramaniam et al., 2001; Fan and Dong,
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2002; Jakoby et al., 2002; Kim and Delaney, 2002; Després et al., 2003; Johnson et al., 2003). TGA transcription factors can bind to both positive and negative cis-elements in the PR-1 promoter (Lebel et al., 1998) and act as either positive or negative regulators of PR-1 gene expression (Pontier et al., 2001; Zhang et al., 2003; Rochon et al., 2006; Kesarwani et al., 2007). Moreover, NPR1 is required for removal of the suppressor protein SNI1, which negatively affects PR expression, possibly through chromatin modifications (Li et al., 1999; Mosher et al., 2006). Besides regulating PR genes, NPR1 was also shown to target the transcription of genes that function in the protein secretory pathway. Expression of these proteins ensures proper processing of PR transcripts and secretion of PR proteins (Wang et al., 2005). Thus, multiple positive and negative integrators play a role in fine-tuning SA-dependent defense signaling.
Secondly, we assessed whether chromatin remodeling plays a role in the SA-mediated downregulation of JA-responsive genes. DNA is packaged with histone proteins into chromatin, which physically restricts the accessibility of the genome to regulatory proteins, such as transcription factors. The chromatin configuration can be altered to allow or prevent access of the transcription machinery by covalent modifications of the exposed N-terminal histone tails in the nucleosome. Histone acetylation is mediated by the activity of histone acetyltransferases (HATs) and is often associated with increased gene activity. Histone deacetylation, mediated by histone deacetylases (HDACs), and methylation are generally correlated with transcriptional repression (Grant, 2001; Pfluger and Wagner, 2007). Thus, gene expression can be regulated at the level of histone modifications. Previously, a HDAC was found to interact with the JA regulatory protein COI1 (Devoto et al., 2002). The expression of this gene, as well as another HDAC, HDA19, was shown to be upregulated by JA and the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC) (Zhou et al., 2005). Overexpression of HDA19 enhanced expression of several JA- and ET-responsive genes and conferred increased resistance to the necrotrophic pathogen Alternaria brassicicola. In view of the repressive nature of a HDAC, it is likely that HDA19 influences transcription of these genes indirectly (Zhou et al., 2005). Chromatin immunoprecipitation analysis demonstrated that induction of SA-responsive PR-1 gene expression is associated with an increase in histone acetylation at the PR-1 promoter in Arabidopsis and tobacco (Butterbrodt et al., 2006; Mosher et al., 2006), suggesting involvement of histone modifications in the activation of SA-responsive genes too.
Finally, to investigate transcriptional regulation of SA/JA cross-talk, we performed whole-genome transcript profiling, followed by an in silico analysis of the promoter sequences of all MeJA-inducible genes that are sensitive to SA-mediated suppression, to identify common regulatory motifs that may be involved in SA/JA cross-talk. A previous study on coregulated genes comprising PR-1 in SAR-expressing Arabidopsis revealed an overrepresentation of the WRKY binding motif (Maleck et al., 2000). Since then, several WRKY genes have been shown to be involved in the SA signaling pathway (Wang et al., 2006). Similarly, enrichment of the MYC2 binding site in promoters of ISR-primed genes led to the identification of the MYC2 transcription factor as an essential regulatory
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component in the expression of this induced resistance mechanism (Van der Ent, 2008). Hence, promoter analysis might aid in the identification of putative transcription factors involved in SA/JA cross-talk.
R E S U LT S
I n v o l v e m e n t o f S A R s i g n a l i n g c o m p o n e n t s i n S A / J A c r o s s - t a l k
Previously, we demonstrated that, like SAR, SA-mediated suppression of JA signaling involves redox modulation and activation of the regulatory protein NPR1 (Spoel et al., 2003; Chapter 2 and 3). To investigate to what extent the SA signaling pathway leading to SAR and SA/JA cross-talk overlap, we analyzed a number of well-characterized mutants that are known to be affected in SAR, and determined the effect of the mutations on SA/JA cross-talk. Five-week-old Arabidopsis plants were treated with 0.1 mM MeJA or a combination of 1 mM SA and 0.1 mM MeJA. At 24 h after treatment, the expression level of the JA-responsive PDF1.2 gene was assessed by northern blot analysis and quantified using a Phosphor imager. MeJA treatment resulted in PDF1.2 transcription in all genotypes analyzed (set at 100% for each genotype in Figure 4.1A). In the combination treatment, SA suppressed the level of MeJA-induced PDF1.2 in wild-type Col-0 plants by 74% (Figure 4.1A). In mutant npr1-1 and transgenic SA-degrading NahG plants, SA did not suppress PDF1.2 expression, confirming our previous findings (Spoel et al., 2003; Chapter 2 and 3). Besides npr1-1, only the TGA quadruple mutant tga2-1 tga3-1 tga5-1 tga6-1 behaved significantly differently from wild-type Col-0 with respect to SA-mediated suppression of MeJA-induced PDF1.2 (Kruskal-Wallis test, p<0.01). The TGA quadruple mutant tga2-1 tga3-1 tga5-1 tga6-1 is impaired in SA-responsive PR-1 induction and SAR (Kesarwani et al., 2007). That this mutant did not show suppression of MeJA-induced PDF1.2 expression by SA either, suggests that TGA transcription factors also play a role in SA/JA cross-talk.
All other SAR-related mutants tested displayed wild-type levels of SA/JA cross-talk. For mutant sid2-1, which is impaired in SA biosynthesis (Nawrath and Métraux, 1999), this was expected because exogenous application of SA rescues the SA minus phenotype. A mutation in NIMIN1 did not affect SA/JA cross-talk. The same held for the single mutants sec61α, dad1, and bip2, and the double mutants sec61α bip2 and dad1 bip2, which were shown to be involved in NPR1-dependent PR protein secretion during SAR (Wang et al., 2005). The transcription factor WRKY70 did not affect SA/JA cross-talk either, since exogenous application of SA suppressed MeJA-induced PDF1.2 to wild-type levels in mutant wrky70-1.
The sni1 npr1-1 double mutant was originally identified in a mutant screen for suppression of the mutant npr1 phenotype (Li et al., 1999). In sni1 npr1-1, SA-inducible PR-1 gene expression and SAR are restored, suggesting that wild-type SNI1 acts as a repressor of PR gene expression. Because the sni1 npr1-1 double mutant has no functional NPR1 protein, we expected that this mutant would still be impaired in SA/JA cross-talk.
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Indeed, mutant sni1 npr1-1 appeared to behave significantly differently from the Col-0 wild-type (p<0.05). However, in half of the experiments, sni1 npr1-1 did show a reduction in PDF1.2 expression upon combined treatment with SA and MeJA, compared to MeJA alone, resulting in an average level of SA/JA cross-talk of 45%. These results suggest that the sni1 mutation restores not only SA-inducible PR-1 expression and SAR in the npr1-1 mutant background, but also SA/JA cross talk in some cases. However, we recently observed that enhanced levels of ET can overrule the NPR1-dependency of SA/JA cross-talk, resulting in wild-type levels of SA-mediated suppression of MeJA-induced PDF1.2 in the npr1-1 mutant background (A. Leon-Reyes and C.M.J. Pieterse, unpublished results). To test whether enhanced production of ET by the sni1 npr1-1 mutant could explain the occasionally observed NPR1-independent SA/JA cross-talk in this mutant, we measured ET levels in untreated npr1-1 and sni1 npr1-1 mutants. Indeed, the sni1 npr1-1 double mutant emitted substantially higher levels of ET than npr1-1 (Figure 4.1B), which may explain the observed NPR1-independent SA/JA cross-talk in this double mutant.
H i s t o n e m o d i f i c a t i o n s a r e n o t i n v o l v e d i n S A / J A c r o s s - t a l k
Histone modifications have been demonstrated to play a role in the regulation of SA- and JA-responsive gene expression (Zhou et al., 2005; Butterbrodt et al., 2006; Mosher et al., 2006). Here, we tested whether SA-induced histone modifications may play a role in the SA-mediated suppression of MeJA-induced PDF1.2 expression. Five-week-old Col-0 and npr1-1 plants were treated with SA, MeJA, or a combination of both chemicals, and the expression of SA-responsive PR-1 and MeJA-responsive PDF1.2 marker genes was verified
Figure 4.1. Quantification of SA-mediated suppression of MeJA-induced PDF1.2 expression in SAR signaling mutants.(A) Northern blot analysis of PDF1.2 gene expression in several SAR-related mutants after treatment with 0.1 mM MeJA or a combination of 0.1 mM MeJA and 1 mM SA. Leaf tissue was harvested 24 h after chemical treatment. Equal loading of RNA samples was checked using a probe for 18S rRNA. Signal intensities were quantified using a Phosphor imager. PDF1.2 transcript levels in the single MeJA treatments were set to 100%. Asterisks indicate significant differences from Col-0 wild-type, as determined by Kruskal-Wallis test (double asterisk; p<0.01, single asterisk; p<0.05).(B) ET production in Arabidopsis mutants sni1 npr1-1 (closed squares) and npr1-1 (open circles). The represented values are means (±SE) for ten untreated plants that were measured at different time points over a 77-h time interval. At all time points, the ET emission differed significantly between sni1 npr1-1 and npr1-1 (Student’s t test; p<0.05).
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(Figure 4.2A). As expected, SA induced PR-1 and suppressed MeJA-induced expression of PDF1.2 in Col-0 plants. Both induction of PR-1 and suppression of PDF1.2 by SA was blocked in mutant npr1-1, confirming that the plant material displayed NPR1-dependent SA/JA cross-talk. This plant material was subjected to chromatin immunoprecipitation (ChIP) analysis, using an antibody directed against acetylated histone H3 (AcH3), which is a marker for a more transcriptionally permissive state (Strahl and Allis, 2000; Pfluger and Wagner, 2007). RT-Q-PCR analysis was performed on the immunoprecipitated and non-immunoprecipitated input DNA with primers designed to amplify fragments of the PR-1 and PDF1.2 promoter. As a control, primer sets were included that amplify promoter fragments of two constitutively expressed marker genes, GAPDH and UBQ10 (Czechowski et al., 2005). The amount of immunoprecipitated DNA was calculated relative to the input DNA, and for the control treatments of Col-0 and npr1-1 the values were set to 1 (Figure 4.2B). Figure 4.2B shows that SA treatment resulted in an increase of AcH3
Figure 4.2. Chromatin immunoprecipitation (ChIP) analysis of histone H3 acetylation at the PR-1 and PDF1.2 promoters in Col-0 and npr1-1.(A) Northern blot analysis of PDF1.2 and PR-1 expression in Col-0 and npr1-1 leaves treated with 1 mM SA, 0.1 mM MeJA, or a combination of both chemicals. Leaf tissue was harvested 24 h after chemical treatment. Equal loading of RNA samples was checked using a probe for 18S rRNA.(B) ChIP analysis of Col-0 and npr1-1 treated with 1 mM SA, 0.1 mM MeJA, or a combination of both chemicals. Chromatin samples were subjected to immunoprecipitation using an AcH3 antibody. The immunoprecipitated DNA was analyzed for the enrichment of PR-1, PDF1.2, UBQ10, and GAPDH promoter sequences by Q-RT-PCR. The fold increase of immunoprecipitated DNA was calculated relative to the input DNA.
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at the PR-1 promoter, confirming previous findings with the SA analog benzothiadiazole S-methyl ester (BTH) as the inducing agent (Mosher et al., 2006). This increase was absent in npr1-1, suggesting that SA-mediated acetylation of histone H3 at the PR-1 promoter is NPR1-dependent. However, the combined treatment with SA and MeJA did not result in AcH3 enrichment at the PR-1 promoter in Col-0 while the PR-1 gene was normally expressed (Figures 4.2A and 4.2B), indicating that basal levels of AcH3 are sufficient for PR-1 expression.
In contrast to the PR-1 promoter, AcH3 association at the PDF1.2 promoter did not surmount the fluctuation observed at the promoters of the two constitutively expressed genes, GAPDH and UBQ10 (Figure 4.2B). Hence, the negative effect of SA on MeJA-induced expression of PDF1.2 did not correlate with this histone modification, suggesting that chromatin remodeling is not involved in the regulation of SA/JA cross-talk. Conclusive evidence should be provided by additional experiments and include analysis with antibodies directed against multiple histone modifications.
M i c r o a r r a y a n a l y s i s o f S A - a n d M e J A - r e s p o n s i v e g e n e e x p r e s s i o n
To gain insight into the regulation and complexity of SA/JA cross-talk at the whole-genome level, we performed a transcript profiling approach to 1) identify additional MeJA-responsive genes whose expression is affected by SA; 2) identify SA-responsive genes whose expression is affected by MeJA; 3) investigate the importance of NPR1 in SA/JA cross-talk; and 4) identify cis-acting elements involved in the regulation of SA/JA cross-talk. Three independent cross-talk experiments were performed with 5-week-old Col-0 and npr1-1 plants that were treated with SA, MeJA, or a combination of both chemicals. Leaf tissue was harvested approximately 28 h after chemical induction. The expression of the marker genes PR-1 and PDF1.2 was assessed in each biological replicate by northern blot analysis (Figure 4.3). In all three experiments, SA induced PR-1 expression and suppressed MeJA-induced expression of PDF1.2 in Col-0 plants. Both induction of PR-1 and suppression of PDF1.2 by SA was blocked in mutant npr1-1, confirming that the plant material displayed NPR1-dependent SA/JA cross-talk.
The transcript profile of each independent experiment was analyzed using Affymetrix ATH1 whole-genome GeneChips representing approximately 23,750 Arabidopsis genes (Redman et al., 2004). After hybridization, expressed genes were identified using GeneChip Operating Software (GCOS), which uses statistical criteria to generate a “present” or “absent” call for genes represented by each probe set on the array. The fact that each of the three experiments was conducted at a different time of the year and in different growth chambers contributed significantly to the variation in gene expression observed between the biological replicates. However, by identifying conserved gene expression patterns within the three biological replicates, genes were selected that are likely to contribute to the biological significance of the phenomenon studied. To identify a robust set of MeJA-responsive genes, we selected genes that were statistically significantly up- or downregulated in MeJA-treated plants compared to the mock-treated control (Student’s t test, p<0.05).
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In addition, the expression level had to be detectable (present call generated by GCOS) in all three MeJA-treated samples (for the upregulated genes) or in all three control treatments (for the downregulated genes). These selection criteria were met by 731 genes that were significantly upregulated upon MeJA treatment (Supplemental Table S1). Among these were genes involved in JA biosynthesis (AOS, OPR3, LOX2), JA signal transduction (ERF1, ERF4, JAZ1, JAZ2, JAZ5, JAZ7, JAZ9), and JA-dependent defenses (PDF1.2, Thi2.1, VSP1, HEL, ß-1,3-glucanase). In addition, a group of 807 genes was significantly downregulated by MeJA (Supplemental Table S1). For SA-responsive genes, a similar selection procedure was followed, resulting in 705 SA-upregulated genes (including PR-1, ICS1, PAL3) and 698 SA-downregulated genes (Supplementary Table S1).
Tr a n s c r i p t o m e a n a l y s i s o f S A / J A c r o s s - t a l k g e n e s
To select for MeJA-induced genes that were suppressed by SA, we identified MeJA-upregulated genes that were significantly repressed by the combined treatment with SA and MeJA, compared to MeJA alone. In addition, we selected MeJA-downregulated genes that were significantly upregulated by SA and MeJA, compared to MeJA alone. These selection criteria resulted in the identification of 123 MeJA-inducible genes that are suppressed by SA, and 135 MeJA-downregulated genes that are induced by SA (Student’s t test; p<0.05; Figures 4.4A and 4.4B; Supplemental Table S2). On average, 17% of all MeJA-responsive genes were affected by SA/JA cross-talk, demonstrating that this type of pathway cross-talk is specific for only a subset of the MeJA-responsive genes. Using slightly less stringent selection criteria for the identification of SA/JA cross-talk genes (Student’s t test; p<0.1), another 98 and 80 MeJA-responsive genes were suppressed and induced by SA, respectively, demonstrating that, depending on the stringency of the selection, up to 28% of MeJA-responsive genes were affected by SA (Figures 4.4A and 4.4B; Supplemental Table S2). Among the MeJA-inducible genes that were suppressed by SA were those encoding defense-related
Figure 4.3. Northern blot analysis of SA/JA cross-talk in three biological replicates.Northern blot analysis of PDF1.2 and PR-1 gene expression in Col-0 and npr1-1 after treatment with 1 mM SA, 0.1 mM MeJA, or a combination of both chemicals. The three biological replicates consistently showed SA-mediated suppression of MeJA-induced PDF1.2 expression in wild-type Col-0. This suppression was absent in mutant npr1-1 plants. Leaf tissue was harvested 28 h after chemical treatment. Equal loading of RNA samples was checked using a probe for 18S rRNA. RNA from these three biological replicates was used for whole-genome transcript profiling using Affymetrix ATH1 GeneChips.
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proteins PDF1.2a, PDF1.2b, and HEL, and the defense-related transcription factor ERF1, confirming previous findings (Spoel et al., 2003; Chapter 2; Ndamukong et al., 2007; Spoel et al., 2007). Several other MeJA-inducible genes with defense-related functions that were suppressed by SA included a gene from the defensin-like family, thionin Thi2.1, a putative thionin, a β-1,3-glucanase gene, and a disease resistance gene (Supplemental Table S2).
Selection of SA/JA cross-talk genes in the reciprocal direction revealed that 11% of the 1403 SA-responsive genes were significantly affected by MeJA (Student’s t test; p<0.05; this was 21% with p<0.1; Supplemental Table S2), demonstrating that SA/JA cross-talk occurs in both directions. However, in depth analysis of the selected SA-responsive genes that are antagonized by MeJA is beyond the scope of this study and will be the focus of future research.
N P R 1 - d e p e n d e n c y o f S A / J A c r o s s - t a l k
NPR1 has been identified as an essential regulator of SA-mediated suppression of JA-responsive genes, such as PDF1.2, VSP2, and LOX2 (Spoel et al., 2003; Chapter 2). To investigate the general importance of NPR1 in the regulation of SA/JA cross-talk, we determined the NPR1-dependency of the expression patterns identified from the whole-genome profiles. To compare the SA/JA cross-talk genes in Col-0 and npr1-1, we focused on the 123 MeJA-inducible genes that were significantly suppressed by SA in Col-0 (Supplemental Table S2), and thus followed a similar expression pattern as PDF1.2. Of the 123 selected SA/JA cross-talk genes, only 45 were also induced by MeJA in the npr1-1 background (Student’s t test; p<0.05). The remaining 78 genes were discarded from the selection as their expression levels were not significantly enhanced. This may be because MeJA-responsive expression is truly NPR1-dependent, or just did not reach the level of statistical significance due to the relatively large biological variation in the plant material of the three independent experiments. Amongst the remaining robust set of genes with a MeJA-responsive expression profile in the npr1-1 background was PDF1.2, confirming the northern blot data shown in Figure 4.3. Of the 45 selected MeJA-responsive genes that were all significantly suppressed by SA in the Col-0 background, 27 genes (60%) were not significantly suppressed by SA in the npr1-1 background (Student’s t test; p<0.05; Supplemental Table S3;). Again, PDF1.2 was amongst this selection. These results suggest that the SA-mediated suppression of MeJA-inducible genes is dependent on NPR1 in approximately 60% of the cases.
Figure 4.4. MeJA-responsive genes of which the expression is antagonized by SA. (A) The pie chart represents the proportion of MeJA-upregulated genes that are suppressed by SA (Student’s t test; p<0.05, 123 genes in bright green; 0.05<p<0.1, 98 genes in dark green) and that are not significantly suppressed by SA (510 genes in red). The log2 fold-changes in the expression of the 123 SA/JA cross-talk genes in the three biological replicates are depicted in the heat map (Saeed et al., 2003).(B) The pie chart represents the number of MeJA-downregulated genes whose repression is relieved by SA (Student’s t test; p<0.05, 135 genes in bright red; 0.05<p<0.1, 80 genes in dark red) and that are not significantly induced by SA (592 genes in green). The log2 fold-changes of the 135 SA/JA cross-talk genes in the three biological replicates are depicted in the heat map (Saeed et al., 2003).
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F u n c t i o n a l a n a l y s i s o f S A / J A c r o s s - t a l k g e n e s
Using the Gene Ontology (GO) tool at The Arabidopsis Information Resource (TAIR) (http://www.arabidopsis.org) (Rhee et al., 2003), we classified the SA/JA cross-talk genes according to their functional categories. To evaluate the relevance of a given functional category, the percentage of SA-antagonized MeJA-responsive genes (Supplemental Table S2) belonging to the defined functional group was compared to the relative representation of the respective functional category in the whole genome. The dominant biological processes that are overrepresented in the SA/JA cross-talk gene set are the categories “response to stress”, “electron transport or energy pathways”, “response to abiotic or biotic stimulus”, and “signal transduction”, suggesting that SA/JA cross-talk predominantly affects genes that play a role in these plant responses (results not shown). For the categories related to biotic stress responses and signal transduction, this was to be expected. The functional category “electron transport and energy pathways” is a less obvious functional category involved in SA/JA cross-talk, and could be related to redox signaling. However, the number of genes assigned to this category was quite low, making it difficult to draw conclusions about their functional significance. Another overrepresented category in the SA/JA cross-talk genes, which contained a larger number of genes, comprised genes encoding chloroplast-targeted proteins, involved in chlorophyll constitution and photosynthesis, such as chlorophyllase AtCLH2, uroporphyrinogen decarboxylases HEME1 and HEME2, and photosystem- and light harvesting complex-associated genes. These genes were suppressed by MeJA, but induced in the SA+MeJA combination treatment (Supplemental Table S2). This confirms previous studies that demonstrated antagonistic interactions between JA and light-inducible and photosynthesis-related genes (Wierstra and Kloppstech, 2000; Zhai et al., 2007). Apparently, SA antagonizes this JA-mediated effect, suggesting a positive correlation between SA and light-related signaling, in agreement with previous findings (Schenk et al., 2000; Genoud et al., 2002; Zeier et al., 2004).
E x p r e s s i o n o f S A / J A c r o s s - t a l k g e n e s
u p o n p a t h o g e n o r i n s e c t a t t a c k
Cross-talk between plant defense signaling pathways is thought to play a role in the fine-tuning of the defense response. Therefore, we hypothesized that the set of SA/JA cross-talk genes identified in this study would be enriched for genes that respond to pathogen or insect attack. Previously, we analyzed the transcriptome of Arabidopsis upon attack by different pathogens and insects (De Vos et al., 2005). To investigate whether the selected MeJA-responsive, SA-antagonized cross-talk genes are enriched for genes that respond during biotic stress, we compared the SA/JA cross-talk genes with the gene sets that were previously demonstrated to be responsive to the bacterial leaf pathogen Pseudomonas syringae pv tomato DC3000/avrRpt2, the necrotrophic fungus Alternaria brassicicola, tissue-chewing caterpillars of Pieris rapae (small cabbage white), or cell content-feeding Frankliniella occidentalis (Western flower thrips) (De Vos et al., 2005). These four attackers have in common that they all provoke an increase in JA biosynthesis and JA-responsive gene expression in Arabidopsis
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(De Vos et al., 2005). First, we selected all attacker-responsive genes that were identified as responsive to MeJA in our study, after which the number of overlapping genes for each attacker was set at 100%. Subsequently, we selected the MeJA- and attacker-responsive genes whose expression was significantly affected by SA (Student’s t test: p<0.05 or p<0.1). Figure 4.5 shows pie-charts with the proportion of SA/JA cross-talk genes among the MeJA-, or MeJA/attacker-responsive genes. As described above, 28% of all MeJA-responsive genes in our study were sensitive to SA/JA cross-talk (Student’s t test; p<0.1). The proportion of SA/JA cross-talk genes among the MeJA/P. syringae- and MeJA/F. occidentalis-responsive genes did not differ from that in the total pool of MeJA-responsive genes. However, the proportion of SA/JA cross-talk genes among the MeJA/P. rapae-responsive genes (14%) was significantly smaller than that in the total pool of MeJA-responsive genes (χ2 test; p<0.05), indicating that the JA-dependent defense response that is triggered by this herbivore is relatively insensitive to antagonism by SA. Conversely, the proportion of SA/JA cross-talk genes among the MeJA/A. brassicicola-responsive genes (41%) was higher than that in the total pool of MeJA-responsive genes (28%), suggesting that the SA-mediated antagonism on JA-responsive gene expression is biased towards necrotroph-responsive genes.
P r o m o t e r a n a l y s i s o f S A / J A c r o s s - t a l k g e n e s
To gain further insight into the molecular mechanism of SA/JA cross-talk, we performed an in silico analysis of the promoter sequences of the selected MeJA-inducible genes that, like the PDF1.2 marker gene, were suppressed by SA in the SA+MeJA combination treatment. Functional cis-regulatory elements in plant promoters are typically found within the first kilobase (kb) upstream of the ATG translation start site (Rombauts et al., 2003). Therefore, we scanned the 1-kb regions upstream of the 5’-UTRs of the 123 MeJA-inducible genes that were suppressed by SA (Supplemental Table S2), using the visualization tool of the web-based application Athena (http://www.bioinformatics2.wsu.edu/cgi-bin/Athena/cgi/home.pl) (O’Connor et al., 2005). The Athena program identified two motifs as being significantly enriched in the promoters of these genes: the I box motif (GATAAG), and
Figure 4.5. Proportion of SA/JA cross-talk genes among attacker-responsive genes. The outer left pie chart represents the total number of MeJA-responsive genes identified in this study, and the proportion of genes that is antagonized by SA (Student’s t test: p<0.05, 17% in yellow; 0.05<p<0.1, 12% in light yellow) or that is not significantly affected by SA (blue). The other pie charts represent MeJA-responsive genes that are also responsive to the JA-inducing attackers P. syringae, F. occidentalis, P. rapae, or A. brassicicola. Lists of attacker-responsive genes are taken from De Vos et al., (2005). Depicted is the percentage of MeJA-responsive genes that are antagonized by addition of SA (p<0.05, yellow; 0.05<p<0.1, light yellow) and that is not significantly affected by SA (blue).
Pierisrapae
MeJA Pseudomonassyringae
Alternariabrassicicola
Frankliniellaoccidentalis
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the GCC-box motif ((A)GCCGCC), suggesting a putative role for these motifs in SA/JA cross-talk.
The I box motif is a cis-acting element that is found in the promoters of light-regulated and circadian clock-controlled plant genes (Borello et al., 1993). However, this motif was also found to be overrepresented in MeJA-inducible genes that were not suppressed by SA. Therefore, we employed the promoter bootstrapping program POBO, that allows a three-way comparison between two clusters of coregulated genes and the genomic background, to visualize the overrepresentation of the I box motif (Kankainen and Holm, 2004). The I box motif was found to be even more overrepresented in SA/JA cross-talk genes than in the MeJA-inducible genes that were not suppressed by SA (Figure 4.6A). Therefore, we further investigated the involvement of this promoter element in the regulation of SA/JA cross-talk. The promoter of the PDF1.2 gene contains a single I box at position –188 to –183 relative to the ATG start codon. Using site-directed mutagenesis, we knocked out this I box motif (GATAAG changed into GAATTC), and fused the mutated PDF1.2∆Ibox promoter to the ß-glucuronidase (GUS) reporter gene. Four independent transgenic PDF1.2∆Ibox::GUS lines were treated with SA, MeJA, or a combination of both chemicals and evaluated for GUS and endogenous PDF1.2 expression. Figure 4.6B shows the relative GUS and PDF1.2 transcript levels in Col-0 and a representative transgenic line. Clearly, the MeJA-induced activation of PDF1.2 and PDF1.2∆Ibox was equally sensitive to suppression by SA, indicating that the I box motif is not an essential regulatory element in the SA/JA antagonism.
The GCC box provides a binding site for members of the family of AP2/ERF transcription factors (Fujimoto et al., 2000). Mutation of the GCC box in the PDF1.2 promoter demonstrated that this motif is essential for induction of the PDF1.2 gene by
Figure 4.6. The I box motif in the PDF1.2 promoter is not essential for SA/JA cross-talk. (A) Frequency distribution of the I box motif in the promoter sequences of MeJA-inducible and SA/JA cross-talk genes. Occurrence of the I box motif was determined in the 1-kb sequences upstream of the 5’-UTR of the genes using POBO bootstrapping analysis (Kankainen and Holm, 2004). The promoters of the MeJA-inducible genes that were not affected by SA (dashed black) and the MeJA-inducible genes that were suppressed by SA (dashed grey) were compared to randomly selected promoter sequences from the Arabidopsis genome (solid black). (B) Northern blot analysis of PDF1.2 and PDF1.2∆Ibox::GUS gene expression. Five-week-old plants were treated with 1 mM SA, 0.1 mM MeJA, or a combination of both chemicals. Leaf tissue was harvested 24 h after chemical treatment. Equal loading of RNA samples was checked using a probe for 18S rRNA. Signal intensities were quantified using a Phosphor imager. PDF1.2 and GUS transcript levels in the single MeJA treatments were set to 100%.
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MeJA and ET (Brown et al., 2003). Therefore, this motif provides an attractive target for SA-mediated regulation of cross-talk, either through interference with binding of positive regulators of JA-responsive gene expression, or through direct competition for binding sites. Expression of several ERF transcription factor genes was found to be affected by SA/JA cross-talk. ERFs that were induced by MeJA and suppressed by the addition of SA are ERF4, ERF1, ERF107, and ERF008. ERFs that were repressed by MeJA and upregulated by addition of SA are ERF014, ERF034, and ERF119 (Supplemental Table S2; Nakano et al., 2006). Since mutation of the GCC box abolishes MeJA-inducible PDF1.2 expression (Brown et al., 2003), an antagonistic effect of SA cannot be readily determined. Future research using the GCC box fused to a minimal promoter and a reporter gene should elucidate the importance of this motif in SA-mediated suppression of JA-responsive gene expression.
D I S C U S S I O N
Plant defense signaling pathways consist of intricate regulatory networks rather than linear signal transduction pathways. Through cross-talk between stress response pathways, plants are able to tailor their defense reaction to the particular attacker encountered. Elucidation of the mechanisms underlying pathway cross-talk will contribute significantly to our understanding of the complexity of plant defense regulation (Pieterse and Dicke, 2007; Robert-Seilaniantz et al., 2007; Koornneef and Pieterse, 2008). Here, we employed a pharmacological approach to mimic the antagonistic interaction between SA- and JA-dependent defense signaling pathways and investigated the role of several putative signal transduction components in the regulation of SA/JA cross-talk.
Since the SA signaling steps leading to SAR and SA/JA cross-talk involve both redox changes (Mou et al., 2003; Chapter 3) and activation of NPR1 (Spoel et al., 2003; Chapter 2), we first tested the role of further SAR signaling components in SA/JA cross-talk. We showed that mutations in genes encoding NIMIN1 and proteins from the secretory pathway, which were previously shown to play a role in SA-based resistance (Wang et al., 2005; Weigel et al., 2005), are not required for SA-mediated downregulation of MeJA-inducible expression of PDF1.2 (Figure 4.1A). These results indicate that the SA signaling pathways leading to SAR and SA/JA cross-talk diverge downstream of NPR1. This corroborates previous findings that nuclear localization of NPR1, which is required for SA-induced expression of PR-1 and SAR, is not essential for the antagonistic effect of SA on MeJA-induced expression of PDF1.2 (Spoel et al., 2003; Chapter 2).
Mutation of the WRKY70 transcription factor, which was previously shown to function as a node of convergence between SA and JA signaling (Li et al., 2004; Li et al., 2006), did not affect SA-mediated suppression of MeJA-induced PDF1.2 expression (Figure 4.1A). This demonstrates that, although the balance between SA and JA pathways is altered in this mutant (Li et al., 2006), the molecular mechanism leading to SA/JA cross-
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talk is still functional. SA/JA cross-talk was impaired in the tga2-1 tga3-1 tga5-1 tga6-1 quadruple mutant (Figure 4.1A), suggesting that TGA transcription factors play a role in the SA-mediated suppression of PDF1.2. Previously, Ndamukong et al. (2007) tested the TGA triple mutant tga2-1 tga5-1 tga6-1 for SA/JA cross-talk with similar results. How TGAs exert their antagonistic effect on JA-responsive genes, such as PDF1.2 is unknown. TGA factors have been shown to interact with NPR1 in the nucleus to activate PR-1 gene expression (Després et al., 2000; Fan and Dong, 2002). However, in view of the cytosolic role of NPR1 in SA/JA cross-talk, an indirect interaction of NPR1 with TGAs seems more likely. Although TGA factors can bind to the TGACG element in the PDF1.2 promoter both in vitro and in vivo (Spoel et al., 2003; Chapter 2; Ndamukong et al., 2007), removal of this promoter element did not abolish SA-mediated suppression of the PDF1.2 gene (Spoel et al., 2003; Chapter 2). Therefore, TGAs are likely to antagonize PDF1.2 expression indirectly, perhaps by regulating the genes that encode the transcriptional regulators of PDF1.2.
Analysis of the sni1 npr1-1 double mutant revealed that, in some cases, SA is able to suppress MeJA-inducible PDF1.2 in the npr1-1 background (Figure 4.1A), suggesting that the sni1 mutation overrules the NPR1-dependency of the SA-mediated suppression of this gene. Since we had indications that high ET levels can overrule the NPR1-dependency of SA/JA cross-talk in Col-0 wild-type plants (A. Leon-Reyes and C.M.J. Pieterse, unpublished results), we further investigated the NPR1-independent SA/JA cross-talk by measuring ET levels emitted by the sni1 npr1-1 mutant. Indeed, we found that sni1 npr1-1 emitted 50% more ET than npr1-1 (Figure 4.1B). Hence, the enhanced ET emission by sni1 npr1-1 may be sufficient to overcome the NPR1-dependency of SA-mediated suppression of PDF1.2. Mutations in the SNI1 protein cause decreased leaf size and altered leaf texture (Li et al., 1999), which may relate to the enhanced ET emission.
Our approach to investigate whether chromatin remodeling is involved in the regulation of SA/JA cross-talk did not point to an important role for this process. Histone acetylation increases the accessibility of the transcriptional machinery to promote gene expression, and acetylation of histone H3 is considered to be a marker for a transcriptionally active state (Strahl and Allis, 2000; Pfluger and Wagner, 2007). The level of AcH3 at the PDF1.2 promoter was comparable in the wild-type and mutant npr1-1 background, and did not change considerably in response to chemical treatment (Figure 4.2B). Hence, our results suggest that histone modifications are not involved in SA/JA cross-talk. However, conclusive evidence should be provided by additional experiments and include analysis with antibodies directed against multiple histone modifications. In our ChIP experiments we did observe an SA-mediated increase in AcH3 at the SA-responsive PR-1 promoter, which confirmed previous findings (Butterbrodt et al., 2006; Mosher et al., 2006). This increase was absent in the npr1-1 background, suggesting that histone modifications are necessary to initiate PR-1 transcription. However, the combined treatment of SA and MeJA strongly reduced AcH3 association at the PR-1 promoter in Col-0, compared to SA treatment alone, without affecting PR-1 expression (Figure 4.2B). Therefore, the chromatin
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structure around the PR-1 promoter appears to be sufficiently relaxed to allow access to the transcriptional machinery. This notion is supported by data on the epigenetic regulation of the WRKY70 transcription factor (Alvarez-Venegas et al., 2007). It was suggested that the chromatin structure of WRKY70 target genes, such as PR-1, is sufficiently open to allow a fast response to stimuli. Consequently, by epigenetic regulation of a single transcription factor, the expression of a large number of genes can be affected, thus significantly increasing the regulatory potential of chromatin remodeling (Alvarez-Venegas et al., 2007).
In order to gain insight into transcriptional changes induced by SA and MeJA treatment, we monitored gene expression of all 23,750 Arabidopsis genes, using the Affymetrix ATH1 GeneChip. The effect of SA and (Me)JA on gene expression has been analyzed in several microarray studies in Arabidopsis and sorghum (Schenk et al., 2000; Salzman et al., 2005). In addition, global expression phenotyping of signaling-defective mutants of SA and JA pathways has been exploited to investigate the network of regulatory interactions among different defense signaling pathways (Glazebrook et al., 2003). These expression profiling studies revealed one-way and mutual antagonism as well as synergistic effects between SA- and JA-dependent signaling pathways. We identified 731 genes that were significantly induced by MeJA, 123 of which were significantly downregulated by SA (Figure 4.4A; Supplemental Tables S1 and S2). Sixty percent of these SA/JA cross-talk genes were suppressed by SA in an NPR1-dependent manner. In silico analysis of the 1-kb promoter region of the 123 MeJA-inducible genes that were suppressed by SA revealed that the I box motif (GATAAG) and the GCC box motif ((A)GCCGCC) were significantly overrepresented, suggesting that these elements are involved in the regulation of the SA/JA antagonism. However, site-directed mutagenesis of the I box motif in the PDF1.2 promoter did not alter the response to either SA, MeJA, or both chemicals, demonstrating that the I box motif is not essential for cross-talk (Figure 4.6B). In contrast, the GCC box is an interesting candidate for cross-talk regulation, as this element is essential for MeJA-responsiveness of the PDF1.2 promoter (Brown et al., 2003).
Previously, PDF1.2 promoter-deletion constructs fused to the GUS reporter gene were tested for their ability to show SA/JA cross-talk. All constructs tested were susceptible to SA-mediated suppression, except for the construct lacking the GCC box and the first 304 bp upstream of the ATG start codon (which includes the I box) (Spoel et al., 2003; Chapter 2). This supports the hypothesis that SA-mediated suppression of PDF1.2 expression is targeted through the GCC box. Members of the family of AP2/ERF transcription factors bind to the GCC box and have been shown to function in plant defense signaling (Fujimoto et al., 2000; Berrocal-Lobo et al., 2002; Berrocal-Lobo and Molina, 2004; McGrath et al., 2005). Several ERFs are present in the group of MeJA-induced genes that are suppressed by SA and also in the group of MeJA-repressed genes that are induced by SA (Supplemental Table S2). Thus, SA may indirectly suppress MeJA-responsive GCC box-containing genes by affecting the expression of genes that encode transcription factors that target the GCC box in JA-responsive genes. However, not all identified cross-talk genes contain a GCC box. Previously, a tomato ERF, Pti4, was shown to bind to both GCC box- and non-GCC
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box-containing promoters and influence their expression, which increases the regulatory potential of this ERF transcription factor (Chakravarthy et al., 2003).
In addition to the antagonistic effect of SA on MeJA-inducible genes, and antagonistic effect of SA was also noted on MeJA-repressed genes. The group of 135 MeJA-repressed genes of which the suppression was relieved by SA contained a large number of genes that were predicted to encode chloroplast-targeted proteins. These genes have functions related to photosynthesis and chlorophyll constitution, indicating that MeJA downregulates light-related gene expression, which is consistent with previous observations (Schenk et al., 2000; Wierstra and Kloppstech, 2000; Zhai et al., 2007). Biosynthesis of SA and JA originates in the chloroplast (Wildermuth et al., 2001; Wasternack, 2007). It was hypothesized that the similar compartmentalization of steps in SA and JA production may facilitate antagonism between these signaling molecules. The mutant ssi2 is disturbed in its plastidial fatty acid composition, which results in overexpression of SA-responsive PR-1 and suppression of JA-responsive PDF1.2 (Kachroo et al., 2003), supporting a possible role for the chloroplast in mediating SA/JA cross-talk.
In conclusion, we demonstrated that 1) the SA signaling pathways leading to SAR and SA-mediated suppression of JA-responsive gene expression are partly divergent, 2) TGA transcription factors play a role in the regulation of SA/JA cross-talk, 3) chromatin remodeling is unlikely to play a dominant role in the regulation of SA/JA cross-talk, 4) SA/JA cross-talk acts on both induced and repressed genes, 5) SA/JA cross-talk is only partly dependent on NPR1, and 6) the GCC box in the promoters of JA-responsive genes is a candidate target for SA/JA cross-talk.
M AT E R I A L S A N D M E T H O D S
C u l t i v a t i o n o f p l a n t s
Seeds of Arabidopsis thaliana mutants npr1-1, tga2-1 tga3-1 tga5-1 tga6-1, sni1 npr1-1, sec61α, dad1, bip2, sec61α bip2, and dad1 bip2 were kindly provided by Xinnian Dong (Duke University, Durham, USA). Mutant nimin1-1 seeds were kindly provided by Ralf Weigel (Georg-August University, Göttingen, Germany), and mutant sid2-1 by Christiane Nawrath (University of Fribourg, Fribourg, Switzerland). These seeds, and wild-type Col-0 (N1092), transgenic NahG, and the SALK insertion line wrky70-1 (SALK_025198) were sown in quartz sand. Two weeks later, seedlings were transferred to 60-mL pots containing a sand-and-potting soil mixture (5:12 v/v) that was autoclaved twice for 20 min with a 24-h interval. Plants were cultivated in a growth chamber with an 8-h day (200 μE/m2/s at 24°C) and 16-h night (20°C) cycle at 70% relative humidity for another 3 weeks. Plants were watered every other day and received half-strength Hoagland solution (Hoagland and Arnon, 1938) containing 10 μM Sequestreen (CIBA-Geigy, Basel, Switzerland) once a week.
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C h e m i c a l i n d u c t i o n
Induction treatments were performed by dipping the leaves of 5-week-old plants in an aqueous solution containing 0.015% (v/v) Silwet L-77 (Van Meeuwen Chemicals BV, Weesp, The Netherlands), supplemented with 1 mM SA (Mallinckrodt Baker, Deventer, The Netherlands), or 0.1 mM MeJA (Serva, Brunschwig Chemie, Amsterdam, The Netherlands), or a combination of both chemicals. Control plants were treated with 0.015% Silwet L-77 only. MeJA was added to the medium from a 1000-fold stock solution in 96% ethanol. Solutions without MeJA were supplemented with equal amounts of ethanol. Leaf tissue was harvested 24-28 h after induction treatment and immediately frozen in liquid nitrogen.
R N A e x t r a c t i o n a n d n o r t h e r n b l o t a n a l y s i s
Total RNA was extracted as described previously (De Vos et al., 2005). For northern blot analysis, 15 μg RNA was denatured using glyoxal and dimethyl sulfoxide (Sambrook et al., 1989), electrophoretically separated on a 1.5% agarose gel, and blotted onto Hybond-N+ membrane (Amersham, ‘s-Hertogenbosch, The Netherlands) by capillary transfer. The electrophoresis and blotting buffer consisted of 10 and 25 mM sodium phosphate (pH 7.0), respectively. Northern blots were hybridized with gene-specific probes for PR-1, PDF1.2 and GUS (ß-glucuronidase) as described previously (Pieterse et al., 1998). After hybridization with α-32P-dCTP-labeled probes, blots were exposed for autoradiography and signals quantified using a BioRad Molecular Imager FX (BioRad, Veenendaal, The Netherlands) with Quantity One software (BioRad, Veenendaal, The Netherlands). To check for equal loading, the blots were stripped and hybridized with a probe for 18S rRNA. The AGI numbers of the genes studied are At2g14610 (PR-1) and At5g44420 (PDF1.2). The probe for 18S rRNA was derived from an Arabidopsis cDNA clone (Pruitt and Meyerowitz, 1986). All gene expression analyses were repeated 2-15 times with comparable results. Statistical analysis was performed using a Kruskal-Wallis test with post-hoc correction for multiple comparisons.
E t h y l e n e m e a s u r e m e n t
Leaf rosettes of 5-week-old uninduced npr1-1 and sni1 npr1-1 plants were detached from the roots, weighed, and placed individually in 35-mL gas-tight serum flasks (n=10) that were subsequently incubated under climate chamber conditions. At intervals over a 77-h time period, 1-mL gas samples were withdrawn through the rubber seal. Ethylene emission was determined by gas chromatography as described previously (De Laat and Van Loon, 1982).
C h r o m a t i n i m m u n o p r e c i p i t a t i o n
Leaves of Col-0 and npr1-1 plants were mock-treated or treated with SA, MeJA, or a combination of both chemicals as described above. Chromatin immunoprecipitation (ChIP) analysis was performed as described by Ndamukong et al. (2007). Immunoprecipitation was carried out using an AcH3 antibody that specifically recognizes acetylated histone H3
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(06-599; Upstate, Lace Placid, USA). After purification, DNA was resuspended in 50 µL (immunoprecipitated DNA) or 150 µL (input control) of milliQ water.
Q u a n t i t a t i v e r e a l - t i m e P C R a n a l y s i s
Q-RT-PCR analysis was performed in optical 96-well plates with a MyIQTM Single Color Real-Time PCR Detection System (Bio-Rad, Veenendaal, The Netherlands), using SYBR® Green to monitor dsDNA synthesis. Each reaction consisted of 1.5 μL immunoprecipitated or input DNA, 0.5 μL of each of the two gene-specific primers (10 pmol/μL), and 5 μL 2x IQ SYBR® Green Supermix reagent (Bio-Rad, Veenendaal, The Netherlands) in a final volume of 18 µL. Primer sequences used were: PDF1.2 Fw1 (5’ TTC AGT AAT AGG TGT GTC CCA GG 3’) and PDF1.2 Rv1 (5’ GCG GCT GGT TAA TCT GAA TGG 3’), PR-1 Fw (5’ TCG GTC CCT AGA GTT TTT CAA 3’) and PR-1 Rv (5’ CCG CCA CAT CTA TGA CGT AAG 3’), UBQ10 Fw (5’ TTG CCA ATT TTC AGC TCC AC 3’) and UBQ10 Rv (5’ TGA CTC GTC GAC AAC CAC AA 3’), and GAPDH Fw (5’ GCA AAG CTC ATT GGC TGT CA 3’) and GAPDH Rv (5’ GGA AAC TAA TGG CGC TTG GA 3’). The following PCR program was used: 95°C for 6 min; 40 cycles of 95°C for 25 s, 60°C for 35 s, and 72°C for 30 s. C
T (threshold cycle) values were calculated using Optical
System Software, version 1.0 for MyIQTM (Bio-Rad, Veenendaal, The Netherlands). The amount of immunoprecipitated DNA was calculated relative to the input control using the 2-ΔC
T method. The CT values of input and immunoprecipitated DNA were averaged before
performing the ΔCT calculation, and the variance estimated from the replicate C
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was carried through to the final calculation of relative quantities using standard propagation of error methods. The error was estimated by calculating the 2-ΔC
T term using ΔC
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standard deviation and ΔCT minus the standard deviation (Livak and Schmittgen, 2001).
Next, the amount of immunoprecipitated DNA was corrected for dilution factors, and control-treated samples were set at 1.
S a m p l e p r e p a r a t i o n a n d m i c r o a r r a y d a t a c o l l e c t i o n
For isolation of RNA, whole rosettes from Col-0 and npr1-1 plants were mock-treated or treated with SA, MeJA, or a combination of both as described above. Leaf tissue was harvested at approximately 28 h and immediately frozen in liquid nitrogen. RNA was prepared from three independent biological experiments, each consisting of three to ten plants per treatment, as described above, and purified using RNeasy Plant Mini Kit columns (Quiagen Benelux BV, Venlo, The Netherlands). RNA samples were analyzed for quality by capillary electrophoresis, using an Agilent 2100 Bioanalyzer system. Synthesis of cRNA probes, hybridization to ATH1 Affymetrix GeneChips, and collection of data from the hybridized GeneChips were carried out by ServiceXS (Leiden, The Netherlands) and the Affymetrix service station of Leiden University Medical Center, where they passed all internal quality checks. Hybridizations with labeled cRNAs were conducted with Arabidopsis ATH1 full-genome GeneChips (Affymetrix, Santa Clara, USA), containing a
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total of 22,810 probe sets representing approximately 23,750 Arabidopsis genes (Redman et al., 2004).
E x p r e s s i o n p r o f i l i n g a n d p r o m o t e r a n a l y s i s
GeneChip Operating Software (GCOS; Affymetrix, Santa Clara, USA) was used to globally normalize the expression data on each GeneChip to an average of 200 so that hybridization intensity of all 24 chips was equivalent. In addition, expressed genes were identified by GCOS, which uses statistical criteria to generate a “present” or “absent” call for genes represented by each probe set on the array. For analysis of differentially expressed genes, log2-transformed expression values of three independent biological experiments were compared between treatments using Student’s t test. Furthermore, the expression level in the induced treatment had to be significantly detectable (present call generated by GCOS) in all three biological replicates. False discovery rate (FDR) correction was applied to account for testing of multiple genes. All q values were <0.06 and therefore acceptable for inclusion in the analysis. To identify overrepresented promoter elements in clusters of coregulated genes, the visualization tool of the web-based application Athena was employed (http://www.bioinformatics2.wsu.edu/cgi-bin/Athena/cgi/ home. pl) (O’Connor et al., 2005) using default settings. In addition, the promoter bootstrapping program POBO was used (Kankainen and Holm, 2004) with the following parameter settings: number of pseudoclusters: 1000, number of promoters in the pseudoclusters: 123.
C o n s t r u c t i o n o f I b o x k n o c k o u t l i n e s
The 1.2-kb PDF1.2 promoter fragment was amplified by PCR from genomic DNA of Col-0 plants using the PDF1.2 Fw2 (5’ GCG AAT TCA TGC ATG CAT CGC CGC ATC G 3’) and PDF1.2 Rv2 (5’ CGC TCG AGA TGA TTA TTA CTA TTT TGT TTT C 3’) primers. The PDF1.2 promoter fragment was first cloned into the pCR-Blunt II-TOPO vector for direct insertion of blunt-end PCR products into a plasmid vector (Invitrogen, Breda, The Netherlands). The I box motif (5’ GATAAG 3’) was mutagenized to an EcoRI recognition sequence (5’ GAATTC 3’) to facilitate identification of mutagenized transformants. Site-directed mutagenesis was carried out by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, USA) according to the manufacturer’s protocol. Primers ΔIbox Fw (5’ CAA CAA ACA AAA AGC AAG ATG AAT TCT TTT GAT ATT GGC TAC GGG 3’) and ΔIbox Rv (5’ CCC GTA GCC AAT ATC AAA AGA ATT CAT CTT GCT TTT TGT TTG TTG 3’) were designed that introduced the desired mutation. Transformants were selected by digestion with EcoRI. After sequence verification, the mutated PDF1.2∆Ibox promoter fragment was ligated into the pGREENII 0229-GUS binary vector (Hellens et al., 2000), using the SpeI and PstI recognition sites. The plasmid was transformed into Agrobacterium tumefaciens strain C58 (pMP90) (Koncz and Schell, 1986). Col-0 plants were transformed using the floral dip method as described by Clough and Bent (1998), and surface-sterilized seeds of transformants were selected on Murashige and Skoog medium (1962) supplemented with 1% (w/v) sucrose, 0.6% (w/v)
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8 4 • Towards the ident i f i ca t ion of nove l key p layer s of c ros s - ta lk
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plant agar, pH 5.7, and 20 mg/L DL-phosphinothricin (Duchefa Biochemie BV, Haarlem, The Netherlands) as a selection marker.
S U P P L E M E N TA RY M AT E R I A L
S u p p l e m e n t a l Ta b l e S 1 .
MS Excel file with normalized expression levels, fold-change information, AGI numbers and TIGR annotation of the selected MeJA- and SA-responsive genes.
S u p p l e m e n t a l Ta b l e S 2 .
MS Excel file with normalized expression levels, fold-change information, AGI numbers and TIGR annotation of the selected SA/JA cross-talk genes.
S u p p l e m e n t a l Ta b l e S 3 .
MS Excel file with normalized expression levels, fold-change information, AGI numbers and TIGR annotation of the selected NPR1-dependent and –independent SA/JA cross-talk genes.
A l l s u p p l e m e n t a r y m a t e r i a l c a n b e d o w n l o a d e d f r o m :
http://www.bio.uu.nl/~fytopath/GeneChip_data.htm
A C K N O W L E D G M E N T SThe authors would like to thank Ruth Joosten and Reinier Snetselaar for technical assistance. We thank Jurriaan Ton, Saskia van Wees, Bas van Breukelen, and Paul Westers for useful discussions and help with the statistical analysis. This research was supported by grants 813.06.002 and 865.04.002 of the Earth and Life Sciences Foundation (ALW), which is subsidized by The Netherlands Organization of Scientific Research (NWO).
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C H A P T E R 5
Development of a reporter system
to identify regulators of cross-talk
between sal icylate and jasmonate
signaling pathways in Arabidopsis
Annemart Koornneef, Adriaan Verhage, Antonio Leon-Reyes, Reinier Snetselaar, L.C. Van Loon, and Corné M.J. Pieterse
Graduate School Experimental Plant Sciences, Plant-Microbe Interactions,
Institute of Environmental Biology, Faculty of Science, Utrecht University,
P.O. Box 800.56, 3508 TB Utrecht, The Netherlands
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Chapter 5 • 8 7
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Development of a reporter system
to identify regulators of cross-talk
between sal icylate and jasmonate
signaling pathways in Arabidopsis
A B S T R A C T
The signaling hormones salicylic acid (SA) and jasmonic acid (JA) are regulators of inducible defenses in Arabidopsis. SA has an antagonistic effect on JA signaling, a process called “cross-talk”, which is believed to provide a fine-tuning mechanism for activating optimal defense responses against particular attackers. SA-mediated suppression of the JA-responsive PDF1.2 promoter was exploited for setting up a genetic screen aiming at the isolation of signal transduction mutants that are impaired in this cross-talk mechanism. The PDF1.2 promoter was fused to the herbicide resistance gene BAR to allow for life/death screening of a population of mutagenized transgenic plants. Non-mutant plants should survive herbicide treatment when methyl jasmonate (MeJA) is applied, but the combined treatment of SA and MeJA with the herbicide should be lethal. Conversely, crucial SA/JA cross-talk mutants should survive the combination treatment. Effective suppression of the PDF1.2 promoter by SA was observed at the mRNA level. However, the suppression of the BAR resistance gene did not result in suppression of herbicide resistance in either plate- or soil-grown plants. A screening method based on quantitative differences in the expression of a reporter gene may be better suited to identify SA/JA cross-talk mutants.
I N T R O D U C T I O N
Arabidopsis thaliana has been adopted as a model organism for biologists worldwide, rendering a wealth of well-characterized mutants (Meinke et al., 1998). The Arabidopsis genome was sequenced in 2000, and at present projects are underway to determine the function of all ~25.500 genes by 2010 (Somerville and Koornneef, 2002). How these genes are regulated is a further question to be addressed. It has become increasingly clear that gene regulation through signal transduction pathways is subject to regulatory networks, rather than linear pathways. Therefore, multiple factors can contribute to the final outcome of a gene-regulated
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process. The major focus of future Arabidopsis research will be to understand the dynamic properties of the genes and networks that control plant functioning. For the dissection of such complex pathways, the identification of mutants in regulatory components remains essential.
In the regulation of plant defense signaling networks, the phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) have been found to play essential roles in orchestrating defense responses against a wide variety of attackers (Howe, 2004; Pozo et al., 2004; Grant and Lamb, 2006; Van Loon et al., 2006; Von Dahl and Baldwin, 2007). Their effectiveness against different types of pathogens generally differs depending on the type of interaction: pathogens with a biotrophic lifestyle are resisted mainly through SA-dependent defense reactions, whereas necrotrophic pathogens and herbivorous insects are predominantly suppressed by JA- and ET-dependent defenses (Kessler and Baldwin, 2002; Glazebrook, 2005). Depending on the attacker encountered, a highly specific blend of phytohormones is produced (De Vos et al., 2005). Cross-communication between defense signaling pathways allows the plant to fine-tune its defense response, and to preserve energy by regulated activation of costly defenses. This is exemplified by the cross-communication between SA- and JA-dependent defense signaling pathways. These routes are considered mutually antagonistic (Pieterse et al., 2001; Kunkel and Brooks, 2002; Glazebrook et al., 2003; Rojo et al., 2003; Bostock, 2005; Beckers and Spoel, 2006; Koornneef and Pieterse, 2008), although exceptions have been noted (Schenk et al., 2000; Mur et al., 2006).
Suppression of JA responses by induction of SA-responsive defenses has been described in numerous studies and is operative among all Arabidopsis accessions studied, demonstrating that this is a highly conserved regulatory phenomenon (Pieterse et al., 2001; Spoel et al., 2003; Chapter 2 and 3). However, the underlying molecular mechanism remains to be elucidated. Previously, SA-activated NPR1 was found to be a key regulator in the suppression of JA-responsive genes (Spoel et al., 2003; Chapter 2). In addition, several other factors have been identified as molecular players in SA/JA cross-talk, including the transcription factor WRKY70 and the glutaredoxin GRX480 (Li et al., 2004; Ndamukong et al., 2007). However, their positions and interactions in the ‘cross-talk signaling network’ are mostly unclear.
In plant signal transduction research, mutants are often the key to unraveling important steps in the signaling pathway of interest. So far, no mutant screens have been performed with the specific aim to unravel the molecular mechanism underlying SA/JA cross-talk. Hence, an approach specifically targeting cross-talk mutants will be highly instrumental in the identification of signaling components that act downstream of SA-activated NPR1 in the antagonism on JA signaling in Arabidopsis. This type of mutant screen has the advantage that it provides a non-biased selection method. It allows for specific cross-talk regulators to be identified, whereas most previous cross-talk mutants were selected based on altered SA-responsive phenotypes. Characterization of such cross-talk mutants will contribute significantly to the unraveling of the antagonism between SA- and JA-dependent defense signaling pathways. In the present chapter, we describe attempts to develop a system
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Chapter 5 • 8 9
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that allows efficient screening of mutants in which the suppression of JA-induced gene expression by SA is abolished.
R E S U LT S
E x p e r i m e n t a l s e t - u p
The SA/JA antagonism is manifested when Arabidopsis plants are exposed simultaneously to SA and methyl jasmonate (MeJA). Whereas MeJA treatment alone induces JA-responsive genes, such as PDF1.2, the combined treatment of SA and MeJA results in suppression of this gene (Spoel et al., 2003; Chapter 2). We have shown previously that this mechanism is robust (Chapter 3) and can, therefore, be exploited to develop a mutant screen. However, screening of mutants disturbed in the suppression of JA-responsive genes by SA application is laborious. Therefore, we set out to develop a mutant screen based on selection for herbicide resistance. In order to generate Arabidopsis plants that are resistant to the broad-spectrum herbicide Finale (other trade names include Basta, Ignite, Liberty) in a JA-dependent manner, we devised a construct of the BIALOPHOS RESISTANCE (BAR) gene under transcriptional control of the JA-responsive PDF1.2 promoter. The promoter of the plant defensin PDF1.2 is well-characterized and its expression can be induced by pathogen challenge, JA, and ET (Penninckx et al., 1996; Manners et al., 1998; Penninckx et al., 1998; Brown et al., 2003). It is commonly used as a marker gene for JA-dependent defenses, as well as a parameter for SA-antagonized JA responses (Penninckx et al., 1996; Spoel et al., 2003; Chapter 2; Ndamukong et al., 2007; Spoel et al., 2007). The BAR gene encodes a phosphinothricin acetyltransferase, which detoxifies phosphinothricin (PPT), the active ingredient in the herbicide Finale. The toxicity of this herbicide is based on blockage of glutamine synthetase, resulting in a rapid build-up of ammonia, a decline in photosynthesis, and death of the plant within a few days after herbicide treatment (De Block et al., 1987).
By placing the herbicide resistance gene under the control of the JA-responsive promoter, one should be able to control plant survival by applying Finale, with or without MeJA. In the absence of Finale, these plants will function normally. However, application of Finale will kill these plants, unless PDF1.2::BAR is activated by exogenous application of MeJA. Inclusion of SA in the chemical treatment will suppress MeJA-induced activation of the PDF1.2 promoter and, thus, these plants will retain their sensitivity to Finale. Hence, treatment of PDF1.2::BAR plants with a mixture of MeJA and SA, followed by application of Finale will be lethal as a result of the cross-talk between SA and JA signaling. Ethyl methane sulfonate (EMS) mutagenesis of this transgenic line and screening of the M2 population after MeJA and SA and Finale treatment should yield cross-talk mutants that are resistant to the herbicide, indicating loss of SA/JA cross-talk (Figure 5.1). Mutants that survive a second round of selection should be tested for a functional SA signal transduction pathway to exclude indirect effects on SA/JA cross-talk due to non-responsiveness to the SA treatment. The corresponding gene(s) can then be mapped, cloned, and further
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characterized. The mutants should be tested further for resistance against microbial pathogens and herbivorous insects.
C l o n i n g o f t h e P D F 1 . 2 : : B A R c o n s t r u c t
a n d s e l e c t i o n o f t r a n s f o r m a n t s
The 35S promoter of Cauliflower mosaic virus (CaMV 35S) was excised from the pCAMBIA 3200 binary vector by partial digestion with XhoI and full digestion with EcoRI, and replaced by a 1.2-kb PDF1.2 promoter fragment. Wild-type Arabidopsis Col-0 plants were transformed with this construct using the floral dip method (Clough and Bent, 1998). Transformants were selected on medium supplemented with 20 μM MeJA to induce the BAR gene, and 20 mg/L PPT. Surviving plants were transferred to soil and selfed to produce homozygous offspring. Segregation analysis of the T2 generation was used to determine the number of inserts in independent transformants. We selected line 15 of PDF1.2::BAR for further analysis. Figure 5.2 shows the presence of a 268-bp BAR fragment in genomic DNA amplified from this line. Non-transformed Col-0 DNA was included as a negative control. A fragment of a ß-TUBULIN gene was amplified to check for intactness of the genomic DNA. Growth of T2 plants on medium supplemented with 20 μM MeJA and 20 mg/L PPT yielded 206 healthy and 79 dead plants, consistent with a 3:1 segregation ratio (χ2 = 1.124, p=0.289), indicative of a single insertion event in this transgenic line.
D e t e r m i n i n g o p t i m a l c o n d i t i o n s f o r S A - m e d i a t e d
s u p p r e s s i o n o f P D F 1 . 2 : : B A R
The selected PDF1.2::BAR line was tested for expression of cross-talk by a foliar drench with SA, MeJA, or a combination of both chemicals. Thereupon, the expression patterns of the SA-responsive PR-1 and MeJA-responsive PDF1.2 marker genes and the MeJA-responsive BAR gene were assessed. The BAR gene was expected to behave like the intrinsic PDF1.2 gene and, thus, to be induced by MeJA treatment and suppressed by additional treatment with SA. Figure 5.3A shows that the PR-1 and PDF1.2 marker genes
Figure 5.1. Experimental set-up for an SA/JA cross-talk mutant screen.Transgenic PDF1.2::BAR plants survive Finale treatment when the BAR resistance gene under control of the PDF1.2 promoter is induced by MeJA, but die after combined treatment with SA, MeJA, and Finale due to SA/JA cross-talk. Conversely, a cross-talk mutant disrupted in the suppression of JA signaling by SA survives the combined treatment of SA, MeJA, and Finale.
PDF1.2::BAR
cross-talk mutant
- - +-
SA
Finale®
MeJA + ++ + +
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Chapter 5 • 9 1
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were induced by SA and MeJA treatment, respectively, and that PDF1.2 was suppressed upon simultaneous application of SA and MeJA. The expression of the BAR gene was comparable to that of PDF1.2 in all treatments, except for a slightly higher basal expression. We tested whether this basal BAR expression affected herbicide resistance in this line, and found that the PDF1.2::BAR line was fully resistant to Finale in the absence of MeJA (Figure 5.3B). A similar constitutive herbicide resistance was found in other transformants tested (results not shown). However, the PDF1.2::BAR line had remained responsive to the SA and MeJA chemical treatments (Figure 5.3A). Importantly, application of SA suppressed the basal BAR expression, similar to the suppression of basal PDF1.2 expression by SA (Figure 5.3A). Thus, a combined treatment of SA and Finale could be expected to be sufficient to kill these transgenic plants in the absence of the PDF1.2-inducer MeJA. This would simplify the mutant selection procedure by eliminating one component necessary to perform the mutant screen.
PDF1.2::BAR seedlings were grown in the absence of MeJA for 10 days on Murashige and Skoog (1962) (MS) medium supplemented with 0.1 mM SA and 20 mg/L PPT. Col-0 and CaMV 35S::BAR plants served as negative and positive controls, respectively. All Col-0 plants were killed by the inclusion of PPT in the growth medium, whereas CaMV 35S::BAR plants grew normally. However, inclusion of SA in the growth medium caused
Figure 5.2. PCR amplification of the BAR gene in transgenic PDF1.2::BAR line 15.The BAR gene was amplified with BAR-specific primers, yielding a band of 268 bp. Primers for ß-TUBULIN (1.2 kb) were included as a control for intact genomic DNA. Non-transformed Col-0 was included as a negative control.
Mn; B-Koornneef; B 07-943; 05-02.ai
Col-0PD
F1.2
::BAR
BAR
TUB
Figure 5.3. SA- and MeJA-responsive gene expression and herbicide tolerance in PDF1.2::BAR.(A) PR-1, PDF1.2, and BAR expression in Col-0 and PDF1.2::BAR plants after foliar drench with 1 mM SA, 0.1 mM MeJA, or a combination of both chemicals. Leaf tissue was harvested 24 h after chemical treatment. Equal loading of RNA samples was checked using a probe for 18S rRNA.(B) Plant survival after Finale treatment of 5-week-old Col-0 and PDF1.2::BAR plants. Photographs were taken 1 week after herbicide treatment.
Mn; B-Koornneef; B
18S
PDF1.2PR-1
BAR
MeJASA
- - + +- - ++
- - + +- + - +
BA
PDF1.2::BARPDF1.2::BAR
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9 2 • Deve lopment of a reporter sy s tem to ident i fy c ros s - ta lk regu lator s
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stunting and inhibition of root growth (Figure 5.4). PDF1.2::BAR plants were expected to no longer show PPT resistance upon simultaneous application of PPT and SA because of the suppression of basal BAR expression by SA (Figure 5.3A). Therefore, they should behave as non-transformed Col-0 plants. However, in the presence of SA, no difference between the CaMV 35S::BAR and PDF1.2::BAR plants was detected (Figure 5.4). The growth-inhibitory effect of SA was evident, but no additional harmful effect of the toxic PPT was observed. It must be concluded that a screen for SA/JA cross-talk mutants with PDF1.2::BAR seedlings grown on MS medium is not feasible.
To investigate whether the SA-mediated suppression of BAR expression, which was observed in the PDF1.2::BAR transformants (Figure 5.3A), would prove lethal upon herbicide treatment of soil-grown plants, we proceeded to test the PDF1.2::BAR reporter line growing in potting-soil. Possibly, a single treatment with SA might circumvent the problem of growth retardation observed with plate-grown plants germinated on SA-containing medium. Col-0, PDF1.2::BAR, and CaMV 35S::BAR plants were grown for 3 weeks before receiving a 10-mL soil drench with 0, 1, or 5 mM SA. One day later, leaf tissue samples were harvested from the 0 and 5 mM SA-treated plants to assess marker gene expression, and the remaining plants were sprayed with Finale. Plant survival over the course of the next few days was recorded. The gene expression patterns of PR-1, PDF1.2, and BAR in all three genotypes are shown in Figure 5.5A. The soil drench with SA strongly induced the SA-responsive marker gene PR-1 in all three genotypes. PDF1.2 was not induced by any of the treatments, showing that the constitutive BAR gene expression in the PDF1.2::BAR line was not due to any JA-related stress response. The BAR gene in the PDF1.2::BAR line was expressed to a lower basal level than in the
Figure 5.4. Plate-grown PDF1.2::BAR plants survive SA and PPT treatment.Ten-day-old seedlings of Col-0, PDF1.2::BAR, and CaMV 35S::BAR grown on MS medium supplemented with 20 mg/L PPT, with or without 0.1 mM SA.
Finale®
Finale®
+SA
PDF1.2::BAR CaMV 35S::BAR
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Chapter 5 • 9 3
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CaMV 35S::BAR line, and its expression was completely suppressed by the application of SA (Figure 5.5A). In contrast, the SA treatment induced BAR expression to an even higher level in the CaMV 35S::BAR line compared to the control treatment. This is most likely due to the SA-responsive as-1 element that is present in the CaMV 35S promoter (Qin et al., 1994). Thus, the induction conditions were successful in suppressing BAR expression in the PDF1.2::BAR line. However, plant survival did not correlate with BAR gene expression level. As shown in Figure 5.5B, most Col-0 control plants had died from the herbicide treatment 5 days after Finale application. Application of SA had an additive toxic effect. As expected, Finale had no harmful effect on the CaMV 35S::BAR line in any of the treatments. Unfortunately, the PDF1.2::BAR line was not sensitive to Finale
either, not even after application of 5 mM SA, which strongly suppressed the expression of PDF1.2::BAR. As the strong reduction in BAR gene expression in response to SA in the PDF1.2::BAR line was not accompanied by a reduction in plant survival, this PDF1.2::BAR line proved unsuitable for the desired cross-talk mutant screen.
D e v e l o p m e n t o f a n a l t e r n a t i v e m u t a n t s c r e e n i n g s t r a t e g y
Because suppression of PDF1.2 expression by SA was apparently insufficient to allow screening for cross-talk mutants on the basis of plant survival, a screening method that is able to visualize quantitative differences might be better suited. The GUS reporter system allows relatively quick screening of gene expression patterns by histochemical staining of plant
Figure 5.5. Soil-grown PDF1.2::BAR plants survive SA and Finale treatment.(A) PR-1, PDF1.2, and BAR expression in Col-0, PDF1.2::BAR, and CaMV 35S::BAR plants after a soil drench with 0 or 5 mM SA. Leaf tissue was harvested 24 h after chemical treatment. Equal loading of RNA samples was checked using a probe for 18S rRNA.(B) Survival of Col-0, PDF1.2::BAR, and CaMV 35S::BAR plants after a soil drench with 0, 1, or 5 mM SA and treatment with Finale 1 day later. Photographs were taken 5 days after Finale treatment.
-Finale®
+Finale®
18S
PDF1.2
PR-1
BAR
SA (mM)
SA (mM)
A
B
5
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F1.2
::BAR
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AR
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CaMV 35S::BAR
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tissue. Previously, we demonstrated that the suppression of JA-responsive PDF1.2 by SA can be visualized by histochemical staining for GUS expression (Spoel et al., 2003; Chapter 2). Therefore, we sought to optimize the induction conditions for the PDF1.2 gene by fusing its promoter to the GUS reporter gene. EMS mutagenesis of a PDF1.2::GUS reporter line would be expected to yield putative cross-talk mutants, which are characterized by a blue staining, even after combined treatment with SA and MeJA. Single leaves of individual plants can be harvested and assessed for GUS staining activity. Mutants that show increased blue staining compared to the non-mutagenized parental line upon application of a soil drench with both SA and MeJA should then be retested and further analyzed. This screening method is more laborious than the herbicide-based mutant screen, but allows detection of quantitative differences in gene expression. An experimental set-up is shown in Figure 5.6. Soil drench with a control solution, 1 mM SA, 0.1 mM MeJA, or a combination of SA and MeJA was provided to induce SA and JA responses, and trigger cross-talk, respectively. As control lines, constitutive GUS-expressor PG15 and SA-responsive PR-1::GUS were included. The MeJA-inducible line PDF1.2::GUS showed clear blue staining upon MeJA treatment, which was completely suppressed upon simultaneous application of SA and MeJA. PG15 plants were stained equally in all four treatments, whereas the PR-1::GUS line was induced only in the treatments involving SA. Hence, application of this approach should lead to a workable screening method for the identification of cross-talk mutants.
Figure 5.6. GUS activity in PDF1.2::GUS and PR-1::GUS plants.Histochemical staining for GUS expression in 4-week-old PR-1::GUS, PDF1.2::GUS, and the constitutive GUS-expressor PG15 after soil drench with water, 1 mM SA, 0.1 mM MeJA, or a combination of both chemicals. Leaf tissue was harvested after 24 h and immersed in GUS staining solution. PG15 shows constitutive blue staining, whereas PR-1::GUS is induced only upon SA and SA+MeJA treatment. PDF1.2::GUS is induced by MeJA treatment, but the combined application of MeJA and SA results in complete suppression of blue staining, demonstrating the antagonism between SA and JA.
PG15
PDF1.2::GUS
PR-1::GUS
MeJASA
- - + +- + - +
Mn; B-Koornneef; B
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D I S C U S S I O N
The use of forward genetic screens has been successful in identifying and characterizing many plant genes that have essential roles in the regulation of plant defense responses (Dong, 2001; Glazebrook, 2001; Pieterse et al., 2002). Here, we attempted to develop an experimental set-up for a mutant screen to identify regulators in the cross-communication between SA- and JA-dependent defense signaling routes. We exploited the MeJA-inducibility of the PDF1.2 promoter to construct transgenic plants with MeJA-inducible resistance to the broad-spectrum herbicide Finale. Simultaneous application of SA and MeJA suppresses PDF1.2 gene expression, compared to MeJA treatment alone. Therefore, simultaneous application of SA, MeJA, and Finale should kill transgenic plants that contain the BAR herbicide resistance gene under transcriptional control of the PDF1.2 promoter. It was expected that EMS mutagenesis of this transgenic line would yield mutants that are disturbed in their cross-talk between SA and JA and, thus, survive the combined treatment with SA, MeJA, and Finale. However, the induction conditions employed for the PDF1.2::BAR line revealed a constitutive herbicide resistance of this line (Figure 5.3B). This could be due to positional effects of the transgene or to our selection procedure on medium with both MeJA and PPT, which might have favored transgenic plants showing some constitutive expression of the BAR gene.
A previously conducted genetic screen for constitutive Thi2.1-expressing mutants successfully employed the BAR resistance gene to select for EMS-treated Thi2.1::BAR plants that constitutively expressed the BAR gene (Hilpert et al., 2001). This transgenic line was constructed in such a way that plants survived herbicide treatment only when MeJA was included in the treatment to activate the MeJA-responsive Thi2.1 promoter. Thus, the induction conditions for the PDF1.2::BAR and the Thi2.1::BAR lines were based on the same principle. However, the Thi2.1::BAR lines were first selected for kanamycin resistance, and then tested for MeJA-inducibility of the BAR gene, whereas the PDF1.2::BAR lines were directly selected for PPT resistance.
Application of SA to the PDF1.2::BAR line was successful in suppressing BAR gene expression, similar to the downregulation of basal PDF1.2 expression by SA (Figures 5.3A and 5.5A). However, it did not result in death of the plants, as shown in Figures 5.4 and 5.5B. It seems, therefore, that even very low levels of BAR expression are sufficient to confer herbicide resistance. A similar observation was made when transgenic tobacco lines expressed the BAR gene under the control of the constitutive CaMV 35S promoter (De Block et al., 1987). The expression of the BAR gene varied significantly between independent transgenic lines, but lines with even the lowest expression levels were fully resistant to herbicide treatment. Alternatively, the stability of the BAR protein might be such that herbicide tolerance is conferred by BAR protein that is produced prior to SA application. However, even when SA and PPT were continuously present in the growth medium on which PDF1.2::BAR seeds had been sown, SA could not suppress the PPT resistance (Figure 5.4). Thus, the low BAR expression which remains after suppression
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by SA appears to be sufficient to confer the full herbicide tolerance that was observed. Suppression of JA-responsive genes by SA can be variable, as demonstrated previously among Arabidopsis accessions that showed suppression levels between 45 and 96% (Chapter 3). Thus, although screening for plant survival can be relatively straightforward, it did not appear to be optimal for screening for cross-talk mutants between the SA and JA signaling pathways.
In hindsight, a genetic screen based on a reporter system that allows detection of a quantitative trait might be more successful. A drawback of such an approach is the occurrence of subtle intermediate phenotypes, which require extensive re-testing. We developed such an alternative screening method based on histochemical staining for GUS expression in a PDF1.2::GUS reporter line. Soil-drench application with MeJA induces GUS expression under transcriptional control of the PDF1.2 promoter, whereas the combined treatment with MeJA and SA results in downregulation of the GUS reporter gene. Reduction in blue staining could be visualized by comparing the single MeJA treatment with the combination of SA and MeJA (Figure 5.6). EMS mutagenesis of this transgenic line should render plants that retain blue staining upon soil drenching with SA and MeJA, and these can then be tested further for disrupted SA/JA cross-talk. This screening strategy will be employed in future research. The cross-talk mutants that will be identified are to be tested for resistance against microbial pathogens and herbivorous insects as well. Presumably, the trade-off between SA-dependent pathogen resistance and JA-dependent insect defense that has been described in the literature (Thaler et al., 1999; Felton and Korth, 2000; Pieterse et al., 2001; Bostock, 2005) will be absent in these mutants. If these mutants do not show a decline in overall plant fitness, they might provide an attractive tool for breeding crops with improved resistance properties.
M AT E R I A L S A N D M E T H O D S
P l a n t m a t e r i a l a n d c u l t i v a t i o n
Seeds of Arabidopsis thaliana transgenic lines PDF1.2::GUS and PR-1::GUS in the Col-0 background were kindly provided by Johan Memelink (Leiden University, The Netherlands) and Julia Plotnikova (Massachusetts General Hospital, Boston, USA), respectively. These lines, together with the accession Col-0 and transgenic CaMV 35S::BAR and PDF1.2::BAR, as well as the constitutive ß-glucuronidase (GUS)-expressing line PG15 (Spoel et al., 2003; Chapter 2) were sown on quartz sand or on a sand-and-potting soil mixture (5:12 v/v) that was autoclaved twice for 20 min with a 24-h interval. Two weeks later the sand-grown seedlings were transferred to 60-mL pots containing the autoclaved sand-and-potting soil mixture. Plants were cultivated in a growth chamber with an 8-h day (200 μE/m2/s at 24°C) and 16-h night (20°C) cycle at 70% relative humidity for another 1-3 weeks. Plants were watered every other day and received half-strength Hoagland solution (Hoagland and Arnon, 1938) containing 10 μM Sequestreen (CIBA-Geigy, Basel, Switzerland) once
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a week. Experiments with plate-grown plants were performed on Murashige and Skoog (1962) (MS) medium supplemented with 1% (w/v) sucrose, 0.6% (w/v) plant agar, pH 5.7 (see below).
C l o n i n g o f t h e B A R c o n s t r u c t a n d p l a n t t r a n s f o r m a t i o n
The 1.2-kb fragment of the PDF1.2 promoter was amplified by PCR from genomic DNA of Col-0 plants using the PDF1.2 Fw3 (5’ GCG AAT TCA TGC ATG CAT CGC CGC ATC G 3’) and PDF1.2 Rv3 (5’ CGC TCG AGA TGA TTA TTA CTA TTT TGT TTT C 3’) primers, with added EcoRI and XhoI recognition sequences at the 5’ and 3’ ends, respectively. The PDF1.2-promoter fragment was cloned into the XhoI and EcoRI sites of pCAMBIA 3200. Using partial digestion with XhoI and full digestion with EcoRI, the pre-existing CaMV 35S-promoter fragment was deleted and replaced by the PDF1.2-promoter fragment. After sequence verification, the binary vector was transformed into Agrobacterium tumefaciens strain AGLO. Col-0 plants were transformed using the floral dip method as described by Clough and Bent (1998), and surface-sterilized seeds of transformants were selected on MS medium supplemented with 1% (w/v) sucrose, 0.6% (w/v) plant agar, pH 5.7, 20 μM MeJA (Serva, Brunschwig Chemie, Amsterdam, The Netherlands), and 20 mg/L DL-phosphinothricin (PPT; Duchefa Biochemie, Haarlem, The Netherlands). The presence of the BAR gene in the transformants was confirmed by PCR, using BAR-specific primers BAR Fw (5’ ACT TCA GCA GGT GGG TGT AGA G 3’) and BAR Rv (5’ ATC GTC AAC CAC TAC ATC GAG AC 3’). Primers for the ß-TUBULIN gene (At5g44340) were used as a control for intact genomic DNA (TUB Fw: 5’ AAT ACG TCG GCG ATT CTC CG 3’, and TUB Rv: 5’ CGT CAA GTC CAG TGT CTG TG 3’). PCR fragments were run on a 1% agarose gel with a 100-bp DNA ladder (Promega Benelux BV, Leiden, The Netherlands).
C h e m i c a l i n d u c t i o n
Induction treatments were performed by a foliar drench of 5-week-old soil-grown plants in an aqueous solution containing 0.015% (v/v) Silwet L-77 (Van Meeuwen Chemicals BV, Weesp, The Netherlands), supplemented with 1 mM SA (Mallinckrodt Baker, Deventer, The Netherlands), 0.1 mM MeJA, or a combination of both chemicals. MeJA was added to the medium from a 1000-fold stock solution in 96% ethanol. Solutions without MeJA were supplemented with equal amounts of ethanol. Control plants were treated with 0.015% Silwet L-77 only. Leaf rosettes were harvested 24 h after induction treatment and immediately frozen in liquid nitrogen.
For herbicide selection conditions, surface-sterilized seeds were sown on MS medium supplemented with 1% (w/v) sucrose, 0.6% (w/v) plant agar, pH 5.7, and 20 mg/L PPT, with or without 0.1 mM SA. Seedlings were allowed to grow for 10 days before assessing survival. Alternatively, 3-week-old plants grown in a sand-and-potting soil mixture received a 10-mL soil drench with 1 or 5 mM SA and were sprayed the next day with an aqueous solution containing 0.015% (v/v) Silwet L-77 with or without Finale SL14 (Bayer
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Cropscience BV, Mijdrecht, The Netherlands) (150 mg/L PPT). For assessing GUS activity, 4-week-old plants received a soil drench with water, 1 mM SA, 0.1 mM MeJA, or a combination of both chemicals. The following day, leaves were harvested and assayed for GUS activity by histochemical staining.
R N A e x t r a c t i o n a n d n o r t h e r n b l o t a n a l y s i s
Total RNA was extracted as described previously (De Vos et al., 2005). For northern blot analysis, 10-15 μg RNA was denatured using glyoxal and dimethyl sulfoxide (Sambrook et al., 1989), electrophoretically separated on a 1.5% agarose gel, and blotted onto Hybond-N+ membrane (Amersham, ‘s-Hertogenbosch, The Netherlands) by capillary transfer. The electrophoresis and blotting buffer consisted of 10 and 25 mM sodium phosphate (pH 7.0), respectively. Northern blots were hybridized with gene-specific probes for PR-1, PDF1.2, and BAR as described previously (Pieterse et al., 1998). After hybridization with α-32P-dCTP-labelled probes, blots were exposed for autoradiography and signals analyzed using a BioRad Molecular Imager FX (BioRad, Veenendaal, The Netherlands) with Quantity One software (BioRad, Veenendaal, The Netherlands). To check for equal loading, the blots were stripped and hybridized with a probe for 18S rRNA. The AGI numbers for the genes studied are At2g14610 (PR-1) and At5g44420 (PDF1.2). The probe for 18S rRNA was derived from an Arabidopsis cDNA clone (Pruitt and Meyerowitz, 1986).
G U S a s s a y
GUS activity was assessed by transferring a single leaf from individual seedlings to a GUS staining solution (1 mM X-Gluc, 100 mM NaPi buffer, pH 7.0, 10 mM EDTA, and 0.1% (v/v) Triton X-100). After vacuum infiltration and overnight incubation at 37°C, the seedlings were destained by repeated washes in 70% ethanol and evaluated visually for staining intensity.
A C K N O W L E D G M E N T SWe thank Hans van Pelt and Ido Vlaardingerbroek for technical assistance. This research was supported by grants 813.06.002 and 865.04.002 of the Earth and Life Sciences Foundation (ALW), which is subsidized by The Netherlands Organization of Scientific Research (NWO).
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C H A P T E R 6
General discussion
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T R A D E - O F F S B E T W E E N P L A N T D E F E N S E S I G N A L I N G PAT H WAY S
Plants have adapted to thrive under conditions where they are vulnerable to abiotic and biotic stresses. Morphological adaptations, such as a thick cuticula, thorns, needles and trichomes, aid the plant in coping with harsh climate conditions and attack by deleterious organisms (Agrios, 2005). Moreover, various inducible defense mechanisms can be deployed to fend off attackers and protect the plant against invasion by harmful organisms. The phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are important stress signals involved in the activation of defense pathways that are effective against various classes of attackers (Durrant and Dong, 2004; Howe, 2004; Pozo et al., 2004; Lorenzo and Solano, 2005; Grant and Lamb, 2006; Van Loon et al., 2006; Von Dahl and Baldwin, 2007). SA-mediated defense responses are generally effective against pathogens with a biotrophic lifestyle, whereas JA together with ET mediates defenses against herbivorous insects and necrotrophic pathogens (Thomma et al., 2001; Glazebrook, 2005).
Upon attack by insect herbivores or pathogenic micro-organisms, the plant reacts by producing a specific blend of stress hormones that is eventually translated into an appropriate defense reaction (De Vos et al., 2005). However, a plant often has to cope with simultaneous or subsequent attack by multiple aggressors and it, therefore, requires regulatory mechanisms that integrate, prioritize, and coordinate plant defenses. Cross-communication between signaling networks is thought to provide a fine-tuning mechanism that allows prioritization of appropriate defense reactions. Hence, trade-offs between different defense signaling pathways may occur. The best-studied example of pathway cross-talk is the mutual antagonism between SA- and JA-dependent defense signaling. As a result of negative cross-talk between SA and JA, activation of the SA response can render a plant more susceptible to attackers that are resisted through JA-dependent defenses, and vice versa. Indeed, many examples of trade-offs between SA-dependent and JA-dependent defense reactions have been described (Pieterse et al., 2001; Bostock, 2005; Beckers and Spoel, 2006).
In order to investigate cross-talk between SA and JA signaling pathways, the interaction of a plant with multiple biotic threats has to be studied. This will provide useful insights into the prioritization of the elicited defense reactions and allows one to place experimental data in an ecological context. However, attackers may have direct effects on each other, which can hamper detection of specific plant-mediated responses. Additionally, variation that is introduced by experimental set-ups with multiple biotic agents increases significantly with the number of attackers employed. Therefore, activation of defense signaling pathways by chemical application can be useful to understand the molecular mechanisms underlying cross-talk regulation. Although it is impossible to capture the detailed events that occur upon plant-attacker interactions, pharmacological studies can be valuable in elucidating general patterns in signal cross-talk.
The main objective of this thesis was to elucidate the molecular mechanism underlying the interaction between SA- and JA-dependent defense signaling. The model plant
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Arabidopsis thaliana provides an excellent model system to study plant-attacker interactions, since a wealth of information and well-characterized mutants are available. A predominantly pharmacological approach was used, applying SA and methyl jasmonate (MeJA) at various concentrations and with different kinetics (Chapter 3). In addition, we monitored SA- and JA-responsive gene expression upon biological induction by a biotrophic pathogen, Hyaloperonospora parasitica, and by two necrotrophs, Alternaria brassicicola and Botrytis cinerea, and two insect herbivores, Frankliniella occidentalis and Pieris rapae (Chapter 3). We demonstrated that the JA-responsive genes induced by the necrotrophic pathogens and herbivorous insects were suppressed by application of SA or by the SA-inducing H. parasitica. Furthermore, mutant and transcriptome analyses were employed to identify regulatory components involved in SA/JA cross-talk (Spoel et al., 2003; Chapter 2 and 4).
K I N E T I C S O F S A / J A C R O S S - TA L K
It is generally assumed that traits that increase plant fitness will have been preserved through evolution and natural selection. Genetic dissection of naturally occurring variation in Arabidopsis provides the opportunity to study a trait of interest in an ecologically significant context (Koornneef et al., 2004). We found that all Arabidopsis accessions tested showed suppression of the JA-responsive PDF1.2 marker gene when simultaneously treated with SA and MeJA. In contrast, expression of the SA-responsive PR-1 marker gene was not consistently affected by SA and MeJA treatment, demonstrating a one-way antagonistic effect (Chapter 3). Several studies have reported on a differential responsiveness of Arabidopsis accessions to the plant hormones SA and (Me)JA (Rao et al., 2000; Kliebenstein et al., 2002; van Leeuwen et al., 2007). Nevertheless, our study demonstrates that the molecular mechanism of SA/JA cross-talk is conserved among accessions.
In nature, timing of elicitation is likely to be crucial for prioritization of defense pathways. Therefore, we investigated the kinetics of SA/JA cross-talk by dose-response studies and time course and time interval analyses in Chapter 3. Simultaneous treatment with SA and MeJA always resulted in suppression of MeJA-responsive genes without affecting SA-responsive gene expression. This suppression occurred within hours after chemical application and lasted up to several days. In addition, very low doses of SA that did not induce any observable PR-1 expression were sufficient to suppress MeJA-responsive PDF1.2. Previous dose-response studies have shown that low concentrations of SA and JA can have transient synergistic effects, whereas later time points and higher concentrations revealed antagonistic effects (Mur et al., 2006). We further monitored the dynamics of SA/JA cross-talk by increasing the time interval between the SA and MeJA treatments. When SA was applied more than 30 h before MeJA, SA was incapable of suppressing MeJA-responsive PDF1.2 expression, even though at this point PR-1 mRNA levels were still detectable. Conversely, application of SA after MeJA always resulted in PDF1.2 suppression,
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comparable to the effect of simultaneous treatment. Thus, the antagonistic effect of SA on JA signaling appears to be restricted to the 30-h time interval following its application.
The ecological consequences of dose-dependent and temporally separated chemical treatments were investigated by Thaler et al. (2002), who demonstrated that the performance of Spodoptera exigua caterpillars on tomato increased upon treatment with the SA analog benzothiadiazole S-methyl ester (BTH), which suppressed the effect of JA. This antagonistic effect was most consistent at high concentrations of BTH (1.2 mM), even when applied 2 days apart from JA, and when dual-elicitation treatments with low concentrations of BTH (0.5 mM) and JA were used. Hence, for low concentrations of SA to antagonize JA signaling, simultaneous elicitation with JA is required (Thaler et al., 2002; Chapter 3).
Recently, Spoel et al. (2007) demonstrated an antagonistic effect of SA-eliciting virulent Pseudomonas syringae on subsequent infection of Arabidopsis by A. brassicicola. Infection of one leaf half with P. syringae, followed by inoculation of the other leaf half with A. brassicicola, enhanced spore production and disease symptom development of the latter. The increased susceptibility to A. brassicicola was absent in the sid2 mutant, demonstrating that this effect is dependent on endogenous SA. Prior inoculation with P. syringae suppressed A. brassicicola-induced JA-responsive gene expression, and this was partly dependent on NPR1. However, this was observed only when leaf halves from the same leaf were infected with P. syringae and A. brassicicola, and not when different leaves were infected. This lack of SA/JA antagonism was attributed to below-threshold levels of SA in systemic leaves. This contradicts our findings in Chapter 3 that low levels of SA, which do not trigger observable PR-1 expression, are still capable of suppressing MeJA-induced PDF1.2 expression. In the systemic leaves of P. syringae-infected Arabidopsis, PR-1 expression was observed (Spoel et al., 2007), indicative of an effective SA response. Contributing to the complexity, only virulent P. syringae triggered suppression of JA-responsive gene expression, whereas two avirulent strains that produced similarly high levels of SA and induced SA-responsive genes, did not suppress A. brassicicola-triggered PDF1.2 expression. This is difficult to reconcile with suppression of JA responses by SA. Thus, there must be unknown factors besides concentration, timing, and type of elicitation that influence the outcome of SA/JA cross-talk.
M O L E C U L A R M E C H A N I S M O F C R O S S - TA L K
The most consistent outcome of SA/JA cross-talk was observed upon simultaneous elicitation of the SA and JA signaling pathways. Therefore, simultaneous application of SA and MeJA was used to further unravel the molecular mechanism underlying cross-talk. We aimed at elucidating the SA-mediated signal transduction components that confer the suppressive effect, and at identifying the molecular targets in the JA signaling pathway.
In Chapter 5, a reporter system was designed to identify essential SA/JA cross-talk regulators in a mutant screen. However, SA did not always fully suppress MeJA-responsive
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gene expression, making selection based on plant survival phenotype unreliable. Therefore, a screening method that allows detection of quantitative differences in gene expression is required. Though laborious, a mutant screen using the PDF1.2 promoter fused to a suitable reporter gene would meet this criterion and could help further dissect SA/JA cross-talk signal transduction pathways.
S A s i g n a l i n g c o m p o n e n t s
There are several steps in the SA signaling pathway that could be important in mediating cross-talk. Pathogen attack leads to accumulation of SA and confers a redox change in the plant tissue that mediates PR gene expression through the regulatory protein NPR1. In an uninduced state, NPR1 is present as an inactive oligomer, formed through disulfide bonds between conserved cysteine residues. A change in the cellular redox state that occurs upon activation of SAR reduces the disulfide bonds and releases the active NPR1 monomer (Mou et al., 2003). We found that the window of opportunity of SA to suppress MeJA-responsive gene expression correlated with a transient change in the amount of the redox compound glutathione. Moreover, the glutathione biosynthesis inhibitor BSO blocked the suppressive effect of SA, suggesting that redox modulation is involved in induction of SA/JA cross-talk (Chapter 3). Furthermore, the inhibitory effect of SA on MeJA-responsive gene expression was found to be absent in mutant npr1-1 plants, demonstrating a role for this protein in the regulation of SA/JA cross-talk (Spoel et al., 2003; Chapter 2). However, while nuclear localization of NPR1 monomers is required for SA-responsive gene expression (Kinkema et al., 2000), the suppression of MeJA-responsive genes was shown to rely on a cytosolic function of NPR1 (Spoel et al., 2003; Chapter 2). Indeed, mutation of the nuclear localization signal of NPR1 resulted in a protein that cannot induce PR gene expression, but still shows inhibition of JA signaling (Dong, 2004). Mutations in two protein-protein interaction domains, the ankyrin repeat domain and the BTB/POZ domain in npr1-1 and npr1-2, respectively, both abolished SA/JA cross-talk (Cao et al., 1997; Spoel et al., 2003; Chapter 2). Hence, these domains must be important in mediating SA/JA cross-talk, possibly by sequestering positive regulators or interacting with negative factors of JA signaling in the cytosol.
Although NPR1 is conserved across plant species with homologues in rice, tobacco, apple, and orange (Durrant and Dong, 2004), reports on the regulatory role of NPR1 in SA/JA cross-talk differ between species. While NPR1 appears to have a similar function in mediating SA/JA cross-talk in Arabidopsis and rice (Spoel et al., 2003; Chapter 2; Yuan et al., 2007), a study on NPR1-silenced wild tobacco revealed that silencing of NPR1 resulted in suppression of JA responses (Rayapuram and Baldwin, 2007). It was proposed that the increased SA levels in NPR1-silenced tobacco plants suppressed JA-mediated defenses against herbivores, while in wild-type plants NPR1 suppresses SA production and SA/JA cross-talk (Rayapuram and Baldwin, 2007). These results suggest a diverse regulatory role of NPR1 in cross-talk.
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We screened several additional mutations in the SAR signal transduction pathway to uncover further potential cross-talk regulators. Although most of these mutations did not affect SA-mediated suppression of MeJA-responsive PDF1.2 expression, mutation of TGA transcription factors abolished PDF1.2 suppression (Chapter 4; Ndamukong et al., 2007). Hence, these transcription factors are essential components for both SAR expression and SA/JA cross-talk (Zhang et al., 2003). It is unlikely that TGA factors bind directly to promoters of JA target genes, such as PDF1.2, to suppress transcription, since deletion of the TGA binding site in the PDF1.2 promoter did not abolish SA-mediated suppression (Spoel et al., 2003; Chapter 2). However, TGA transcription factors might positively or negatively influence the expression of upstream signaling components, such as transcription factor genes that directly regulate JA-responsive genes, which eventually leads to PDF1.2 suppression.
Mutation of both NPR1 and the repressor protein SNI1 in the sni1 npr1-1 double mutant restores SA-inducible PR-1 expression (Li et al., 1999). SA/JA cross-talk is also partly recovered in this double mutant, which indicates that suppression of JA-responsive gene expression by SA may also occur independently of NPR1 (Chapter 4). High levels of ET were measured in the sni1 npr1-1 mutant as compared to the npr1-1 single mutant, and ET may well have a modulating role in cross-talk. Treating Arabidopsis with the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC) not only boosted PR-1 expression (De Vos et al., 2006) and PDF1.2 induction (Penninckx et al., 1998), but also increased the suppressive effect of SA on PDF1.2 expression in the npr1-1 mutant (S.H. Spoel, A. Leon-Reyes, and C.M.J. Pieterse, unpublished results). Thus, ET appears to influence the antagonism between SA and JA signaling in a NPR1-independent manner.
J A - d e p e n d e n t t a r g e t s
In our pharmacological assays and in other studies, SA and its functional analogs were shown to suppress the expression of both JA biosynthesis and JA response genes (Peña-Cortés et al., 1993; Doares et al., 1995; Harms et al., 1998; Van Wees et al., 1999; Spoel et al., 2003; Chapter 2). However, a common target of SA-mediated suppression of JA signaling remains to be identified. Based on work on tomato, Doares et al. (1995) suggested that SA targets JA signaling downstream of JA biosynthesis. Indeed, analysis of mutants defective in JA biosynthesis, but not in JA responsiveness, confirmed that SA/JA cross-talk is regulated downstream of JA biosynthesis (A. Leon-Reyes, T. Ritsema, and C.M.J. Pieterse, unpublished results). A putative target of SA/JA cross-talk is presented by the recently identified JAZ repressor proteins (Chini et al., 2007; Thines et al., 2007). Interference with the proteasome-mediated degradation of JA repressors would result in inhibition of JA signal transduction. Wang et al. (2007) recently demonstrated that SA can suppress auxin signaling by stabilizing auxin repressor proteins. A similar mechanism may be involved in SA-mediated suppression of JA-dependent signaling.
In order to study cross-talk regulation at the transcriptional level, we used Affymetrix ATH1 whole genome GeneChips and searched for MeJA-responsive genes that were
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antagonized by SA (Chapter 4). Expression of many genes was suppressed by SA, and promoter analysis of these coregulated genes revealed an overrepresentation of the I box and GCC box motifs. Site-directed mutagenesis of the I box motif in the PDF1.2 promoter did not affect MeJA-inducibility or abolish SA/JA cross-talk, demonstrating that this promoter element is not essential for suppression of MeJA-responsive PDF1.2 expression (Chapter 4). The GCC box, however, is an essential element in the PDF1.2 promoter that confers MeJA responsiveness (Brown et al., 2003). Mutation of this motif abolishes PDF1.2 induction, and, therefore, the effect of SA cannot be tested by mutational analysis of the GCC box. A minimal promoter containing only the GCC box motif fused to a reporter gene should prove whether this element is sufficient to confer the suppressive effect of SA. If the GCC box is indeed required for SA/JA cross-talk, identification of binding factors should aid in unraveling the regulatory mechanism underlying this phenomenon.
The antagonistic effect of SA on MeJA-responsive gene expression was determined for both MeJA-induced and MeJA-repressed genes. Application of SA reduced, respectively, the induction and the repression of these genes by MeJA. A number of the MeJA-suppressed genes encoded chloroplast-localized proteins implicated in light-regulated processes (Chapter 4). Colocalization of steps in the biosynthesis of SA and JA in the chloroplast may facilitate signal interactions, as has been suggested for the ssi2 mutant that is disturbed in plastidial fatty acid composition and shows altered SA- and JA-related phenotypes (Kachroo et al., 2003). Furthermore, the accumulation of SA, as well as induction of SA-responsive genes, has been shown to be light-dependent (Genoud et al., 2002; Karpinski et al., 2003; Zeier et al., 2004). It has been argued that glutathione levels are coupled to photosynthetic activity and can function as a transducing signal from the chloroplast to the cytosol. Resulting cytosolic redox changes, which affect redox-sensitive proteins, such as NPR1, can thus provide a link between chloroplast, cytosol, and nucleus (Mullineaux and Rausch, 2005).
On the level of marker gene expression we found a one-way antagonistic effect of SA on MeJA-responsive genes. However, the microarray data revealed that a significant portion of SA-responsive genes was antagonized by MeJA treatment, indicating mutually antagonistic cross-talk (Chapter 4). Annotation of the antagonized SA-responsive genes did not demonstrate any clearly overrepresented functional category. Further analysis of these cross-talk genes may reveal clues about their functioning.
P L A N T H O R M O N E C R O S S - TA L K
Upon pathogen or insect attack, plants produce a specific blend of phytohormones that shapes the outcome of the defense signaling network. The complex interplay between hormone-regulated signaling pathways allows fine-tuning of plant defenses. The antagonistic interaction between SA- and JA-dependent defense signaling pathways described in this thesis has been documented repeatedly in plant defense signaling research. Although a few molecular players in SA/JA cross-talk have been identified, translation of molecular
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mechanisms to predictability of trade-offs between pathogen and insect resistance remains complicated. Parameters, such as timing and concentration, contribute to the complexity of the signal interplay. Moreover, pathway cross-talk between multiple plant hormones besides SA and JA may modulate the outcome of plant-attacker interactions. For instance, a dual role in defense responses has been ascribed to ET. It has been described to promote disease resistance in concerted action with JA responses, but also to aggravate disease by accelerating senescence (Van Loon et al., 2006). Together with abscisic acid (ABA), ET also mediates cross-talk between two branches in the JA signaling pathway through the transcription factors MYC2 and ERF1. ET and JA positively influence ERF1 expression, whereas MYC2 is induced by ABA and JA. Overexpression of ERF1 suppresses MYC2-regulated genes and vice versa, thus demonstrating the antagonistic relationship between these transcription factors (Anderson et al., 2004; Lorenzo et al., 2004).
Gibberellin has been suggested to induce susceptibility to necrotrophs by inducing SA-dependent resistance responses that are active against biotrophs (Robert-Seilaniantz et al., 2007). In contrast, gibberellin induces trichome formation synergistically with JA, a response that is inhibited by SA (Traw and Bergelson, 2003). Similarly, microarray analysis of BTH-treated plants revealed downregulation of several genes involved in the gibberellin pathway (Wang et al., 2007).
Auxin is often produced after pathogen attack and promotes susceptibility to biotrophs (Robert-Seilaniantz et al., 2007). SA was shown to repress auxin responses by stabilization of auxin-response repressors as part of the plant defense response (Navarro et al., 2006; Wang et al., 2007). Moreover, auxin induces the expression of JA biosynthesis genes, suggesting that this hormone can contribute to SA/JA cross-talk (Tiryaki and Staswick, 2002; Robert-Seilaniantz et al., 2007). Thus, even among the multitude of interactions between various phytohormones, SA/JA cross-talk appears to be a recurring theme. Hence, unraveling the signal transduction pathways leading to the SA/JA antagonism is a first step in understanding the complexity of the induced defense response and in providing a deeper insight into how plants cope with enemies with differing modes of attack.
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Summary
Plants are equipped with an array of defense mechanisms to protect themselves against herbivorous insects and microbial pathogens. Some are pre-existing while others are only activated upon insect attack or pathogen invasion. However, induced defense responses entail fitness costs. Therefore, plants possess elaborate regulatory mechanisms that efficiently coordinate activation of attacker-specific defenses. A major focus in plant defense signaling research is to uncover key mechanisms by which plants tailor their responses to different attackers, and to investigate how plants cope with simultaneous interactions with multiple aggressors.
The phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play a major role in the regulation of induced defense mechanisms against partially distinct classes of attackers. In response to a particular attacker, the plant produces a specific blend of these phytohormones, which leads to an appropriate defense reaction. Biotrophic pathogens that require living plant tissue for growth and proliferation are generally suppressed through activation of SA-dependent defense mechanisms. Necrotrophic pathogens that feed on dead plant tissue and insect herbivores are resisted mainly through JA/ET-dependent reactions.
SA- and JA/ET-dependent defense signaling pathways cross-communicate, providing the plant with a regulatory tool to fine-tune its defense reaction to the attacker encountered. In Arabidopsis thaliana, pharmacological experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PDF1.2, indicating that the SA pathway can be prioritized over the JA pathway (Chapter 2). This antagonistic effect of SA on JA signaling is a robust phenomenon. It is conserved among Arabidopsis accessions, and JA-responsive gene expression is readily suppressed by SA for several days, even when triggered by very low doses of SA. Time interval studies revealed that SA has a window of opportunity to suppress methyl jasmonate (MeJA)-responsive gene expression, and that this time interval correlates with the SA-induced redox change in the plant tissue. Thus, redox modulation is likely to play a central role in the regulatory mechanism underlying SA/JA cross-talk (Chapter 3).
Redox modulation is involved in the activation of the key regulatory protein NPR1, which is an essential SA signal transduction component. Analysis of the Arabidopsis mutant
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npr1 revealed that the antagonistic effect of SA on JA signaling requires the NPR1 protein. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for suppression of JA-responsive genes, indicating that cross-talk between SA- and JA-dependent signaling pathways is modulated through a function of NPR1 in the cytosol (Chapter 2).
Analysis of several other mutants in SA-dependent defense signaling demonstrated that TGA transcription factors are required for both SA-dependent gene expression and SA-mediated suppression of JA signaling (Chapter 4). However, TGA factors are unlikely to exert their effect through direct interaction with the promoter of JA-responsive genes, such as PDF1.2, as removal of the TGA binding site in this promoter did not affect cross-talk (Chapter 2).
Transcriptome analysis identified a large number of MeJA-inducible genes whose expression is antagonized by SA. Promoter analysis of these SA-suppressed, MeJA-inducible cross-talk genes revealed enrichment of the I box and the GCC box motif. Site-directed mutagenesis of the I box motif in the PDF1.2 promoter did not affect suppression of this gene by SA, indicating that the I box is not essential in mediating cross-talk (Chapter 4). The GCC box remains an attractive candidate for SA/JA cross-talk regulation. This motif is required for MeJA-responsiveness in the PDF1.2 promoter, and could thus be a target for SA-mediated suppression. Future research should reveal whether the GCC box is sufficient to mediate SA/JA cross-talk.
Finally, we designed a reporter system to identify essential cross-talk regulators through an unbiased mutagenesis screen. The MeJA-responsive PDF1.2 promoter was fused to a herbicide resistance gene, to allow for MeJA-inducible herbicide tolerance. EMS mutagenesis of this transgenic line would lead to the identification of mutants that survive the SA/JA cross-talk and herbicide treatments. However, the SA/JA antagonism proved insufficient to fully suppress herbicide tolerance (Chapter 5). A mutant screen that allows detection of quantitative differences in gene expression would be better suited for identification of cross-talk mutants.
Collectively, this work provides novel insight into how plants regulate their defense response upon attack by multiple aggressors, and may prove valuable for the development of novel strategies for crop protection.
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Samenvatting
Planten kunnen worden aangetast door vele soorten ziekteverwekkers en insecten. Om zich hiertegen te verweren hebben planten uiteenlopende strategieën ontwikkeld. Effectieve afweer kan worden bewerkstelligd door verdedigingsmechanismen die continu aanwezig zijn, of door mechanismen die pas geïnduceerd worden na een aanval. Het aanschakelen van afweergeschut vergt veel energie en kan ten koste gaan van groei en reproductie van de plant. Daarom is het van belang dat er gecoördineerde activering van afweer plaatsvindt die specifiek gericht is tegen het type belager waarmee de plant geconfronteerd wordt. Een belangrijke vraag in het onderzoek naar verdedigingsmechanismen van planten is dan ook: hoe zijn planten in staat om de signalen die geïnduceerd worden na aanval door ziekteverwekkers en insecten dusdanig te integreren dat dit leidt tot een verdedigingsrespons die specifiek werkzaam is tegen de betrokken aanvaller?
De plantenhormonen salicylzuur (SA), jasmonzuur (JA) en ethyleen (ET) spelen een belangrijke rol bij de coördinatie van geïnduceerde afweer tegen verschillende typen aanvallers. Zodra de plant een belager herkent, verhoogt hij de productie van één of meer van deze signaalmoleculen. Verhoogde productie van SA is veelal effectief tegen biotrofe pathogenen die groeien ten koste van levende planten. Daarentegen worden necrotrofe pathogenen, die leven op dood weefsel, en insectenvraat onderdrukt door verdedigingsmechanismen die worden geactiveerd door verhoogde productie van JA en ET. Onderzoek naar de rol van SA, JA en ET heeft aangetoond dat deze drie signaalmoleculen interacteren in een complex netwerk van signaal-transductieroutes die gezamenlijk bepalend zijn voor de inductie van de afweerrespons tegen het pathogeen of insect dat de plant belaagt. De interactie tussen de verschillende signaal-transductieroutes heet ‘cross-talk’.
Onderzoek aan de modelplant Arabidopsis thaliana (zandraket) heeft aangetoond dat SA een remmend effect heeft op de expressie van genen die worden geïnduceerd door JA, zoals PLANT DEFENSIN (PDF1.2) (hoofdstuk 2). De robuustheid van dit verschijnsel blijkt uit het feit dat alle Arabidopsis accessies deze SA/JA cross-talk laten zien. Bovendien treedt het antagonistische effect binnen een paar uur op en zijn zeer lage concentraties SA effectief. Wel moet SA toegediend worden binnen 30 uur voor activering van de van JA afhankelijke genexpressie. Binnen dit tijdsbestek zorgt SA ervoor dat er een redoxverandering in het
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plantenweefsel plaatsvindt. Dit suggereert dat de redox staat van de cel een belangrijke rol speelt bij de regulering van SA/JA cross-talk (hoofdstuk 3).
Als gevolg van de door SA geïnduceerde redoxverandering vindt activering plaats van het regulerende eiwit NPR1. NPR1 speelt een essentiële rol bij de van SA afhankelijke afweer. Mutatie van NPR1 in Arabidopsis heeft laten zien dat het antagonistische effect van SA op de van JA afhankelijke afweer eveneens door dit eiwit wordt gereguleerd. Hoewel NPR1 in de kern aanwezig moet zijn om zijn rol te vervullen bij door SA geïnduceerde afweer, is dit niet het geval voor het reguleren van SA/JA cross-talk. Dit impliceert dat NPR1 een cytosolische rol vervult in de regulatie van cross-talk tussen van SA en van JA afhankelijke afweer (hoofdstuk 2).
Naast NPR1 spelen TGA transcriptiefactoren een belangrijke rol, zowel bij door SA geïnduceerde afweer als bij SA/JA cross-talk (hoofdstuk 4). Het is niet aannemelijk dat deze transcriptiefactoren direct binden aan de promoters van door JA induceerbare genen, aangezien verwijdering van het TGA bindingselement in de promoter van het PDF1.2 gen geen effect had op de remming door SA van de expressie van dit gen (hoofdstuk 2).
Met behulp van microarrays is het genexpressie profiel van Arabidopsis vastgesteld in reactie op behandeling met de plantenhormonen SA en methyl JA (MeJA). Van een grote groep genen die geïnduceerd werd door behandeling met MeJA bleek de expressie onderdrukt te worden door toevoeging van SA. Analyse van de regulerende elementen in de promoters van deze groep genen toonde een oververtegenwoordiging aan van twee motieven: de I box en de GCC box. Mutatie van de I box in de promoter van het PDF1.2 gen had geen effect op de onderdrukking van PDF1.2 door SA, hetgeen erop duidt dat dit element niet essentieel is voor de regulatie van SA/JA cross-talk (hoofdstuk 4). De GCC box blijft echter een interessante kandidaat. Dit motief zorgt ervoor dat de promoter van het PDF1.2 gen kan reageren op MeJA. SA zou dus zijn antagonistische effect via dit promoter element kunnen uitoefenen. Onderzoek moet uitwijzen of de GCC box voldoende is voor SA/JA cross-talk.
Om essentiële regulatoren van SA/JA cross-talk te identificeren werd een screeningsmethode ontwikkeld door gebruik te maken van mutagenese van transgene planten. Daarbij werd de door MeJA induceerbare promoter van het PDF1.2 gen gekoppeld aan een gen dat de plant resistent maakt tegen een herbicide. In deze planten kan herbicide tolerantie worden geactiveerd door behandeling met MeJA. Mutagenese van deze transgene planten zou moeten leiden tot het voorkomen van SA/JA cross-talk mutanten, die behandeling met SA, MeJA en het herbicide overleven. Helaas bleek het onmogelijk om herbicide resistentie volledig te onderdrukken door behandeling met SA (hoofdstuk 5). Een selectiemethode die berust op kwantitatieve verschillen in genexpressie zou daarom beter geschikt zijn voor het ontwikkelen van een cross-talk mutanten screen.
Het in dit proefschrift beschreven onderzoek heeft hiermee ons inzicht vergroot in de coördinerende mechanismen die ten grondslag liggen aan de natuurlijke afweerreacties van planten die gericht zijn tegen verschillende typen belagers.
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Dankwoord
In feite ben ik in 2001 al begonnen bij de leerstoelgroep Fytopathologie, die tegenwoordig bestaat onder de naam Plant-Microbe Interacties. Ik liep toen stage bij Corné en maakte kennis met het onderwerp ‘cross-talk’. De sfeer in de groep beviel me erg goed, en ik was dan ook blij toen Corné me een aio baan aanbood. Hoewel, ik wist niet zeker wat ik wilde gaan doen, maar in geval van twijfel beslist Corné voor me. En ik denk dat het een goede beslissing is geweest. Dankjewel Corné, voor je begeleiding, peptalk, geduld en het bewonderen van mijn nieuwe outfits! Ook kon ik altijd zeer waarderen dat je op de hoogte was van de nieuwste shock-docs, en anders kon je de smeuïge verhalen wel aanvullen met familie-info. Bovendien wil ik mijn tweede promotor, Kees, bedanken voor zijn kritische blik en vlotte commentaar op mijn manuscripten. Daarnaast was je altijd beschikbaar voor discussies over mijn onderzoek, wat heel motiverend was.
Bedankt Fyto’s voor de goede en ontspannen werksfeer. Adriaan, Antonio, Christos, Dieuwertje, Hans, Ientse, Marieke, Peter, Roeland, Rogier, Saskia, Sjoerd en Tita: jullie zijn geweldige collega’s! Ook de oud-collega’s mogen niet ontbreken: Jur, Ruth, Mohammad, Vivian, Martin, Maria, Mareike en Bas, bedankt!
Dan een woord aan mijn paranimfen, Sjoerd en Adriaan. Sjoerd, we hebben bijna gelijktijdig onze aio-trajecten doorlopen en daardoor samen veel meegemaakt: labstress als ik een Q-PCR moest inzetten of wanneer jij een northern blot moest draaien (ieder zo zijn eigen talent), maar ook congressen in Denemarken, Mexico en China, waar op de een of andere manier altijd wel een jacuzzi bij kwam kijken! Adriaan, mijn student, collega, buurman en paranimf. Toen ik je ontmoette was ik doodsbenauwd om een student te begeleiden, maar het bleek al snel dat je een goede aanwinst was voor het lab en zeker ook voor de gezelligheid. Zelfs Overvecht is er een stuk fleuriger op geworden!
Collega’s zijn bovendien ideale sportkompanen, en ik denk dat ik zonder jullie niet erg vaak naar de sportschool was gegaan. Vivian, het begon met onze zwemhalfuurtjes in de vroege ochtend, die door het optreden van de billenman helaas steeds minder aanlokkelijk werden. Je werd afgelost door Jur, die te snel voor me zwom, maar met wie het me toch meer om het douchen ging. Jur, ik wil jou ook bedanken voor de koffiegesprekken die al dan niet over werkgerelateerde zaken gingen, en voor de ‘ontspannen’ samenwerking tijdens
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de ChIP experimenten. Na het zwemmen was er de (twee)wekelijkse fitness en poweryoga sessie samen met Tita, waar ik meestal onder luid protest mee naartoe ga. Tita, bedankt voor de prettige samenwerking tijdens de redox proeven. Hopelijk zetten we die binnenkort voort in Amsterdam! Hans, Ientse, Ruth en Antonio, bedankt voor het fotograferen van gebleekte rozetten, het aanleveren van zaad en voor de hulp bij de experimenten. Ook wil ik de studenten noemen die tijdens hun stage bijgedragen hebben aan het onderzoek dat beschreven staat in dit proefschrift: Adriaan, Floor en Reinier, ontzettend bedankt!
Tijdens mijn onderzoeksperiode heb ik heel wat afgemekkerd, en gelukkig kon ik hiervoor, behalve bij collega’s, ook altijd terecht bij vrienden en familie. Vooral de mensen die zelf ook in de promotiestress zitten of zaten konden een hart onder de riem steken of mee-mekkeren. Ik denk hierbij natuurlijk aan Susanne (nog eventjes!), Leonie, Anne Chris en Jerôme. Maar ook de niet-biologen boden een luisterend oor: Lonneke, Conny, Sanne, Salwa, Nanda en mijn broer Wietse, bedankt!
Daniel, jij hebt mij gedurende het merendeel van mijn promotieonderzoek meegemaakt, waarvan een half jaar als ‘directe collega’. We zijn tot de conclusie gekomen dat we maar niet samen moeten werken, maar samenwonen klinkt als een goed plan! Dankjewel voor je liefde, steun en geduld.
Rest mij nog mijn ouders te bedanken. Maarten, je had altijd tijd om naar mijn problemen te luisteren en ook om stukken door te lezen. Het kwam zeker goed uit dat je de ‘Arabidopsis-goeroe’ bent, hoewel je ook door kon blijven gaan, terwijl ik geen zin meer had om over werk te praten. Elly, jij hebt daar veel geduld mee gehad, en bij jou kon ik dan weer terecht met Arabidopsis-ongerelateerde zaken. Heel erg bedankt voor jullie steun!
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Curriculum vitae
Annemart Koornneef werd geboren op 4 september 1980 te Wageningen. In 1998 behaalde zij haar Gymnasium diploma aan de Christelijke Scholengemeenschap ’t Streek te Ede. In datzelfde jaar werd aangevangen met de studie Biologie aan de Universiteit Utrecht. Tijdens de doctoraalfase werden twee onderzoeksstages vervuld. Allereerst bij de leerstoelgroep Fytopathologie, waar onder begeleiding van Dr. ir. Corné Pieterse werd gewerkt aan ‘Cross-talk between defense signaling pathways, the role of a TGACG motif in a JA-responsive promoter’. De opleiding werd vervolgd in de Verenigde Staten met een stage aan Cincinnati Children’s Hospital Medical Center, ditmaal onder begeleiding van Dr. Marie-Dominique Filippi en Prof. dr. David Williams met als titel: ‘The role of POR1 and PAK1 in Rac2-mediated chemotaxis and superoxide production in neutrophils’. In december 2003 studeerde zij af, om vervolgens direct aan de slag te gaan als aio bij de toenmalige leerstoelgroep Fytopathologie van de Universiteit Utrecht, die tegenwoordig bestaat onder de naam Plant-Microbe Interacties. Daar werd onder begeleiding van Prof. dr. ir. Corné Pieterse en Prof. dr. ir. Kees van Loon tot januari 2008 het onderzoek uitgevoerd dat in dit proefschrift is beschreven.
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L i s t o f pub l i ca t ions • 1 3 1
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List of publications
Koornneef, A., Leon-Reyes, A., Ritsema, T., Verhage, A., Den Otter, F.C., Van Loon, L.C., and Pieterse, C.M.J. Unraveling the kinetics of cross-talk between salicylate and jasmonate signaling pathways in Arabidopsis, submitted.
Koornneef, A., Verhage, A., Leon-Reyes, A., Snetselaar, R., Van Loon, L.C., and Pieterse, C.M.J. Towards a reporter system to identify regulators of cross-talk between salicylate and jasmonate signaling pathways in Arabidopsis, submitted.
Van der Ent, S.*, Koornneef, A.*, Ton, J., and Pieterse, C.M.J. (2008). Induced resistance - orchestrating defence mechanisms through cross-talk and priming. In: Annual Plant Reviews - Molecular Aspects of Plant Disease Resistance (J.E. Parker, ed), Blackwell, in press. * Equal contribution.
Pieterse, C.M.J., Koornneef, A., Léon Reyes, A., Ritsema, T., Verhage, A., Joosten, R., De Vos, M., Van Oosten, V., and Dicke, M. (2008). Cross-talk between signaling pathways leading to defense against pathogens and insects. In: Biology of Molecular Plant-Microbe Interactions, Vol. 6 (M. Lorito, S. Woo, and F. Scala, eds), The International Society for Molecular Plant-Microbe Interactions, St. Paul, MN., in press.
Koornneef, A., and Pieterse, C.M.J. (2008). Cross talk in defense signaling. Plant Physiol. 146, 839-844.
De Vos, M., Van Zaanen, W., Koornneef, A., Korzelius, J.P., Dicke, M., Van Loon, L.C., and Pieterse, C.M.J. (2006). Herbivore-induced resistance against microbial pathogens in Arabidopsis. Plant Physiol. 142, 352-363.
Pieterse, C.M.J., Van Pelt, J.A., Verhagen, B.W.M., De Vos, M., Van Oosten, V.R., Van der Ent, S., Koornneef, A., Van Hulten, M.H.A., Pozo, M.J., Ton, J., Dicke, M., and Van Loon, L.C. (2006). Molecular mechanisms involved in induced resistance signaling in Arabidopsis. In: Biology of Molecular Plant-Microbe Interactions, Vol. 5 (F. Sanchez, C. Quinto, I.M. Lopez-Lara, and O. Geiger, eds), The International Society for Molecular Plant-Microbe Interactions, St. Paul, MN., pp. 188-194.
Carstanjen, D., Yamauchi, A., Koornneef, A., Zang, H., Filippi, M.D., Harris, C., Towe, J., Atkinson, S., Zheng, Y., Dinauer, M.C., and Williams, D.A. (2005). Rac2 regulates neutrophil chemotaxis, superoxide production, and myeloid colony formation through multiple distinct effector pathways. J Immunol. 174, 4613-4620.
Pieterse, C.M.J., Van Pelt, J.A., Van Wees, S.C.M., Ton, J., Verhagen, B.W.M., Léon-Kloosterziel, K., Hase, S., De Vos, M., Van Oosten, V., Pozo, M., Spoel, S., Van der Ent, S., Koornneef, A., Chalfun-Junior, A., Resende, M.L.V., and Van Loon, L.C. (2005). Indução de resistência sistêmica por rizobactérias e comunicação na rota de sinalização para uma defesa refinada. Revisão de Patologia de Plantas 13, 277-319.
Spoel, S.H., Koornneef, A., Claessens, S.M.C., Korzelius, J.P., Van Pelt, J.A., Mueller, M.J., Buchala, A.J., Métraux, J.-P., Brown, R., Kazan, K., Van Loon, L.C., Dong, X., and Pieterse, C.M.J. (2003). NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell 15, 760-770.
