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Abscisic acid is essential for rewiring of jasmonic acid-
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dependent defenses during herbivory
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Irene A Vos
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, Adriaan Verhage
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, Lewis G Watt
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, Ido Vlaardingerbroek
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,
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Robert C Schuurink
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, Corné MJ Pieterse
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, Saskia CM Van Wees
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Plant-Microbe Interactions, Department of Biology, Faculty of Science, Utrecht
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University, P.O. Box 80056, 3508 TB Utrecht, the Netherlands
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Department of Plant Physiology, Swammerdam Institute for Life Sciences,
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University of Amsterdam, P.O. Box 94215, 1090 GE Amsterdam, the Netherlands
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Address correspondence to s.vanwees@uu.nl
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Short title: ABA differentially affects JA signaling
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The author responsible for distribution of materials integral to the findings presented
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in this article in accordance with the policy described in the Instructions for Authors
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(www.plantcell.org) is Saskia Van Wees (s.vanwees@uu.nl).
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not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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Abstract
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Jasmonic acid (JA) is an important plant hormone in the regulation of defenses
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against chewing herbivores and necrotrophic pathogens. In Arabidopsis thaliana, the
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JA response pathway consists of two antagonistic branches that are regulated by
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MYC- and ERF-type transcription factors, respectively. The role of abscisic acid
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(ABA) and ethylene (ET) in the molecular regulation of the MYC/ERF antagonism
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during plant-insect interactions is still unclear. Here, we show that production of ABA
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induced in response to leaf-chewing Pieris rapae caterpillars is required for both the
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activation of the MYC-branch and the suppression of the ERF-branch during
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herbivory. Exogenous application of ABA suppressed ectopic ERF-mediated PDF1.2
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expression in 35S::ORA59 plants. Moreover, the GCC-box promoter motif, which is
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required for JA/ET-induced activation of the ERF-branch genes ORA59 and PDF1.2,
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was targeted by ABA. Application of gaseous ET counteracted activation of the
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MYC-branch and repression of the ERF-branch by P. rapae, but infection with the
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ET-inducing necrotrophic pathogen Botrytis cinerea did not. Accordingly, P. rapae
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performed equally well on B. cinerea-infected and control plants, whereas activation
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of the MYC-branch resulted in reduced caterpillar performance. Together, these data
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indicate that upon feeding by P. rapae, ABA is essential for activating the MYC-
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branch and suppressing the ERF-branch of the JA pathway, which maximizes
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defense against caterpillars.
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Introduction
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In nature plants are a food source for over one million herbivorous insect species
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(Howe and Jander, 2008). The evolutionary arms race between plants and their
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herbivorous insect enemies has led to a highly sophisticated defense system in
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plants that can recognize wounding and oral secretion of the insects and respond
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with the production of nutritive value-diminishing enzymes, toxic compounds, or
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predator-attracting volatiles (Kessler and Baldwin, 2002; Lawrence and Koundal,
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2002; Wittstock et al., 2004; Chen et al., 2005; Mithöfer and Boland, 2012; Dicke,
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2016). Conversely, insects can estimate the quality and suitability of the plant as a
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food source by contact chemoreceptors on the insect mouthparts, antennae and tarsi
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(Howe and Jander, 2008; Appel and Cocroft, 2014; Dicke, 2016). Because plant
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defenses are costly, they are often only activated in case of insect or pathogen
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attack and not constitutively expressed (Walters and Heil, 2007; Vos et al., 2013a).
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The induced immune response is shaped by the induced production of diverse plant
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hormones. The quantity, composition and timing of the hormonal blend tailors the
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defense response specifically to the attacker at hand, thereby prioritizing effective
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over ineffective defenses and minimizing fitness costs (De Vos et al., 2005; Pieterse
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et al., 2012; Vos et al., 2013a; Vos et al., 2015).
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Infestation with chewing herbivores or infection with necrotrophic pathogens
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triggers the production of the plant hormone jasmonic acid (JA), and its bioactive
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derivative JA-Ile (Creelman et al., 1992; Penninckx et al., 1996). Binding of JA-Ile to
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the JA receptor complex consisting of the F-box protein COI1 and a JAZ repressor
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protein (Xie et al., 1998; Yan et al., 2009; Sheard et al., 2010), leads to degradation
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of JAZ proteins via the 26S proteasome pathway (Chini et al., 2007; Thines et al.,
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2007). Without JA, JAZ proteins repress JA-responsive gene expression by binding
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to transcriptional activators, such as MYC2, EIN3 and EIL1 (Pauwels and Goossens,
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2011; Song et al., 2014b; Caarls et al., 2015). When JA accumulates the JAZ
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proteins are degraded thereby releasing transcription factors that can activate JA-
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regulated genes.
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Within the JA pathway, two distinct, antagonistic branches of transcriptional
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regulation are recognized; the MYC-branch and the ERF-branch. Feeding by
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chewing herbivores activates the MYC-branch (Verhage et al., 2011; Vos et al.,
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2013b). This branch is controlled by the basic helix-loop-helix leucine zipper
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transcription factors MYC2, MYC3 and MYC4 leading to transcription of hundreds of
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JA-responsive MYC-branch regulated genes, including VSP1 and VSP2 (Anderson
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et al., 2004; Lorenzo et al., 2004; Fernández-Calvo et al., 2011; Niu et al., 2011).
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Furthermore, previous studies have indicated that ABA plays a co-regulating role in
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the activation of the MYC-branch (Anderson et al., 2004; Bodenhausen and
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Reymond, 2007; Sánchez-Vallet et al., 2012; Vos et al., 2013b). For example, in the
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ABA-deficient mutant aba2-1, expression of the JA-responsive gene VSP1 was
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reduced upon feeding by caterpillars of Pieris rapae (small cabbage white) compared
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to wild-type Col-0 plants (Vos et al., 2013b). In contrast to the herbivore-induced
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MYC-branch, the ERF-branch is activated upon infection with necrotrophic
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pathogens. The transcription factors EIN3 and EIL1 and the ERF transcription
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factors ERF1 and ORA59 activate a large set of JA-responsive ERF-branch
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regulated genes, including PDF1.2 (Caarls et al., 2015). The expression of ERF1,
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ORA59 and PDF1.2 is impaired in both JA- and ethylene (ET)-insensitive mutants,
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indicating that joint activation of the JA and ET pathways is necessary for full
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expression of the ERF-branch (Penninckx et al., 1998; Lorenzo et al., 2003; Pré et
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al., 2008; Broekgaarden et al., 2015).
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It has been shown that the ABA co-regulated MYC-branch and the ET co-
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regulated ERF-branch of the JA pathway antagonize each other. For example, upon
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infestation with P. rapae caterpillars, the MYC-branch is activated, while the ERF-
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branch is suppressed (Verhage et al., 2011; Vos et al., 2013b). In myc2 mutant
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plants, ORA59 and PDF1.2 expression was highly upregulated after feeding by P.
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rapae, indicating that in wild-type plants, MYC2 represses ORA59 and PDF1.2
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expression after feeding by P. rapae (Verhage et al., 2011; Vos et al., 2013b).
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Additionally, exogenously applied ABA had a positive effect on expression of the
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MYC-branch after feeding by P. rapae (Vos et al., 2013b) and caused suppression of
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PDF1.2 induction after exogenous application of JA (Anderson et al., 2004).
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Recently, it was shown that the MYC-branch transcription factors MYC2, MYC3 and
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MYC4 interact with the ERF-branch transcription factors EIN3 and EIL1 and that they
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repress each other’s transcriptional activity (Song et al., 2014a).
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These antagonistic effects between the MYC- and ERF-branch on gene
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expression levels also have an effect on plant resistance. ABA-deficient mutants
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have been reported to be more susceptible to herbivory (Thaler and Bostock, 2004;
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Bodenhausen and Reymond, 2007; Dinh et al., 2013) and more resistant to
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necrotrophic pathogens (Anderson et al., 2004; Sánchez-Vallet et al., 2012).
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Conversely, ET insensitive mutants are in general more susceptible to necrotrophic
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pathogens and more resistant to herbivorous insects compared to wild-type plants
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(Van Loon et al., 2006; Broekgaarden et al., 2015). Hence, the interplay between the
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MYC- and the ERF-branch may allow the plant to activate a specific set of JA-
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responsive genes that is required for an optimal defense against the attacker
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encountered (Pieterse et al., 2012).
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To study the role of ABA and ET in the molecular regulation of the MYC/ERF
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balance in Arabidopsis thaliana (hereafter Arabidopsis) upon attack by P. rapae, we
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analyzed hormone signaling mutants for their gene expression response, hormone
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production and defense against P. rapae. We provide evidence that after P. rapae
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infestation ABA accumulation plays an essential modulating role in the activation of
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the MYC-branch, possibly by activating the MYC2, MYC3 and MYC4 transcription
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factors. Concomitantly, ABA can suppress the ERF-branch independently of the
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MYC transcription factors, by targeting the GCC-box, which is present in the
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promoters of ORA59 and PDF1.2. Furthermore, activation of the MYC-branch, either
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by application of JA or ABA or by using the ein2-1 mutant, resulted in a negative
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effect on caterpillar performance, whereas activation of the ERF-branch by infection
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with the necrotrophic pathogen Botrytis cinerea did not.
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Results
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ABA- and ET-dependency of JA-dependent defense gene expression upon P. rapae
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feeding
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The JA-dependent transcriptional response of Arabidopsis to P. rapae feeding is
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predominantly regulated through activation of the MYC-branch of the JA pathway
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and concomitant suppression of the ERF-branch (Verhage et al., 2011). Here, we
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investigated whether ABA and ET have a role in the differential expression of the
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MYC- and the ERF-branch during induction of JA-dependent defense signaling by P.
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rapae feeding. Expression of the MYC-branch marker gene VSP2 and the ERF-
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branch marker gene PDF1.2 was monitored in wild-type Col-0, MYC2-impaired
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mutant jin1-7 (hereafter called myc2), MYC2, MYC3, MYC4 triple mutant myc2,3,4,
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ABA biosynthesis mutant aba2-1 and ET response mutant ein2-1. First-instar P.
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rapae caterpillars were allowed to feed for 24 h on the different Arabidopsis
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genotypes, after which they were removed. Comparable to Col-0, ein2-1 plants
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showed strong P. rapae-induced transcription of VSP2 at 24 h and 30 h (Figure 1).
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.CC-BY-NC-ND 4.0 International licenseavailable under a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which wasthis version posted August 28, 2019. ; https://doi.org/10.1101/747345doi: bioRxiv preprint