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The Calmodulin-Binding Transcription Factor SIGNAL RESPONSIVE1 is a Novel Regulator of Glucosinolate Metabolism and Herbivory Tolerance in Arabidopsis K. Laluk1,5,6, K.V.S.K. Prasad2,5, T. Savchenko3, H. Celesnik1, K. Dehesh3, M. Levy4, T. Mitchell-Olds2 and A.S.N. Reddy1,* 1

Department of Biology, Colorado State University, Fort Collins, CO 80523, USA Institute for Genome Sciences and Policy, Department of Biology, Duke University, Durham, NC 27708, USA 3 Department of Plant Biology, University of California, Davis, CA 95616, USA 4 Department of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, PO Box 12 Rehovot 76100, Israel 5 These authors contributed equally to this work. 6 Present address: Plant Science Department, California State Polytechnic University, Pomona, CA 91768, USA. *Corresponding author: E-mail, [email protected]; Fax, +1-970-491-0649. 2

Keywords: Arabidopsis AtSR1 Glucosinolates Herbivory Wound responses.

Abbreviations: Ca2+, calcium; CaM, calmodulin; GS, glucosinolate; I3M, indol-3-ylmethyl; JA, jasmonate; 4MSOB, 4-methylsulfinylbutyl; 8MSOO, 8-methylsulfinyloctyl; 3MSOP, 3-methylsulfinylpropyl; qRT–PCR, quantitative reverse transcription–PCR; SA, salicylate; Wt, wild-type.

Introduction Arabidopsis SIGNAL RESPONSIVE1 (AtSR1 or CAMTA3) encodes a calmodulin (CaM)-binding transcription factor

involved in the mediation of both biotic and abiotic stress responses (Galon et al. 2008, Doherty et al. 2009, Du et al. 2009). AtSR1 negatively regulates plant defense responses to biotrophic and necrotrophic pathogens, mainly because its loss of function results in significantly increased levels of endogenous salicylate (SA) (Galon et al. 2008, Du et al. 2009). Overall, it is well established that jasmonate (JA) and ethylene (ET) synergistically regulate plant defense responses against necrotrophic infection, and generally function in direct antagonism with SA-mediated responses to biotrophic pathogens (Glazebrook 2005). Thus, the observed resistance of the atsr1 mutant to both pathogen types (Galon et al. 2008) suggests that AtSR1 may represent an uncharacterized branch of the defense response pathway that is able to over-ride the signaling antagonism between JA/ET- and SA-regulated responses. Alternatively, AtSR1 may serve as a novel link between pathways currently unassociated with hormone-mediated defense and/or plant immune responses. In an effort to unravel its complex function(s) in Arabidopsis stress responses, we examined the role of AtSR1 in plant resistance to insect herbivory. SA-dependent responses are known to be involved in regulatory cross-talk with those mediated by JA (Beckers and Spoel 2006, Mur et al. 2006, Howe and Jander 2008, Browse 2009). As JA and its methyl ester are the primary regulators of plant defense against insect herbivory, we hypothesized that AtSR1 may have a function in Arabidopsis feeding tolerance based on its involvement in the regulation of SA responses (Chehab et al. 2008). In addition, stress-induced cellular calcium (Ca2+) changes have been implicated in regulating responses to many biotic stresses including insect herbivory (Maffei et al. 2007, Reddy et al. 2010). Insect feeding, as well as wounding, elicits specific intracellular Ca2+ signatures thought to regulate plant defense through Ca2+-mediated signaling responses (Maffei et al. 2007). Phosphorylation of the transcription

Plant Cell Physiol. 53(12): 2008–2015 (2012) doi:10.1093/pcp/pcs143, available online at www.pcp.oxfordjournals.org ! The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

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Plant Cell Physiol. 53(12): 2008–2015 (2012) doi:10.1093/pcp/pcs143 ! The Author 2012.

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The Arabidopsis Ca2+/calmodulin (CaM)-binding transcription factor SIGNAL RESPONSIVE1 (AtSR1/CAMTA3) was previously identified as a key negative regulator of plant immune responses. Here, we report a new role for AtSR1 as a critical component of plant defense against insect herbivory. Loss of AtSR1 function impairs tolerance to feeding by the generalist herbivore Trichoplusia ni as well as wound-induced jasmonate accumulation. The susceptibility of the atsr1 mutant is associated with decreased total glucosinolate (GS) levels. The two key herbivory deterrents, indol-3-ylmethyl (I3M) and 4-methylsulfinylbutyl (4MSOB), showed the most significant reductions in atsr1 plants. Further, changes in AtSR1 transcript levels led to altered expression of several genes involved in GS metabolism including IQD1, MYB51 and AtST5a. Overall, our results establish AtSR1 as an important component of plant resistance to insect herbivory as well as one of only three described proteins involved in Ca2+/CaM-dependent signaling to function in the regulation of GS metabolism, providing a novel avenue for future investigations of plant–insect interactions.

AtSR1 involvement in wound and herbivory responses

Results AtSR1 is required for tolerance to Trichoplusia ni feeding Previously, AtSR1 was found to be a critical negative regulator of Arabidopsis defense responses since its loss of function results in significantly increased levels of endogenous SA (Galon et al. 2008, Du et al. 2009). Based on the known cross-talk between SA- and JA-mediated signaling (Glazebrook 2005, Beckers and Spoel 2006, Browse 2009), as well as the significant up-regulation of AtSR1 in response to methyl-JA (Yang and Poovaiah 2002), a critical regulator of defense against insect feeding (Howe and Jander 2008), we assayed for AtSR1 function in plant responses to herbivory using AtSR1 knock-out and overexpression lines (Fig. 1A). Four days post-incubation with larvae of the generalist herbivore Trichopulsia ni, atsr1 plants were severely defoliated (Fig. 1B), supporting significantly higher larval weight gains compared with those of wild type(Wt) fed insects (Fig. 1C). Similarly, in detached leaf assays, loss of AtSR1 function resulted in larval weight gains nearly three times greater than those of insects feeding on Wt plants only 12 h post-incubation (Supplementary Fig. S1), indicating that AtSR1 is required for feeding tolerance in Arabidopsis. Constitutive expression of AtSR1 in atsr1 plants (AtSR1;atsr1) restored Wt levels of resistance, thus verifying that the susceptibility of the mutant is a direct result of loss of AtSR1 function (Fig. 1; Supplementary Fig. S1).

AtSR1 expression modulates glucosinolate accumulation in Arabidopsis Arabidopsis IQ-DOMAIN1 (IQD1) encodes a CaM-binding protein involved in the positive regulation of total GS levels during biotic stress responses, with a gain-of-function IQD1 mutation resulting in increased GS accumulation as well as enhanced resistance to T. ni feeding (Levy et al. 2005). Since GSs and their breakdown products are well-described herbivory deterrents (Howe and Jander 2008) and, like IQD1, AtSR1 is a CaM-binding protein functioning in tolerance to insect herbivory, we analyzed GS levels in 25-day-old (Fig. 1D) and fiveweek-old (Supplementary Table S2) Wt, atsr1 and AtSR1;atsr1 plants to determine if the increased susceptibility of the mutant to T. ni feeding is associated with altered GS content. In general, atsr1 showed a significant reduction in both aliphatic and aromatic GS levels compared with Wt plants (Supplementary Table S1). Levels of 3-methylsulfinylpropyl (3MSOP), 8-methylsulfinyloctyl (8MSOO) and, to a greater degree, 4-methylsulfinylbutyl (4MSOB) and indol-3-ylmethyl (I3M) were all significantly decreased in the mutant (Fig. 1D, Supplementary Fig. S2, Supplementary Table S1), suggesting that AtSR1 functions in GS metabolism. GS levels were restored to those of the Wt in AtSR1;atsr1 #1 plants, further indicating that the altered GS content in the mutant is a direct result of loss of AtSR1 function. Additionally, despite having an almost 2.5-fold difference in AtSR1 expression between AtSR1;atsr1 #1 and #2, no statistically significant differences in GS accumulation were observed between them. However, relative to the Wt, GS levels in AtSR1;atsr1 #2 plants were lower overall, suggesting that a degree of AtSR1 expression is a determinant factor in regulation of Arabidopsis GS content. When coupled with its herbivory phenotype, these results suggest atsr1 susceptibility to T. ni feeding is probaby a result of altered GS content, a notion further supported by restoration of GS levels as well as feeding tolerance in plants expressing high levels of AtSR1.


factor HsfB2a by Ca2+-dependent protein kinases led to activated expression of plant defensin PDF1.2, a marker of JA-regulated responses including defense against necrotrophic infection and insect herbivory (Reymond and Farmer 1998, Kunkel and Brooks 2002). Several CaM isoforms have also been shown to be induced by wounding (Bergey and Ryan 1999, Yamakawa et al. 2001, Phean et al. 2005, Jung et al. 2010), yet the downstream events associated with Ca2+/ CaM-mediated signal transduction are still largely undefined. Among the few characterized components of Ca2+-regulated responses to insect herbivory, the CaM-binding protein IQ-DOMAIN1 (IQD1) was found to be a critical regulator of resistance to Myzus persicae and Trichoplusia ni feeding, functioning in the positive regulation of glucosinolate (GS) accumulation (Levy et al. 2005). Similarly, WRKY53, a target of CaM and CaM-like proteins, modulates tolerance to insect feeding through regulation of GS hydrolysis (Miao and Zentgraf 2007, Popescu et al. 2007). Here, we define new functions for AtSR1 as a key regulator of GS levels and critical component of plant defense against insect herbivory in Arabidopsis. In addition to IQD1 and WRKY53, our results make AtSR1 one of only three described proteins involved in Ca2+/ CaM-mediated signaling to function in the regulation of GS metabolism.

Loss or constitutive expression of AtSR1 alters wound-induced JA accumulation AtSR1 is a negative regulator of SA biosynthesis; its loss of function results in >6-fold increases in free and conjugated SA levels (Du et al. 2009). Both positively affect GS biosynthesis, with the former also contributing to its negative regulation depending on the context (Mikkelsen et al. 2003, Mewis et al. 2005). Mechanical damage mimics many aspects of insect feeding, and wound-induced JA responses share significant overlap with those induced by herbivory (Kessler and Baldwin 2002, Mithofer et al. 2005, Howe and Jander 2008). Therefore, we wounded atsr1 plants with serrated forceps and assayed SA, JA and ABA levels to determine if the altered GS content in the atsr1 mutant is related to its disrupted hormone regulation. As previously reported, without treatment, atsr1 plants


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Fig. 2 Loss of AtSR1 function alters wound-induced JA accumulation. Basal (–) and wound-induced (+) (A) SA and (B) JA accumulation in Wt, atsr1 and AtSR1;atsr1 plants.

accumulated significantly higher levels of SA (Du et al. 2009). After wounding, SA levels in the mutant remained elevated whereas both basal and wound-induced SA accumulation were restored to the Wt level in AtSR1;atsr1 lines (Fig. 2A). In contrast, whereas basal JA accumulation was not affected, induced JA levels were significantly lower than those of the Wt in both the atsr1 and AtSR1;atsr1 lines (Fig. 2B). This suggests that both high and low AtSR1 expression can negatively

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impact wound-induced JA accumulation. Additionally, since constitutive AtSR1 expression only disrupts induced JA levels and still restores Wt responses to insect feeding, atsr1 susceptibility to herbivory is likely to be a joint effect of altered SA and JA levels leading to a negative regulation of GS metabolism. Basal and wound-induced levels of ABA were not affected by the atsr1 mutation (Supplementary Fig. S3), suggesting the specificity of AtSR1 function.



Fig. 1 The atsr1 mutant shows enhanced sensitivity to insect herbivory associated with decreased GS levels. (A) Expression of AtSR1 in wild type, atsr1 and AtSR1;atsr1 plants (B) Representative defoliation of Wt, atsr1, and AtSR1;atsr1 (OE) plants 0 and 4 days post-feeding (dpf ). (C) Mean percent increases in larval weight 4 dpf. (D) GS levels in 25 day-old Wt, atsr1 and AtSR1;atsr1 plants.


The atsr1 mutant has significantly altered expression of genes associated with GS metabolism


Previously, transcriptome analysis determined that expression of WRKY53 is significantly up-regulated in the atsr1 mutant (Galon et al. 2008). WRKY53 encodes a CaM-binding transcription factor that functions in the regulation of GS hydrolysis (Miao and Zentgraf 2007, Popescu et al. 2007). WRKY53 interacts with EPITHIOSPECIFYING SENESCENCE REGULATOR (ESR) and the two antagonistically regulate leaf senescence as well as the conversion of GS to nitriles and isothiocyanates in Arabidopsis (Miao and Zentgraf 2007). In an effort to define the role of AtSR1 in GS metabolism further, we assayed the mutant for altered wound-induced expression of WRKY53, ESR and other genes related to GS biosynthesis and hydrolysis. As previously reported (Galon et al. 2008), basal expression of WRKY53 and WOUND-RESPONSIVE 3 (WR3), a positive control for wound treatment, was significantly higher in atsr1 plants as compared with the Wt (Fig. 3A; Supplementary Fig. S4). Mechanical wounding led to further increases in WRKY53 and WR3 transcript accumulation in the mutant, with levels of both nearly four times greater than that observed in Wt plants following induction. Additionally, basal expression levels of WRKY53 and WR3 were higher in each of the constitutive lines but similar to Wt levels following wounding. Basal expression of ESR was also dramatically altered (25-fold increase) in AtSR1;atsr1 #2 plants, yet these levels were not affected by loss of AtSR1 function (Supplementary Fig. S4). However, the basal expression levels of MYB51, encoding an R2R3 MYB transcription factor that activates indole GS biosynthesis genes (Gigolashvili et al. 2007), were elevated in all three transgenic lines, with the degree of increased expression inversely proportional to AtSR1 transcript level (Fig 3A). This same pattern of expression was observed for four other genes encoding MYB transcription factors: MYB34, MYB28, MYB29 and MYB76. MYB34 regulates indole GS homeostasis whereas the latter three are involved in the coordinated regulation of aliphatic GS accumulation (Gigolashvili et al. 2008) (Fig. 3A, B). As I3M was the most significantly changed GS in atsr1 plants, we therefore quantified expression of AtST5a, which is positively regulated by MYB51 and encodes a desulfoglucosinolate sulfotransferase, the main converter of desulfo-I3M to I3M during indole GS biosynthesis (Gigolashvili et al. 2007). Basal AtST5a levels were marginally changed in all three transgenic lines relative to the Wt, with changes following the same inverse pattern as that observed for MYB gene expression (Fig. 3C). In response to wounding, AtST5a expression was significantly lower in all three lines, with the most and least significant decreases occurring in AtSR1;atsr1 #2 and atsr1 plants, respectively. Basal expression of two additional genes encoding desulfoglucosinolate sulfotransferases involved in the final step of GS biosynthesis, AtST5b and AtST5c, was not affected by loss of atsr1 function (Gigolashvili et al. 2007, Malitsky et al. 2008) (Fig. 3C). However, in response to

wounding, AtST5b and AtST5c transcript levels were significantly increased and decreased, respectively, compared with that of Wt plants (Fig. 3C). Expression of both genes was higher than in the Wt in AtSR1;atsr1 #2 plants both before and after wounding, yet transcript levels did decrease following treatment. Moreover, basal and induced levels of AtST5c were lower than in the Wt in AtSR1;atsr1 #1 transgenics whereas AtST5b expression was higher both before and after treatment. Wound-induced expression of IQD1, a positive regulator of GS levels (Levy et al. 2005), was only negatively affected by the atsr1 mutation and its constitutive expression in AtSR1;atsr1 #2 plants (Fig. 3D). In contrast, UGT74B1 levels were significantly higher in both atsr1 and AtSR1;atsr1 #2 following wounding, with the latter exhibiting an increase in basal expression as well (Fig. 3D). UGT74B1 is an S-glycosyltransferase involved in the formation of desulfoGSs that are converted to GSs following sulfation by AtST5a, AtST5b or AtST5c (Grubb and Abel 2006).

Discussion Though several proteins involved in Ca2+ sensing have been associated with plant responses to wounding and insect herbivory, little is known about the relationship between Ca2+ signaling and the regulation of GS metabolism (Levy et al. 2005). Based on current literature, Arabidopsis IQD1 and WRKY53 are the only described proteins involved in Ca2+/CaM-mediated signaling to function in the regulation of GS biosynthesis and hydrolysis, respectively (Levy et al. 2005, Miao and Zentgraf 2007, Popescu et al. 2007). Our results establish the Ca2+/CaMbinding transcription factor AtSR1 as a novel component of plant resistance to insect herbivory, probably through modulation of GS accumulation. Loss of AtSR1 function impairs feeding tolerance to the generalist herbivore T. ni, with mutant plants supporting significantly higher larval weight gains as early as 12 h after incubation. The sensitivity of the mutant to T. ni feeding is associated with significantly decreased total GSs, most notably reduced levels of I3M and 4MSOB which are known herbivory deterrents (Howe and Jander 2008). Based on gene expression analysis, the reduced I3M content in atsr1 plants does not appear to be a result of disrupted desulfo-I3M metabolism as AtST5a expression was not significantly altered in the mutant background. In conjunction with AtST5b and AtST5c, AtST5a catalyzes the conversion of desulfo-I3M to I3M (Gigolashvili et al. 2007), while the former two prefer (Met)-derived substrates and the latter those derived from tryptophan (Malitsky et al. 2008). Though AtST5c is responsible for the majority of I3M conversion, it is of note that loss of AtSR1 function results in significantly higher and lower wound-induced AtST5b and AtST5c expression, respectively. It has been previously reported that the atsr1 mutant accumulates high levels of endogenous SA (Du et al. 2009). This increase probably leads to depleted levels of the SA precursor chorismate, which is the same metabolite as required for tryptophan biosynthesis (Tzin an Galili 2010). Thus, the lack


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Fig. 3 The atsr1 mutant has significantly altered expression of genes associated with GS metabolism. Basal and wound-induced expression of genes involved in/related to (A) indole GS metabolism, (B) aliphatic GS accumulation, (C) sulfation of desulfoglucosinolates and (D) desulfo-/GS levels in Wt, atsr1 and AtSR1;atsr1 plants.

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display increased leaf senescence similar to that observed in the atsr1 mutant, suggesting that WRKY53 and AtSR1 may have additional functional interaction (Miao and Zentgraf 2007, Du et al. 2009). Intriguingly, MYB51 overexpression leads to increased GS accumulation and reduced insect herbivory (Gigolashvili et al. 2007); however, atsr1 plants display the opposite phenotypes despite high MYB51 transcript levels in the mutant background. This could be explained if AtSR1 is affecting MYB51 function in GS biosynthesis at the protein level, either directly or indirectly through interaction(s) with positive and/or negative regulators; however, their exact relationship requires further experimental analyses. It is also possible that the influence of AtSR1 expression on MYB51 transcription is connected to its regulatory effects on chorismate, a precursor of SA, as well as phenylalanine-, tyrosine- and tryptophan-derived GS biosynthesis rather than direct involvement in the GS pathway (Tzin and Galili 2010). Alternatively, AtSR1 may function jointly with IQD1 to integrate Ca2+ signaling and GS metabolism, with regulation of MYB51 serving as a mechanism for fine-tuning GS accumulation (Levy et al. 2005). As Ca2+ and CaM have been shown to differentially regulate JA-independent/dependent woundinduced signaling (Leon et al. 1998) and JA can induce intraand/or extracellular Ca2+ mobilization (Sun et al. 2010), this would also account for AtSR1 function in SA/JA-mediated responses. Overall, our results define AtSR1 as a critical regulator of Arabidopsis GS accumulation and provide a novel avenue for exploring the molecular mechanisms underlying SA- and JA-mediated wound responses as well as further defining the role of Ca2+/CaM-dependent signaling in plant resistance to herbivory. However, the exact role of AtSR1 in GS regulation, biosynthesis and/or metabolism still requires further examination. In the future, it will beneficial to analyze the effects of AtSR1 on specialist insect feeding for comparison with those on T. ni herbivory and to determine how AtSR1 function relates to IQD1 and MYB51 expression as well as hormone signaling integration. While this paper was in revision, Qiu et al. (2012) have reported that atsr1 plants are susceptible to another insect (Bradysia impatiens). Their study has shown that SR1 regulates both JA-dependent and -independent wound signaling pathways. However, they have not shown a role for SR1 in regulating GS metabolism.


of observed correlation between the reduced levels of I3M in atsr1 plants and AtST5a expression could be a result of decreased amounts of tryptophan-derived desulfo-I3M leading to decreased AtST5a activity based on substrate preference. Decreased AtST5a activity may also explain the observed up-regulation of AtST5b and MYB28 in atsr1 as the plant tries to overcompensate by augmenting methionine-derived GS biosynthesis. The effects of increases or decreases in AtSR1 expression on SA biosynthesis may also explain why constitutive expression does not lead to higher GS accumulation than that observed in Wt plants. Alternatively, the function of AtSR1 or aspects of its role in regulating GS biosynthesis and/ or metabolism may be governed by some type of feedback regulation. As, based on our analyses, transcript accumulation of various GS-related genes appears to be inversely related to the amount of AtSR1 present, it seems plausible that the contribution of AtSR1 to GS regulation may be dependent on specific expression thresholds. Both loss of AtSR1 function and its constitutive expression lead to significantly lower levels of wound-induced JA accumulation, which could explain the differential expression of ESR and WRKY53, both JA-regulated genes, observed in AtSR1;atsr1 plants. As constitutive AtSR1 expression only disrupted induced JA accumulation yet restored feeding tolerance as well as Wt SA and GS levels in the mutant, it appears that atsr1 susceptibility to herbivory is likely to be a joint effect of altered SA and JA leading to a negative regulation of GS biosynthesis and/or catabolism. This is consistent with published reports showing that GS accumulation is dependent on functional JA as well as SA signaling responses (Mikkelsen and Halkier 2003, Mewis et al. 2005). Thus, the decreased tolerance of atsr1 plants to T. ni feeding could be a reflection of SA-mediated suppression of JA-dependent oxylipin signaling required for Wt GS accumulation and herbivory responses (Mewis et al. 2005). Similar to AtSR1, loss-of-function mutation in HYPERSENSITIVE RESPONSE-LIKE LESIONS1 (HRL1) results in increased levels of endogenous SA as well as reduced total GS levels and enhanced susceptibility to insect feeding (Devadas et al. 2002, Mewis et al. 2005, Du et al. 2009). HRL1 is also a negative regulator of plant defense against pathogen infection involved in the modulation of synergistic interactions between the SA and JA/ET signaling pathways (Devadas et al. 2002). Based on the shared phenotypes resulting from their loss of function, AtSR1 and HRL1 may have overlapping or similar function in the regulation of Arabidopsis defense responses (Devadas et al. 2002, Mewis et al. 2005, Du et al. 2009). The notion that AtSR1 may act as a novel regulator of synergistic cross-talk between SA- and JA-regulated responses is further supported by the altered levels of these hormones in the mutant as well as its putative role in the negative regulation of WRKY53 expression. WRKY53 and ESR antagonistically regulate pathogen resistance and senescenc, with their interaction governed by the SA and JA equilibrium (Miao and Zentgraf 2007). It is also of note that plants overexpressing WRKY53

Materials and Methods Plant lines, growth conditions and treatments Plants were grown in soil under fluorescent light at 21 ± 2 C with a 16 h light/8 h dark cycle. Isolation of the atsr1 mutant alleles and generation of transgenic AtSR1;atsr1 lines were described previously (Du et al. 2009). For wounding, samples were taken 90 min post-perforation with serrated forceps, with plants spatially separated to prevent cross-exposure of released volatiles. For herbivory assays, pre-weighed second instar larvae


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of T. ni were placed on individual rosettes of 5-week-old plants. Immediately after inoculation, 4 inch transparent plastic cylinders topped with muslin were placed over individual plants to control insect movement and allow for free gas exchange. Plants were randomized to minimize the effects of microenvironment variation, with feeding tolerance scored as measures of larval weight gain and percentage leaf damage. Feeding tolerance was also assessed by placing 6–8 leaves from 5-week-old plants in Petri dishes lined with filter paper soaked in half-strength Murashige and Skoog (MS) medium. Each leaf set was inoculated with a single larva, and larval weights were recorded at regular time intervals for a minimum of 10 leaf sets per genotype. Experiments were repeated at least three times with similar results.

Plant hormone extractions and quantification were carried out as previously described (Engelberth et al. 2003, Schmelz et al. 2003, Chehab et al. 2008) with dihydro-JA, deuterated SA (CDN Isotopes Inc.) and deuterated ABA (Icon Services) used as internal standards. Volatiles were analyzed by gas chromatography–mass spectrometry (GC-MS) using an Agilent Technologies 5973 network mass selective detector operated in electronic ionization mode with a DB-5MS column (30 m 0.25 mm, 0.25 mm). Constitutive levels of GSs were determined as previously described (Mikkelsen and Halkier 2003) using sinigrin as an internal standard. HPLC separation of GSs was carried out on a reverse phase C-18 column (Zorbax Eclipse XDB C18, 4.6 150 mm, 5 mm) and eluents monitored by diode array detection between 190 and 360 nm (2 nm intervals). Experiments were repeated at least three times with similar results.

RNA extraction and expression analyses RNA extraction, cDNA synthesis and quantitative reverse transcription–PCR (qRT–PCR) were carried out as previously described (Dhawan et al. 2009) using gene-specific primers with Arabidopsis Actin2 as an endogenous reference for normalization. Expression levels were calculated by the comparative cycle threshold method (Applied Biosystems). For wound-induced expression, samples were taken 90 min post-perforation with serrated forceps. All experiments were repeated at least three times with similar results. Primers used are listed in Supplementary Table S2.

Statistical analysis Data were analyzed by Tukey–Kramer pairwise comparisons (P = 0.05) using JMP Pro 9 software, with error bars in all figures representing standard error of the mean. Letters in graphs indicate statistically significant differences amongst values.

Supplementary data Supplementary data are available at PCP online.

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This work was supported by the National Science Foundation [5333470 to A.S.N.R.], the Binational Agricultural Research and Development Fund [5357100 to A.S.N.R. and M.L.]; the National Institute of Health [R01 GM086496 to T.M.-O.].

Acknowledgments We would like to thank Dr. Irene Day for her comments on this manuscript.

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Extraction and quantification of plant hormones and metabolites

Funding


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2015