Combined biotic stresses trigger similar transcriptomic responses but contrasting resistance against a chewing herbivore in Brassica nigra

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1 Bonnet et l. BMC Plnt Biology (217) 17:127 DOI /s RESEARCH ARTICLE Open Aess Combined bioti stresses trigger similr trnsriptomi responses but ontrsting resistne ginst hewing herbivore in Brssi nigr Christelle Bonnet 1, Steve Lssueur 1, Cmille Ponzio 2, Riet Gols 2, Mrel Dike 2 nd Philippe Reymond 1* Abstrt Bkground: In nture, plnts re frequently exposed to simultneous bioti stresses tht tivte distint nd often ntgonisti defense signling pthwys. How plnts integrte this informtion nd whether they prioritize one stress over the other is not well understood. Results: We investigted the trnsriptome signture of the wild nnul ruifer, Brssi nigr, in response to eggs nd terpillrs of Pieris brssie butterflies, Brevioryne brssie phids nd the bteril phytopthogen Xnthomons mpestris pv. rphni (Xr). Pretretment with egg extrt, phids, or Xr hd wek impt on the subsequent trnsriptome profile of plnts hllenged with terpillrs, suggesting tht the seond stress domintes the trnsriptionl response. Nevertheless, P. brssie lrvl performne ws strongly ffeted by egg extrt or Xr pretretment nd depended on the site where the initil stress ws pplied. Although egg extrt nd Xr pretretments inhibited inset-indued defense gene expression, suggesting sliyli id (SA)/jsmoni id (JA) pthwy ross tlk, this ws not stritly orrelted with lrvl performne. Conlusion: These results emphsize the need to better integrte plnt responses t different levels of biologil orgniztion nd to onsider lolized effets in order to predit the onsequene of multiple stresses on plnt resistne. Keywords: Brssi nigr, Brevioryne brssie, Combined stresses, Herbivory, Pieris brssie, Trnsriptome, Xnthomons mpestris pv. rphni (Xr) Bkground Bioti nd bioti stresses impose strong pressure on plnts in nture. When ombined, stresses suh s het, drought or high light intensity hve profound effets on rop performne nd yields [1]. Plnts hve developed speifi mehnisms to preisely detet environmentl hnges nd respond to omplex stress onditions to minimize dmge nd onserve suffiient resoures for growth nd reprodution. Over the yers, reserh hs foused minly on responses to single stress in severl * Correspondene: Philippe.Reymond@unil.h 1 Deprtment of Plnt Moleulr Biology, University of Lusnne, Biophore Building, 115 Lusnne, Switzerlnd Full list of uthor informtion is vilble t the end of the rtile model plnts inluding Arbidopsis [2 5]. However, there is growing reognition for the need to onsider the effets of multiple stresses t the moleulr level nd t higher levels of biologil orgniztion [6 9]. Suh n pproh is ruil s we need to know how plnts dpt to novel environmentl ftors in the ontext of o-ourring stresses [1]. Inset herbivory is mjor bioti stress under nturl onditions. Therefore, plnts hve evolved sophistited onstitutive nd induible defenses to resist or redue the effets of inset ttk [11]. Severl studies hve shown tht plnts subjeted to bioti stress or nutritionl limittion differentilly ffet the performne nd behvior of insets [12 17]. In ddition to insets, plnt The Author(s). 217 Open Aess This rtile is distributed under the terms of the Cretive Commons Attribution 4. Interntionl Liense ( whih permits unrestrited use, distribution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Publi Domin Dedition wiver ( pplies to the dt mde vilble in this rtile, unless otherwise stted.

2 Bonnet et l. BMC Plnt Biology (217) 17:127 Pge 2 of 14 pthogens re mjor thret to plnt growth nd survivl, but lso impt on the oloniztion by nd performne of herbivores feeding on pthogen-infeted plnts [18, 19]. As biotrophi nd nerotrophi phytopthogens exhibit distint infetion pthwys, they indue different plnt responses [2]. Their effets on plnts my influene the phytohemil environment of the inset ttker in different wys. Moreover, pthogen effets on plnt resistne to insets will depend on the biology of the herbivore, e.g., whether it is phloem feeder or hewing lrv [19]. For exmple, the nerotrophi pthogen Botrytis inere inhibited the development, feundity nd survivl rte of the phid Aphis fbe in Vii fb, wheres the biotrophi fungus Uromyes viie-fbe enhned phid performne [21]. Most interestingly, the effet of ombined B. inere nd U. viie-fbe stress on phid performne seemed to flutute depending on the order of infetion [21]. In ontrst, B. inere pretretment hd no signifint effet on further performne of Pieris rpe terpillrs in Arbidopsis [22]. Tomto plnts hllenged by Pseudomons syringe redued Spodopter exigu growth, wheres tomto mosi virus inresed terpillr performne but deresed phid oloniztion [23]. Mndu sext lrve feeding on Niotin ttenut plnts treted with the bteril quorum-sensing N-ylhomoserine ltone were signifintly hevier thn on untreted plnts. This effet ws ttributed to n inhibition of plnt defenses ginst herbivores [24]. Oviposition by Pieris brssie inhibited growth of P. syringe strins in Arbidopsis [25]. Furthermore, P. brssie lrve showed poor performne on P. syringe-infeted Arbidopsis plnts, suggesting tht inset eggs inhibit plnt defenses for the benefit of their progeny [25]. In summry, the outome of bioti pretretment on herbivore performne is diffiult to predit nd depends primrily on the severity nd durtion of the infetion, ttk strtegies of the pthogens nd herbivores involved, nd the plnt speies tht is ttked. Indued defenses re ontrolled by phytohormones. Biotrophi pthogens, whih obtin nutrients from living tissues tivte minly the sliyli id (SA) pthwy, while nerotrophs obtining nutrients from ded host tissues nd hewing herbivores tivte espeilly the jsmoni id (JA) nd ethylene (ET) pthwys [26]. These pthwys regulte the expression of defense genes tht provide speifi resistne to the ttker. The existene of ntgonism between the SA nd JA pthwys is well estblished [26]. It is thought to modulte prioritiztion of defense llotion towrds different ttkers [27, 28] but is lso the trget of defense mnipultion by plnt pthogens nd inset herbivores [26, 29 33]. Stimultion of the SA pthwy ttenuted plnt response to generlist herbivores, i.e. herbivores feeding on plnt speies in different plnt fmilies, but hd no effet on the speilist P. brssie, whih primrily feeds on plnt speies within the Brssiee fmily [34, 35]. At the moleulr level, tretments with SA or pthogens tht enhne SA levels redued the expression of the ntiherbivore VSP2 in Arbidopsis [36, 37]. Pthwy ross tlk my thus represent ruil omponent of plnt responses to ombined stresses. Trnsriptome nlyses hve been onduted to better understnd plnt responses to multiple stresses. For instne, one study nlyzed trnsriptomi differenes in ten eotypes of Arbidopsis hllenged by single or dul () bioti stress ombintions. The uthors onluded tht the mjority of hnges in gene regultion in response to ombined stresses were not preditble using expression profiles from single tretments [38]. Drought or flooding pretretment signifintly modified the trnsriptome signture of Solnum dulmr plnts infested with S. exigu [16]. Simultneous ttk by spfeeding nd hewing herbivores in N. ttenut triggered trnsriptionl response tht ws distint from those in response to single ttkers [39]. An overview of 33 different ombined stresses reveled tht eh tretment seems to generte unique response, refleting the plnt s bility to speifilly dpt to hnging nd omplex environment [8]. The sme onlusion ws rehed for the impt of ombined stresses t the metbolomi nd proteomi level, with severl unique metbolites nd proteins umulting fter multiple stresses but not fter single stresses [8, 4]. However, Arbidopsis plnts hllenged by both nemtodes nd drought responded primrily to drought [41]. Thus, plnt responses to multiple stresses re interonneted nd result in omplited nd unpreditble outomes. More studies on plnt responses to ombined stress onditions re ritil to understnd the effets of these intertions. This requires nlysis t multiple levels, trnsriptionl nd hormonl responses, defense ompound umultion nd eologil onsequenes, using different plnt speies. Here, we investigted the effets of ombined biotis stresses on plnt trnsriptomi hnges, hnges in plnt hormones nd metbolites nd inset performne, using nd eologilly relevnt system. We seleted the wild nnul ruifer, Brssi nigr, subjeted to feeding by nturlly ssoited lepidoptern pest, terpillrs of the lrge bbge white P. brssie, lone or in ombintion with seond stress. Combined stresses onsisted of pretretment with P. brssie egg extrt, the bbge phid Brevioryne brssie, or the nerotrophi bteril phytopthogen Xnthomons mpestris pv. rphni (Xr), followed by terpillr herbivory. All stresses used here our nturlly on B. nigr in the field [42, 43]. Wheres

3 Bonnet et l. BMC Plnt Biology (217) 17:127 Pge 3 of 14 plnt defenses ginst hewing lrve re primrily regulted by the JA pthwy, eggs nd phids tivte primrily the SA pthwy [35, 44 46], nd defense ginst Xr is medited by SA, JA nd ET [47]. Given the known mutul ntgonisti tions of these signling pthwys, we were expeting signifint effets of primry stress on the responses to P. brssie lrve. Intertions of B. nigr-ttker intertions re well-investigted t the eologil level under field onditions where multiple ttkers our [43, 48, 49]. However, muh less is known bout the mehnisti spets of the responses of B. nigr plnts to single s ompred to ombined stresses. This is the topi of the present study. Results Effet of ombined stresses on trnsriptionl responses to herbivory We used whole-genome Arbidopsis CATMA mirorrys [5, 51] to ssess gene expression hnges in B. nigr. Previous studies hve shown tht Arbidopsis mirorrys n be suessfully used to study trnsriptionl responses of Brssi olere or B. nigr [52, 53]. After 1 dy of feeding by P. brssie lrve on B. nigr, 218 genes were signifintly upregulted (log 2 >.585, P <.5) nd 49 genes were signifintly downregulted (log2 >.585, P <.5) (Additionl file 1: Tble S1). Gene ontology (GO) serh of the upregulted genes reveled highly signifint enrihment of terms inluding response to wounding (GO:9611), response to stress (GO:695), response to jsmoni id stimulus (GO:9753), response to bioti stimulus (GO:967), response to hitin (GO:12), defense response (GO:6952), jsmoni id biosynthesis (GO:9695), oxylipin biosynthesis (GO:3148), seondry metboli proess (GO:19748). Downregulted genes were enrihed in terms like photosynthesis (GO:15979), ellulr metboli proess (GO:44237), nitrogen metbolism (GO:34631, GO:44271), hloroplst (GO:597, GO:44434) (Additionl file 2: Tble S2). This trnsriptionl signture onfirms results from previous studies on the response to terpillr herbivory in other plnt speies, whih hve identified ruil role for the jsmonte pthwy in induing nti-inset defense genes nd observed downregultion of photosynthesis-relted genes [45, 54 59]. Beuse we used Arbidopsis mirorrys, some more distntly relted B. nigr defense genes my hve been missed in the hybridiztion proedure. A more exhustive list of inset-responsive genes will wit trnsriptome nlyses by RNA sequening one B. nigr referene genome is vilble. Then, to investigte how bioti pretretment my ffet P. brssie-indued trnsriptome hnges, we hllenged B. nigr plnts with P. brssie egg extrt, the bteril pthogen Xr, or B. brssie phids before dding P. brssie lrve for 24 h. As ontrol experiments, we subjeted B. nigr plnts to eh single stress. Strikingly, n expression-bsed lustering nlysis of ll experiments showed tht trnsriptomes from the three ombined stress tretments were grouped with the trnsriptome of P. brssie lrvl tretment, wheres trnsriptomes from egg extrt, the bteril pthogen, or phid single tretments were lerly seprted (Fig. 1). Indeed, from the list of 218 uregulted nd 49 downregulted genes fter herbivory lone, 26 (94%), repetively 43 (88%), were still similrly regulted fter egg extrt pretretment, 155 (72%), respetively 38 (78%), fter pthogen pretretment, nd 21 (92%), respetively 46 (94%), fter phid pretretment, inditing tht the bioti pretretments pplied hd wek effet on the subsequent trnsriptionl response to herbivory (Fig. 1b, ). Anlysis of the 5 top up- nd downregulted genes fter single tretment with terpillrs showed tht expression of 48, respetively 42 genes, did not differ signifintly between single or ombined stress with egg extrt. Similrly, 36 upregulted nd 46 downregulted genes were not expressed differently between herbivory nd ombined stress with phids (Additionl files 3, 4: Figures S1, S2). However, in the se of pthogen pretretment, 22 of the top-5 genes showed signifintly redued indution, inluding known JA-regulted genes like LOX3, CORI3, nd OPR3, suggesting tht bteril infetion inhibits defense ginst herbivory (Additionl file 3: Figure S1). A ombintion of stresses my tivte genes tht re normlly not regulted during single stresses. To identify speifi signture of ombined stress, we serhed for genes tht were signifintly indued or repressed only in the three dul-stress tretments (egg extrt/terpillrs, pthogen/terpillrs/, or phids/terpillrs). There were respetively 7, 23, nd 52 upregulted genes nd 16, 15 nd 13 downregulted genes meeting these riteri. Strikingly, omprison of these ombinedstress-speifi genes indited tht only one gene ws ommonly regulted in egg extrt/terpillrs nd pthogen/terpillrs while other genes were speifilly regulted by eh ombintion of stresses (Fig. 2). A GO serh of the ombined-stress responsive genes did not revel enrihment of ny prtiulr or onserved biologil proess (Additionl files 5, 6: Figures S3, S4). These results indite tht there is no typil trnsriptionl signture of ombined stress but tht eh ombintion tivtes reltively smll number of dditionl genes. Effet of ombined stresses on lrvl performne nd plnt defense ompounds Performne of P. brssie terpillrs ws lso mesured in terms of weight gin on plnts pretreted with egg extrt or the pthogen nd on untreted plnts.

4 Bonnet et l. BMC Plnt Biology (217) 17:127 Pge 4 of 14 A B % of up-regulted genes C % of down-regulted genes EE Xr Aphids Xr + P. b. EE + P. b. P. b. Aphids + P. b. EE + P.b. Xr + P.b. Aphids + P.b. When terpillrs were feeding freely on entire leves pre-treted with egg extrt or the pthogen, their weight gin ws signifintly redued ompred to tht on ontrol plnts (liner mixed model (LMM), P [egg extrt] =2*1 16 nd pthogen, P [pthogen] =2*1 8 ) (Fig. 3)..2 EE + P.b. Xr + P.b. Aphids + P.b. Fig. 1 Expression profiles in response to single nd ombined stresses in Brssi nigr plnts. Correspondene nlysis of expression profiles inluding ll indued or repressed genes in t lest one experiment (.585 < log 2 rtio >.585, P <.5,n =961). Clustering nd node length lultions were performed with MultiExperiment Viewer using Person s orreltion. b Proportion of P. brssie-upregulted genes tht re lso upregulted during ombined stress. Eh br segment (yellow, green, blue) represents different ombined stress. The proportion of genes speifilly indued by P. brssie is shown in ornge. The number of genes regulted by herbivory (upregulted, n = 218; downregulted, n = 49) is set to 1%. () ProportionofP. brssie-downregulted genes lso downregulted during ombined stress. P.b., P. brssie lrve; EE, P. brssie egg extrt; Xr, Xnthomons mpestris pv. rphni; Aphids, Brevioryne brssie To test whether ltered inset performne on pretreted plnts orrelted with hnges in defense signls nd metbolites, we quntified SA, JA, nd gluosinolte (GS) onentrtions. GS re potent defense ompounds in brssieous plnts, effetive ginst generlist insets, tht umulte in response to herbivory [6 64]. However, results from whole-lef nlyses showed tht onentrtions of JA nd totl GS were not signifintly different in leves tht were pretreted with egg extrt or the pthogen followed by terpillr feeding nd in leves exposed to terpillr feeding lone (Two-wy ANOVA, P [JA, egg extrt] =.62,P [JA, pthogen] =.17,P [GS, egg extrt] =.46,P [GS, pthogen] =.41) (Additionl file 7: Figure S5). For SA, the presene or bsene of terpillr feeding did not lter the signifint umultion in response to egg extrt or pthogen tretment (Two-wy ANOVA, P [SA, egg extrt] =.2, P [SA, pthogen] =.49) (Additionl file 7: Figure S5). Sine the differene in lrvl performne between untreted nd pretreted plnts ws not esily explined by trnsriptomi dt, by hnges in defense hormonl signling, or by GS umultion in whole B. nigr leves, we deided to study the effet of pretretment reltive to the feeding site of the terpillrs. The rtionle ws tht lolly indued hnges within the lef my ount for the observed effets. Noteworthy, reent study on mize hs reported sttistilly signifint higher onentrtion of defense metbolites, i.e. 1,4-benzoxzin-3-ones, in young leves ompred to old ones nd tht this ws negtively orrelted with inset performne [65]. We modified the experimentl design by onstrining lrve in lip ges lolly or distlly, reltive to the pretretment site. Cterpillr weight gin ws signifintly redued fter egg extrt pretretment, but only when terpillrs were fored to feed on the pretretment site (LMM, P [lol] =.29; P [distl] =.37) (Fig. 3b). The effet ws similr to the whole-lef response (Fig. 3). In ontrst, terpillrs performed signifintly better on pthogen-pretreted site thn on ontrol leves (LMM, P = 9*1 7 ) but their weight ws not different when fored to feed distlly from the pretretment site (LMM, P =.22), suggesting for instne lol suppression of defenses by bteril effetors (Fig. 3b). This result ws different from the result of the whole-lef experiment, where terpillr performne ws redued on pthogen-pretreted leves (Fig. 3). Thus, the respetive loliztion of pthogen pretretment nd terpillr feeding site lerly impted the effet of the pthogen on inset performne. To further orrelte inset performne nd site of tretment with defense ompound nd signling hormone umultion, we quntified SA, JA, nd GS onentrtions in lef tissues olleted from untreted plnts, nd from pretreted plnts t the site where the

5 Bonnet et l. BMC Plnt Biology (217) 17:127 Pge 5 of 14 A upregulted Egg extrt + P. brssie Xr + P. brssie Aphids + P. brssie B downregulted Egg extrt + P. brssie Xr + P. brssie Aphids + P. brssie C upregulted EE + P.brssie Xr + P. brssie D downregulted EE + Pieris Xr + P. brssie Aphids + P. brssie Aphids + P. brssie Fig. 2 Number of upregulted nd b downregulted genes in response to single nd ombined stresses. Number of genes differentilly regulted fter ombined P. brssie feeding nd P. brssie egg extrt (yellow), Xnthomons mpestris pv. rphni (green) ndbrevioryne brssie (blue) re indited. Number of genes speifilly regulted by P. brssie feeding (ornge) nd speifilly regulted by ombined stress (grey) islso indited., d Distribution of genes speifilly regulted by ombined stress shows very little overlp pretretment stress ws pplied. In plnts tht were lso exposed to terpillr feeding, lef tissues were olleted from the res where the terpillrs were onstrined, so t the site of pretretment or t site distl from the pretretment pplition (Fig. 4). In the experiment with egg extrt, JA umulted only in response to herbivory. Egg-extrt pretretment itself did not use JA umultion nd did not lter terpillr-indued JA onentrtion (ANOVA, F = 13,31, P =.1) (Fig. 4b). In ontrst, pretretment with the pthogen resulted in 1-fold inrese of JA, wheres JA onentrtions in lef tissues exposed to terpillr feeding lone were not signifintly different from those found in ontrol plnts (ANOVA, F = 68.85, P = 5*1 7 ) (Fig. 4b). JA ws indued eqully (5-fold) when terpillrs were feeding distlly from the site where the pthogen ws pplied, nd when terpillrs were feeding t the sme site (Fig. 4b). Egg extrt nd pthogen pretretments signifintly indued SA, only t the tretment site. Moreover, there ws no hnge in SA onentrtions in response to herbivory, nd terpillrs did not ffet egg extrt- or pthogenindued SA onentrtions (ANOVA, F [egg extrt] =18.37, P [egg extrt] = 2*1 5, F [pthogen] = 66.18, P [pthogen] = 5*1 8 ) (Fig. 4). Thus, B. nigr leves respond lolly to bioti hllenges by umulting distint JA or SA onentrtions depending on the bioti stress. Furthermore, the SA response to ombined stresses did not differ from the hormonl response to single stresses, wheres it did for JA. As with whole-lef experiments, totl GS onentrtions did not hnge signifintly between ontrol nd treted plnts (ANOVA, F [egg extrt] = 2.71, P [egg extrt] =.58, F [pthogen] =.66, P [pthogen] =.68) (Fig. 4d). Among the 13 gluosinoltes tht were identified nd quntified (Additionl file 8: Tble S3), sinigrin ontributed 91% to 96% of the totl GS ontent in different tretments. Consistent with lk of GS umultion fter bioti stress in B. nigr, we observed tht expression of 22 out of 27 GS biosynthesis genes ws not signifintly enhned in response to single or ombined stresses (Additionl files 1, 9: Tble S1, Figure S6). This ontrsts with the oordinted indution of genes involved in ll steps of GS biosynthesis in Arbidopsis fter herbivory [64].

6 Bonnet et l. BMC Plnt Biology (217) 17:127 Pge 6 of 14 A Pretretment P. b. lrve Lrvl weight (mg) B CTL EE *** 3 *** CTL EE CTL Xr Pretretment Xr Lrvl weight (mg) Clip ge P. b. lrve CTL * EE lol CTL EE distl EE or Xr lol CTL EE or Xr distl *** Xr lol Xr distl Fig. 3 Impt of pretretment on inset performne. Lrvl weight of P. brssie feeding on 5-week-old B. nigr plnts pretreted for 3 dys with P. brssie egg extrt or Xnthomons mpestris pv. rphni (Xr) ws mesured fter 7 dys of feeding. Vlues (± SE) re the men of independent experiments (P. brssie: CTL/EE, n = 5; CTL/Xr, n = 4). The totl number of lrve is indited in eh olumn. Signifint differenes between ontrol nd pretretment re indited (liner mixed model, ***P <.1).b Lrvl weight of P. brssie feeding on 5-week-old B. nigr plnts pretreted for 3 dys with P. brssie egg extrt (EE) or Xnthomons mpestris pv. rphni (Xr) ws mesured fter 4 dys. Lrve pled in lip ges were feeding on the treted site (lol) or djent to the treted site (distl). Vlues (± SE) re the men of four (CTL/EE) or three (CTL/Xr) independent experiments. The totl number of lrve is indited in eh olumn. Signifint differenes between ontrol nd pretretment re indited (liner mixed model, ***P <.1, **P <.1,*P <.5) Effet of ombined stresses on SA/JA ross tlk Sine exposure to egg extrt, pthogen tretment nd terpillr feeding triggered SA nd JA umultion to different extents, we deided to investigte the known SA/JA ntgonism in response to ombined stresses. We designed QPCR primers for B. nigr sequenes relted to VSP2 nd MYC2, whih re JA- nd herbivoryregulted genes [56], nd for PR2 nd SAG13, whih re SA- nd egg-regulted genes [66]. In single stress tretments, BnVSP2 nd BnMYC2 expression were signifintly upregulted in tissues exposed to terpillr feeding, but not in tissues treted with egg extrt or the pthogen (Two-wy ANOVA, P [VSP2, egg extrt] =.1, P [VSP2, pthogen] <.1, P [MYC2, egg extrt] =.4, P [MYC2, pthogen] <.1) (Fig. 5). Contrstingly, BnPR2 nd BnSAG13 were signifintly upregulted in tissues treted with egg extrt or the pthogen, but not in tissues exposed to terpillr feeding (Two-wy ANOVA, P [PR2, egg extrt] <.1, P [PR2, pthogen] <.1, P [SAG13, egg extrt] <.1, P [SAG13, pthogen] <.1) (Fig. 5). Interestingly, for ombined stresses we found tht both egg extrt nd pthogen pretretments led to signifintly redued indution of inset-responsive BnVSP2 nd BnMYC2. Combined stresses lso redued the indution of egg extrt- or pthogen-responsive BnSAG13, wheres BnPR2 indution ws only inhibited by egg extrt pretretment (Fig. 5). These results suggest tht under dul-stress onditions ombined umultion of SA nd JA in response to either egg extrt or pthogen pretretment followed by herbivory negtively ffets speifi JA-nd SA-responsive genes. We thus observed onsistent nd reiprol SA/JA ross tlk in B. nigr, in response to tretment with egg extrt or the pthogen followed by terpillr feeding.

7 Bonnet et l. BMC Plnt Biology (217) 17:127 Pge 7 of 14 A Pretretment Clip ge P. b. lrve B JA (ng / g FW) Smple b CTL EE or Xr CTL EE P - EE/P EE P b b P d EE or Xr lol b EE or Xr distl CTL Xr P - Xr/P Xr P d b C SA (ng / g FW) D Totl GS (mg / g FW) Lol Distl Lol Distl b 2 b b 15 b 1 b 5 CTL EE P - EE/P EE P CTL Xr P - Xr/P Xr P Lol Distl Lol Distl CTL EE P - EE/P EE P CTL Xr P - Xr/P Xr P Lol Distl Fig. 4 Quntifition of defense signls nd gluosinoltes. Experimentl design. Quntifition of jsmoni id (JA) b, sliyli id (SA) nd totl gluosinoltes (GS) d in single nd ombined stress. Lef diss (blue irle) were olleted on 5-week-old B. nigr plnts pretreted for 3 dys with P. brssie egg extrt (EE) or Xnthomons mpestris pv. rphni (Xr) nd further hllenged with P. brssie (P) lrve feeding for 24 h t the site or distl to the site of tretment. Controls onsisted of untreted plnts (CTL) or plnts exposed to single tretment. -, smple distl from the ombined tretment. Vlues (± SE) re the men of three independent experiments. Letters indite signifint differene between tretments (ANOVA followed by Tukey s honest signifint differene test, P <.5) Lol Distl Disussion Exposure to two or more bioti stresses n either be more detrimentl thn single stress or, onversely, hve n ttenuting effet. The bility of plnts to reognize nd respond to ombined nd speifi stresses ppers thus to be importnt, espeilly if stresses, suh s pthogens nd herbivores, trigger different plnt defense pthwys. Few studies hve been onduted on wholegenome responses under multiple stress onditions, nd gene expression studies often foused on the plnt model Arbidopsis. Here, B. nigr, whih is lso brssieous plnt speies, ws used to investigte how plnts respond to ombined stresses. Surprisingly, trnsriptomi responses of B. nigr to different pretretments followed by P. brssie herbivory reveled tht the first stress hs only wek impt on trnsriptionl responses

8 Bonnet et l. BMC Plnt Biology (217) 17:127 Pge 8 of 14 A Pretretment Clip ge B Reltive expression BnVSP2 b CTL EE P EE/P 25 BnPR2 b CTL EE P EE/P P. b. lrve Smple.6 b BnVSP C CTL Xr P Xr/P BnPR2 b b CTL Xr P Xr/P EE or Xr BnMYC2 2.5 b P b CTL EE P EE/P BnSAG13 CTL EE P EE/P EE/P or Xr/P BnMYC2 b CTL Xr P Xr/P BnSAG13 b CTL Xr P Xr/P Fig. 5 Expression of JA- nd SA-relted genes. Experimentl design. b Expression of B. nigr genes ws mesured by QPCR nd normlized to the housekeeping gene BnSAND. Lef diss (blue irle) were olleted on 5-week-old B. nigr plnts pretreted for 3 dys with P. brssie egg extrt (EE) or Xnthomons mpestris pv. rphni (Xr) nd further hllenged with P. brssie (P) feeding for 24 h t the site of tretment. Untreted plnts (CTL) or plnts exposed to single stress were inluded. Mens (± SE) of three tehnil replites re shown. These experiments were repeted t lest twie with similr results. Different letters indite signifint differenes (two-wy ANOVA followed by Tukey s honest signifint differene test, P <.5) to the seond stress. In ddition, no genes ommon to ombined stress ould be identified. It ws reently found tht the effet of previous exposure to B. inere or to drought only slightly hnged Arbidopsis trnsriptionl response to P. rpe feeding, suggesting tht plnts prioritize response to the seond stress [22]. Similrly, Arbidopsis trnsriptome fter P. brssie feeding ws not ffeted by pre-exposure to P. brssie eggs, lthough it ws impted by old pretretment [67]. However, detiled time-ourse nlysis reveled tht pre-exposure shifts the timing of terpillr-indued responses. Plnts responded fster to P. rpe if they were preeded by drought or B. inere tretment [59], inditing tht timing of the response needs to be onsidered. Atkinson nd oworkers [41] postulted tht during multiple ttk, plnts respond preferentilly to the most dmging stress (see lso [68]). In Arbidopsis hllenged by drought nd/ or nemtodes, 96% of differentilly regulted genes were shred between the ombined stress nd wter stress, wheres only 2% overlpped with nemtode feeding [41]. We hypothesize tht B. nigr prioritized response to terpillr feeding rther thn to phids, eggs or bteri for the benefit of its own fitness, s P. brssie terpillrs re known to be vorious feeders on brssieous plnts; these terpillrs re florivorous when rehing the seond nd subsequent instrs, thus reduing fitness diretly [69]. Also t the level of the metbolome, hnges in B. nigr plnts exposed to B. brssie phids nd/or P. brssie terpillrs were the strongest in response to feeding by terpillrs both when feeding lone or together with the phids on the sme lef [68]. It would be interesting to perform reiprol experiments to see if the trnsriptome of P. brssie-pretreted plnts is dominted by the signture of seond bioti stress or if plnts prioritize the response to herbivory over other stresses. A reent trnsriptome study on Arbidopsis plnts infeted by Botrytis inere with or without prior herbivory suggests tht the first hypothesis is more likely [59]. Another testble hypothesis is tht plnts respond to the most severe stress, irrespetive of the order of ttk. We observed tht pretretment with the biotrophi pthogen Xr hd mesurble effet on the P. brssie trnsriptome. Indeed,. 3% of inset-indued genes, inluding JA-regulted genes, were signifintly less indued in response to the ombined stress, but t the sme time weight-gin of P. brssie terpillrs ws redued on pthogen-infeted plnts when feeding on the entire lef or when restrited to feed distlly from the site where the pthogen-pretretment ws pplied. Sine Xr single tretment triggered SA umultion, we hypothesize tht

9 Bonnet et l. BMC Plnt Biology (217) 17:127 Pge 9 of 14 SA/JA ross tlk ws responsible for this ttenution of gene expression. Our trgeted nlysis of the JA mrkers BnVSP2 nd BnMYC2 onfirmed this observtion, but the inset performne ssy lso indited tht ttenution of these genes does not hve negtive onsequenes for plnt defense ginst P. brssie. Cterpillr weight gin ws lso redued on plnts pretreted with n egg extrt. SA-responsive genes, inluding PR1, were lerly upregulted by egg extrt tretment inditing tht the SA pthwy ws tivted by eggs, like in Arbidopsis [35, 66]. This pprent bsene of SA/JA ross tlk t the whole-genome level my be explined by reltively less strong response to P. brssie eggs. Indeed, we found muh higher SA umultion fter Xr pthogen thn fter P. brssie egg extrt tretment. In ddition, whole-lef nlysis my hve diluted lolized response sine we deteted lolized suppression of BnVSP2 nd BnMYC2 expression fter egg extrt tretment. Cross tlk between defense signling pthwys is known to strongly modulte the outome of ombined bioti stresses [26]. Here, we lso showed tht pthogen nd egg extrt pretretments inhibited both indution of JA- nd SA-regulted genes in response to dditionl feeding by P. brssie terpillrs, suggesting tht SA/JA ross tlk redued the trnsription of some genes onsidered importnt in plnt defense ginst insets. Although single Xr infetion led to strong SA nd JA umultion, it is noteworthy tht only SA-regulted BnPR2 nd BnSAG13 were indued but not JA-regulted BnVSP2 nd BnMYC2. Moreover, SA/JA ross tlk effets were most pronouned on BnVSP2 nd BnMYC2 trnsript levels in response to ombined Xr/herbivory stresses, inditing tht SA strongly influened the JA pthwy in this prtiulr ontext. Depending on the hormonl ontext, umultion of defense signl is thus not neessrily orrelted with the indution of downstrem genes. Conversely, pthogen- nd eggextrt indution of SA-signling relted genes BnPR2 nd BnSAG13 ws inhibited by terpillr feeding, suggesting tht the reiprol JA/SA ross tlk ws lso operting. The onsequene of suh ross tlk on suseptibility to Xr infetion ws not tested but would be n interesting topi for future reserh. Thus, JA nd SA tivtion nd their mutulisti ntgonisti effets my depend on the strength or the nture of the tretment. For exmple, study in Arbidopsis reported synergisti or ntgonisti effet on JA- nd SA-indued genes if plnts were treted with low or high onentrtions of eh hormone, respetively [7]. In ddition, plnt responses to herbivory re known to be dynmi nd my depend on the smpling time [59, 71]. It will thus be interesting in future experiments to see if our observtions on SA/JA ross tlk t single time point robustly underlie the outome of the ombined intertions. In onlusion, lthough the emerging piture is tht of domintion of the most reent stress on the trnsriptionl response [22, 59], it would be interesting to onfirm this hypothesis by extending the rnge of reiprol ombintions of bioti nd bioti stresses, inluding time-ourse nlyses. We found tht inset performne differed between tretments, suggesting tht plnt resistne sttus fter ombined stresses is diffiult to predit bsed on trnsriptome, defense hormone profiles or defense pthwy ross tlk. Indeed, we observed tht inset performne fter P. brssie egg-extrt pplition ws deresed. In Arbidopsis, we previously showed tht P. brssie egg deposition hd no effet on performne of the speilist P. brssie [35]. Other studies with Arbidopsis nd B. nigr reveled tht P. brssie performed less well or eqully in the presene of eggs, depending on the speies identity of the egg donor [67, 72 74]. Similr results were found for P. brssie feeding on other wild brssieous speies, i.e. Brssi olere, Morindi morindioides nd Sinpsis rvensis exposed to P. brssie eggs [43]. On the ontrry, the generlist S. littorlis performed better on plnts lredy treted with P. brssie egg extrt or fter nturl oviposition [35, 75] but no effet ws found for eggs of the generlist Mmestr brssie on subsequent M. brssie lrvl performne [72]. Thus, whether inset eggs indue plnt defenses is ontext-speifi. Similrly, whether there is n effet of exposure to biotrophi pthogen is ontext speifi [25, 76] nd my lso depend on the virulene level of the pthogen [77]. Furthermore, P. brssie terpillrs feeding freely on Xr-pretreted leves gined less weight thn terpillrs feeding on n untreted lef. Thus, t the whole-lef level, Xr pretretment impted plnt defense responses similrly to the egg extrt pretretment, lthough the underlying mehnism might hve been different. A study on Cpsium nnuum L. reported n enhned performne of S. exigu lrve on plnts infeted with X. mpestris pv. vesitori [76]. In ontrst, performne of P. brssie lrve ws redued on Arbidopsis infeted with Pseudomons syringe pv. tomto [25]. Agin, inset performne on plnts infeted with phytopthogens seems to be vrible. Surprisingly, ompred to whole-lef biossys, inset performne ssys yielded somewht ontrsting onlusions using lip ges to restrit feeding by terpillrs on speifi sites on lef. Wheres egg-extrt tretment impted lrve similrly regrdless whether they were feeding freely on the whole lef or lolly in lip ge, P. brssie terpillrs feeding on Xr-infiltrted lef re were lrger thn those onstrined to

10 Bonnet et l. BMC Plnt Biology (217) 17:127 Pge 1 of 14 feed on non-infeted zone or on n untreted lef. This observtion ould be explined by our finding of lol inhibition of JA-dependent defense gene expression fter Xr pretretment, lthough restrited feeding on egg-extrt pretreted tissues did not result in enhned inset performne. Hene, other ftors likely ontribute to lolized effet. Xr infetion triggered lol umultion of both SA nd JA while P. brssie eggextrt tretment only triggered SA umultion. Inset herbivores tend to void defended lef res [78], whih they ould not do under the onstrined lip-ge onditions. Indeed, when given the hoie we notied tht P. brssie lrve voided egg-treted nd Xrinfeted zones (Additionl file 1: Figure S7). This is intriguing with regrd to the opposite performne of lrve when feeding on egg-treted or Xr-infeted zones. This finding of lrvl seletive feeding deserves further investigtion. Differentil tivtion of the SA nd JA signling pthwys my ffet metbolite omposition. However, we did not observe differentil GS umultion between tretments. A similr finding ws observed fter ombined ozone nd P. brssie tretment in B. nigr, lthough lrve grew less well on ozone-pretreted plnts [79]. Contrsting results were reported in the study by Ponzio et l. [68] where the totl GS onentrtion signifintly inresed in response to feeding by P. brssie terpillrs. Furthermore, indution of GS under dul stress onditions with terpillrs nd B. brssie phids depended on the density of the phids. The differene in terpillr densities per lef, i.e. Thirty in the Ponzio et l. study [68] nd 1 here, my explin this disrepny. As postulted previously [8], other defense ompounds my ply ruil role in influening P. brssie performne on Brssieous plnt speies. The reent identifition of flvonoid ompounds tht negtively impt P. brssie terpillr performne in Arbidopsis supports this onlusion [81, 82]. In ddition, sine ET is n importnt regultor ssoited with Xr in Arbidopsis [47], lol ET signling my be involved in the lol effet of Xr on P. brssie performne. Moreover, plnt nutritionl qulity t the tretment site ould lso ply role. It my negtively orrelte with inset performne on eggtreted sites but positively on Xr-treted sites. For instne, lef rbohydrte ontent ws found to be ontrolled by JA nd medited plnt suseptibility to n dpted herbivore in Niotin ttenut [83]. Conlusions Our trnsriptome nlysis of B. nigr in response to ombined stress tretments reveled tht the seond stress domintes the trnsript signture, lthough pretretments lerly impted how plnts resisted n herbivore ttk. Mesurement of defense-signling hormones nd trnsript levels of defense mrker genes in response to multiple ttk by different stresses do not neessrily predit the plnt s defense response in strightforwrd fshion. Future studies should inlude more mrker genes representing different steps long the moleulr sequene of events. Our results show tht under onditions of multiple stress the plnt responds highly speifilly to eh stress ombintion. Contrsting responses strongly suggest tht we need to better integrte responses t different levels of biologil orgniztion, to onsider lol versus distnt plnt responses within lef, nd to mesure the umultion of rnge of (defense) metbolites determining nutritionl qulity when trying to orrelte plnt trits with inset performne. Methods Biologil mteril Seeds of Brssi nigr were olleted from wild popultion in Wgeningen (The Netherlnds) [43]. Plnts were grown in soil in growth hmbers (16 h light, t 25 C dy, 22 C night, 6% reltive humidity) under white fluoresent light (17 μmol m 2 s 1 ). Seeds were strtified for 3 dys t 4 C fter sowing. The soil ontined 65% humus, 1% snd, 15% perlite nd 1% silt. Growth onditions were the sme in the different biossys desribed below. Xnthomons mpestris pv. rphni (Xr) (formerly lssified s X. mpestris pv. rmorie) ws obtined from the Plnt-Mirobe Intertions group of Utreht University (The Netherlnds) nd ws originlly quired from the Deprtment of Plnt Pthology t Ohio Stte University (USA). The pthovr identity ws onfirmed by pthogeniity ssys nd PCR. Bteri were grown in 1 ml of liquid King B ulture medium (2 g / l peptone (Sigm-Aldrih), 1.5 g / l dipotssium hydrogen phosphte, 1.5 g / l mgnesium sulfte hepthydrte, 12 g /l gr, t finl ph of 7.2) supplemented with rifmpiin (25 μg/ ml) nd grown in shker t 28 C, 2 rpm, during 48 h. Xr ulture ws entrifuged t 7 rpm during 2 min. The superntnt ws disrded nd the pellet ws wshed nd re-suspended in 1 mm MgCl 2 nd entrifuged gin t 7 rpm during 2 min. The superntnt ws disrded nd the pellet diluted in 1 mm MgCl 2 nd djusted to n OD 6 of.7 to obtin onentrtion of 1 7 fu/ml. Pieris brssie ws rered on Brussels sprout plnts (Brssi olere vr. gemmifer) in1m 3 ges in greenhouse (25 ± 5 C, 6 ± 5% RH, 16/8 h light-drk yle) t Lusnne University (Switzerlnd). Eggs were removed mnully from the plnts nd rushed with pestle in Eppendorf tubes. After entrifugtion (15 g, 3 min), the superntnt (egg extrt) ws stored t 2 C. Brevioryne brssie phids were rered on B. olere vr. gemmifer in greenhouse (22 ± 3 C, 65 ± 5%

11 Bonnet et l. BMC Plnt Biology (217) 17:127 Pge 11 of 14 RH, 16/8 h light-drk yle) t Wgeningen University (The Netherlnds), where ll experiments with phids were lso performed. B. nigr plnts were grown in pet soil (Lentse potgrond no. 4, Lent, The Netherlnds). The pest speies P. brssie nd B. brssie, nd B. nigr plnts were olleted in the wild in The Netherlnds. This omplies with ntionl legisltion s The Netherlnds llows free ess to its biodiversity under the Ngoy Protool. Corret identifition of B. nigr ws onfirmed by Dr. E. H. Poelmn (Deprtment of Plnt Sienes, Wgeningen University, The Netherlnds). Seeds of B. olere vr. gemmifer were obtined ommerilly from Semenes Zollinger (1897 Les Evouettes, Switzerlnd) or Syngent Seeds (2678 LV De Lier, The Netherlnds). Plnt tretments The overll experimentl design is summrized in Tble S4 (Additionl file 11: Tble S4). Plnts were 5 weeks old when exposed to the vrious tretments. Pretretments with egg extrt nd the pthogen were pplied to the three youngest fully developed leves of three plnts. Aphids were pplied to single lef, i.e. the first fully developed lef (nine plnts in totl), ording to design tht hs been used in previous experiments with the sme study system [84, 85]. For P. brssie egg-extrt tretment, 12 2 μl of egg extrt were dded to eh of the three leves nd inubted for 72 h. This tretment ws equivlent to tretments previously pplied to Arbidopsis nd orresponds to pproximtely 1 12 egg bthes per lef, eh bth onsisting of 2 3 eggs [35, 66]. Leves of untreted plnts were used s ontrols. For infetion with the bteril pthogen, X. mpestris pv. rphni, eh of the three tretment leves ws subjeted to three infiltrtions of 1 7 fu/ml using 1 ml needleless syringe nd inubted for 72 h. Eh infiltrtion zone represented irle of 1.5 m 2. In ontrol plnts, the sme number of 1 mm MgCl 2 infiltrtions ws performed. For tretment with B. brssie phids, 1 nymphs were pled on the youngest fully developed lef on eh of nine plnts, whih were inubted for 48 h. Aphids were not onstrined but remined on the lef on whih they hd been introdued. Tretment with terpillrs onsisted of the introdution of 1 neonte terpillrs on the three leves tht hd reeived pretretment (ombined stresses) or on three leves similr in development of len plnts. Thirty neonte terpillrs were introdued on the single phid-treted lef or single lef of len plnt. Cterpillrs were llowed to feed for 24 h. All experiments were repeted independently five or more times t intervls of severl weeks. Inset performne ssys on plnts pretreted with egg extrt or pthogen Five-week-old B. nigr plnts were pled in m plsti tents (Bugdorm ompny) in growth hmber (2 ± 1 C, 65 ± 1% reltive humidity, 1/14 h light-drk yle, 1 μmol m 2 s 1 ). For inset biossys performed on entire leves, ten neonte terpillrs were pled on eh of the three pretreted leves or on three leves of len plnts with totl of 3 terpillrs per plnt. Cterpillr weight ws mesured fter 7 dys of feeding. For biossys investigting lol vs. distl effets of pretretment, five neonte terpillrs were pled in lip ge ( mm, Bio- Quip Produts, USA) on eh of three pretreted leves with totl of 15 lrve per plnt either t the sme site or site distl from where the pretretment ws pplied. Plnts were pretreted s desribed bove with egg extrt or the pthogen nd inubted for 3 dys (see Fig. 4 for experimentl design). Cterpillr weight ws mesured fter 4 dys. For ll experiments, inset reovery ws similr between tretments. Eh tretment ws done on three different plnts for eh biologil replite. All experiments were repeted independently three or more times t intervls of severl weeks. Hormone nd gluosinolte nlysis Lef tissues tht were smpled for hormone (SA nd JA) nd GS nlysis were exposed to egg extrt, the pthogen, nd/or terpillr feeding s desribed bove. Entire leves (experiments with no onstrint on terpillr feeding) or 2.4 m lef diss (experiments with onstrined terpillr feeding) were hrvested nd frozen in liquid nitrogen. Extrtion, UHPLC-QTOFMS mesurement nd dt nlysis were onduted s desribed erlier [86, 87]. Three independent biologil replites were nlyzed for eh tretment. Trnsriptome nlyses Following tretment, entire leves were hrvested, flshfrozen in liquid nitrogen nd stored t 8 C. RNA extrtion, probe lbeling, hybridiztion onto Arbidopsis CATMAv4 mirorrys, nd dt nlyses hve been published previously [51, 56, 88]. For dt nlysis, we used n expression threshold of log 2 >.585 nd <.585, nd n undjusted P-vlue of.5. FDR vlues re shown in supplementry dt for further evlution. GO enrihment nlysis ws performed with AgriGO singulr enrihment nlysis using hypergeometri test [89]. Quntittive PCR Reltive gene expression ws mesured ording to previously published proedures [35, 9]. Briefly, 5

12 Bonnet et l. BMC Plnt Biology (217) 17:127 Pge 12 of 14 nnogrms of totl RNA were trnsribed to DNA using M-MLV reverse trnsriptse (Invitrogen) nd oligo dt primers ording to ommeril instrutions. DNA synthesis ws done in triplites. QPCR nlysis ws performed in finl volume of 25 μl ording to the Brillint III Fst SYBR Green instrution mnul (Agilent). B. nigr primers (Additionl file 12: Tble S5) were designed on onserved sequenes identified by multiple lignments of genes from different speies of the Brssi fmily. Sequenes were obtined from the Brssi dtbse ( Eh primer hs Tm of 6 C nd gives n mplions length between 1 nd 25 bp in the onserved prt of the DNA strnd. Primer effiienies were evluted by five-step dilution regression. Eh mplion produed single bnd nd ws onfirmed by Snger sequening. For normliztion, the BnSAND gene ws used s housekeeping gene. Similr to Arbidopsis SAND gene [91], its expression ws stble ross experiments. Additionl files Additionl file 1: Tble S1. Gene expression rtios (log 2 ) for ll biologil replites. (XLSX kb) Additionl file 2: Tble S2. GO nlysis of P. brssie-regulted genes. (PDF 49 kb) Additionl file 3: Figure S1. Expression of the top-5 upregulted genes in response to P. brssie feeding nd ombined stresses. The highest signifintly upregulted genes (log 2 >.585, P <.5) were extrted from mirorry dt (ornge brs) nd plotted with vlues from ombined stresses. (A) Egg extrt/p. brssie lrve (yellow brs), (B) Xnthomons mpestris pv. rphni/p. brssie lrve (green brs), nd (C) Brevioryne brssie/p. brssie lrve (blue brs). Signifint differenes between single nd ombined stress re indited (Student s t-test, ***P <.1, **P <.1, *P <.5). (PDF 1871 kb) Additionl file 4: Figure S2. Expression of the top-5 downregulted genes in response to P. brssie feeding nd ombined stresses. The highest signifintly downregulted genes (log 2 <.585, P <.5) were extrted from mirorry dt (ornge brs) nd plotted with vlues from ombined stresses. (A) Egg extrt/p. brssie lrve (yellow brs), (B) Xnthomons mpestris pv. rphni/p. brssie lrve (green brs), nd (C) Brevioryne brssie/p. brssie lrve (blue brs). Signifint differenes between single nd ombined stress re indited (Student s t-test, ***P <.1, **P <.1, *P <.5). (PDF 1875 kb) Additionl file 5: Figure S3. GO nlysis of genes speifilly upregulted by ombined stress. GO terms signifintly enrihed with eh ombined stress re shown seprtely. Length of the brs shows the perentge of regulted genes in the respetive GO tegories. (PDF 951 kb) Additionl file 6: Figure S4. GO nlysis of genes speifilly downregulted by ombined stress. GO nlysis of genes speifilly downregulted by ombined stress. GO terms signifintly enrihed with eh ombined stress re shown seprtely. Length of the brs shows the perentge of regulted genes in the respetive GO tegories. (PDF 918 kb) Additionl file 7: Figure S5. Quntifition of defense signls nd gluosinoltes. (A) Experimentl design. (B) Quntifition of jsmoni id (JA), sliyli id (SA) nd totl gluosinoltes (GS) in single nd ombined stress in whole 5-week-old B. nigr leves. Plnts were pretreted for 3 dys with P. brssie egg extrt (EE) or Xnthomons mpestris pv. rphni (Xr) nd further hllenged with P. brssie (P) lrve for 24 h. Controls (CTL) onsisted of untreted plnts or plnts exposed to single tretment. Vlues (± SE) re the men of three independent experiments. Letters indite signifint differene between tretments (two-wy ANOVA followed by Tukey s honest signifint differene test, P <.5). (PDF 15 kb) Additionl file 8: Tble S3. Gluosinolte ontent in B. nigr leves. (PDF 37 kb) Additionl file 9: Figure S6. Expression of gluosinolte biosynthesis genes in response to P. brssie feeding nd ombined stresses. Vlues were extrted from mirorry dt. P. brssie lrve (ornge brs), egg extrt/p. brssie lrve (yellow brs), Xnthomons mpestris pv. rphni/p. brssie (green brs), Brevioryne brssie/p. brssie (blue brs). Signifint differenes between single nd ombined stress re indited (Student s t-test, ***P <.1, **P <.1, *P <.5). (PDF 982 kb) Additionl file 1: Figure S7. Feeding behvior of P. brssie lrve in response to ombined stresses. Neonte lrve were llowed to feed freely for 2 dys (P. brssie) on 5-week-old B. nigr plnts pretreted for 3 dys with egg extrt (A) or Xnthomons mpestris pv. rphni (B). Representtive imges from three biologil replites re shown. Sle br = 1 m. (PDF 9284 kb) Additionl file 11: Tble S4. Overll experimentl design. (PDF 48 kb) Additionl file 12: Tble S5. List of primers used for QPCR. (PDF 51 kb) Aknowledgements We thnk Blise Tissot for mintenne of the plnts nd Johnn Weber for help with mirorry nlyses. Funding The Swiss Ntionl Siene Foundtion (grnt 313A_ nd EUROCORES progrm EuroVOL grnt 31VL3_ to PR) nd the Erth nd Life Sienes Counil (ALW) of the Netherlnds Orgnistion for Sientifi Reserh (NWO) (EUROCORES progrm EuroVOL grnt to MD) supported this work. Avilbility of dt nd mterils Mirorry dt hve been submitted to the ArryExpress dtbse ( under ession number E-MTAB-559. Authors ontributions CB, CP, RG, MD nd PR designed the experiments. CB, SL nd CP performed the experiments. CB nd PR nlyzed the dt. CB, MD, RG nd PR wrote the pper. All uthors hve red nd pproved this mnusript. Ethis pprovl nd onsent to prtiipte Not pplible. Consent for publition Not pplible. Competing interests The uthors delre tht they hve no ompeting interests. Publisher s Note Springer Nture remins neutrl with regrd to jurisditionl lims in published mps nd institutionl ffilitions. Author detils 1 Deprtment of Plnt Moleulr Biology, University of Lusnne, Biophore Building, 115 Lusnne, Switzerlnd. 2 Lbortory of Entomology, Wgeningen University, P.O. Box 16, 67 AA Wgeningen, The Netherlnds. Reeived: 6 Jnury 217 Aepted: 1 July 217 Referenes 1. Chew IH, Hllidy KJ. A stress-free wlk form Arbidopsis to rops. Curr Opin Biotehnol. 211;22: Bry EA. Plnt responses to wter defiit. Trends Plnt Si. 1997;2:48 54.