Feeding on Leaves of the Glucosinolate Transporter Mutant gtr1gtr2 Reduces Fitness of Myzus persicae

Transcription

1 J Chem Ecol (2015) 41: DOI /s Feeding on Leves of the Glucosinolte Trnsporter Mutnt gtr1gtr2 Reduces Fitness of Myzus persice Svend Roesen Mdsen 1 & Grit Kunert 2 & Michel Reichelt 2 & Jonthn Gershenzon 2 & Brr Ann Hlkier 1 Received: 9 June 2015 /Revised: 22 Septemer 2015 /Accepted: 29 Septemer 2015 /Pulished online: 28 Octoer 2015 # The Author(s) This rticle is pulished with open ccess t Springerlink.com Astrct As phids re pest on vrious crops worldwide, etter understnding of the interction etween phids nd plnt host defenses is required. The green pech phid (Myzus persice) feeds on vriety of plnt species, including the model plnt Aridopsis thlin (Aridopsis), in which glucosinoltes function s mjor prt of the chemicl defense. Severl studies hve shown tht glucosinoltes ply role in interctions etween Aridopsis nd the green pech phid. In this work, we used recently identified Aridopsis glucosinolte trnsporter mutnt (gtr1gtr2 dko), with ltered glucosinolte content in the vsculture, to investigte the role of defense compound trnsport in phid infesttion. By monitoring phid performnce on cged leves nd nlyzing glucosinoltes in lef tissue nd phloem sp, s well s inside phids, we exmined if chnge in sptil distriution of glucosinoltes within lef influences phid performnce. Bsed on reduced glucosinolte content in the phloem sp of the trnsporter mutnt, we hypothesized tht phids would perform etter on gtr1gtr2 dko leves compred to WT. Unexpectedly, phids performed poorly on gtr1gtr2 dko leves. Our dt suggest tht higher glucosinolte content in tissues surrounding the phloem of the doule trnsporter Electronic supplementry mteril The online version of this rticle (doi: /s ) contins supplementry mteril, which is ville to uthorized users. Brr Ann Hlkier h@plen.ku.dk 1 2 DynMo Center of Excellence, Deprtment of Plnt nd Environmentl Sciences, Fculty of Science, University of Copenhgen, 40 Thorvldsensvej, DK-1871 Frederikserg C, Denmrk Mx Plnck Institute for Chemicl Ecology, Jen, Germny mutnt my ply role in reducing phid performnce on this genotype. Keywords Glucosinoltes. Aphid performnce. Sptil glucosinolte distriution. Trnsporter mutnt. Cged leves Introduction Aphids constitute mjor pest on crops worldwide (Ahuj et l. 2010). The green pech phid (Myzus persice) feeds on the sugr-rich phloem of wide rry of plnt species, including the model plnt Aridopsis thlin (Aridopsis) (de Vos et l. 2007; Louis 2012). In Aridopsis, mjor prt of the chemicl defense is constituted y methionine (liphtic) nd tryptophn (indole)-derived glucosinoltes (Hlkier nd Gershenzon 2006; Klieenstein et l. 2001), which upon plnt injury re hydrolyzed rpidly y myrosinses into multitude of physiologiclly ctive products (Hlkier nd Gershenzon 2006). Severl experiments hve shown glucosinoltes to ffect interctions etween Aridopsis nd the green pech phid. Glucosinolte levels were noted to influence host plnt choice, with phids preferring to feed on Aridopsis plnts with reduced glucosinolte content (Levy et l. 2005). In other studies, phid reproduction ws correlted negtively with the content of liphtic nd indole glucosinoltes in Aridopsis mutnts (Mewis et l. 2005, 2006). Altered glucosinolte levels in response to green pech phid infesttion lso hve een reported, leit with conflicting trends: In one study, oth liphtic nd indole glucosinolte content incresed in Aridopsis rosettes fter one week of phid feeding (Mewis et l. 2006), wheres decrese of lmost ll glucosinoltes in Aridopsis rosettes ws oserved fter three dys of phid exposure in nother experiment (Kim

2 976 J Chem Ecol (2015) 41: nd Jnder 2007). In the ltter study, totl glucosinolte content in individul cged leves infested with phids ws either unchnged or incresed, lrgely due to locl ccumultion of 4-methoxyindol-3-ylmethyl glucosinolte (4MI3M) nd 8- methylsulfinyloctyl glucosinolte (8-MSO) (Kim nd Jnder 2007). While the enzymes involved in glucosinolte iosynthesis hve een identified (Jensen et l. 2014; Sondery et l. 2010), less is known out trnsport of these compounds. Recently, two glucosinolte-specific importers, GTR1 (NPF2.10) nd GTR2 (NPF2.11), were identified in Aridopsis (Lern et l. 2014; Nour-Eldin et l. 2012). Asence of liphtic nd indole glucosinoltes in seeds of the gtr1gtr2 doule knockout (dko) mutnt long with vsculr locliztion of these trnsporters demonstrted tht GTR1 nd GTR2 re essentil for long-distnce trnsport of glucosinoltes to the seeds, most likely y trnsporting glucosinoltes into phloem compnion cells (Nour-Eldin et l. 2012). Investigtion of long-distnce root-shoot/shoot-root trnsport of glucosinoltes in three-week-old wild-type (WT) nd gtr1gtr2 dko Aridopsis plnts reported strong ccumultion especilly of long-chin, liphtic glucosinoltes (minly 8-methylsulfinyloctyl (8-MSO)) in rosettes of the glucosinolte trnsporter mutnt (Andersen et l. 2013). Furthermore, 8-MSO nd its precursor 8-MTO were found collectively to e 6-fold enriched in the xylem sp of the gtr1gtr2 dko mutnt compred to WT (Mdsen et l. 2014). Although green pech phids re phloem feeders, their feeding pttern cn e divided into three min phses: pthwy phse, phloem phse, nd xylem phse (Louis 2012). During the phloem phse, the phid is feeding minly from phloem sieve element. The pthwy phse includes intrcellulr smpling of epidermis nd mesophyll cells on the wy to the phloem. Xylem phse refers to the time when the insect is ingesting xylem sp, nd it is thought to fcilitte the uptke of wter needed to dilute the high sucrose content of the phloem sp, or to compenste for wter loss during periods without feeding. In Aridopsis, phids re le to void the glucosinolte myrosinse defense since they inflict only minor wounding on the plnt while feeding (Brth nd Jnder 2006), s lso evidenced y the fct tht intct glucosinoltes hve een detected in the honey-dew of phids (Kim nd Jnder 2007). This suggests tht the negtive correltion etween glucosinolte levels nd preferred feeding nd fecundity is relted to intct glucosinoltes, nd not their hydrolysis products. A etter understnding of the interction etween phids nd plnt host defenses is required, s these insects re worldwide pests on crops. Since glucosinoltes re trnsported vi the phloem nd xylem sps tht phids feed from, investigtion of phid performnce on the gtr1gtr2 dko with ltered glucosinolte content in the vsculture my improve our understnding of the defensive role of glucosinoltes in plntphid interctions. In this study, we used the gtr1gtr2 dko mutnt to investigte whether chnge in sptil distriution of glucosinoltes within lef influences phid performnce. We show tht green pech phid fecundity nd survivl rte re dversely ffected when fed only on the gtr1gtr2 dko mutnt compred to WT. Methods nd Mteril Plnt Growth Conditions Seeds of Aridopsis WT (Col-0) nd mutnts with T-DNA insertions in oth GTR1 (At3g47960) nd GTR2 (At5g62680) (gtr1gtr2 dko - for more detils, see (Nour-Eldin et l. 2012)) were sown on soil in 10 cm pots nd cold-strtified t 4 C for 2 d. Then, the plnts were grown in climte chmer under short dy conditions (L:D 10:14 h, C, nd % reltive humidity) for 3.5 wk. until they reched the rosette stge. After this, plnts were trnsferred to long dy conditions (L:D 16:8 h) for hlf week to dpt to conditions necessry for thriving phids. Lef Phloem Exudte Collection Leves (5 replictes) were cut on the se of the rosette nd put into plstic vil (petiole down) contining 300 μl 20 mm EDTA with 5 mm sodium phosphte uffer (ph 6), nd incuted for 1 h in closed drk ox with wet tissues in the ottom to increse humidity (Jnder et l. 2004). EDTA ws employed to ind C 2+ ions nd prevent sieve tue occlusion. However, the contents of cut lef cells could lso lek into the smple. Afterwrds, leves were trnsferred to new plstic vils contining 300 μl tp wter (petiole dipped in wter efore inserting into vil to rinse off EDTA uffer), nd incuted in the sme drk ox for 4.5 h for phloem smpling. Leves were weighed for normliztion. Wter smples including phloem exudtes were nlyzed for metolites. Glucosinolte Anlysis in Phloem nd Single Aphids Aphids (15 replictes) were weighed (round 0.5 mg) nd extrcted with 0.05 ml of 100 % methnol contining 25 pmol of pr-hydroxyenzyl glucosinolte (pohb) s n internl stndrd. The phloem smples were spiked with the sme internl stndrd. Chromtogrphy ws performed on n Agilent 1200 HPLC system (Agilent Technologies). Seprtion ws chieved on Zorx Eclipse XDB-C18 column ( mm, 1.8 μm, Agilent). An API 5000 tndem mss spectrometer (Applied Biosystems) equipped with Turospry ion source ws operted in negtive ioniztion mode. The ion spry voltge ws mintined t 4500 ev. The turo gs temperture ws set t 700 C. Neulizing gs ws set t 60 psi, curtin gs t 25 psi, heting gs t 60 psi, nd collision gs t 10 psi. Multiple rection monitoring (MRM) ws used to monitor nlyte prent ion product

3 J Chem Ecol (2015) 41: ion: see (Tle 1) for detils. Both Q1 nd Q3 qudrupoles were mintined t unit resolution. Anlyst 1.5 softwre (Applied Biosystems) ws used for dt cquisition nd processing. Intct glucosinoltes were quntified reltive to the signl of the internl stndrd pohb pplying the experimentlly determined response fctors listed in (Tle 1). Sugr Anlysis of Phloem Exudtes Sugrs in the phloem smples were nlyzed directly y LC-MS/MS fter 1:10 (v/ v) dilution in wter. Chromtogrphy ws performed on n Agilent 1200 HPLC system (Agilent Technologies, Boelingen, Germny). Seprtion ws chieved on n HILIC-HPLC-column (pher NH2 Polymer; 15 4,6 mm, 5 μm, Supelco). Wter nd cetonitrile were employed s moile phses A nd B, respectively. The elution profile ws: min, 80 % B in A; min, % B in A; min, % B in A; nd min, 80 % B in A. The moile phse flow rte ws 1.0 ml/min. Column temperture ws mintined t 25 C. Liquid chromtogrphy ws coupled to n API 3200 tndem mss spectrometer (Applied Biosystems, Drmstdt, Germny) equipped with Turospry ion source operted in negtive ioniztion mode. The instrument prmeters were optimized y infusion experiments with pure stndrds (D-(+)-glucose, D-( )-fructose, sucrose, ll Sigm-Aldrich). The ion spry voltge ws mintined t 4500 ev. The turo gs temperture ws set t 600 C. Neulizing gs ws set t 50 psi, curtin gs t 20 psi, heting gs t 60 psi, nd collision gs t 5 psi. Multiple rection monitoring (MRM) ws used to monitor nlyte prent ion product ion: m/z (collision energy (CE) -10 V; declustering potentil (DP) -25 V) for D-(+)-glucose; m/z (CE -12 V; DP -25 V) for D-(-)-fructose; m/z (CE -46 V; DP -55 V) for sucrose. Both Q1 nd Q3 qudrupoles were mintined t unit resolution. Anlyst 1.5 softwre (Applied Biosystems, Tle 1 Detils of nlysis of intct glucosinoltes (Gls) y LC-MS/ MS (HPLC 1200 (Agilent Technologies)-API 5000 (Applied Biosystems)) in negtive ionistion mode. Given re the prent-toproduct ion trnsitions used to quntify ech compound. LC conditions: Drmstdt, Germny) ws used for dt cquisition nd processing. Individul sugrs in the smple were quntified y externl stndrd curves generted with dilution series of uthentic stndrds. Amino Acid Anlysis of Phloem Exudtes Undiluted phloem smples (245 μl) were mixed with 13C nd 15 N lelled mino cid mix (Algl mino cids 13C,15 N, Isotec, Mimisurg, US) (5 μl) to concentrtion of 10 μg/ml. Amino cids in the diluted extrcts were nlyzed directly y LC-MS/MS. The nlysis method ws modified from protocol previously descried y Jnder et l. (2004). Chromtogrphy ws performed on n Agilent 1200 HPLC system (Agilent Technologies, Boelingen, Germny). Seprtion ws chieved on Zorx Eclipse XDB-C18 column ( mm, 1.8 μm, Agilent Technologies, Germny). Formic cid (0.05 %) in wter nd cetonitrile were employed s moile phses A nd B, respectively. The elution profile ws: 0 1 min, 3 % B in A; min, % B in A; min 100 % B; nd min 3 % B in A. The moile phse flow rte ws 1.1 ml/min. Column temperture ws mintined t 25 C. Liquid chromtogrphy ws coupled to n API 3200 tndem mss spectrometer (Applied Biosystems, Drmstdt, Germny) equipped with Turospry ion source operted in positive ioniztion mode. The instrument prmeters were optimized y infusion experiments with pure stndrds (mino cid stndrd mix, Fluk, St. Louis, MO, USA). The ionspry voltge ws mintined t 5500 ev. Turo gs temperture ws set t 700 C. Neulizing gs ws set t 70 psi, curtin gs t 35 psi, heting gs t 70 psi, nd collision gs t 2 psi. Multiple rection monitoring (MRM) ws used to monitor nlyte prent ion product ion: MRMs were chosen s descried previously (Jnder et l. 2004), except for Arg (m/z ) nd Lys (m/z ). Both Q1 nd Q3 qudrupoles were mintined t unit resolution. Anlyst flow rte 800 μl/min, formic cid 0.05 % (A), cetonitrile (B): 95 % A (0.5 min), % A (3.5 min), 60 0 % A (0.1 min), 0 % A (1.9 min), 0 95 % A (0.1 min), 95 % A (2.4 min). DP, declustering potentil; CE, collision energy Compound Q1 Mss (D) Q3 Mss (D) DP CE Molr response fctor 3-methylsulfinylpropyl-Gls methylsulfinylutyl-Gls methylsulfinylpentyl-Gls methylsulfinylheptyl-Gls methylsulfinyloctyl-Gls methylthioutyl-Gls Indolyl-3-methyl-Gls Methoxy-indolyl-3-methyl-Gls Methoxy-indolyl-3-methyl-Gls pr-hydroxyenzyl Gls

4 978 J Chem Ecol (2015) 41: softwre (Applied Biosystems) ws used for dt cquisition nd processing. Linerity in ioniztion efficiencies ws verified y nlyzing dilution series of stndrd mixtures (mino cid stndrd mix, Fluk plus Gln, Asn nd Trp, lso Fluk). The concentrtion of the individul lelled mino cids in the mix hd een determined y clssicl HPLCfluorescence detection nlysis fter pre-column derivtiztion with ortho-phthldildehyde-mercptoethnol using externl stndrd curves mde from stndrd mixtures (mino cid stndrd mix, Fluk plus Gln, Asn nd Trp, lso Fluk). Individul mino cids in the smple were quntified y the respective 13C, 15 N leled mino cid internl stndrd, except for tryptophn nd sprgine: Tryptophn ws quntified using 13C, 15 N-Phe pplying response fctor of 0.42, wheres sprgine ws quntified using 13C, 15 N-Asp pplying response fctor of 1.0. Glucosinolte nd Glucosinolte Brekdown Product Anlysis of Lef Extrcts Collected leves (11 replictes) (~100 mg) were freeze-dried until constnt weight nd ground to fine powder. Glucosinoltes were extrcted with 1 ml of 80 % methnol solution contining 0.05 mm intct p- hydroxyenzyl glucosinolte s internl stndrd. After centrifugtion, extrcts were loded onto DEAE Sephdex A 25 columns, nd the flow-through of the smples ws collected. Bound glucosinoltes were treted with rylsulftse for desulftion (Sigm-Aldrich) overnight. The desulfo glucosinoltes were eluted with 0.5 ml wter nd were seprted using high performnce liquid chromtogrphy (Agilent 1100 HPLC system, Agilent Technologies) on reversed phse C-18 column (Nucleodur Sphinx RP, mm, 5 μm, Mchrey-Ngel, Düren, Germny) with wter (A)-cetonitrile (B) grdient (0 1min,1.5%B;1 6min,1.5 5 % B; 6 8min,5 7 % B; 8 18 min, 7 21%B;18 23 min, % B; min, % B; min 100 % B, nd min 1.5 % B; flow 1.0 ml/min). Detection ws performed with photodiode rry detector, nd peks were integrted t 229 nm. We used the following response fctors: liphtic glucosinoltes 2.0, indole glucosinoltes 0.5 (Burow et l. 2006) for quntifiction of individul glucosinoltes. Flow through frctions (21 23 replictes) from the DEAE Sephdex A 25 columns were collected nd diluted in rtio 1:4 (v:v) with wter. Diluted extrcts were nlyzed y LC- MS using n Agilent 1100 HPLC system (Agilent Technologies, Wldronn, Germny) coupled to Bruker Esquire 6000 ion trp mss spectrometer (Bruker Dltonics, Bremen, Germny) operted in lternting ioniztion mode in the rnge m/z (cpillry exit voltge, +117; cpillry voltge, +4000; neulizer pressure, 35 psi; drying gs, 11 l/ min; gs temperture, 330 C). Elution ws ccomplished using Nucleodur Sphinx RP column ( mm, 5 μm; Mcherey- Ngel, Düren, Germny). Moile phses were 0.2 % formic cid (v:v) (A) nd cetonitrile (B), strting with 100 % A for 5 min, followed y grdient to 45 % B in 15 min. The sutrction of the mss spectrometer totl ion chromtogrm of WT plnts from tht of different mutnt plnt lines ws done using the softwre pckge Metolite Detect 1.1 (Bruker Dltonics, Bremen, Germny) in order to serch for metolites tht differ etween plnt lines. For reltive quntifiction of pek res of metolites tht differed etween plnt lines, the respective extrcted ion trces were extrcted s follows: positive ioniztion mode: B8- methylsulfinyloctyl mine^ m/z 192; B9-methylsulfinyloctyl nitrile^ m/z 202 (9-methylsulfinylnonyl nitrile =8- methylsulfinyloctyl cynide). Aphid Cging on Leves To test for locl induction of glucosinoltes (nd other metolites) y phids, 4 WT rered dult phids were cged on mture lef (Fig. S3) for3d (11 replictes). Then, cged leves were collected, phids removed, nd leves put in liquid nitrogen for further processing (see ove). Cged leves without phids served s controls. For the phid performnce ssy (15 replictes), 1 dult phid, rered on WT plnt ws cged on lef of WT nd gtr1gtr2 dko plnt, respectively. One dult phid rered on gtr1gtr2 dko plnt lso ws cged on WT nd gtr1gtr2 dko lef. This results in four tretments: WT rered phids tested on WT ((WT)WT) nd on gtr1gtr2 dko plnts ((WT)dKO), nd gtr1gtr2 dko rered phids tested on WT ((dko)wt) nd on gtr1gtr2 dko plnts ((dko)dko). After 3 d, phid offspring were counted. Aphid infested leves were collected, phids were removed, nd leves were put in liquid nitrogen for lter metolite extrction (see ove). Sttisticl Anlysis In order to compre glucosinolte concentrtions in phloem etween WT plnts nd gtr1gtr2 dko plnts, the two smple t-test with the Welch modifiction to ccount for unequl vrinces ws used. To investigte the influence of phids nd plnt genotype on glucosinolte concentrtion in plnt nd phid tissue, we performed two-wy nlysis of vrince (ov) if the vrinces were equl etween tretments nd residuls were normlly distriuted. In cse of heterogeneity, we used the generlized lest squres (gls) method with the restricted mximum likelihood estimtion (REML) from the nlme pckge (Pinheiro et l. 2013) to specify the vrince structure. The optiml vrince structure ws chosen sed on the Akike informtion criterion (AIC). After setting the optiml vrince structure, the significnce of the explntory vriles (phid infesttion nd plnt genotype) ws evluted. Therefore, we stepwise removed the explntory vriles from the model, estimted with the mximum likelihood method (ML), nd compred the more complex model with the simplified model

5 J Chem Ecol (2015) 41: using the mximum likelihood rtio test. The miniml model ws refitted with the REML, nd vlidted for homogeneity of vrinces nd normlity of residuls (Zuur et l. 2009). The sme procedure ws used to look for the influence of rering nd ctul test plnt on the glucosinolte concentrtion in phids. To test which fctor (the rering plnt nd the plnt where the phid ctully fed) influenced offspring production, we performed generlized liner model with negtive inomil error structure (glm.n, MASS lirry (Venles nd Ripley 2002)). Significnce vlues were otined y model simplifiction nd comprison of models using the mximum likelihood rtio test. The concentrtion of 8-MSO derivtives etween WT nd gtr1gtr2 dko plnts ws compred with the Wilcoxon rnk sum test. All tests were performed in R ( org/). Results Amino Acid, Sugr nd Glucosinolte Content in Lef Phloem Sp Since GTR1 nd GTR2 hve een suggested to e involved in phloem loding of glucosinoltes (Nour-Eldin et l. 2012), we nlyzed phloem exudtes from WT nd gtr1gtr2 dko leves for glucosinoltes. We detected reduced levels of ll glucosinoltes in phloem of the trnsporter mutnt compred to WT plnts (Fig. 1). For ll ut two glucosinoltes (5-MSP nd 4MOI3M), this reduction ws significnt (Tle S1). 4-Methylsulfinylutyl (4-MSB) ws the dominting glucosinolte in the phloem for oth genotypes. For mino cids, the concentrtion of lnine in collected gtr1gtr2 dko phloem exudtes ws significntly lower thn in WT, wheres cler trend of reduced mino cid concentrtion ws seen for serine nd glutmine in gtr1gtr2 dko phloem reltive to WT (Fig. S1). Moreover, nlysis of phloem sp for fructose, glucose, nd sucrose showed tht concentrtions of sugrs were significntly lower in gtr1gtr2 dko phloem compred to WT (Fig. S2). Glucosinolte Content in Lef Tissue We exmined whether lef-cged phids induced n increse in locl lef ccumultion of glucosinoltes, nd if such n ltertion in lef glucosinolte levels would e dependent on the GTRs. Four phids were cged on leves for three dys (Fig. S3), fter which leves were nlyzed for metolites. The dominting glucosinolte in leves ws 4-MSB for oth genotypes (Fig. 2), s in phloem sp (Fig. 1). As shown in Fig. 2, ll glucosinoltes were significntly higher in gtr1gtr2 dko leves compred to WT leves, except for 4MOI3M for which the levels were nerly the sme in oth genotypes. Aphid infesttion led to smll ut significnt induction of 7-MSH in WT leves. In Glucosinolte conc. in phloem (signl int. / 30 mg lef) WT gtr1gtr2 dko Totl 3-MSP 4-MSB 5-MSP 7-MSH 4-MTB 8-MSO I3M 4MOI3M 1MOI3M Fig. 1 Glucosinolte concentrtions in phloem sp from wildtype (WT) nd gtr1gtr2 dko leves. Glucosinoltes were nlyzed in phloem sp exudtes. Brs represent mens ± SE (N = 5). indictes sttisticlly significnt different gtr1gtr2 dko glucosinolte concentrtions compred to WT (P <0.05),seeMethods nd Mteril nd (Tle S1). Totl, totl lef glucosinoltes. Glucosinolte revitions: 3-MSP, 3- methylsulfinylpropyl; 4-MSB, 4-methylsulfinylutyl; 4-MTB, 4- methylthioutyl; 8-MSO, 8-methylsulfinyloctyl; 5-MSP, 5- methylsulfinylpropyl; 7-MSH, 7-methylsulfinylheptyl; I3M, indol-3- ylmethyl; 4MOI3M, 4-methoxyindol-3-ylmethyl; 1MOI3M, 1- methoxyindol-3-ylmethyl; n.d., not detected gtr1gtr2 dko leves, only, significnt induction due to phid infesttion lso ws seen for 4-MTB nd 4MOI3M (Tle S2). Feeding on gtr1gtr2 dko Mutnt Leves Chnges Aphid Performnce Due to the lower levels of glucosinoltes in the phloem sp of the trnsporter mutnt, we hypothesized tht the green pech phid would perform etter (produce more Glucosinolte conc. (µmol / g freshweight) 3,0 2,0 1,0 0,3 0,2 0,1 0,0 Totl 3-MSP 4-MSB 5-MSP 7-MSH 4-MTB n.d. 8-MSO I3M 4MOI3M n.d. WT without phids gtr1gtr2 dko without phids WT + phids gtr1gtr2 dko + phids c c 1MOI3M Fig. 2 Glucosinolte concentrtions in wildtype (WT) nd gtr1gtr2 dko leves in the presence nd sence of phids. Leves cged for 3 d with or without four phids were nlyzed for glucosinoltes. Brs represent mens ± SE (N = 11). Different letters indicte sttisticlly significnt different glucosinolte concentrtions (P < 0.05), see Methods nd Mteril nd (Tle S2). Totl, totl lef glucosinoltes. Glucosinolte revitions: 3-MSP, 3-methylsulfinylpropyl; 4-MSB, 4- methylsulfinylutyl; 5-MSP, 5-methylsulfinylpropyl; 7-MSH, 7- methylsulfinylheptyl; 4-MTB, 4-methylthioutyl; 8-MSO, 8- methylsulfinyloctyl; I3M, indol-3-ylmethyl; 4MOI3M, 4-methoxyindol- 3-ylmethyl; 1MOI3M, 1-methoxyindol-3-ylmethyl

6 980 J Chem Ecol (2015) 41: offspring) on gtr1gtr2 dko leves compred to WT. We tested this y cging single phids (rered on either WT or gtr1gtr2 dko plnts) on leves of oth genotypes for three dys, fter which we counted the phid offspring. When rered on WT leves, phid fecundity ws not chnged when phids were then cged on gtr1gtr2 dko or WT leves (Fig. 3). However, when rered on gtr1gtr2 dko leves, the fecundity of phids then cged on gtr1gtr2 dko ws strongly reduced (~4.5 fold) compred to fecundity of phids then cged on WT leves. In fct, mong the four comintions of rering nd lef cging (see Fig. 3), the highest (WT lef; ~14) nd lowest (gtr1gtr2 dko lef; ~3) numer of offspring ws counted from leves with gtr1gtr2 dko-rered phids (Fig. 3). The proportion of dult phids tht died during the performnce experiment (three dys) ws significntly higher when phids hd een rered on gtr1gtr2 dko vs. WT plnts, with the highest proportion found when oth rering nd cging took plce on the trnsporter mutnt (Tle 2). Weight of (living) dult phids did not differ mong the vrious rering/cging comintions (Fig. S4). Glucosinolte Concentrtion in Aphids To exmine whether the reduced glucosinolte content in gtr1gtr2 dko lef phloem (Fig. 1) ws reflected in single phids cged on leves, we nlyzed phid odies for glucosinolte content (Fig. 4). The concentrtion of nerly ll glucosinoltes in phids (except for 8-MSO nd 1MOI3M) ws influenced y which plnt the phid ws cged on. Aphids cged on WT leves generlly 16 Tle 2 Proportion of ded dult phids on cged leves (Rering) nd cged lef Ded/living (% ded) (WT) WT 2/15 (13,3) (gtr1gtr2 dko) WT 5/15 (33,3) (WT) gtr1gtr2 dko 2/15 (13,3) (gtr1gtr2 dko) gtr1gtr2 dko 7/15 (46,7) WT rered totl 4/30 (13,3) gtr1gtr2 dko rered totl 12/30 (40,0) contined higher glucosinolte concentrtions thn phids cged on gtr1gtr2 dko plnts. The concentrtion of few individul glucosinoltes in phids (5-MSP, 7-MSH, I3M, nd 1MOI3M) ws lso influenced y the rering regime of the phids: phids rered on gtr1gtr2 dko plnts contined significntly less of the ove mentioned glucosinoltes thn phids rered on WT plnts (Tle S4). There ws huge reduction in I3M concentrtion when phids were rered nd cged on gtr1gtr2 dkoplntscompredtowhenreredon WT plnts. Accumultion of 8-MSO Glucosinolte Derivtives in gtr1gtr2 dko Leves To investigte other metolites tht could e involved in phid performnce, WT nd gtr1gtr2 dko lef extrcts were nlyzed for glucosinolte-derived metolites. Compred to WT, highly incresed levels (~10-fold) of the 8-MSO derived metolites, 8-methylsulfinyloctyl mine (8-MSO mine) nd 9-methylsulfinylnonyl nitrile (9- MSN nitrile =8-methylsulfinyloctyl cynide (8-MSO Numer of offspring d (WT) WT (dko) WT (WT) dko cd (dko) dko Fig. 3 Myzus persice offspring fter cging on wildtype (WT) nd gtr1gtr2 dko leves. Single phids, either rered on WT or gtr1gtr2 dko plnts, were cged on WT nd gtr1gtr2 dko leves for 3 d fter which their offspring were counted. (WT) WT, phids were rered on WT plnts nd produced offspring on WT leves; (dko) WT, phids were rered on gtr1gtr2 dko plnts nd produced offspring on WT leves; (WT) dko, phids were rered on WT plnts nd produced offspring on gtr1gtr2 dko leves; (dko) dko, phids were rered on gtr1gtr2 dko plnts nd produced offspring on gtr1gtr2 dko leves. Brs represent mens ± SE (N = 8 13 dult phids). Different letters indicte sttisticlly significnt different numers of phid offspring etween the four rering/ cging comintions (P < 0.053), see Methods nd Mteril nd (Tle S3) Glucosinolte conc. in single phids (µmol / g phid) 1,80 1,60 1,40 1,20 1,00 0,80 0,60 0,40 0,20 0,04 0,03 0,02 0,01 0,00 cd (WT) WT (dko) WT (WT) dko (dko) dko Totl 3-MSP 4-MSB 5-MSP 7-MSH 4-MTB 8-MSO I3M 4MOI3M 1MOI3M Fig. 4 Glucosinolte concentrtion in dult phid odies cged on leves. Single phids, either rered on wildtype (WT) or gtr1gtr2 dko plnts, were cged on WT nd gtr1gtr2 dko leves for 3 d fter which glucosinoltes were nlyzed in single phids. (WT) WT, phids rered on WT plnts nd cged on WT leves; (dko) WT, phids rered on gtr1gtr2 dko plnts nd cged on WT leves; (WT) dko, phids rered on WT plnts nd cged on gtr1gtr2 dko leves; (dko) dko, phids rered on gtr1gtr2 dko plnts nd cged on gtr1gtr2 dko leves. Brs represent mens ± SE (N = 8 13 phids). Different letters indicte sttisticlly significnt different glucosinolte concentrtions (P < 0.05), see Methods nd Mteril nd (Tle S4). Glucosinolte revitions re s descried in Fig. 2 c

7 J Chem Ecol (2015) 41: cynide)), were identified in gtr1gtr2 dko leves (Fig. 5, Tle S5) regrdless of phid infesttion (dt not shown). Discussion In this work, we used the recently identified Aridopsis glucosinolte trnsporter doule mutnt (gtr1gtr2 dko) s unique tool to investigte the role of intr-lef glucosinolte distriution nd trnsport in the interction with the green pech phid. Due to their proposed role in phloem loding of glucosinoltes (Nour-Eldin et l. 2012), we expected phloem sp of the gtr1gtr2 dko to exhiit decresed glucosinolte content reltive to WT phloem, which ws indeed the cse (Fig. 1). As previous studies hve indicted, defensive role for glucosinoltes ginst phids (Kim nd Jnder 2007; Kim et l. 2008; Levy et l. 2005; Mewis et l. 2005, 2006, 2012), we hypothesized tht the decrese of glucosinoltes in gtr1gtr2 dko phloem would cuse phids to perform etter on gtr1gtr2 dko leves compred to WT. However, this ws not the cse (Fig. 3). Aphids tested on gtr1gtr2 dko leves produced similr numers of offspring (when rered on WT) or even much fewer offspring (when rered on dko plnts), compred with phids tested on WT plnts. Unexpectedly, phids oth rered nd cged on gtr1gtr2 dko plnts produced the lest offspring of ll rering/cging comintions. Furthermore, the proportion of dult phids tht died during the performnce experiment ws higher when these were rered on gtr1gtr2 dko plnts (40 % for gtr1gtr2 dko vs. 13 % for WT) (Tle 2). On the other hnd, phids rered on gtr1gtr2 dko plnts nd cged on WT produced the most offspring of ll rering/cging comintions (Fig. 3). In Concentrtion (signl int. / mg fresh weight) 1e+5 8e+4 6e+4 4e+4 2e MSO Amine WT 8-MSO Amine dko 9-MSN Nitrile WT 9-MSN Nitrile dko Fig. 5 Concentrtion of 8-MSO derivtives in wildtype (WT) nd gtr1gtr2 dko leves. Methnol extrcts of leves (fter eing cged with one phid for three dys) from WT nd gtr1gtr2 dko were exmined for glucosinolte-derived compounds. 8-methylsulfinyloctyl mine (8-MSO mine) (m/z = 192) nd 9-methylsulfinylnonyl nitrile (9- MSN nitrile =8-methylsulfinyloctyl cynide (8-MSO cynide)) (m/ z = 202) were identified. Error rs re SE (N =21 23). indictes sttisticlly significnt different gtr1gtr2 dko concentrtions compred to WT equivlents (P < 0.001), see Methods nd Mteril nd (Tle S5) contrst to our hypothesis, one cn conclude tht oth survivl nd fecundity were dversely ffected when phids hd een rered AND cged on the trnsporter mutnt. An phid diet is rich in sugrs, ut reltively poor in mino cids, which re importnt for phid growth (de Vos et l. 2007; Louis 2012). Thus, given the importnt role of mino cids for phid nutrition, the generl reduction of mino cid content in gtr1gtr2 dko phloem sp compred to WT (Fig. S1) (rther thn the decrese of sugrs in gtr1gtr2 dko phloem compred to WT (Fig. S2)) my negtively ffect the fecundity of phids rered nd cged on gtr1gtr2 dko plnts. However, phid prolifertion ws comprle when feeding on Aridopsis WT or the p6 mutnt, which hs significnt reduction of mino cid content in phloem sp (Hunt et l. 2010). Thus, lower mino cid vilility my not hve een responsile for reduced phid reproduction on gtr1gtr2 dko leves. Green pech phid reproduction is not significntly ffected y the presence or sence of the TGG1 nd TGG2 myrosinses in Aridopsis (Brth nd Jnder 2006). This indictes tht phids re le to void contct with toxic glucosinolte rekdown products medited y myrosinses during feeding, nd this oservtion is supported y the presence of intct glucosinoltes within phid odies (Kim nd Jnder 2007). Therefore, it cn e hypothesized tht intct glucosinoltes hve defensive role ginst phids in Aridopsis. As expected, sed on phloem sp nlysis, glucosinolte concentrtions within phids cged on gtr1gtr2 dko leves were generlly lower thn in those cged on WT, except for 8- MSO, which hd similr concentrtion in ll phids (Fig. 4). In most cses, the lowest glucosinolte concentrtion ws detected in phids rered on gtr1gtr2 dko/cged on gtr1gtr2 dko, nd the highest concentrtion in phids rered on WT/ cged on WT. As we oserved the lowest numer of offspring (Fig. 3) nd highest mortlity rte (Tle 2) in phids oth rered nd cged on the gtr1gtr2 dko, the lower glucosinolte level in these phids did not fit the hypothesis tht intct glucosinoltes ply n importnt role in the defense ginst phids in Aridopsis. I3M (nd other indole glucosinoltes) nd rekdown products thereof hve een shown to deter phid feeding more efficiently thn liphtic glucosinoltes (Kim nd Jnder 2007; Kim et l. 2008). As we oserved lrge reduction of I3M in phids rered nd cged on the gtr1gtr2 dko compred to the other rering/cging comintions (Fig. 4), it ws further unexpected tht phids on these plnts would perform poorly (Fig. 3). Thus, if glucosinolte levels inside phids rered nd cged on the gtr1gtr2 could not explin their poor performnce, wht could e the reson for this oservtion? Although the totl glucosinolte concentrtion ws significntly lower in gtr1gtr2 dko phloem compred to WT (~2.5- fold) (Fig. 1), the opposite ws true when compring glucosinolte concentrtion in totl lef tissue (~3.5-fold) (Fig. 2),

8 982 J Chem Ecol (2015) 41: where the concentrtion of 8-MSO ws especilly high compred to WT (~18-fold). Thus, in contrst to feeding on gtr1gtr2 dko phloem sp tht is low in glucosinolte content compred to WT, it cn e ssumed tht phids living on gtr1gtr2 dko plnts re encountering higher concentrtions of glucosinoltes (especilly 8-MSO nd 4-MSB) during the pthwy phse (intrcellulr smpling of epidermis nd mesophyll cells (Louis 2012)) compred to phids on WT. Furthermore, recent work crried out in our lortory showed n ccumultion of 8-MSO in lef xylem sp from gtr1gtr2 dko reltive to WT xylem sp (Andersen et l. 2013;Mdsen et l. 2014), indicting tht phids my fce high mounts of this glucosinolte during the xylem phse on gtr1gtr2 dko plnts compred to on WT. Perhps the reduced survivl rte, fecundity, nd glucosinolte content oserved for phids rered nd cged on gtr1gtr2 dko plnts re not result of the reduced glucosinolte concentrtion in the phloem of the mutnt, ut insted reflect tht phids re deterred from feeding s they encounter high concentrtions of glucosinoltes during the pthwy nd xylem phses efore their stylet reches the phloem. Alterntively, the reduced mino cid nd sugr content in phloem from gtr1gtr2 dko leves compred to WT (Figs. S1, S2) my force phids to spend more time in the phloem of this genotype. When phids were rered on gtr1gtr2 dko plnts nd cged on WT leves, we oserved the highest numer of offspring (Fig. 3). This my reflect oost in phid fitness cused y the lower glucosinolte content in lef tissue surrounding the phloem, long with Bnorml^ mino cid nd sugr content in the WT phloem sp. When phids were rered on WT plnts nd cged on gtr1gtr2 dko leves, we oserved similr numer of offspring s phids rered nd cged on WT (Fig. 3). This ws unexpected considering the poor performnce of phids cged AND rered on the gtr1gtr2 dko. This suggests tht phids rered on the WT cn resist the new poor gtr1gtr2 dko conditions discussed ove. We would, however, expect tht if the performnce experiment ws run for longer thn three dys, phid offspring count nd mortlity rte would eventully decrese nd increse, respectively, nd reflect the unfvorle phid environment on the gtr1gtr2 dko lef. These suggest ehviorl chnges could e investigted y using the Electricl Penetrtion Grph (EPG) technique (Louis 2012) in future experiments. The pproximtely 18-fold increse of 8-MSO in gtr1gtr2 dko lef tissue compred to WT long with the previously reported 8-MSO ccumultion in gtr1gtr2 dko lef xylem sp (Andersen et l. 2013; Mdsen et l. 2014), suggests role for this glucosinolte in the poor performnce of phids rered nd cged on the trnsporter mutnt. A defensive role of 8-MSO ginst phids in Aridopsis ws proposed in previous studies, where 8-MSO ccumulted in cged, phid-infested WT leves (Ellerrock et l. 2007; Kim nd Jnder 2007). However, despite the incresed 8-MSO concentrtion in gtr1gtr2 dko leves compred to WT, phids contined similr concentrtions of this glucosinolte, regrdless of rering nd cging (Fig. 4). The potentil defensive function of 8-MSO ginst green pech phids remins to e determined. In contrst to phloem nd lef tissue glucosinolte levels (Figs. 1, 2), 4-MSB ws not the single dominting glucosinolte inside phids, s 8-MSO nd prtly I3M reched similr concentrtions s 4-MSB (Fig. 4). Wheres 4-MSB mde up % of totl glucosinoltes in the phloem nd % in lef tissue, 4-MSB represented only % of totl glucosinoltes inside phids (Fig. S5). On the other hnd, the proportion of 8-MSO inside phids mde up % of totl glucosinoltes, wheres it ws only 6 8 % in phloem nd 5 (WT)-20 % (gtr1gtr2 dko) of totl glucosinoltes in lef tissue (Fig. S5). These shifts in glucosinolte proportions from phloem nd lef tissue to phid my reflect locl ccumultion of e.g., 8-MSO t the site of feeding, s hs een suggested previously (Kim nd Jnder 2007); or selective sequestrtion (e.g., 8-MSO nd 1MOI3M) or discrimintion (e.g., 3-MSP, 4-MSB, nd 5-MSP) of specific glucosinoltes inside the phid, s hs een demonstrted in other crucifer feeding insects, e.g., in the Phyllotret striolt fle eetle (Bern et l. 2014). In contrst to Phyllotret striolt, however, the green pech phid does not, to our knowledge, hror its own myrosinse, rendering the reson for sequestrtion less cler. The difference in glucosinolte proportions inside differently rered/cged phids most likely reflect ltered feeding ehviors of phids living on either the WT or gtr1gtr2 dko, s discussed ove. We investigted whether green pech phids induced locl glucosinolte increse within infested leves (Fig. 2), which hd een demonstrted in previous study where long-chin liphtic nd indole glucosinoltes (4MOI3M) ccumulted in cged Aridopsis WT leves exposed to phids (Kim nd Jnder 2007). We oserved slight increse of 7- MSH in phid infested WT plnts (Fig. 2). The compounds 4- MTB nd 4MOI3M incresed only in phid infested gtr1gtr2 dko. Besides the increse of 7-MSH in WT leves, our dt do not correspond to the results reported previously (Kim nd Jnder 2007). Discrepncies might e explined y differences in experimentl design; for exmple, wheres Kim nd Jnder (2007) plced 20 phids on cged lef for three dys, we plced 4 or 1 phid(s) in our experiments. Differences in plnt growth conditions lso my hve plyed role. Interestingly, metolite nlysis of lef extrcts reveled higher (~10 fold) presence of the 8-MSO derivtives, 8-MSO mine, nd 9-MSN nitrile (simple nitrile) (= 8-MSO cynide), in gtr1gtr2 dko leves compred with WT, regrdless of phid infesttion (Fig. 5). Simple nitriles re well known glucosinolte hydrolysis products, which re considered less toxic ginst some

9 J Chem Ecol (2015) 41: insects thn the corresponding isothiocyntes (Wittstock nd Burow 2010). Their iologicl function is not fully understood, ut role s signling compounds towrd insects hs een proposed. Furthermore, it hs een suggested tht hydrolysis of nitriles would relese the sulfur nd nitrogen toms ound in glucosinoltes (Wittstock nd Burow 2010). Among other glucosinolte-derived compounds, mines derived from liphtic glucosinoltes were identified in Aridopsis mutnts overexpressing the root myrosinse TGG4, nd these compounds were hypothesized to reflect glucosinolte rekdown pthwy in intct plnt tissue (Bednrek et l. 2009). Whether the presence of 8-MSO mine nd 9-MSN nitrile in lef smples merely represent the rekdown of glucosinoltes during smple preprtion, or whether they hold iologicl significnce is n open question. It is intriguing to speculte tht the uild-up of 8-MSO mine nd 9-MSN nitrile in gtr1gtr2 dko leves represents wy to cope with the ccumultion of 8-MSO in this mutnt. But this rises the question of why we did not detect 4-MSB derived compounds, s levels of this glucosinolte ws higher thn 8-MSO in leves (Fig. 2). Whether the 8-MSO derivtives could ffect survivl rte nd fecundity of phids on gtr1gtr2 dko leves remins to e investigted. The gtr1gtr2 dko mutnt represents unique opportunity to exmine the potentil role of glucosinolte trnsport nd locl glucosinolte distriution for phid infesttion. However, s mentioned ove, further studies including e.g., EPG techniques re required efore the role of glucosinoltes in the interction etween Aridopsis nd green pech phids is fully understood. Nevertheless, it ppers cler tht further investigtions of the interply etween this phid nd the gtr1gtr2 dko mutnt will id in elucidting the significnce of defense compound dynmics involved in Aridopsis-phid interctions. Knowledge gined here my e useful in future studies seeking to clrify the impliction of defense compound trnsport in response to insect ttck of plnts. Acknowledgments The grdeners of the Mx Plnck Institute for Chemicl Ecology re thnked for rering plnts with phids, nd Dniel Rosenerger is thnked for help with experiments. Finncil support for the work ws provided y The Dnish Council for Independent Reserch (Grnt ) (Brr Ann Hlkier) nd the Dnish Ntionl Reserch Foundtion for funding DynMo Center of Excellence (Grnt DNRF99) (Brr Ann Hlkier). Author Contriutions Svend Roesen Mdsen performed nd designed experiments. Grit Kunert designed experiments nd performed sttisticl nlyses. Michel Reichelt performed metolite nlyses. Svend Roesen Mdsen, Grit Kunert, Michel Reichelt, Jonthn Gershenzon nd Brr Ann Hlkier wrote the pper sed on drft written y Svend Roesen Mdsen nd Grit Kunert. 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