Aboveground insect infestation attenuates belowgroundAgrobacterium-mediated genetic transformation

Geun Cheol Song1,2, Soohyun Lee1, Jaehwa Hong1,3, Hye Kyung Choi1, Gun Hyong Hong1,2, Dong-Won Bae4,

Kirankumar S. Mysore5, Yong-Soon Park1,6 and Choong-Min Ryu1,2

1Molecular Phytobacteriology Laboratory, Superbacteria Research Center, KRIBB, Daejeon 305-806, South Korea; 2Biosystems and Bioengineering Program, University of Science and

Technology (UST), Daejeon 305-350, South Korea; 3Department of Plant Pathology, Chungnam National University, Daejeon 305-764, South Korea; 4Central Instrument Facility,

Gyeongsang National University, Jinju 660-701, South Korea; 5Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA; 6 Present address: Agricultural

Microbiology Division, National Academy of Agricultural Science, RDA, Wanju 565-851, South Korea

Authors for correspondence:Yong-Soon Park

Tel: +82 63 238 3059Email: yspark2005@gmail.com

Choong-Min RyuTel: +82 42 879 8229

Email: cmryu@kribb.re.kr

Received: 19 May 2014

Accepted: 7 January 2015

New Phytologist (2015) 207: 148–158doi: 10.1111/nph.13324

Key words: Agrobacterium, crown gall,multitrophic interactions, salicylic acid (SA),transformation efficiency, whiteflyinfestation.

Summary

� Agrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be

popularly used for genetic engineering, the influence of aboveground insect infestation on

Agrobacterium induced gall formation has not been investigated.� Nicotiana benthamiana leaves were exposed to a sucking insect (whitefly) infestation and

benzothiadiazole (BTH) for 7 d, and these exposed plants were inoculated with a tumorigenic

Agrobacterium strain. We evaluated, both in planta and in vitro, how whitefly infestation

affects crown gall disease.� Whitefly-infested plants exhibited at least a two-fold reduction in gall formation on both

stem and crown root. Silencing of isochorismate synthase 1 (ICS1), required for salicylic acid

(SA) synthesis, compromised gall formation indicating an involvement of SA in whitefly-

derived plant defence against Agrobacterium. Endogenous SA content was augmented in

whitefly-infested plants upon Agrobacterium inoculation. In addition, SA concentration was

three times higher in root exudates from whitefly-infested plants. As a consequence,

Agrobacterium-mediated transformation of roots of whitefly-infested plants was clearly inhib-

ited when compared to control plants. These results suggest that aboveground whitefly infes-

tation elicits systemic defence responses throughout the plant.� Our findings provide new insights into insect-mediated leaf–root intra-communication and

a framework to understand interactions between three organisms: whitefly, N. benthamiana

and Agrobacterium.

Introduction

Under natural conditions, plants must constantly respond todiverse biotic stresses. Plants have developed defensive machineryto protect themselves against a variety of invading pathogens andinsects (Nimchuk et al., 2003; Hogenhout & Bos, 2011). Percep-tion of pathogen- and damage-associated molecular patterns(PAMPs and DAMPs, respectively) results in rapid activation ofdefence responses and accumulation of reactive oxygen species inlocal tissue (Nimchuk et al., 2003; Hogenhout & Bos, 2011). Inaddition to these rapid responses, the major phytohormones, sali-cylic acid (SA), jasmonic acid (JA) and ethylene (ET), are pro-duced (Glazebrook, 2001; Thomma et al., 2001; Pieterse et al.,2009), which act as key signalling molecules in regulating plantdefence. Salicylic acid predominantly participates in plantdefence against biotrophic and hemibiotropic pathogens andsucking insects. It has been demonstrated that SA and its ana-logues, such as 2,6-dichloroisonicotonic acid (INA) and benzo-

(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH),activate systemic acquired resistance (SAR) to certain pathogensand insects (Ryals et al., 1996; Achuo et al., 2004; Wang et al.,2007; Lee et al., 2012). On the other hand, accumulation of JAand ET occurs in response to necrotrophic pathogens andwounding or herbivore damage (Howe & Jander, 2008).Although some genes are induced by both SA and JA/ET applica-tion, most cases of crosstalk between these two signalling path-ways are antagonistic (Schenk et al., 2000; Kunkel & Brooks,2002; Glazebrook et al., 2003).

Agrobacterium tumefaciens is a soil-borne, Gram-negative bac-terium. It causes crown gall disease in a broad range of dicotyle-donous plants. Agrobacterium species transfer their DNA intoother organisms (Pitzschke & Hirt, 2010); transferred DNA (T-DNA) is subsequently integrated into the host chromosomalDNA resulting in a hyperplastic response, gall formation. Thisprocess of T-DNA transport is controlled by proteins encoded bythe virulence genes on the bacterial tumour inducing (Ti)

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plasmid and chromosome and includes chv and vir genes (Zhuet al., 2000; Tzfira & Citovsky, 2002; Gelvin, 2003). Phenoliccompounds such as acetosyringone, released from woundedplants, activate VirA, which is a membrane-bound sensor, andthis activation regulates the intracellular response regulator, VirG(Wolanin et al., 2002). Specific attachment of Agrobacteriumdepends on the chromosomal genes chvA, chvB and pscA (alsoknown as exoC) which play a role in the synthesis and localizationof periplasmic b-1,2 glucan (McCullen & Binns, 2006). After virgene activation and attachment of Agrobacterium to plant cells,the VirD2 protein along with VirD1 nicks the T-DNA region ofthe Ti plasmid and VirD2 remains attached to the 50 end of theT-DNA. VirB complex, which belongs to the class of type IVsecretion system (T4SS) and consists of at least 12 proteins, andVirD4 are required for transport of VirD2-T-DNA and severalother proteins such as VirE2, VirE3, VirF and VirD5 into thehost cell (Vergunst et al., 2005). VirE2 is a single-stranded DNAbinding protein and presumably coats the T-DNA in the plantcell, thus forming a VirD2-T-DNA and VirE2 complex (T-com-plex) (Vergunst et al., 2000; Cascales & Christie, 2003). In addi-tion, two other virulence proteins, VirE3 and VirF, have beenshown to play a role in T-DNA transport to nucleus and integra-tion (Lacroix et al., 2005). The ability of Agrobacterium to trans-port and integrate part of its DNA into a host genome has beenwidely exploited to express, disrupt,or silence genes in plants(Hellens et al., 2000; Ditt et al., 2001).

Plant responses to Agrobacterium infection result in the releaseof a number of signalling molecules, including plant sugars, phe-nolic compounds, opines, SA, indole-3-acetic acid (IAA), c-amino butyric acid (GABA), quorum-sensing signals, ethyleneand other unidentified signals (Chevrot et al., 2006; Liu &Nester, 2006; Yuan et al., 2007; Anand et al., 2008; Nonakaet al., 2008). Exogenous SA inhibits induction of vir genes andreduces the virulence of Agrobacterium in Arabidopsis thaliana(Yuan et al., 2007) and Nicotiana benthamiana (Anand et al.,2008). Nicotiana benthamiana plants silenced for genes involvedin SA biosynthesis and signalling were more susceptible toAgrobacterium infection (Anand et al., 2008). In addition, exoge-nous application of BTH to N. benthamiana and tomato impairsthe development of crown gall disease caused by Agrobacteriumtumorigenic strain A348 (Anand et al., 2008). All of these resultssuggest that SA plays an important role in plant responses toAgrobacterium infection.

Whitefly (Bemisia tabaci) is a sap-sucking insect widely distrib-uted across the warmer parts of tropical and subtropical regions.Whitefly infestation enhances plant defence responses dependenton the SA and JA/ET-signalling pathways (Kaloshian & Walling,2005). In addition, a novel signalling pathway not regulated bySA or JA may also exist against whitefly infestation (van de Venet al., 2000). We have previously shown that whitefly infestationon leaves significantly enhances plant defence responses in bothleaves and roots against hemibiotropic bacterial spot diseasecaused by infection with Xanthomonas axonopodis pv. vesicatoriaand bacterial wilt caused by Rastonia solanacearum infection(Yang et al., 2011). Root biomass increased significantly in white-fly-infested plants, although it was reduced in plants treated with

BTH (Yang et al., 2011). Transcriptome analysis indicated thatwhitefly infestation on aboveground parts of pepper plants elic-ited SA and JA/ET signalling in both aboveground and below-ground tissues (Park & Ryu, 2014). In addition, auxin-responsive genes and several transporters (including ATP-bindingcassette, peptide, zinc and phosphate transporters) were signifi-cantly upregulated in whitefly-infested roots, suggesting therelease of auxin and nutrients in root exudate by whitefly-infestedplants and implicating the role of upregulated genes in the facili-tation of root biomass (Park & Ryu, 2014).

In this study, we investigated whether whitefly infestation inN. benthamiana leaves changed the susceptibility of above- andbelowground tissues to Agrobacterium infection. Crown gall dis-ease on plants stem and crown regions was significantly attenu-ated by whitefly infestation. At least a two-fold reduction in gallweight and increased stem thickness was seen in whitefly-infested plants in comparison with water treatment. EndogenousSA accumulated only in response to Agrobacterium inoculationin whitefly-infested plants. A reverse genetic approach revealedthat silencing isochorismate synthase 1 (ICS1) in N. benthamianaplants compromised the protective effect of whitefly infestationagainst Agrobacterium infection. Agrobacterium-mediated trans-formation was also inhibited in roots of whitefly-infested plants.In addition, SA concentration was three times higher in rootexudates from whitefly-infested plants when compared tocontrol plants. Our results clearly show that leaf infestation bywhitefly enhances the ability of the SA-dependent defence-signalling pathway to attenuate Agrobacterium-mediated trans-formation in plant roots.

Materials and Methods

Agrobacterium growth

An AT-ammonium sulphate-glucose (ANG) medium containing10.9 g KH2PO4, 0.16 g MgSO4∙7H2O, 0.005 g FeSO4∙7H2O,0.011 g CaCl2∙2H2O, and 0.002 g MnCl2∙4H2O plus 1 gammonium sulphate and 2 g of glucose per litre was used forAgrobacterium growth. Agrobacterium tumefaciens strain C58 wasgrown on solid ANG medium containing 100 lg ml�1 rifampi-cin at 30°C for 2 d, scraped off plates, re-suspended in sterilizeddistilled water (SDW), and adjusted to proper concentration forfurther experiments.

Plant materials and treatments

Seeds of Nicotiana benthamiana Domin. were surface-sterilizedwith 6% sodium hypochlorite, washed four times with SDW andplated on half-strength Murashige and Skoog (0.59 MS) saltssupplemented with 3% sucrose and 0.6% plant agar. Seeds weregerminated and incubated in a growth chamber 25� 2°C underfluorescent light conditions (light : dark 12 : 12 h; c. 7000 luxlight intensity). Seedlings were transferred to soilless pottingmedium (Punong, Co. Ltd, Gyeongju, South Korea). Leaves of3-wk-old plants were separately infested with whitefly (Bemisiatabaci Genn.), or treated with 40 ml of 0.5 mM BTH or 40 ml of

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sterilized water (control) for 7 d in plastic containers(659 709 80 cm).

After plant treatments with whitefly, BTH and water (control)for 7 d, suspensions of A. tumefaciens C58 (OD600 = 2) were inocu-lated by slightly injuring stems with a needle at heights of 2, 4, 6and 8 cm above soil level as described previously (Anand et al.,2007, 2008). In another independent experiment, the crown regionof stems was inoculated with A. tumefaciens C58 (OD600 = 2) asdescribed above. Gall formation was assessed at 15 and 30 d postinoculation (dpi) on stem or crown regions of plants.

Virus-induced gene silencing (VIGS)

For VIGS, 2-wk-old N. benthamiana seedlings were infiltratedwith TRV-based VIGS vectors (pTRV2) containingN. benthamiana homologues of the plant defence-related andplant defence hormone signalling genes NbICS1 and NbCOI1 orwith a vector control pTRV2::00, as described previously (Anandet al., 2007; Senthil-Kumar & Mysore, 2014). The infiltratedplants were monitored for c. 2 wk. Treatments with whitefly,BTH or water (control), followed by inoculation withA. tumefaciens C58 and assessment of gall formation, were con-ducted as described above.

Extraction of plant and bacterial RNA, cDNA synthesis andreal-time quantitative PCR

Stems of N. benthamiana infested with whitefly or treated withwater and inoculated with Agrobacterium C58, together with con-trol stems, were frozen in liquid nitrogen. Total RNA was iso-lated using the RNeasy® plus mini kit according to themanufacturer’s protocol (Qiagen). Bacterial RNA was isolatedusing the same method.

First-strand cDNA was synthesized using 2 lg RNA, oligo dTprimer, dNTP, and Moloney murine leukaemia virus reversetranscriptase (M-MLV RT; enzynomics, Daejeon, South Korea).Quantitative reverse transcription polymerase chain reactions(qRT-PCR) were carried out using a Chromo4 real-time PCRsystem (Bio-Rad). The reaction mixture contained 29 BrilliantSYBR Green qRT-PCR Supermix (Bio-Rad), cDNA, and0.5 lM of each gene-specific primer.

Sequences were amplified using thermocycle parameters:10 min at 95°C, followed by 44 cycles of 30 s at 95°C, 30 s at60°C and 42 s at 72°C. Relative RNA levels were calibrated andnormalized relative to the level of NtACT mRNA (GenBankaccession no. U60489). The primer sets used in this study arelisted in Supporting Information Table S1.

Quantification of amounts of endogenous plant defencehormones

Five replicate samples per treatment were analysed to quantify SAand JA concentrations in response to Agrobacterium in stems ofwhitefly-infested and water-treated plants. The prepared sampleswere quantified using high performance liquid chromatographymass spectrometry (HPLC-MS) 1100 series mass spectrometer

(Agilent, Santa Clara, CA, USA) with a SunfireTM C18(2.19 10 mm) column (Waters, Milford, MA, USA) at a flowrate of 300 l min�1.

The stock solutions of SA and JA were prepared at a concen-tration of 1 mg ml�1 in 100% methanol. Calibration curves werebased on the comparison between the ratio of SA and JA peakarea/internal standard area and the ratio of concentration for SAand JA/internal standard. The limits of detection and quantifica-tion were 3 and 10, respectively, which was determined by a sig-nal-to-noise ratio.

In vitro bioassays

Nicotiana benthamiana seeds were surface-sterilized and germi-nated, as described above. Four-day-old seedlings were trans-ferred to plates (609 15 mm, SPL) containing 26 ml of 0.59MS liquid media. Plates were placed in the plastic container(phytohealth, 1039 78.6 mm, SPL). Whitefly, BTH and watercontrol treatments were applied to plants as described above.Plant growth was monitored for 7 d after treatments and theweight of shoot and root measured. In addition, root exudateswere collected at 7 and 14 d after treatments, when plants were28 and 35 d old, respectively. For each replicate, containing 16plants, c. 80 ml of root exudate was collected from plates. Nomedia contamination was observed in the entire experiment.

Image capture and microscopy

In order to visualize Agrobacterium transformation at the root sur-face, seedlings were inoculated with disarmed A. tumefaciens strainGV2260 carrying the binary vector pBISN1-GUS (Narasimhuluet al., 1996), and observed using a Leica stereo-microscopeS8APO (Leica, Wetzlar, Germany). Representative pictures weretaken using a Hamamatsu digital camera, model Leica DFC295.

Statistical analysis

Analysis of variance of experimental datasets was performed usingJMP software v5.0 (SAS Institute Inc., Cary, NC, USA). Signifi-cant effects of treatment were determined by the magnitude oftheF-value (P = 0.05). When a significant F-test was obtained,separation of means was accomplished by Fisher’s protected leastsignificant difference at P = 0.05.

Results

Whitefly infestation attenuates gall formation byAgrobacterium in stems

It has been recently reported that SA is a key player in attenuatingthe severity of crown gall disease in N. benthamiana (Anand et al.,2008) and that aboveground infestation with whitefly enhancesplant defences in both aboveground and belowground organs ofpepper plants by activating SA-signalling-related gene expression(Yang et al., 2011; Park & Ryu, 2014). This led us to consider

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whether whitefly-triggered, SA-dependent signalling negativelymodulates gall formation by A. tumefaciens in N. benthamiana.To test this, 3-wk-old N. benthamiana plants were infested withwhitefly (55� 7 insects per leaf) or treated with 0.5 mM BTH asa positive control; water was used as a control treatment. Oneweek after initial treatments, we inoculated N. benthamiana stemsat four places with suspensions of a tumorigenic A. tumefaciensC58 (OD600 = 2). These treatments, even after inoculating withAgrobacterium for 30 d, did not have any distinguishable effecton plant height and leaf numbers (data not shown). Gall forma-tion was assessed at 15 and 30 dpi.

Strikingly, the size of galls was smaller in whitefly-infestedplants than in water-treated control plants (Fig. 1a). A c. 3.5-foldreduction of gall weight was recorded in whitefly-infested plantsat both time-points when compared to control plants (Fig. 1b).No obvious physiological difference was observed in stemsamong different treatments (Fig. S1). To prevent any possiblesubtle variation in diameter of stems, which might affect gallweight, we further evaluated gall formation using relative thick-ness (relative thickness (%) = ((b� a)/a)9 100) (Fig. 1c). Inagreement with the gall weight, relative gall thickness at 30 dpiwas 35% lower in whitefly-infested plants than in controls(Fig. 1c). To determine if there were site-specific effects of white-fly infestation on Agrobacterium infection, relative gall thicknesswas analysed at four independent heights (2, 4, 6 and 8 cm abovesoil level) on the stem. Relative gall thickness values were lowerin whitefly-infested plants, regardless of inoculation sites,

compared with water-treated controls (Fig. 1d), clearly indicatingthat whitefly infestation negatively affects gall formation byA. tumefaciens. Similar results were obtained at 15 dpi (Fig. S2).

Differential expression of SA- and JA-dependent genes andaccumulation of endogenous SA and JA in whitefly-infested plants in response to Agrobacterium

In order to examine whether Agrobacterium-mediated gall forma-tion in whitefly-infested plants was affected by signalling path-ways involved in plant defence responses, we measured transcriptlevels of the SA biosynthetic and signalling-related genespathogenesis-related (PR)1a, PR2, phenylalanine ammonia lyase1(PAL1) and PAL2 (Anand et al., 2008) and the JA biosyntheticand signalling-related genes lipoxygenase3 (LOX3),oxophytodienoate reductase3 (OPR3), trypsin protease inhibitor(TPI) and threonine deaminase (TD) (Stitz et al., 2011). Three-week-old N. benthamiana plants were treated with whitefly infes-tation, BTH or water for 7 d, before being inoculated withAgrobacterium for 5 d.

Two genes involved in SA signalling, PR1a and PR2, showedat least a three-fold increase in induction in whitefly-infestedplants (Fig. S3a), but were not significantly activated followinginoculation with Agrobacterium, compared with water controls(Fig. 2a). By contrast, PR1a and PR2 expression was inducedupon Agrobacterium inoculation in BTH pre-treated plants(Fig. 2a). Expression levels of the SA-biosynthetic genes PAL1

(a) (b)

(c) (d)

Whitefly BTH Control

Fig. 1 Assessment gall formation byAgrobacterium tumefaciens in whitefly-infested Nicotiana benthamiana stems.Three-week-old N. benthamiana plants weretreated with whitefly infestation, 0.5 mMbenzo-(1,2,3)-thiadiazole-7-carbothioic acidS-methyl ester (BTH) or water (control) for7 d. Stems were inoculated withA. tumefaciens (OD600 = 2) at four differentplaces, located at 2 cm increments from soillevel to top. (a) A representative picture wastaken at 30 d post inoculation (dpi) ofA. tumefaciens. (b) Individual gall wasweighed at 30 dpi. (c) Schematicrepresentative of relative thickness (RT)calculation RT = ((b� a)/a)9 100% is shownin above graphs. Relative thickness wasassessed at 30 dpi. (d) Relative thicknessmeasured at different heights of the stem.Significant difference between treatmentsand water control: *, P = 0.05. Error bars,� SE of the mean (n = 5).

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and PAL2 doubled in plants infested with whitefly (Fig. S3b) andtheir expression was upregulated 1 d after Agrobacterium inocula-tion (Agro 1 d) when compared to the control plants (Fig. 2b).However, PAL1 and PAL2 were not upregulated following BTHtreatment, but its expression level did increase 5 dpi withAgrobacterium (Fig. 2b). Overall, expression of SA signalling andbiosynthetic genes was similarly affected by whitefly infestation

and biosynthetic genes remained induced upon Agrobacteriuminoculation.

Unlike expression patterns of SA-related genes, expressionof JA-related genes was affected in a pathway-specific man-ner, as expression of JA biosynthetic pathway genes differedfrom that of genes involved in JA-signalling pathways. Forexample, TPI and TD were not induced by whitefly

(a)

(b)

(c)

(d)

Whitefly BTH Control Whitefly BTH Control

Whitefly BTH Control Whitefly BTH Control

Whitefly BTH Control Whitefly BTH Control

Whitefly BTH Control Whitefly BTH Control

Fig. 2 Expression patterns of salicylic acid(SA) and jasmonic acid (JA)-related genes inwhitefly-infested plants followingAgrobacterium tumefaciens inoculation.Three-week-old Nicotiana benthamiana

plants were treated with whitefly or 0.5 mMbenzo-(1,2,3)-thiadiazole-7-carbothioic acidS-methyl ester (BTH) or water (control) for7 d. The stems of these pre-treated plantswere subsequently inoculated withtumorigenic A. tumefaciens (OD600 = 2) afterwounding using a syringe with sharp needle.Samples were harvested for qRT-PCR at thetime-points indicated (Agro 0, Agro 1 d andAgro 5 d). Levels of mRNA expression forSA-signalling genes PR1a and PR2 (a) andSA-biosynthetic genes PAL1 and PAL2 (b).Levels of mRNA expression for JA-signallinggenes TPI and TD (c) and JA-biosyntheticgenes LOX3 andOPR3 (d). Error bars, � SEof the mean. Letters above the bars indicatea significant difference (P = 0.05) betweentreatments.

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infestation (Fig. S3c), whereas expression of LOX3 andOPR3 approximately doubled in levels at 7 d after whiteflyinfestation, relative to expression in water-treated controlplants (Fig. S3d). The amount of TPI transcript increased inwhitefly-infested plants at 1 dpi with Agrobacterium, but therewas no increase in the level of LOX3 and OPR3 expressionat any time-point after Agrobacterium inoculation (Fig. 2c,d).No significant increase in expression levels of ET-relatedgenes occurred at most of the tested time-points in whitefly-infested plants, before or after Agrobacterium inoculation withthe exception of EIN2, which was induced at 5 dpi withAgrobacterium (Fig. S4).

In order to investigate the endogenous plant defence hor-mones in response to whitefly infestation, we analysed the con-tents of SA and JA. Notably, the concentration of endogenousSA doubled in 7 d after whitefly infestation, relative to controlplants (Fig. S5a). An increase in endogenous SA was also seenat 1 dpi with Agrobacterium, compared with the water-treatedcontrols (Fig. 3a). By contrast, although the JA concentrationwas slightly increased following whitefly infestation (Fig. S5b),it was drastically declined after Agrobacterium inoculation(Fig. 3b). These results indicate that whitefly-derived defenceagainst Agrobacterium virulence is dependent on the SA-relatedpathway.

Silencing the SA-biosynthetic gene, ICS1, compromises theplant’s protective effect against Agrobacterium due towhitefly infection

In order to identify plant genes involved in whitefly-inducedplant defence-signalling pathways modulating Agrobacterium-mediated gall formation, we used TRV-mediated virus-inducedgene silencing (VIGS) (Anand et al., 2007) to silence theN. benthamiana ICS1 (SA biosynthetic gene) and coronatine-insentive1 (COI1, a JA signalling gene) genes. The ICS1 andCOI1 silenced N. benthamiana plants were slightly shorter andtaller, respectively, when compared to nonsilenced control plants(data not shown). Plants silenced for these genes were treatedwith whitefly, BTH and water (control) for 7 d, and then imme-diately inoculated with Agrobacterium, as described above. Beforeand after Agrobacterium inoculation no significant difference wasobserved in the same genotype among each treatment (data notshown). A mock control (no TRV inoculation) for VIGS wasincluded. Gall formation was monitored and found to be attenu-ated in whitefly-infested nonsilenced control plants (Fig. S6).

Gall weights were significantly lower in whitefly- and BTH-treated plants than in water-treated plants and empty-vector(pTRV2::00) controls (Fig. 4a). When the ICS1 gene was silenced,the gall weights from plants infested with whitefly were similar togall weights from water-treated controls (Fig. 4b), suggesting therequirement of ICS1 for whitefly-mediated plant protectionagainst Agrobacterium. By contrast, gall formation was still attenu-ated in COI1-silenced plants infested with whitefly (Fig. 4c), sug-gesting the nonrequirement of COI1 in whitefly-induced defencesignalling. Surprisingly, both gene-silenced plants and empty-vec-tor controls produced similar gall weights when treated with BTH(Fig. 4a–c). Taken together, our data suggest that the whitefly-trig-gered SA-dependent pathway plays a key role in attenuatingAgrobacterium-mediated gall formation in plants.

Whitefly infestation impairs gall formation byAgrobacterium on crown tissue

Having examined whether whitefly infestation attenuated gall for-mation by Agrobacterium on stems (Fig. 1), we next investigatedwhether activation of defence responses by whitefly infestation onparts of the plant aboveground systemically and negatively affectedthe formation of galls by Agrobacterium on belowground parts.Three-week-old N. benthamiana plants were infested with whiteflyor treated with BTH or water (control) for 7 d and directlyinjected with A. tumefaciens C58 (OD600 = 2) in belowgroundcrown tissue. Crown galls were harvested at 15 and 30 dpi.

Considerably smaller crown galls occurred on whitefly-infestedand BTH-treated plants in comparison with water-treated con-trols (Fig. 5a). Whitefly infestation had a highly significant effecton crown gall weight at 30 dpi; the mean of gall weight fromwater-treated plants was three times higher (90� 20 mg) thanthat from whitefly-infested plants (37� 11 mg) (Fig. 5b). Therelative thickness of crown was reduced by c. 20% in whitefly-infested plants at 30 dpi when compared with water-treated con-trols (Fig. 5c). Similar results were observed at 15 dpi (Fig. S7).

(a)

(b)

Whitefly Control

Whitefly Control

Fig. 3 Differential accumulation of endogenous salicylic acid (SA) andjasmonic acid (JA) in whitefly-infested plants in response to Agrobacteriumtumefaciens inoculation. Three-week-old Nicotiana benthamiana plants weretreated with whitefly or water (control) for 7 d. The stems of these pre-treated plants were inoculated with A. tumefaciens (OD600 = 2) afterwounding using a syringe with sharp needle. Samples were harvested at Agro0, Agro 1 d and Agro 5 d post inoculation. The concentration of endogenousSA (a) and JA (b) was measured at these time points. Letters above the barsindicate a significant difference (P = 0.05) between treatments. Error bars,� SE of the mean (n = 5).

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Our data clearly suggest that aboveground whitefly infestationsystemically enhanced the basal level of plant defence responsesto Agrobacterium infection in belowground tissue.

Whitefly infestation attenuates Agrobacterium transforma-tion efficiency

In order to address how Agrobacterium gall formation was modu-lated in whitefly-infested plants, we developed a novel in vitrosystem, summarized in Fig. 6(a). Nicotiana benthamiana seed-lings were grown for 3 wk in liquid culture media before beingtreated with whitefly or BTH or water (control) for a week. Inorder to verify the accumulation of total SA in root exudates fromwhitefly-infested plants, we measured total SA concentration inthe root exudates from the rhizosphere in whitefly-infestedplants. The results showed that the concentration of SA in rootexudates was c. 2.5-fold higher in the rhizosphere of whitefly-infested plants than that of the control plants (Fig. 6b). BecauseSA was endogenously overproduced in root exudates from white-fly-infested plants, the transformation efficiency of Agrobacteriumin roots of plants infested with whitefly was examined. Three-week-old N. benthamiana seedlings were treated with whitefly orBTH or water (control) for 7 d before being inoculated with dis-armed A. tumefaciens strain GV2260 containing a binary vectorwith a b-glucuronidase (gusA)-intron gene within the T-DNA(pBISN1-GUS). As expected, less intense GUS staining wasobserved in roots of whitefly-infested plants than in roots of con-trol plants (Fig. 6c). This result correlated with a four-fold reduc-tion in transcript level of gusA in whitefly-infested plants(Fig. 6d). Taken together, our results indicate that whitefly

infestation triggers the SA-mediated plant’s systemic defencemachinery, resulting in the reduction of Agrobacterium transfor-mation ability.

Discussion

Before this study, no evidence has been shown regardinginsect-elicited manipulation of the Agrobacterium-mediatedgall disease in plants. In this study, we intriguingly find sev-eral novel aspects: ecological stimulus such as insect infesta-tion manipulates the Agrobacterium-mediated gall formationin plants; higher concentration of salicyclic acid (SA)induced by whitefly infestation dramatically contributes toplant immunity against Agrobacterium infection and thustransformation; and our study provides a new insight into athree-way interaction amongst insect (whitefly), plant andpathogen (Agrobacterium).

Although there is considerable evidence to support the effectsof SA signalling on plant–microbial interactions, only few studieshave clearly demonstrated a direct effect of SA on bacterial, espe-cially Agrobacterium, growth and virulence (Yuan et al., 2007,2008; Anand et al., 2008). The direct effect of SA might dependon exogenous concentration, type of growth media and growthconditions. For example, SA significantly inhibits Agrobacteriumgrowth in acidic minimal media at very low concentrations(5–8 lM) but does not affect growth under neutral conditions(Yuan et al., 2007). Likewise, Anand et al., found that SA inhib-ited Agrobacterium growth in minimal media, but that low SAconcentrations (5–15 lM) did not impede growth in rich media,whereasa much higher concentration (200 lM) did (Anand et al.,

(a)

(b) (c)

Fig. 4 Effect of virus-induced gene silencing(VIGS) of ICS1 and COI1 on gall formationby Agrobacterium tumefaciens in whitefly-infested Nicotiana benthamiana stems. Two-week-old N. benthamiana seedlings wereinfiltrated with Tobacco rattle virus 2-vector(TRV2) vector control (a) or by TRV2harbouring NbICS1 (b) and NbCOI1 (c) andmonitored for 2 wk. The silencedN. benthamiana plants were treated withwhitefly or 0.5 mM benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester(BTH) or water (control) for 7 d and theninoculated with A. tumefaciens (OD600 = 2)at four individual places on the stem. Plantswere incubated separately for 30 d post-inoculation (dpi). Letters above the barsindicate a significant difference (P = 0.05)between treatments. Error bars, � SE of themean (n = 4).

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2008). The negative effect of SA on virulence has also beenreported for few other pathogens (van Spronsen et al., 2003;Stacey et al., 2006). Most studies relied on in vitro assays withexogenous treatment of SA. Our study differs from earlier workby showing that the increase in endogenous SA concentration inplants due to an ecological stimulus (whitefly infestation)influences the virulence of Agrobacterium. Our study is thereforearguably a better approximation of natural events and conditionsthan earlier studies.

Recently, exogenously applied SA has been shown to inhibitthe vir gene mRNA expression (Yuan et al., 2007; Anand et al.,2008). Now, although our data could not convincingly supportthe reduction in transcript levels of vir genes in root exudates ofwhitefly-infested plants, we show that SA is endogenously pro-duced in root exudates from whitefly-infested plants (Fig. 6b).Our results suggest that the SA-mediated defence machinerytriggered by aboveground whitefly infestation may be involvedin attenuating Agrobacterium-mediated plant transformation(Fig. 6c,d). We note that SA is structurally similar to indole-3-acetic acid (IAA) and the phenolic compound, acetosyringone(Liu & Nester, 2006). We speculate that the increased produc-tion of SA after whitefly infestation may suppress the transforma-tion strategy by Agrobacterium via an antagonistic effect onacetosyringone. Furthermore, elevated production of IAA hasbeen observed in Arabidopsis (Efetova et al., 2007) and Ricinuscommunis (Veselov et al., 2003) in response to Agrobacteriuminfection. We also observed an elevated level of IAA in whitefly-infested plants after Agrobacterium inoculation; this may alsoaffect Agrobacterium virulence (Fig. S8). Unlike SA, endogenousIAA did not accumulate in whitefly-infested plants, but its con-centrations increased dramatically in response to Agrobacteriuminfection. Therefore, we suggest that SA accumulation triggeredby whitefly infestation is central to the subsequent reduction inAgrobacterium-mediated virulence, and that the rise in IAA con-centrations following Agrobacterium infection acts in conjunctionwith SA to attenuate gall formation in plants.

It has also been proposed that herbivore-associated molecularpatterns (HAMPs) are applicable to plant–insect interactions.Diverse elicitors and HAMPs have been characterized in insectherbivores, including modified lipids such as fatty acid-aminoacid conjugates (FACs; Alborn et al., 1997), glucose oxidase(Diezel et al., 2009), beta-glucosidase (Mattiacci et al.,1995), in-ceptins (Schmelz et al.,2007) from lepidopterans, and sulphur-containing fatty acid caeliferins from a grasshopper (Alborn et al.,2007). More specifically, infestation by silverleaf whitefly, aphloem-feeding insect, causes an upregulation in SA-signallingbut not JA/ET-signalling pathways of Arabidopsis (Zarate et al.,2007). This indicates that a nymph effector may enhance SA pro-duction locally and systemically; a precedent for such an idea isthe example of insect effectors modulating nicotine productionand volatile biosynthesis (Musser et al., 2002; Bede et al., 2006).An intriguing finding of the present study is that we show a corre-lation between whitefly-triggered endogenous SA accumulationand attenuation of Agrobacterium-mediated crown gall disease.We speculate that HAMPs/elicitors/effectors released by whiteflyactivate SA production and lead to systemically induced resis-tance in plants. Accumulation of SA in whitefly-infested plantsmay result in more rapid recognition of Agrobacterium PAMPafter infection, resulting in increased resistance and attenuationof crown gall formation by Agrobacterium.

Although the mechanism by which root exudates are secretedis still poorly understood, our knowledge of multitrophic interac-tions in the rhizosphere mediated by chemicals secreted fromroots has recently advanced (Badri et al., 2008). These chemicalsare released in root exudates by plants in response to chemical

(a)

(b)

(c)

Fig. 5 Crown gall disease on whitefly-infested plants. Three-week-oldNicotiana benthamiana plants were treated with whitefly or 0.5mMbenzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) orwater (control) for 7 d before crown tissues were inoculated withAgrobacterium tumefaciens (OD600 = 2). (a) Smaller crown galls appearedin whitefly and BTH-treated plants than on water-treated controls. Arepresentative picture was taken at 30 d post inoculation (dpi). Asdescribed in Fig. 1, individual gall weight (b) and increased stem thickness(c) were seen in control plants at 30 dpi. Letters above the bars indicate asignificant difference (P = 0.05) between whitefly and BTH treatment andwater control. Error bars, � SE of the mean (n = 5).

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and physical stimuli (Gleba et al., 1999). Here, we have focusedon potential mechanisms by which root exudates from whitefly-infested plants mediate changes in pathogen virulence. We sug-gest that whitefly infestation triggers production of endogenousSA followed by increased secretion of phytochemicals via root ex-udates. This suggestion is supported by the fact that exogenousapplication of SA, methyl jasmonate (MeJA) and nitric oxide(NO) induces both the accumulation of a wide range of second-ary metabolites (Zhao et al., 2005) and an increase in root exuda-tion of phytochemicals (Badri & Vivanco, 2009). For example,exogenous SA and MeJA treatments result in the secretion ofindole glucosinolates and camalexin by Arabidopsis plants (Badriet al., 2008). Our results suggest that SA accumulated in rootexudates in whitefly-infested plants clearly attenuatesAgrobacterium-mediated gall disease.

Earlier studies have shown that whitefly infestation inhibitsgrowth of aerial tissues but results in an increase of root biomassin pepper plants (Yang et al., 2011; Park & Ryu, 2014). Thesephysiological aspects of infestation also occur in N. benthamiana(Y. S. Park et al., unpublished). Surprisingly, growth of aerial tis-sues is promoted in N. benthamiana plants grown in root exudatefrom whitefly-infested plants (Y. S. Park et al., unpublished), sug-gesting that whitefly infestation can regulate plant physiology aswell as resistance to the Agrobacterium infection. Further studiesare needed to elucidate the full role of root exudates from

0

0.1

0.2

0.3

0.4

0.5

0.6Whitefly BTH Control

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Whitefly BTH Control

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*

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SA (μ

g m

l–1)

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ativ

e ex

pres

sion

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gus

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ParafilmPetri Dish26 ml, 1/10 MS

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In the root exudates

Treatments: Whitefly, BTHand Control

(a)

Fig. 6 Effect of root exudates from whitefly-infested plants on Agrobacterium virulencegene expression and transformationefficiency. (a) Schematic representation ofthe experimental setup. (b) Three-week-oldNicotiana benthamiana plants grown in0.19MS liquid media were treated withwhitefly or water (control) for 7 d.Endogenous total salicylic acid (SA)production was measured in root exudatesfrom whitefly-infested plants. (c)Visualization of root segments transformedwith A. tumefaciens disarmed strain GV2260carrying the binary vector pBISN1-gusA. (d)Expression levels of gusA in root segments.Significant difference between treatments: *,P = 0.05. Error bars, � SE of the mean(n = 5).

AbovegroundBelowground

(b)(a) (c)

A. tumefaciens

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Plant cell

Fig. 7 A model depicting the mechanism by which a sucking insect(whitefly) infestation on leaves attenuates crown gall disease caused byAgrobacterium tumefaciens. (a) A plant under infestation by a suckinginsect whitefly in aboveground tissue. (b) Whitefly-infested plant producesendogenous salicylic acid (SA) and elicits plant systemic defence signallingin above- and below-ground tissues. (c) SA released from root exudatesnegatively affects the transformation efficiency of A. tumefaciens thusreducing the severity of crown gall disease.

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whitefly-infested plants in modulating plant physiology and plantdefence responses.

In conclusion, although Agrobacterium-mediated transforma-tion of plant host genomes has been extensively studied, ourunderstanding of precise plant host defence responses againstAgrobacterium is still not complete. Here, we show a novel aspectof plant defence whereby infestation with whitefly modulates thevirulence and pathogenicity of Agrobacterium infection; theresults are summarized in Fig. 7. Our findings therefore suggestthat whitefly-induced resistance determinants have potential asbio-control agents, enhancing plant immunity to diseases causedby Agrobacterium and perhaps other pathogens.

Acknowledgements

We are grateful for the financial support from the IndustrialSource Technology Development Program of the Ministry ofKnowledge Economy (TGC0281011) of Korea, the Next-Gener-ation BioGreen 21 Program (SSAC grant #PJ008170), and theKRIBB initiative program, South Korea.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Assessment of stem thickness.

Fig. S2Gall formation by A. tumefaciens in whitefly-infestedN. benthamiana stems at 15 dpi.

Fig. S3Defence-related gene expression in response to whiteflyinfestation.

Fig. S4 Expression patterns of ethylene related genes in whitefly-infested plants following A. tumefaciens inoculation.

Fig. S5Quantification of SA and JA concentrations after whiteflyinfestation.

Fig. S6 A. tumefaciens-mediated gall formation as no VIGS con-trol in whitefly-infested N. benthamiana stems.

Fig. S7 Examination of crown gall formation by Agrobacteriuminoculation at 15 dpi.

Fig. S8Quantification of endogenous indole-3-acetic acid(IAA) in whitefly-infested plants in response to A. tumefaciensinoculation.

Table S1 Primers used for qRT-PCR analyses

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