genetic and physiological analysis of the relationship between partial resistance to clubroot and...

Genetic and physiological analysis of the relationshipbetween partial resistance to clubroot and tolerance totrehalose in Arabidopsis thaliana

Antoine Gravot1, Louis Grillet2, Geoffrey Wagner3, Melanie Jubault3, Christine Lariagon3, Cecile Baron3,

Carole Deleu1, Regine Delourme3, Alain Bouchereau1 and Maria J. Manzanares-Dauleux2

1Universite de Rennes 1, UMR 118, F–35042 Rennes, France; 2Agrocampus Ouest, UMR 118, F–35042 Rennes, France; 3INRA, UMR 118, F–35042

Rennes, France

Author for correspondence:Antoine Gravot

Tel: + 33 223 485139Email: [email protected]

Received: 3 February 2011

Accepted: 25 March 2011

New Phytologist (2011) 191: 1083–1094doi: 10.1111/j.1469-8137.2011.03751.x

Key words: Arabidopsis thaliana, clubroot,partial resistance to pathogens,Plasmodiophora brassicae, primarymetabolism, quantitative trait loci, trehalose.

Summary

• In Arabidopsis thaliana the induction of plant trehalase during clubroot disease

was proposed to act as a defense mechanism in the susceptible accession Col-0,

which could thereby cope with the accumulation of pathogen-synthesized treha-

lose. In the present study, we assessed trehalose activity and tolerance to trehalose

in the clubroot partially resistant accession Bur-0.

• We compared both accessions for several trehalose-related physiological traits

during clubroot infection. A quantitative trait loci (QTLs) analysis of tolerance to

exogenous trehalose was also conducted on a Bur-0xCol-0 RIL progeny.

• Trehalase activity was not induced by clubroot in Bur-0 and the inhibition of

trehalase by validamycin treatments resulted in the enhancement of clubroot

symptoms only in Col-0. In pathogen-free cultures, Bur-0 showed less trehalose-

induced toxicity symptoms than Col-0. A QTL analysis identified one locus

involved in tolerance to trehalose overlapping the confidence interval of a QTL for

resistance to Plasmodiophora brassicae. This colocalization was confirmed using

heterogeneous inbred family (HIF) lines.

• Although not based on trehalose catabolism capacity, partial resistance to club-

root is to some extent related to the tolerance to trehalose accumulation in Bur-0.

These findings support an original model where contrasting primary metabolism-

related regulations could contribute to the partial resistance to a plant pathogen.

Introduction

Clubroot is a plant disease caused by the obligate biotrophprotist Plasmodiophora brassicae, which affects allBrassicaceae including Arabidopsis thaliana. This disease isassociated with worldwide agronomic losses with significanteconomic incidences (Dixon, 2009). The life-cycle of thesoil-borne P. brassicae consists of two phases. In the primaryphase events are confined to the root hairs whereas the sec-ondary phase occurs in both the cortex and the stele of thehypocotyl and roots of infected plants. In the secondaryphase, multinucleate plasmodia cause cell hypertrophy(abnormal cell enlargement) and cell hyperplasia (uncon-trolled cell division) leading to the development of tumors(clubs) that obstruct nutrient and water transport (Ingram& Tommerup, 1972). Successful management of clubroot

disease relies on an integrative combination of strategies,including adapted cropping practices as well as chemical,biological and cultivar control methods (Donald & Porter,2009). Clubroot resistance under oligogenic controlrecently introduced into commercial cultivars of cabbage,cauliflower and oilseed rape crops led to complete resist-ance, but could be overcome by rapidly evolving pathogenpopulations, after large-scale use (Diederichsen et al.,2009). Polygenic resistance is regarded as potentially ‘harderto break through’ and should thus be considered for thelong-term management of host plant resistance (Brun et al.,2010).

Consistent information is now available about the geneticarchitecture of quantitative partial resistance to clubroot indifferent Brassica species (Manzanares-Dauleux et al., 2000;Rocherieux et al., 2004; reviewed in Diederichsen et al.,

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2009). In Arabidopsis, a screen of 57 accessions identifiedthree genotypes – Bur-0, Tsu-0 and Kn-0 – harboring par-tial resistance to clubroot (Alix et al., 2007). Four additivequantitative trait loci (QTLs) contributing to resistance tothe P. brassicae eH isolate were identified in the progeny ofthe Bur-0 (resistant) · Col-0 (susceptible) cross (Jubaultet al., 2008b). However, mechanisms underlying partialresistance remain poorly understood and there is no reporton the nature and ⁄ or function of genes underlying partialresistance QTLs. This gap in knowledge contrasts with ourincreasing understanding of the physiopathological mecha-nisms involved in the infection of susceptible hosts byP. brassicae. The involvement of auxin and cytokinins inthe development of symptoms has been widely investigated.‘Omics’ and targeted metabolic profiling approachesassociated with phenotyping of Arabidopsis mutants oroverexpressor lines have recently contributed to refine adetailed model for hormonal control of clubroot develop-ment (reviewed by Ludwig-Muller & Schuller, 2008).

Several studies have also focused on the large and charac-teristic perturbations in carbohydrate metabolism whichoccur in Brassicaceae during clubroot infection. Thedevelopment of galls is associated with a continuous accu-mulation of starch, glucose and fructose in roots, a processthat is related to an enhanced carbon remobilization fromleaves (Keen & Williams, 1969; Ludwig-Muller et al.,2009). Among the metabolic traits involved in the inter-action, trehalose, a disaccharide generally found only intrace amounts in higher plants, reaches strikingly high levelsin clubs during the secondary phase, when the secondaryplasmodia are developing in cortical cells. The pathogenwas proposed to synthesize this trehalose – a very commontransitory storage molecule in microbes, fungi and insects –and indeed a P. brassicae transcript coding for trehalose-6-phosphate synthase (TPS) accumulates in developing gallsduring infection (Brodmann et al., 2002). This disaccha-ride, however, does not accumulate exclusively within theplasmodial compartment, as it was also measured at sub-stantial levels in leaves of root-infected plants (Brodmannet al., 2002).

Endogenous trehalose is assumed to be involved in osmo-protection mechanisms in desiccation-tolerant organisms.Biotechnological approaches for enhancing tolerance todrought stress have attempted to engineer the artificialaccumulation of trehalose in transgenic crops or associatesymbionts (Romero et al., 1997; Garg et al., 2002; Karimet al., 2007; Suarez et al., 2008). However, and paradox-ically, in plants that do not naturally accumulateendogenous trehalose (most higher plants), treatment withexogenous trehalose causes phytotoxic effects (Veluthambiet al., 1981). This trehalose toxicity is linked to accumula-tion of starch and anthocyanins and root growth inhibition(Wingler et al., 2000; Schluepmann et al., 2003), both ofwhich also typically occur during clubroot disease. The

initial discovery that exogenous trehalose treatment leads togrowth impairment and starch accumulation is now inter-preted as a retrocontrol exerted by artificially accumulatedtrehalose on the trehalose-6-phosphate (T6P) pathway,leading to T6P accumulation (Schluepmann et al., 2004).Indeed, T6P is a molecule maintained at very low levels inplants, but whose variation has a strong regulatory effect onprimary metabolism (Kolbe et al., 2005; Lunn et al., 2006;Zhang et al., 2009; Smeekens et al., 2010).

In the Col-0 accession, trehalase activity and transcrip-tion of the unique trehalase encoding gene are both inducedduring the earliest steps of P. brassicae infection. This mech-anism is assumed to reduce trehalose accumulation inclubroot-infected plant tissues and therefore would beinvolved in defense (Brodmann et al., 2002) even thoughthis accession is highly susceptible to clubroot. However, itis unknown if this mechanism could play a more prominentrole in naturally occurring resistant plant accessions. Toelucidate this point, we investigated a possible relationshipbetween trehalose metabolism and partial resistance to club-root in Bur-0. We assessed trehalose and trehalase activitylevels during clubroot infection and the effect of the treha-lase inhibitor validamycin on clubroot symptoms in Col-0and Bur-0. In vitro experiments were performed to quanti-tatively compare the respective sensitivity to treatmentswith exogenous trehalose in Bur-0 and Col-0. A QTLapproach was then used to identify relationships betweengenetic factors involved in tolerance to trehalose and resist-ance to clubroot.

Materials and Methods

Plant material and genetic map construction

Col-0 and Bur-0 accessions of A. thaliana which are suscepti-ble and partially resistant to clubroot respectively (Alix et al.,2007) were used in clubroot tests and for the evaluation ofthe tolerance to exogenous trehalose treatments. The Bur-0 ·Col-0 recombinant inbred line (RIL) population wasobtained from the Versailles Resource Centre for A. thaliana(VNAT, INRA, France, http://dbsgap.versailles.inra.fr/vnat);the production of this progeny is described in detail inSimon et al. (2008). Of the 347 available RILs, 250 wereused for QTL mapping: 164 RILs that constitute the corepopulation (Simon et al., 2008) and 86 other RILs, whichwere randomly selected. The linkage map was created using87 single nucleotide polymorphisms (SNP) genotyped withSNPlex technology (Applied Biosystems, Life TechnologyCorporation, Carlsbad, California, USA); details are givenin Jubault et al. (2008a). The genetic map was estab-lished using MAPMAKER ⁄ EXP 3.0 with the Kosambi mappingfunction.

Heterogeneous inbred family (HIF) lines 10351 and13351 were also provided by the Versailles Resource Centre

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for A. thaliana (VNAT, INRA, France). These near-isogenic lines were derived from the RIL accession 351 ofthe Bur-0 · Col-0 segregating population, through theexploitation of residual heterozygosity. The HIF lines10351 and 13351 exhibit Bur-0 and Col-0 alleles, respec-tively, in the region of the clubroot resistance QTLAt-Pb5.1 reported in Jubault et al. (2008b) (between themarkers c5_04011 and c5_10428, see the SupportingInformation, Fig. S2). For clarity, 10351 and 13351 arethereafter called 351-Bur and 351-Col.

Clubroot tests

Susceptibility to clubroot was estimated by resistance tests at21 d post inoculation (dpi) as described in Jubault et al.(2008a), using the isolate eH, the accessions Bur-0 and Col-0 and the HIF lines 351-Bur and 351-Col. For each replicate(three to eight biological replicates depending on experi-ments), data was expressed as the means of the evaluation of12 plants. Susceptibility to clubroot was quantified by gallarea evaluation (Ga in mm2) using image analysis. As Bur-0is a fast-growing accession with large rosette and hypocotyls,we expressed the size of the gall relative to the surface ofleaves, roughly evaluated by the square of the longest leaflength (maximal leaf length2 = leaf area index = La, in cm2).The ratio Ga : La was multiplied by 5000 to give theGa : La index to obtain values in the range of those obtainedwith the classical disease index. For this purpose, every plantwas photographed with a scale and image analyses were per-formed using IMAGEJ software (Rasband, W.S., ImageJ, U. S.National Institutes of Health, Bethesda, Maryland, USA,http://imagej.nih.gov/ij/, 1997–2011). Tissues from thoseplants was then collected and pooled for additional biochem-ical characterization. Roots were washed, and then rootfragments of 3 cm from the root crown were sampled andfrozen in liquid nitrogen before freeze drying and metabolitequantification. In some experiments, validamycin wasapplied by inoculation of 1 ml of 20 lM validamycin A(Duschefa, http://www.duchefa.com) aqueous solution inthe soil, near each plant hypocotyl. These applicationsstarted from 7 dpi, up to the estimation of symptoms at21 dpi. Data for clubroot resistance in the Bur-0 · Col-0RIL population – evaluated by a classical disease indexmethod – were from Jubault et al. (2008b).

In vitro plant growth and trehalose treatments

Surface-sterilized seeds were germinated on agar (1%) solid-ified Hoagland’s medium. After 3 d of stratification at 4�C,plates were incubated in a culture chamber under 130 lEillumination, 80% hygrometry with a 16 h ⁄ 8 h photope-riod at 22�C ⁄ 20�C. In a first approach, seeds of parentallines, Col-0 and Bur-0, were germinated on trehalose-freemedium for 4 d and then transferred aseptically to trehalose

(or saccharose in the control plates) containing media at40 mM or 80 mM. Plantlets were then photographed afteran additional culture of 10 d. Next, trehalose toxicity symp-toms were evaluated in parental lines germinated directlyon trehalose-containing media, through the analysis of rootgrowth inhibition and accumulation of anthocyanins inplantlets in response to trehalose, using various trehaloseconcentrations in the medium (20–80 mM). Root growthwas followed by ink-dot labeling at 7 and 10 d post stratifi-cation (dps). Depending on experiments, root growth wasevaluated directly on plates or using image analysis andImageJ software. The proportion of root growth inhibitionwas calculated from the number of roots which grew< 1 mm in the interval between 7 and 10 dps. This variablewas then referred to as TIRGI (trehalose induced rootgrowth inhibition). Plant leaf rosettes were cut at 10 dps,weighed and frozen in liquid nitrogen, and then freeze-dried before metabolite extractions for the quantificationof anthocyanins and trehalose. Anthocyanin accumulation(expressed as OD532 ml)1 mg DW)1) at 10 dps on 40 mMexogenous trehalose was referred to as the variable TIAA(trehalose induced anthocyanin accumulation). Evaluationof tolerance in RIL and HIF lines was performed usingdirect sowing on media containing 40 mM trehalose.

Metabolite extractions and quantifications

Freeze-dried samples were ground to a fine powder using aball mill and then metabolites were extracted as follows:samples were incubated for 15 min with agitation in 400 llmethanol and 400 lM adonitol (internal standard);(200 ll of chloroform was added, followed by 5 min agita-tion; 400 ll of ultrapure water was added, tubes werevortexed vigorously for 1 min then centrifuged for 10 min(14 000 g), leading to separation of a lower apolar phasecontaining chlorophyll and an upper polar phase containingwater ⁄ methanol and metabolites. A 400-ll sample fromthis upper phase was combined with 100 ll HCl 0.3 M toreveal purple anthocyanin derivatives. Anthocyanins werequantified by measuring OD532 in these acidified extractsand data were expressed as OD532 ml)1 mg DW)1. A 100ll sample of the remaining nonacidified methanolic extractswas evaporated under vacuum for subsequent analysis ofmetabolites. Trehalose and glucose were quantified follow-ing a procedure adapted from the metabolic profilingprocedure described in Lugan et al. (2009). The dry residuewas dissolved in 50 ll of 20 mg ml)1 methoxyaminehydrochloride in pyridine at 30�C for 90 min. Afterwards,50 ll of N,O-bis(trimethylsisyl)trifluoroacetamide (BSTFA)was added and samples were incubated at 37�C for 30 min.One microliter of the mixture was injected in a Trace 2000GC-flame ionization detector (FID) (Thermo-FisherScientific, Waltham, CA, USA) fitted with an AS2000Autosampler (Thermo-Fisher Scientific), a J&W DB5

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30 m · 0.32 mm · 0.25 lm column and a FID. The gra-dient temperature was: 5 min at 70�C, 5�C min)1 until220�C, 2�C min)1 until 260�C, 20�C min)1 until 300�Cand finally 5 min at 300�C.

Real-time RT-PCR experiments

The RNA extractions and DNase treatments were per-formed as described in Jubault et al. (2008a). First-strandcomplementary DNA (cDNA) synthesis was performed ina 20 lL total reaction volume using 250 ng DNAse-digested total RNA, oligo(dT)12–18 primers (Invitrogen,http://www.invitrogen.com), 1 mM dNTPs, 1· first strandbuffer (Invitrogen), 20 mM dithiothreitol (DTT;Invitrogen), 40 U RNaseOUT recombinant ribonucleaseinhibitor (Invitrogen) and 200 U SUPERSCRIPT IIreverse transcriptase (Invitrogen) by incubating for 2 h at42�C. The reaction was terminated by incubation for15 min at 70�C. The relative transcript abundance wasquantified using Light Cycler 480 Cyber GREEN I Master(Roche Applied Science) and two technical replicates foreach of two biological replicates were performed usingthe Light Cycler 480 (Roche Applied Science, http://www.roche-applied-science.com). A PCR-amplification ofTRE1 and PP2AA3 was performed on 1 ⁄ 10 diluted cDNAwith the primers described in Table 1 at a final concen-tration of 0.5 lM, using the following cycle parameters:95�C 5 min, 95�C 15 s ⁄ 60�C 30 s. Negative controls werecarried out using water instead of cDNA template. Relativequantification of gene expression was calculated followingPfaffl (2001) using protein phosphatase 2A subunit PDF2encoding gene (At1g13320) as the reference (Czechowskiet al., 2005). We checked that this reference gene exhibitedlow variation (delta CT < 2) among the samples analysed.

Trehalase activity

Trehalase activity was assayed based on the methoddescribed in Kendall et al. (1990). Enzymes were extractedfrom 100 mg of liquid nitrogen-ground biological materialwith 400 ll of 0.2 M potassium phosphate buffer, pH 5.8.After centrifugation, the protein content was quantified inthe supernatant using the Bradford procedure. Trehalaseassays were started with the addition of trehalose (or waterfor background) to a final concentration of 10 mM. Afterincubation for 1 h at 30�C, 100 ll were sampled thenboiled for 10 min and centrifuged. Glucose concentrations

were measured in the supernatants using GC-FID asdescribed earlier. The amount of glucose released from tre-halase activity was inferred taking into account thebackground glucose content in control reactions.

Starch quantification

Starch was quantified in c. 5–6 mg samples of ground dryplant matter. Samples were first incubated in 80% ethanolfor 1 h at room temperature (RT), centrifuged and thesupernatant containing soluble sugars was discarded. Thiswashing step was repeated once. Pellets were air-dried, thenloosened from the bottom of microtubes using a spatulaand resuspended in 150 ll MOPS (3-(N-morpholino)propanesulfonic acid)) 50 mM pH 7.5 with 5 ll of ther-mostable amylase (Sigma) at 4 mg ml)1. Samples wereincubated at 100�C for 6 min then cooled on ice. Then200 ll of sodium acetate buffer 0.2 M pH 4.8 and 35 U ofamyloglucosidase (Sigma) were added and the samples wereincubated for 3 h at 50�C to quantitatively digest starch.Finally, samples were centrifuged and glucose was quanti-fied in supernatants using the Glucose (HK) Assay Kit(Sigma).

Statistical methods and QTL analysis

The data obtained from each test were statistically analysedusing a generalized linear model (PROC GLM of StatisticalAnalysis System (SAS) software, SAS Institute Inc., 2000).The number of independent biological replicates, the num-ber of plants used for each replicate and statistical tests arespecified in figure legends. The QTL detection was per-formed by composite interval mapping (CIM) using QTL

CARTOGRAPHER version 2.5 (Wang et al., 2010) followingthe procedure described in Jubault et al. (2008b).

Results

Comparison of trehalose contents and trehalaseactivities in infected Col-0 and Bur-0 roots

Inoculation of 7-d-old plantlets with eH spore suspensionsresulted, 21 d later, in typical pronounced clubroot symp-toms in the Col-0 accession, contrasting (as expected) withthe low-level symptoms observed in Bur-0 (Fig. 1a,b). Smallamounts of trehalose were detected in roots of uninoculatedplants of both accessions at 21 dpi. At this stage, trehalose

Table 1 List of primers used for Real-time PCR experiments

Locus (gene name) PrimersAmpliconlength (bp)

At4G24040 (TRE1) TRE1-for 5¢-ATGGGATTCTCCGAATGGAT-3¢ TRE1-rev 5¢-TGCAATATCCTCTG-CCATCTC-3¢ 103At1g13320 (PP2AA3) PP2AA3-for 5¢-TAACGTGGCCAAAATGATGC-3¢ PP2AA3-rev 5¢-GTTCTCCACAACCGCTTGGT-3¢ 61

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levels were strongly enhanced in both infected roots of Col-0and Bur-0, although to a lesser extent for the latter (Fig. 1c).Expression of the unique trehalase encoding gene TRE1 wasmonitored using real-time PCR. TRE1 expression wasinduced at similar levels in both genotypes at both 14 dpiand 21 dpi (Fig. 1d). Enzymatic trehalase activity wasassayed in root sample extracts, through the evaluation ofresulting glucose following an in vitro incubation with alarge amount of trehalose substrate. Inoculation-dependentenhancement of trehalase activity was only observed in crudeextracts of Col-0 infected roots (Fig. 1e), and they remainedat basal levels in inoculated Bur-0 roots. Trehalase activity ininfected Col-0 roots was found to be within the range of thatdescribed by Brodmann et al. (2002).

Effect of validamycin on clubroot symptoms in Col-0and Bur-0

Validamycin is a trehalase inhibitor with a soil half-life ofonly few days, as reported by Xu et al. (2009). A solution of

validamycin (20 lM in water) was applied at 7, 10, 14, 17and 20 dpi to the soil near the hypocotyls of inoculated(eH isolate) and uninoculated plants. This treatment didnot have an obvious visible effect on plant growth anddevelopment of uninoculated controls. For inoculatedplants of the susceptible accession Col-0, application of vali-damycin resulted in enhanced clubroot symptoms evaluatedat 21 dpi by the Ga : La index (Table 2). By contrast, inthe partially resistant Bur-0 accession, validamycin treat-ment did not induce significant changes in clubrootsymptoms.

Evaluation of tolerance to exogenous trehalose inBur-0

Experiments were performed to evaluate trehalose toxicitysymptoms in Bur-0 and Col-0. In a first approach we germi-nated seedlings on trehalose-free media and then transferredplantlets to media containing 40 mM or 80 mM trehalose.A treatment with 80 mM sucrose was used as control. At

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Fig. 1 Clubroot symptoms and trehalose metabolism in Bur-0 and Col-0. (a) and (b) Illustration of clubroot symptoms at 21 d post inoculation(dpi) in Bur-0 and Col-0 ecotypes after inoculation by the eH isolate of Plasmodiophora brassicae. (c) Trehalose content, (d) Real-time PCRquantification of TRE1 gene expression levels, using PP2AA3 as reference gene (open bars, 7 dpi; light tinted bars, 14 dpi; dark tinted bars,21 dpi). (e) Trehalase activity in roots of Bur-0 and Col-0 following inoculation (I) or in uninoculated controls (NI). Data are means of six (c) ortwo (d,e) independent biological replicates, each one consisting of a pool of root samples from 12 plants. Asterisks indicate a significantdifference between two treatments according to Student tests (P < 0.05). Bars, 1 cm.




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40 mM trehalose, after 10 d, first symptoms of toxicitywere apparent only for Col-0 plantlets (Fig. 2). At 80 mMtrehalose, plantlets of both accessions were stunted. Col-0plantlets, however, exhibited symptoms of higher toxicity(curled leaves with high anthocyanin content and very shortroot systems), while for Bur-0 plants, leaves remained greenand their root system was more developed (Fig. 2). Sucrosetreatment did not lead to growth inhibition, giving an indi-cation that trehalose effect was not related to any osmoticshock. A second experiment was then carried out, withseedlings germinated in the presence of 20–60 trehalose

concentrations. A GC-FID analysis in samples from plant-lets at 10 d post stratification (dps) revealed an accumulationof trehalose. This accumulation increased with the concen-tration of trehalose in the medium (Fig. 3a). The meantrehalose content was slightly higher in Col-0 than in Bur-0after growth on 40 mM. A more pronounced contrast wasobserved for the 60 mM treatments, as the Col-0 plantletswere strongly stunted (data not shown) with high content ofanthocyanins, while Bur-0 seedlings were still green andapparently alive. We also determined root-associated andleaf-associated criteria to quantify trehalose toxicity symp-toms. The most relevant criteria were the percentage of rootgrowth inhibition between 7 dps and 10 dps (Fig. 3b), andanthocyanin accumulation in plantlets (Fig. 3c). Applicationof these criteria suggested that Bur-0 exhibited increasedtolerance to exogenously supplied trehalose for 60 mM and40 mM trehalose treatments, and even for 20 mM whenconsidering only anthocyanin accumulation. Plotting treha-lose toxicity symptom indicators against plant trehalosecontents showed that, for a plant trehalose content higherthan 10 lmol g DW)1, Bur-0 exhibited less symptomsthan Col-0 plantlets (Fig. 4).

QTL analysis for tolerance to exogenous trehalose

Trehalose sensitivity was evaluated in 230 RecombinantInbred Lines (RILs) issued from the cross Bur-0 · Col-0(Simon et al., 2008) using both root (TIRGI) and leaf-associated (TIAA) criteria. Trehalose toxicity was evaluatedthrough direct germination tests at 40 mM trehalose,because it was assumed that at this concentration RILs withhigher tolerance than Bur-0 or greater sensitivity thanCol-0 could be potentially identified. This analysis resultedin valuable data for 182 RILs for TIRGI and 178 RILs forTIAA. Both traits showed in the RIL progeny a continuousdistribution pattern, suggesting a quantitative and polygeniccontrol of the trehalose tolerance (Fig. S1).

Two QTLs on chromosome 5 were detected using theroot-associated criterion for the estimation of tolerance totrehalose. The QTL TIRGI-At5.1 (LOD = 3.4, the allelefor higher tolerance to trehalose was derived from Bur-0)was associated with the marker c5_05319 and a confidenceinterval between 10.2 cM and 21.6 cM (Fig. 5 andTable S1). This marker was previously identified as associ-ated with the clubroot partial resistance QTL Pb-At5.1(Jubault et al., 2008b). According to the FLAGdb++ v4.5database (Samson et al., 2004), we found that the confi-dence interval for TIRGI-At5.1 includes 771 gene identitiesfrom At5g13130 to At5g20320. Among them, we did notidentify any gene that would have an obvious functionalrelationship with trehalose metabolism. The second QTL,TIRGI-At5.2 (LOD = 2.8), was mapped to the bottom ofchromosome 5, but it did not colocalize with At-PbAt5.2.TIRGI-At5.1 and TIRGI-At5.2 accounted for 8.8% and

Table 2 Effect of validamycin treatment during infection on club-root symptoms

Ga : Lapathologicalindex

Col eH 44.9 ± 7.3Col eH + VAL 68.7 ± 12.2a

Bur eH 31.3 ± 4.2Bur eH + VAL 30.3 ± 7.0

Means are estimated from three independent experiments, each oneconsisting of the evaluation of 12 infected plants (eH isolate) at21 dpi with or without validamycin (see the Materials and Methodssection). The Ga : La pathological index reflects the ratio betweengall extent and rosette leaf area and was evaluated using imageanalysis as described in the materials and methods section. Resultsare reported ± SE.aSignificant effect of the validamycin treatment according toStudent’s pair t-test (P < 0.05).

Bur-0 Col-0

Trehalose

40 mM

Trehalose

80 mM

Sucrose

80 mM

Fig. 2 Trehalose toxicity symptoms in Bur-0 and Col-0 in thetransfer technique. Seeds of accessions Bur-0 (a,c) and Col-0 (b,d)were germinated on trehalose-free Hoagland’s media for 4 d thentransferred to media supplemented with trehalose 40 mM, 80 mMor sucrose 80 mM (control) for an additional 10-d period. Scale barsrepresent 1 cm.

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5.6%, respectively, of the total variation. Three QTLswere detected using the leaf-associated criterion to evaluatetrehalose-induced stress: the major effect QTL TIAA-At1(chromosome 1, LOD = 4.98, explaining 11.7% of thetotal phenotypic variation) colocalized with the locusPAP1 ⁄ MYB75 (At5g56650) previously reported to be

involved in the control of anthocyanin biosynthesis inresponse to sugar status (see the Discussion section). Inaddition, two low effect-QTLs TIAA-At5.1 (LOD = 2.7)and TIAA-At5.2 (LOD = 2.9) were found on chromosome5, with confidence intervals recovering those found with theTIRGI method. The RIL plantlets were also analysed fortrehalose content after treatment with 40 mM trehalose.No QTLs for trehalose content could be detected using thisapproach (data not shown).

In parallel, QTLs controlling clubroot partial resistancewere recalculated in the present study using phenotypic datafrom Jubault et al., 2008b, but using exactly same RIL sub-set as those used for TIRGI and TIAA QTL identification.The resulting confidence interval for Pb-At5.1 between8.6 cM and 15.6 cM (Table S1) was found to includegenes from At5g10580 to At5g16520. The cross-interval

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Fig. 4 Relationships between trehalose content in plantlets andtoxicity symptoms in Bur-0 (squares) and Col-0 (circles) accessions.Toxicity was estimated from root growth inhibition (a) andanthocyanin content (b). Both figures were constructed using datapresented in Fig. 3. Error bars represent standard errors.

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Fig. 3 Trehalose toxicity symptoms in Bur-0 (open bars) and Col-0(closed bars) in the direct germination technique. Seeds weregerminated directly on trehalose supplemented media. (a) Trehalosecontent in plantlets at 10 dps. (b) Root growth inhibition as apercentage of roots exhibiting growth < 1 mm between 7 d poststratification (dps) and 10 dps. (c) Anthocyanin content in plantletsat 10 dps. Data are means of two (a,c) or four (b) biologicalreplicates, each one consisting of eight plantlets. Error bars representstandard errors. Asterisks indicate a significant difference betweentwo genotypes according to Student’s tests (P < 0.05).




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defined by At-TIRGI5.1 and At-Pb5.1 includes a subset of358 genes between At5g13130 and At5g16520.

Evaluation of clubroot symptoms and tolerance totrehalose in HIF lines

The HIF couple 351-Col ⁄ 351-Bur was obtained from theVersailles Resource Center for A. thaliana. These lines werederived from the RIL 351, which showed residual hetero-zygosity between the markers c5-04011 and c5-10428, aregion that includes the genetic markers within the Pb-At5.1and TIRGI-At5.1 confidence intervals (Fig. S2). Thisgenetic material is well suited to get a validation of the locali-zation and effects of the two QTLs that had been statisticallyinferred from the QTL analysis on the RIL segregatingpopulation. Clubroot symptoms were quantified at 21 dpifor 351-Col and 351-Bur lines. The 351-Col HIF line dis-played stronger clubroot symptoms – more galls and higherlevels of starch accumulation in infected roots – when com-pared with the 351-Bur at 21 dpi (Fig. 6a and Table 3). Invitro cultures also revealed that trehalose-induced rootgrowth inhibition was lower for the 351-Bur line than for351-Col (Fig. 6b). Experiments with HIF lines confirmedthat an allelic variation in the genomic region between c5-04011 and c5-10428 markers, contributes to the control ofboth tolerance to trehalose and partial resistance to clubroot.

Discussion

Trehalose accumulates in infected roots at levels thatcan affect plant physiology

In this work, we have shown that differences in tolerance totrehalose between two genotypes, are linked to differences

in susceptibility to clubroot. In clubroot-infected tissues,trehalose is accumulated at levels that could modify theplant metabolism. Indeed, in the susceptible Col-0 acces-sion, pathogen infection resulted in the root accumulationof trehalose at concentrations (> 20 lmol g DW)1) that,when resulting from exogenously supplied trehalose treat-ments, are similar those above which first toxicitysymptoms can be detected, especially at the amount ofanthocyanin accumulation (Figs 1c, 4). Trehalose was alsoaccumulated in substantial amounts in infected Bur-0 roots,although to a lower extent. This contrast between Col-0and Bur-0 is not the consequence of higher trehalase activityin Bur-0, because trehalase activity is not induced duringthe infection in this accession. Instead, lower amounts oftrehalose in infected roots are more likely the consequenceof a slower development of the pathogen in Bur-0.Plasmodiophora brassicae was indeed proposed to essentiallysynthesize the trehalose which accumulates in clubroot-infected plants and potentially affect plant metabolism(Brodmann et al., 2002). As root samples from infectedBur-0 plants contained a substantial proportion of gall-freeroots, absolute quantification of trehalose relative to wholeroot sample DW probably underestimated the trehalosecontent in authentically infected areas.

Induction of trehalase is a basal defense mechanism inthe susceptible accession Col-0

When considering Col-0, our results corroborate the previ-ous work of Brodmann et al., 2002: in parallel withtrehalose accumulation, trehalase activity was highly inducedin infected roots both at the transcriptional (AtTRE1) andenzymatic level. In addition, our study revealed that the inhi-bition of trehalase activity during clubroot infection (by

Ch1 Ch2 Ch3 Ch4 Ch5

c1_005930.0c1_022125.8c1_029927.1c1 041769 6

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c2_008930.0c2_023655.2c2_042635.5c2_055886.12 066559 2

c3_008850.0c3_019013.2c3_029685.6c3_041417.0

c4_006410.0c4_021336.9c4_038337.0c4 048777 3

c5_005760.0c5_015874.8c5_029006.7c5 0401110 9

Pb-A

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c1_041769.6c1_0559314.5c1_0838519.3

c1_0978225.7

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c2_066559.2c2_0765013.5

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c2 1243537 1

c3_0514110.2c3_0663114.0c3_0804219.2

c3_0974831.3c3 1099636.8

c4_048777.3c4_0562913.3c4_0692322.6c4_0754925.7c4_0774026.5c4_0893031.0c4_1060935.8

c5_0401110.9c5_0531913.6

c5_0682022.6c5_0744224.3c5_0856328.4

5 1042838 8

At5.1

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c1_13869c1_1563444.5

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c2_1525249.1c2_1683754.8c2_1760656.7c2 1875361 8

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c3_1667753.9c3_1818058.4

c4_1187838.8c4_1317141.0c4_1481944.3c4_1576546.5c4_1768452.7

Pb-A

t4

c5_1042838.8c5_1269940.8c5_1361442.2c5_1476646.8

c5 1757061 2

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I-At5.2

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Pb-A

t5.2

c1_2479587.9c1_2569891.3

c1_2699398.5c1_28454103.3c1_28667103.5c1_29898108.6

c5_2499788.3

c5_2667199.0

Fig. 5 The quantitative trait locus (QTL) map for clubroot partial resistance and tolerance to trehalose. Arabidopsis additive QTLs controllingpartial resistance to P. brassicae (Pb-At, white bars), trehalose-induced root growth inhibition (TIRGI-At, closed bars) and trehalose-inducedanthocyanin accumulation (TIAA-At, hatched bars) in the Bur-0 · Col-0 RIL population. Each marker name consists of its chromosomenumber and physical position in kb. The bar length is in proportion to the one-LOD likelihood confidence interval.

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treatment with validamycin) resulted in enhanced symptomsin Col-0. This suggests that trehalase induction is a basaldefense mechanism, that is, a disease resistance activated by avirulent pathogen on a susceptible host, in agreement withthe definition of Jones & Dangl (2006). In clubroot disease,trehalase induction would thus restrict the deleterious effectof trehalose in plant, as proposed by Brodmann et al.(2002), and trehalose can be seen as a pathogen-effector,using the broad definition of Hogenhout et al. (2009).

There are two indications that the observed effect of vali-damycin treatment is directly related to the inhibition ofplant trehalase AtTRE1, and not an artifact caused by theinhibition of Plasmodiophora trehalase enzymatic activity.First, during the cortical infection steps, P. brassicae is

found as an intracellular inclusion, whereas plant trehalaseTRE1 is targeted to plant plasma membranes with an apo-plastic active site (Frison et al., 2007). We can thus expectthat validamycin would first come into contact with plantextracellular enzymes before perturbing P. brassicae treha-lose-related enzymes. Second, contrary to what weobserved, the inhibition of pathogen trehalase activity isexpected to reduce its fitness. For example, validamycin isused in agriculture as a fungicide for the control ofRhizoctonia associated plant diseases (Bai et al., 2006). Inthat case, however, the fungi do not form cellular inclusionsinside plant cells. Nevertheless, additional work is nowrequired to confirm our view through complementaryapproaches, including, for example, the downregulation ofTRE1 expression in Col-0 and HIF lines.

Partial resistance to clubroot in Bur-0 is not associatedwith enhanced trehalase activity but could be linked totolerance to trehalose

In contrast to the situation observed for Col-0, the accumula-tion of trehalose in infected Bur-0 roots was not accompaniedby the induction of trehalase activity, suggesting that in thispartially resistant accession, apoplastic catabolism of treha-lose by the plant enzyme TRE1 is not a major component ofplant resistance. Corroborating this idea, clubroot symptomswere not enhanced in Bur-0 when trehalase activity was inhib-ited by validamycin. The discrepancy between the inductionof TRE1 RNA and enzyme levels in Bur-0 suggests that, atleast at the transcriptional level, early plant responses to theinfection partly overlap in the two accessions.

Bur-0 exhibits a significant level of tolerance to exoge-nous trehalose, using both leaf and root-associated stresscriteria (TIAA and TIRGI respectively) and various pheno-typing conditions (direct germination or transferexperiments), suggesting a whole-plant level mechanism.The ability of Bur-0 to tolerate exogenous applications oftrehalose could be the result of reduced trehalose absorp-tion ⁄ translocation kinetics or higher trehalose catabolism inthis accession. This hypothesis would fit with the contrasted

0

10

20

30

40

50

60

70

80

Bur-0 Col-0 351-Bur 351-Col

Roo

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wth

inhi

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)

0

20

40

60

80

100

120

140

160

Bur-0 Col-0 351-Bur 351-Col

Ga/

La p

atho

logi

cal i

ndex

(a)

(b)

*

*

**

Fig. 6 Heterogeneous inbred family (HIF) validation of quantitativetrait loci (QTLs) Pb-At5.1 and TIRGI-At5.1. Parental lines and 351-Bur and 351-Col HIF lines, displaying allelic variation on a region ofchromosome 5 that overlaps Pb-At5.1 and TIRGI-At5.1. The QTLconfidence intervals were evaluated for susceptibility to clubrootusing the Ga : La pathological index (ratio between gall extent androsette leaf area; a) and for tolerance to trehalose through theevaluation of trehalose-induced root growth inhibition (b). Meanswere estimated from eight (a) or three (b) independent replicates,each one resulting from the analysis of 12 individual plants. Errorbars represent standard errors. Asterisks indicate a significantdifference between two treatments according to Student tests(P < 0.05).

Table 3 Effect of the Bur ⁄ Col allelic variation between 351-derivedheterogeneous inbred families (HIFs) onto root starch accumulationat 21 dpi for inoculated (I) or uninoculated (NI) plants

Genotype Inoculation

Root starchat the end oflight period(lg mg DW)1)

Root starchat the end ofdark period(lg mg DW)1)

351-Bur I 108 ± 15 109 ± 14351-Bur NI 61 ± 4 76 ± 11351-Col I 144 ± 21 146 ± 15351-Col NI 69 ± 11 55 ± 12

Data are from three biological replicates, each one consisting in 12plants. Results are reported ± SE.




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accumulations of trehalose when comparing both accessionsafter trehalose treatments, although mostly for the highestdose – 60 mM (Fig. 3). Indeed, at 20 mM and 40 mMtrehalose in the medium, accumulations of trehalose inplants are only slightly different (although statistically sig-nificant) and cannot account fully for the contrast betweenstress symptoms in Bur-0 and Col-0. Fig. 4 further illus-trates the idea that for a given trehalose accumulation levelin plant, Bur-0 exhibited less toxicity symptoms thanCol-0. A constitutive mechanism of tolerance to the accu-mulation of trehalose would therefore contribute to thetolerance to trehalose in Bur-0. Accordingly, we did notdetect any QTL for trehalose accumulation in tissue usingthe same experimental samples that allowed the detection ofQTL controlling TIAA and TIRGI. We can then reason-ably assume that TIRGI-At5.1 (the detected QTL thatexhibits the highest additive effect, Table S1) does notdepend on differential trehalose accumulation. Variationamong Arabidopsis ecotypes for trehalose sensitivity has notbeen previously described, but the abi4 (aba insensitive 4,At2g40220) mutant (Col-0 genetic background) is tolerantto trehalose (Ramon et al., 2007). This phenotype wasattributed to a defect in ABI4-directed starch synthesis, acellular signaling mechanism that would be specificallyinvolved in trehalose toxicity. One hypothesis could havebeen that Bur-0 displays an abi4-like metabolic phenotype.However, the abi4 mutation leads to glucose tolerancewhereas Bur-0 is highly sensitive to glucose in germinationtests (Fig. S3), suggesting that different physiological pro-cesses underlie tolerance in those two genotypes.

A plausible model is therefore emerging from our results,where trehalase induction does not act as a defense mecha-nism to delay clubroot symptoms in Bur-0, despitesubstantial trehalose accumulation in infected roots, and pre-sumably in line with the fact that this accession exhibits ahigher tolerance to trehalose. If correct, this hypothesis neces-sarily involves at least some overlaps between the geneticarchitectures of tolerance to trehalose and clubroot resist-ance. In the idea of testing this hypothesis, we took advantageof the availability of a Bur-0 · Col-0 segregating populationto perform a QTL analysis of the tolerance to trehalose.

TIAA-At1 co-localizes with the anthocyanin-controlling locus PAP1

The major QTL controlling anthocyanin accumulation inresponse to trehalose (TIAA-At1) is located on chromosome1. This QTL did not colocalize with any QTL controlling theextent of root growth under trehalose treatment, or with pre-viously detected QTL for clubroot resistance. The confidenceinterval for this QTL contains the gene encoding PAP1(PRODUCTION OF ANTHOCYANIN PIGMENT1),the MYB75 transcription factor previously identified as apositive regulator of anthocyanin accumulation in response

to sucrose and originally identified through a QTL approach(Teng et al., 2005). Although it is plausible that allelic varia-tion on MYB75 is underlying TIAA-At1, this would alsosuggest that Bur-0 and Col-0 display contrasted anthocyaninaccumulation in response to sugar status, whatever the stress.This would mean that there is a significant risk that this vari-able offers a biased estimation of stress intensity and is not asrelevant as expected for the evaluation of trehalose tolerance.

Tolerance to trehalose is genetically linked to a QTLfor clubroot resistance

In addition to the specific case of TIAA-At1, the two otherQTLs detected, TIAA-At5.1 and TIAA-At5.2, colocalizewith the two QTLs TIRGI-At5.1 and TIRGI-At5.2, whichcontrol trehalose-induced root growth inhibition. Ourgenetic analysis therefore infers that these two genomicregions exert a control on tolerance to trehalose, whether weuse a root or a leaf-associated phenotyping method. This sug-gests that the results are somewhat robust, even if anthocyaninaccumulation was – a posteriori – not the best toxicity markerto study trehalose response (see the previous paragraph).

TIRGI-At5.1 and TIAA-At5.1 are located near the mar-ker c5-05319, and the confidence intervals for these twoQTLs overlap with QTL Pb-At5.1, which controls clubrootresistance, as described by Jubault et al. (2008b). These sta-tistically significant results inferred from the phenotypesobserved in a RIL population were confirmed by the pheno-types of HIF lines. This colocalization brings a substantialsupport to our initial hypothesis about a possible linkbetween tolerance to trehalose and resistance to clubroot.

One explanation of the toxic effects of exogenous treha-lose in plants is the induction of a massive allocation ofcarbon into starch synthesis, in leaves (Wingler et al.,2000). We have shown that starch is accumulated more inclubroot-infected 351-Col roots than in 351-Bur. It wouldtherefore be interesting now to utilize those HIF lines toinvestigate if higher starch accumulation in infected roots ofthe 351-Col line is the consequence or the cause of thehigher susceptibility to clubroot of this line.

The other QTLs controlling root growth inhibition inresponse to trehalose (TIRGI-At5.2) and anthocyanin accu-mulation (TIAA-At5.2) colocalize in a region devoid ofQTL for clubroot partial resistance. Because of their smalleffect on tolerance to trehalose, this lack of colocalizationdoes not dismiss our previous conclusion. Small-effectQTLs for clubroot resistance in this region could have beenmissed in Jubault et al. (2008b) study.

Tolerance to trehalose shows parallels with previouslydescribed atypical carbohydrate metabolism in Bur-0

Bur-0 is an atypical accession when considering severalaspects, including for example flowering time, growth rate

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and carbohydrate metabolism (Werner et al., 2005; Crosset al., 2006). An attractive hypothesis would be that specificfeatures of carbohydrate metabolism in Bur-0 are partlyrelated to an idiosyncratic fine-tuning of primary meta-bolism control by the T6P ⁄ trehalose pathway. Acomprehensive analysis (in appropriate HIF pairs) of carbo-hydrate metabolism and trehalose or T6P-controlledtranscriptional networks will be of great interest to gain abetter understanding of cellular mechanisms underlyingtolerance to trehalose and clubroot resistance. Furtherpositional cloning is also required to clearly assess if thosephenotypes are under the control of the same genetic poly-morphism and to obtain insight into the putativeunderlying coding sequence.

Conclusion

Our initial goal was to test if high trehalase activity couldcontribute to clubroot partial resistance in Bur-0. Ourexperimental data infirmed this hypothesis, but revealed asurprising level of tolerance to exogenous trehalose in thisaccession, that appears to be at least in part associated to abetter ability to tolerate trehalose accumulation. A linkbetween this metabolic feature and clubroot resistance issupported by a colocalization between genetic factors con-trolling clubroot resistance and tolerance to trehalose. Theseresults open new perspectives on the long-known outstandingtrehalose accumulation in clubroot infected plants, and callsfor taking into account the genetic determinants of primarymetabolism features in the study of partial quantitativeresistances to plant pathogens.

Acknowledgements

The authors thank Christine Camilleri (Versailles Geneticsand Plant Breeding Laboratory, Arabidopsis thalianaResource Centre, INRA, France) for providing seeds of RILand HIF lines, Vincent Bouguennec, Morgane Havard,Laurent Charlon and Pascal Glory for their technical assis-tance, and Francois Lahrer for the critical reading of themanuscript.

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Supporting Information

Additional supporting information may be found in theonline version of this article.

Fig. S1 Frequency distribution of the trehalose toxicityevaluated through the TIRGI (trehalose induced rootgrowth inhibition) and TIAA (trehalose induced rootgrowth inhibition) traits in the Bur-0 · Col-0 RIL popula-tion.

Fig. S2 Illustration of the genome structure in recombinantinbred line (RIL) 351.

Fig. S3 Effect of high glucose supply on the germinationand growth of Bur-0 and Col-0.

Table S1 Full ANOVA model including additive quantita-tive trait loci (QTLs) controlling partial clubroot resistance,TIAA (trehalose induced anthocyanin accumulation) andTIRGI (trehalose induced root growth inhibition)

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting informationsupplied by the authors. Any queries (other than missingmaterial) should be directed to the New Phytologist CentralOffice.

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