Physiological and Molecular Plant Pathology (2001) 58, 149±158doi:10.1006/pmpp.2001.0323, available online at http://www.idealibrary.com on

Lipopolysaccharides from Burkholderia cepacia contribute toan enhanced defensive capacity and the induction ofpathogenesis-related proteins in Nicotianae tabacum

HELEN S. COVENTRY and IAN A. DUBERY*

Department of Biochemistry, Rand Afrikaans University, P.O. Box 524, Auckland Park, 2006, South Africa

(Accepted for publication March 2001 and published electronically 26 April 2001)

responses [

0885-5765/0

* To whoiad@na.rau.

Lipopolysaccharides (LPS) from the outer cell wall of Gram-negative bacteria can in¯uence the outcome ofcertain plant±pathogen interactions. LPS from an endophytic strain of Burkholderia cepacia, were puri®edand characterized by denaturing electrophoresis. A protective e�ect of LPS from Burkholderia cepacia on theNicotianae tabacum±Phytophthora nicotianae interaction was found when plants were infected with zoospores ofthe pathogen. Progressive development of Black-shank disease symptoms occurred in the control plantswhile plants pre-treated with 100 mg mlÿ1 LPS remained una�ected. The LPS were found to possessactivity as elicitors of plant defense responses in tobacco where the induction of PR-proteins wasinvestigated by selective low pH extraction and electrophoretic analyses. Membrane permeability studiesshowed a dose dependent increase in permeability and of loss of cell viability due to the increasing toxice�ect of higher concentrations (200±1000 mg mlÿ1) of LPS. The optimum concentration for PR-proteininduction was found to be 75-100 mg mlÿ1, where the e�ect on cell permeability was minimal butinduction was optimal. Time studies of 0±4 days, with 100 mg mlÿ1 LPS added to cell suspensions and leafdiscs, showed an increase in intensity of protein bands with Mrs of 6

.5, 15, 17, 23, 33 and 35. These valuescorrespond to PR-proteins from classes VI, IV, I, V, III and II, respectively. Experiments were extended toinclude whole plant and leaves to compare the results obtained from the cell suspension and leaf discs andwere found to be similar with regard to the time and dose-dependent induction of PR-proteins. PR-proteins extracted from the leaves following bacterial inoculation of the roots indicated a systemic responsewhich was also observed in upper leaves following treatment of lower leaves. The results are indicative of anenhanced defensive capacity due to pre-conditioning by the bio-active LPS. *c 2001 Academic Press

Keywords: Burkholderia cepacia; Nicotianae tabacum; Phytophthora nicotianae; endophyte; lipopolysaccharides;

PR-proteins; protection.

INTRODUCTION

Plant disease can be naturally suppressed by plant growthpromoting rhizobacteria and endophytic bacteria[34 , 36]. Apart from direct antagonism against patho-genic organisms, these bene®cial bacteria can induce aform of systemic resistance in plants. The main inducingbacterial component has been suggested to be the outermembrane lipopolysaccharides (LPS), found in the cellwalls of Gram-negative bacteria, but other moleculessuch as siderophores and bacterially produced salicylicacid are also considered as candidates [34].

The role of LPS in plant±pathogen interactions iscomplex; it can prevent the hypersensitive response undercertain conditions, a phenomenon that has been ascribedto increased tolerance or localized induced resistance

20 , 24]. Living, heat-killed pathogenic or

1/040149+10 $35.00/00

m all correspondence should be addressed. E-mail:ac.za

saprophytic bacteria introduced into tobacco leavescould cause the induction of resistance to di�erentpathogens. In addition, puri®ed cell-free fractions, con-taining LPS, exhibited protective activity [20]. LPS wasalso reported to induce systemic resistance in plantsagainst pathogens [17 , 37], but little is known about themechanisms involved. Acquired resistance is explained bythe systemic response of plants after induction by weaklyaggressive strains, avirulent and incompatible forms of thedisease-causing organism. With acquired resistance(SAR), a set of protective genes, including those thatencode for pathogenesis-related proteins, are expressed[14]. In contrast, while induced resistance (ISR) involvesthe activation of the plant's defense mechanisms leadingto systemic protection, it is not associated with theaccumulation of pathogenesis-related proteins [17 , 34 , 38].

Plant cells can be exposed to LPS in planta, as aconsequence of normal bacterial growth or rapid bacterialcell death due to activated defense responses. This raises

the possibility that defense-related gene expression occurs

*c 2001 Academic Press

lyophilized.

according to Ref. [32].

n

as a response to LPS in the interaction between plants andendophytic bacteria [23 , 24]. The spectrum of reactionselicited in plants undergoing a resistance response, eitherdue to host or non-host resistance is complex, but similar.Although emphasis has been placed on gene expressionduring incompatible interactions, defense-related genesare also activated during compatible interactions [33]such as one that would result from a plant±endophyteinteraction [2].

Strains of Burkholderia (Pseudomonas) cepacia have beenused as biocontrol agents against fungal pathogens, butthe mechanism of action was unclear [6]. It has beenpostulated that endophytic bacteria may function aspotential inducers of plant disease resistance [2], and thatendophytic behaviour contributes to the induction ofdefense responses [8 , 34]. In order to investigate themolecular basis of this phenomenon, the LPS fractionfrom the cell wall of the endophytic strain B. cepacia (ASPB 2D) was puri®ed and electrophoretically characterized,and found to induce a resistance response in tobaccoplants against the pathogen Phytophthora nicotianae. Thisreport describes the biological activity of B. cepacia LPS aselicitors of PR-proteins in tobacco. It demonstrates thecoordinated induction of various PR-proteins anddocuments the time and dose dependent responses in

150 H. S. Coventry a

suspension cultured cells and leaf tissues.

MATERIALS AND METHODS

Bacterial strains

B. (Pseudomonas) cepacia strain (ASP B 2D), isolated fromAsparagus o�cinalis, and investigated as a potentialbiocontrol agent against Fusarium oxysporum (Coventry,unpublished), was supplied by the Plant BiotechnologyDivision, AECI Ltd, South Africa. The bacteria, beingnonpathogenic to tobacco, had no adverse e�ect on theplants. The bacteria were cultivated in Nutrient Broth(BioLab) liquid medium, and were incubated at 258C on

a continuous rotary shaker for 8±10 days.

withstand desiccation [18].

Extraction and puri®cation of the bacterial lipopolysaccharides

LPS were extracted from freeze dried bacterial cell wallsusing an adaptation of the phenol±water method wherethe LPS fractionates into the water phase at 658C. Thephase partitioning was repeated twice and the waterphase, containing LPS and traces of RNA, was lyo-philized [40]. To further purify this extract, RNase(Roche), was added and the solution (50 mg enzyme to0.5 g LPS in 20 ml distilled water) and incubated at378C for 2 h. An equal volume of 90% phenol was addedto denature the enzyme and possible adhering proteins.

The solution was vortexed and centrifuged at 12 000 g for

15 min. The upper water layer was removed, dialysedagainst distilled water with three changes of water and

d I. A. Dubery

Characterization of the LPS

The 2-keto-3-deoxyoctonate (KDO), the carbohydrate aswell as the protein content of the LPS were determined.KDO was determined with periodate oxidation [13], thecarbohydrate content with the phenol-sulphuric acidreagent [11], and the protein concentration with theCoomassie G microassay method [4] as well as a solid-phase method with amido black [29].

Puri®ed LPS samples were dissolved in distilled waterand sodium dodecyl sulphate±polyacrylamide gel electro-phoresis (SDS±PAGE) [15] was performed on 10% gelswith 4 M urea added to the separating gel to facilitate theseparation and migration of the large LPS molecules [32].For increased resolution, the electrophoresis was per-formed at constant voltage (90 V) and varying current(440 mA) at 48C. The gels were stained with silver

Preparation of plant and fungal pathogen material

Tobacco plants (Nicotianae tabacum cv. Samsun), germi-nated from seed, were grown in soil under a 16 h/8 hlight±dark cycle in a green house. Tobacco cell suspensioncultures, generated from callus derived from these plants,were cultivated in the dark at 258C on an orbital shaker at90 rpm under sterile conditions. The suspensions weresubcultured weekly. The liquid medium used wasMurashige and Skoog (MS) medium with MS-vitamins,containing 0.9 mM 2±4 D and 0.92 mM kinetin (pH 5.8)[22].

P. nicotianae race one, the causal agent of Black Shankdisease, was obtained from the Tobacco and CottonResearch Institute in Rustenburg, South Africa. Theisolate was maintained on oatmeal-agar medium (pH 4.7)in the dark at 228C [21]. Sterile distilled water was pouredonto the culture followed by chilling to 108C for30±60 min to initiate the germination of the sporangiaby formation of swarmspores. The lid of the Petri dish wasremoved to allow for an adequate oxygen supply which isa critical factor for germination [18]. The water contain-ing the germinated zoospores (5 � 103 mlÿ1) was immedi-ately used to spray onto and infect the plants, as both thesporangia and zoospores are short lived and cannot

The e�ect of LPS on the Nicotianae tabacum±Phytophthora nicotianae interaction

Tobacco plants, growing in sterile soil, were treated three

times with LPS at 3 day intervals in order to induce the

defensive capacity of the plants. A 100 mg mlÿ1 LPSsolution was sprayed on leaves until run-o�. Five daysfollowing the last LPS treatment, both the control andthe LPS treated plants were sprayed and infected with azoospore suspension (5 � 103 mlÿ1) of the pathogen,P. nicotianae, and incubated under humid conditions.The development of the characteristic symptoms of BlackShank disease, wilting and chlorosis of the leaves andnecrotic decay in the stems [18] were scored on a scale of1±5 and recorded over a period of 44 days. The controland test groups consisted of three plants each and the

Lipopolysaccharide

experiment was repeated three times in total.

Cell permeability assay

The spectrophotometric monitoring of membrane per-meability was performed with Evans blue, a nonpermeat-ing or exclusion dye that leaks through rupturedmembranes, and stains the contents of a�ected cells [1].High concentrations of LPS (100, 400, 800 and1000 mg mlÿ1) were used to test for possible phytotoxice�ects. Leaf discs were incubated in Petri dishes with20 ml of 0.25 � MS liquid medium with the LPSsolutions added for 1.5±2.5 days. Leaf discs were removedfrom the medium and submerged in 1 ml of 0.25%aqueous Evans blue for 20 min on an orbital shaker. Cellsuspensions in the early logarithmic phase were sub-cultured into 10 ml aliquots, adjusted to the same LPSconcentrations and incubated at 258C in the dark for 24and 48 h on an orbital shaker at 90 rpm. Cells werestained with 0.25% Evans blue for 5 min [1]. Cells andleaf discs were washed with distilled water until no furtherdye eluted. The trapped dye was released by homo-genization with 0.5% aqueous SDS. Following centri-fugation at 8800 g for 3 min the optical density was

spectrophotometrically determined at 600 nm.

bacteria being non-pathogenic to tobacco.

Cell viability assays

The reduction of 2,3,5-triphenyltetrazolium chloride(TTC) was used as a viability assay for tobacco cells insuspension. Suspensions were pre-incubated with 0, 50,75, 100 and 200 mg mlÿ1 LPS for 24 h and the viabilitysubsequently measured with 0.6% TTC in a 2 : 1mixture of 0.05 M sodium phosphate bu�er and growthmedium at pH 7.5. The amount of formazan produced

was measured in ethanol at 485 nm [31].

Induction of PR-proteins in cultured cells and plants

The tobacco cell suspensions were sub-cultured into50 ml Erlenmeyer ¯asks and inoculated with sterile LPSsolutions from strain ASP-B-2D dissolved in 1 mM CaCl2and 2.5 mM MgCl2 [9]. Concentration studies of

50±500 mg mlÿ1 LPS over 4 days and time studies for

0±4 days using 100 mg mlÿ1 LPS were performed intriplicate in independent experiments. The cells wereincubated at 258C in the dark on an orbital shaker at80 rpm.

Leaf discs were cut using a 10 mm cork borer understerile conditions and rinsed twice in sterile distilled waterfor 30 min in order to dilute any wound metabolites thatmay trigger a wound response. It is known that extensivewounding can induce some of the basic, but not acidicPR proteins [5]. The discs were transferred to Petri dishescontaining LPS solutions in sterile 0.25 � MS liquidmedium and incubated at 258C under a 16 h/8 h light±dark cycle. Concentration studies were performed over 4days using 20±300 mg mlÿ1 LPS, followed by time studiesfor 0±5 days using 100 mg mlÿ1 LPS. Leaf discs werevacuum in®ltrated with the LPS solutions as describedbelow. Ten leaf discs, cut from the same plant, were usedfor each time point or concentration value.

Intact young leaves were removed from plants growingin the greenhouse. The leaves were placed in beakerscontaining the LPS solutions, and were covered to pre-vent excessive evapotranspiration. The LPS concentra-tions used were from 0±200 mg mlÿ1 LPS. The leaveswere incubated at 258C for 4 days under a 16 h/8 hlight±dark cycle.

To investigate systemic induction of PR-proteins, twolower leaves from tobacco plants growing in soil weresprayed with a 100 mg mlÿ1 LPS solution. After 4 daysselective PR-protein [35] and intercellular wash ¯uid(IWF) extractions [27] were done from upper, non-treated leaves as described below. Di�erent plants of thesame developmental stage were used in three separateexperiments.

In order to investigate PR-protein induction by livebacteria, cuttings were made from tobacco plants andallowed to root in 0.25 �MS basal salts medium beforeinduction. B. cepacia culture cells (4 � 105 cells mlÿ1)were added to the liquid medium. After 4 days at 258Cunder a 16 h/8 h light±dark cycle, the leaves of therooted cuttings were used for total and extracellular PR-protein extractions. No lesions or discolouration of theroots or leaves were observed, in accordance with the

induced protection 151

Selective extraction of cellular and apoplastic PR-proteins

The PR-protein extraction procedure was performedusing the selective low pH, phosphate-citrate extractionwith NaCl (0.5 mM) added to avoid non-speci®c ionicinteractions occurring between the proteins and the plantcell wall [16 , 33 , 35].

Tobacco suspension cells were ®ltered and weighed.Cells (1.5±2.0 g) were homogenized with cold extractionbu�er (32 mM Na2HPO4 , 84 mM citric acid, 0.5 M

sucrose, 0.5M NaCl and 0.3% 2-mercaptoethanol,

software, (Version 2.000.03, Syngene, Cambridge, U.K.).

n

pH 2.9) (1 : 1 m/v), using an Ultra-Turrax and kept onice for 0.5 h. Leaf tissues were weighed and ground inliquid nitrogen using a mortar and pestle. Cold extractionbu�er (1 : 1 m/v) was added to the powder (0.8±1.0 g),homogenized and left to stand on ice for 0.5 h. Both theleaf and cell extracts were centrifuged at 48C for 15 minat 12 000 g and ®ltered through miracloth to remove any¯oating particles.

The intercellular washing ¯uid (IWF)-method ofapoplastic ¯uid extraction using in®ltration with anextraction bu�er, (25 mM Tris±HCl, pH 7.8, containing10 mM MgCl2 , 10 mM CaCl2 , 0.5 mM phenylmethyl-sulfonyl¯uoride and 5 mM 2-mercaptoethanol) undergentle vacuum, was performed according to Parent andAsselin [27]. Cytosolic NAD malate dehydrogenase wasused as a marker enzyme to evaluate symplasticcontamination in the IWF. The IWF was straw coloured

152 H. S. Coventry a

and exhibited no marker enzyme activity.

Electrophoretic analysis of PR-proteins

Trichloroacetic acid (TCA) precipitation was used forprotein concentration of the various extracts. Cold TCA(100%) was added to 1 ml of sample supernatants to a®nal concentration of 10% [3]. The solutions werevortexed and left to stand for 30 min at 08C or 15 min atÿ208C. The solutions were centrifuged for 5 min at 58C(10 000 g) in microcentrifuge tubes, and the precipitatesvortexed with 1.5 ml ethanol : ether (1 : 1 v/v) to removeresidual TCA. Following centrifugation, the precipitateswere dissolved in 50 ml SDS treatment bu�er. Equal

volumes of the di�erent samples were loaded on 13% gels

8006004002000

100

80

60

40

20

0

LPS (µg ml�1)

Cel

l via

bili

ty (

%)

(a)

FIG. 1. (a) Lipopolysaccharides puri®ed from B. cepacia, resolvedbanding pattern of the LPS extracted from the strain ASP B 2Dviability and relative membrane permeability in tobacco cell suspeB. cepacia for 24 h. Reduction of TTC to formazan was measured apermeating Evans blue as an indicator of permeability. Followingpores in the membranes was released with 1% sodium dodecyl

fold-inc

and the proteins separated by SDS±PAGE [15]. Proteinwas detected by an ultra-sensitive silver stain methodcapable of detecting 0.01 ng protein mmÿ2 [19]. The lowmolecular weight standards (Amersham, Little Chalfont,U.K.) used throughout this study included the followingproteins: ovalbumin (46 kDa), carbonic anhydrase(30 kDa), trypsin inhibitor (21.5 kDa), lysozyme(14.3 kDa), aprotinin (6.5 kDa), insulin (b) chain(3.4 kDa) and insulin (a) chain (2.35 kDa). Densito-graphic analyses were performed with a UVP imageanalyzer (San Gabriel, CA, U.S.A.) and GeneTools

d I. A. Dubery

RESULTS

Characterization of the LPS

The characterization of the LPS puri®ed from theB. cepacia strain ASP B 2D, gave the following results.The average yield of LPS was 100 mg LPS gÿ1 cells.KDO is an important constituent of LPS and thepresence of KDO can be used as a quantitative marker.The KDO concentration was 4.82 mg KDO mgÿ1 LPSand the carbohydrate concentration 0.87 mg mgÿ1 LPS.The protein concentration was 52.0 mg mgÿ1 (0.2%)LPS with the Coomassie blue method. No protein wasdetected using the solid-phase amido black method that isinsensitive to reducing sugars [29]. The banding patternof the puri®ed LPS is shown in Fig. 1(a). The manyorderly spaced bands represent the LPS molecules havingdi�erent numbers of repeating units in their O-antigen

side chain in addition to the core and lipid A sections of

1000 0 200 400 600 800 1000

1.8

1.6

1.4

1.2

1.0

Cel

l per

mea

bili

ty (

-fol

d)

LPS (µg ml�1)

(b) (c)

on a 10% SDS±PAGE with 4 M urea and silver stained. Theis shown in lanes 1 and 2 (0.17 and 0.25 mg). (b) and (c) Cellnsions after pre-exposure to various concentrations of LPS fromt A485 nm as an indicator of cell viability and the uptake of non-treatment with 0.25% Evans blue, the stain taken up throughsulphate and quanti®ed at A600 nm. Results are expressed asrease.

the molecule. The molecular weight heterogeneityobserved is characteristic of LPS, which varies inmolecular mass from subunits of approximately 20 kDa

Lipopolysaccharide

to several million for micellar aggregates [9].

The e�ect of LPS on cell permeability and viability

In order to evaluate possible contribution of LPS-mediated cytotoxicity towards the induction of aresistance response, the in¯uence of LPS on membranepermeability was investigated as a parameter of cellintegrity and viability. The e�ect of high concentrationsof LPS in both tobacco cell suspensions and leaf discs wasdetermined using the Evans blue method that measuresuptake and retention of the stain by permeabilized ordead cells [1]. High concentrations of LPS had anadverse a�ect on plant cell integrity and there was acontinuous and increasing occurrence of cell permeabilitydue to increasing toxic e�ects [Fig. 1(c)]. Leaf discsexhibited the same sensitivity to the LPS compared tosuspension cultured cells, perhaps due to facilitateduptake through the cut surface areas. The measurableincrease in cell permeability in the concentration rangeused in this study (0±100 mg mlÿ1) was however, verysmall (1.09-fold), and was not associated with a

signi®cant decrease in cell viability [Fig. 1(b)].

The e�ect of LPS on the Nicotianae tabacum±Phytophthora nicotianae interaction

To investigate the induction of acquired/induced resist-ance, plants were pre-treated with 100 mg mlÿ1 LPSbefore being challenged with zoospores of the pathogen,P. nicotianae race one. The progression of the developmentof Black Shank disease symptoms over a period of 44 dayswas observed in the control plants. All the control plantsdeveloped wilting symptoms followed by chlorosis andnecrotic decay in the stems. The LPS-treated plantsdeveloped no necrotic ¯ecks or disease symptoms,appeared to be una�ected and exhibited a normalgrowth rate (Fig. 2). No direct inhibitory e�ects by theLPS were observed when growth of P. nicotianae wasmonitored on potato dextrose agar. This suggests thatdisease suppression was the result of the pre-treatmentwith LPS that triggered a resistance response in theplants. As the mechanisms underlying the pre-condition-ing of plants to respond more rapidly towards pathogenattack are not well understood, the e�ect of puri®ed LPS

was further investigated on cellular level.

Introduction of PR-proteins in cultured cells, leaf discs andplants

In order to investigate the induction of PR-proteins by

LPS from B. cepacia selective extractions at low pH (2.9),

were performed on induced cells and tissues, and theextracts concentrated 20-fold by trichloroacetic acidprecipitation. SDS±PAGE was performed on theextracted proteins in order to visualize both the acidicand basic isoforms of certain PR-proteins.

The results of a concentration study (20, 50, 100, 200,300 and 500 mg mlÿ1), performed over 4 days are shownin Fig. 3. Induction of proteins was evident at 20 mg mlÿ1

LPS, increasing up to 100 mg mlÿ1 LPS. It was alsofound that LPS concentrations of 5200±300 mg mlÿ1

resulted in a decrease in total extracted protein whichcorrelates with the data obtained from the cell per-meability assays. The optimum concentration for PR-protein induction was found to be 75±100 mg mlÿ1 LPS,where the adverse e�ect on cell integrity was minimal.

The results from a time study of 0±4 days withsuspension cultured cells elicited with 100 mg mlÿ1 LPSare presented in Fig. 4(a), showing primarily thevacuolar, basic isoforms of the PR proteins. A relativelyhigh number of proteins were observed in the controls,due to the undi�erentiated state of the cells, the presenceof phytohormones and the ultra-sensitive method ofdetection. Time studies with leaf discs vacuum in®ltratedwith the same concentration of LPS showed similar, butmore pronounced patterns of PR-protein accumulationthat included both basic and apoplastic acidic forms[Fig. 4(b)]. The accumulation of the low Mr proteins inthe 6±30 kDa range is especially prominent and lessbackground was observed compared with the extractsfrom cell suspensions.

Experiments were extended to include whole, detachedleaves to compare the results obtained from the cellsuspension and leaf disc studies, and were found to beconsistent with regard to the time- and dose-dependentinduction of PR-proteins (not shown).

PR-proteins are generally de®ned by their occurrenceas protein bands on gels [36], and increased intensity ofbands with Mrs of 6.5, 15, 17, 23, 33, 35 and 50 wereobserved as shown in Fig. 4(b). Although correspondenceto the di�erent families of PR proteins on the basis ofmolecular masses is insu�cient for speci®c identi®cation,these values do match those of PR-proteins from classesVI (microbial proteinase inhibitors), IV (win-likeproteins), I (PR-1a), V (thaumatins), III (chitinases)and II (b-1,3-glucanases), respectively [14 , 33].

To test for possible systemic induction, lower leaves ofplants were treated with LPS and the induction of PR-proteins monitored in the upper leaves. The proteinconcentrations of the extracts were lower compared tothat obtained with cells and leave discs, perhaps due todilution of the primary and secondary signals. Fig. 5shows the results of upper (systemic) leaves and lowerleaves treated with LPS from plants growing in soil. After4 days higher concentrations of PR-proteins were

induced protection 153

observed in the systemic leaves, suggesting that treatment

FIG. 2. The e�ect of LPS pre-treatment on the infection of tobacco plants by the pathogen P. nicotianae. (a), Plants before pathogeninfection. Progressive disease symptoms are visible at 14 days (b), 25 days (c) and 44 days (d) after initial infection in the untreatedplants, sprayed with 5.103 mlÿ1 germinating zoospores. The LPS treated plants (sprayed three times with 100 mg mlÿ1 solutions),

were not a�ected and showed no symptoms of disease.

C 1 2 3 4 5 6 MR

FIG. 3. Concentration dependent accumulation of vacuolar andapoplastic PR-proteins in leaf discs. Selective low pH extractswere prepared after 4 days following elicitation with LPSconcentrations ranging from 0 (C), 20 (lane 1), 50 (lane 2), 75(lane 3), 100 (lane 4), 200 (lane 5) and 300 mg mlÿ1 (lane 6).Mr refers to the low-molecular weight markers (6.5, 14.3, 21.5,

30 and 46 kDa).

154 H. S. Coventry and I. A. Dubery

of the lower leaves resulted in the generation of asecondary signal molecule(s) as has been proposed forSAR [28]. However, the response will have to beinvestigated over longer periods of time in order todemonstrate a de®nite systemic response to the LPS.

The perception mechanism for LPS seems to be notonly present in the leaves, but also on the root surface.Roots of cuttings were inoculated with live bacteria andthe PR-proteins were extracted 4 days later from upperleaves. The results indicate a systemic response as thebacterial inoculation on the roots and the extraction of theproteins from leaves were spatially separated. Fig. 6(a)and (b) show a systemic increase in PR-proteins after 4days, respectively, in total extracts and IWF extractsprepared from the leaf apoplast [27]. As other bio-activecompounds present on the bacterial surfaces might havecontributed to the response, the experiment was repeated

with 50 mg mlÿ1 LPS and similar results were obtained.

DISCUSSION

The outer parts of the tripartite amphipathic LPSmolecule (which consists of a lipid A, a core region and

an O-antigen region) have a major role in determining

C0 1 2 3 4 Mr C4

C0 1 2 3 4 5 Mr C5

(a)

(b)

FIG. 4. Electrophoretic analysis of accumulated cellular PR-proteins in suspension cultured tobacco cells and leaf discs.Selective low pH extracts were prepared over time periods of 0,1, 2, 3, 4 and 5 days following elicitation with 100 mg mlÿ1 LPS,concentrated 20-fold and separated on 13% SDS±PAGE gelswith silver staining. Equal volumes (15 ml) of the variousextracts were loaded for comparison. (a) Cell suspensions: C0and C4 indicate controls at days 0 and 4, respectively. Bandsshowing an increase in intensity are indicated by arrowheads.Induced proteins are indicated with arrowheads, while the bandmarked with a square indicates a protein constitutively presentin all extracts. Mr refers to the low-molecular weight markers(6.5, 14.3, 21.5, 30 and 46 kDa). (b) Leaf discs: C0 and C5indicate controls at days 0 and 5, respectively. Bands indicatedwith arrowheads correspond toMrs of 6

.5, 15, 17, 23, 33, 35 and50, respectively.

C0 Mr T2 T4 S2 S4

FIG. 5. Accumulation of PR-proteins in upper, non-treatedleaves (S) of tobacco plants following treatment of lower leaves(T) with 100 mg mlÿ1 LPS. Total PR-proteins were extracted 2and 4 days after initial treatment (T2, T4; and S2, S4,respectively) and electrophoretically resolved. The proteinpattern in control tissue is indicated in C0, and Mr refers tothe low-molecular weight markers (6.5, 14.3, 21.5, 30 and

46 kDa).

C T C T

(a) (b)

kDa

46

30

21.5

14.3

6.5

FIG. 6. Accumulation of PR-proteins in leaves of tobaccocuttings in response to inoculation of the roots with B. cepacia.(a) Total cellular PR-proteins extracted from the leaves, and (b)the apoplastic forms present in the IWF. Extractions werecarried out on the leaves at 0 days (C) and 4 days (T) to

investigate a systemic response.

Lipopolysaccharide induced protection 155

n

the surface characteristics and antigenic properties ofbacterial strains. It is also important in the initialinteraction that occurs between the bacterium andplant cell, where it may determine the di�erence betweencompatible and incompatible reactions [30]. The regionof the LPS molecule responsible for active induction ofresistance in plants remains controversial. Although LPSfrom certain Gram-negative bacteria is biologically activein planta [12 , 16], its activity appears to depend on thenature of associated Ca2� and Mg2� cations thatin¯uence the micellation state [9]. The lipid A innercore structure was reported to be required for activity, butnot the O-antigen [9, 24 , 34]. In contrast, it was alsoreported that the immunologically dominant O-antigenchain acts as the inducer of resistance [17]. LPS fromXanthomonas campestris have been shown to exhibit activityas an inducer of defense-related gene expression inBrassica campestris [23], while LPS from certain non-plant pathogenic bacteria appear to be inactive. It ishowever, possible that di�erent plant cells recognizedi�erent structures within bacterial LPS to triggeralterations in plant responses to avirulent pathogens[24]. Puri®ed LPS have been reported to bind to themesophyll cell wall of tobacco cells and to induceultrastructural changes such as vesiculation, also inducedby heat-killed cells [9]. Evidence reported in this paperindicates that the interaction between tobacco cells andhighly puri®ed LPS from B. cepacia results in increasedmembrane permeability at low concentrations and loss ofcell viability at higher concentrations. Increased mem-brane permeability will result in the transmembrane ¯uxof Ca2� ions across the plasmalemma and the intracellu-lar triggering of defense associated cascades.

The results also raise the question of how the LPSmolecules are perceived by the plant. While it has beensuggested that some nonspeci®c elicitors may not requirea receptor based mechanism for their activity, it is alsopossible that recognition processes similar to that involvedin host resistance may be involved [9]. The activation ofthe plant's surveillance system is highly sensitive andspeci®c perception and signal transduction mechanismsexist through which plants can distinguish between ``self''and ``non-self'' to resist microbial pathogen attack. Theconversion of (extra)-cellular signals into activatedintracellular signal transduction cascades, leads tosophisticated and unique defense mechanisms such asthe hypersensitive response and induced/acquired resist-ance in the challenged host plant [26]. Acquired and/orinduced resistance contribute to the overall (nonhost)resistance displayed by plants in nature, where itcontributes to the anti-pathogenic potential or defensivecapacity of plants [33].

An important plant defense response is the productionof pathogenesis-related-proteins that have evolved in all

156 H. S. Coventry a

plant species. PR-proteins are de®ned as plant proteins

that are induced in response to pathogen stress anddi�erent biotic and abiotic elicitors [7 , 14]. Although PR-proteins are de®ned as induced or newly expressedproteins, low level amounts do occur in uninfected controltissues where they may have important developmentalfunctions beyond a role in adaptation to biotic stress [33].In tobacco, the major PR-proteins are acidic, locatedextracellularly, and coordinately expressed upon infec-tion. The basic, vacuolar isoforms are also present inhealthy plants and exhibit di�erent stress expressionpatterns [33]. The coordinate expression of a set of nineclasses of mRNAs encoding tobacco PR-proteins areassociated with the development of acquired resistance[14]. Where the induction of PR-proteins do occur, therelative amounts of individual PR-proteins may di�ergreatly from those seen during a hypersensitive response[33]. Di�erential induction of PR-genes may be the resultof di�erent concentrations of di�erent signal moleculesand the cross-talk between their signal transductioncascades [33].

The contribution of PR-proteins to disease resistancecan vary depending on the plant species and the speci®cbiotic or abiotic agent employed [14 , 39]. The accumula-tion of PR-proteins does not appear to be a prerequisitefor the induction of resistance, but contribute to theprotective state [33 , 39]. The individual PR-proteins mayhave intrinsically small defensive capacities, but thecoordinated induction of the di�erent families of PRsmay confer a level of resistance considerably broader thanany protein by itself [7]. The induced in vivo concentrationranges of PR-proteins are congruent with a protectiverole; induction of their genes in response to pathogens hasbeen well-documented and transgenic plants overexpres-sing these genes or combinations thereof, showedenhanced resistance to pathogens (reviewed in [7 , 14 ,33 , 36 , 38]). Recent results also indicate that increasedbasal levels of PR-gene expression may contribute tononspeci®c resistance in certain host-pathogen inter-actions [10 , 39].

With endophytic bacteria, LPS as components of thecellular surface, can be released in planta where they canact as elicitor molecules and interact with the cells fromthe host. Results obtained in this study indicate that theLPS fraction from the endophyte, B. cepacia (strain ASP B2D) is an e�ective local inducer of PR-proteins in N.tabacum at low concentrations. Results also support apossible role for LPS as an elicitor of a secondary systemicresponse, resulting in induced resistance. It is possiblethat exposure to bio-active LPS results in pre-condition-ing and sensitization of nonhost resistance, which in turnmay be associated with the low-level expression of PR-genes. Results obtained by Newman et al. [25] alsosupports the concept that pre-treatment with LPSconditions plants to the presence of phytopathogenic

d I. A. Dubery

organisms. The exploitation of nonhost resistance to

improve disease resistance within host species is currentlyreceiving much attention and sensitizing a plant torespond more rapidly to pathogen attack by priortreatment/inoculation with plant associated bacteria,endophytes and chemical agents, is considered to beone of the most encouraging options for e�ective

Lipopolysaccharide

management of plant disease [2].

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