host and non-host plant response to bacterial wilt in potato: role of the lipopolysaccharide...

Host and Non-Host Plant Response to Bacterial Wilt in Potato: Role of theLipopolysaccharide Isolated from Ralstonia solanacearum and Molecular

Analysis of Plant–Pathogen Interaction

by Nunzio Espositoa), Olga G. Ovchinnikovab)c), Amalia Baronea), Astolfo Zoinad), Otto Holstb), andAntonio Evidente*a)

a) Dipartimento di Scienze del Suolo, della Pianta, dell�Ambiente e delle Produzioni Animali, Universitadi Napoli Federico II, I-80055 Portici (NA) (phone: þ390812539178; fax: þ39 0812539186;

e-mail: [email protected])b) Division of Structural Biochemistry, Research Center Borstel, Leibniz-Center for Medicine and

Biosciences, D-23845 Borstelc) N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow,

Russian Federationd) Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Universita di Napoli Federico II,

I-80055 Portici (NA)

Ralstonia solanacearum is one of the most devastating phytopathogenic bacteria, in particular itsrace 3. This microorganism is the causal agent of destructive diseases of different crops including tomatoand potato. An important aspect of the interaction between this pathogen, and the host and non-hostplants was its biochemical and molecular basis. Thus, the lipopolysaccharides (LPS) were extracted fromthe R. solanacearum cell wall, purified, and the O-specific polysaccharide (OPS) was isolated andchemically characterized by compositional analyses and NMR spectroscopy. The OPS was constituted oftwo linear polymers of an approximate ratio of 3 : 1, both of which were built up from three rhamnose andone N-acetylglucosamine residues and differed only in the substitution of one rhamnose residue. The LPSinhibited the hypersensitivity reaction (HR) in non-host tobacco plants and induced localized resistancein host potato plants, both of which were pre-treated with the LPS before being inoculated with thepathogen. A cDNA-AFLP approach was used to study transcriptome variation during the resistant andsusceptible interactions. This revealed the presence of metabolites specifically expressed in the S.commersonii-resistant genotypes, which could be involved in the plant–pathogen incompatible reaction.Furthermore, a specific EST collection of the Ralstonia–potato interaction has been built up.

Introduction. – Ralstonia (formerly Burkholderia or Pseudomonas) solanacearum(Smith) causes one of the most devastating bacterial diseases in more than 50 botanicalfamilies, some of which are economically important such as banana, potato, andtomato. The bacterium is found worldwide, mainly in tropical and subtropical areas, butalso in warm-temperate countries and even in some cool-temperate regions [1]. Sinceagrochemicals are costly and poorly effective, and sanitary cropping systems difficult toapply, control strategies of the disease should be based on the production of resistantplant varieties. As for potato (Solanum tuberosum L.), there is a large number of tuber-bearing Solanum species representing a source of noteworthy traits, among theseresistance to pathogens. Several species have already been used to transfer desirablegenes to S. tuberosum, e.g., S. phureja, S. berthaultii, S. tarijense, S. bukasovii [2]. Amongdiploid Solanum species, S. commersonii possesses several desirable traits, including

CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)2662

� 2008 Verlag Helvetica Chimica Acta AG, Z�rich

resistance to environmental stresses. Particularly attractive is its resistance to bacterialwilt caused by R. solanacearum.

To better understand the biochemical and molecular basis of this very complexplant – pathogen interactions concerning resistant/susceptible responses, a multidisci-plinary approach has been undertaken, including chemical, pathogenicity, and tran-scriptomic investigations. Analyses were initiated on the isolation, and structural andbiological characterization of the lipopolysaccharides (LPS) of R. solanacearum.

The LPS protect the bacterial cell from hostile environments, and, in pathogenicbacteria, they represent important virulence factors through their direct interactionwith eukaryotic host cells. Mutants of phytopathogenic bacterial strains, with defects intheir LPS biosynthesis, frequently show reduced virulence, and the number of viablebacteria in plant tissues declines rapidly. Furthermore, such mutants are more sensitiveto agents like particular antibiotics, detergents, and antimicrobial peptides, probablybecause the defective LPS can no longer protect the cell against these agents. It ispossible that killing of mutant bacteria in plant tissues reflects their inability to survivetoxic plant compounds like hydroxycinnamoyl-tyramine [3]. In addition to itsprotective function for the bacteria, the LPS may interact directly with hosts. Thismay, in principle, be mediated by the direct contact of bacteria with host-cell surfaces oras a consequence of a release of LPS from the bacteria. It is not known whether a directcontact of bacterial and host cells is required for LPS-mediated effects, but it has beenshown that a network of functions required for virulence of R. solanacearum isactivated by such a contact [4].

Presumably, interactions leading to modulation of plant gene expression wouldrequire the recognition of LPS by components within or attached to the plantcytoplasmatic membrane. The plant cell wall prevents from the direct contact of themembrane with the bacterial LPS. Moreover, the outermost portion of the LPSmolecule, the O-antigen chain (O-specific polysaccharide, OPS), is not the activemoiety in triggering plant responses [3]. An alternative possibility to LPS perceptionby direct bacteria – cell contact is that a detachment of LPS from the bacterial surface isnecessary. A significant fraction of LPS may be released from bacteria as micelles orblebs during growth [5], and this might be the form in which the LPS interacts witheukaryotic cells. Blebs may also sequester periplasmatic contents and deliver thematerial to other cells by fusion with membranes. Indeed, it has been suggested that thisprocess may represent a widespread alternative route for periplasmatic proteinsecretion [5].

Since, during infection, the recognition of bacterial components by plant plays afundamental role in the outcome of the interaction, it is crucial to understand whichcompounds are differentially involved in plant recognition of bacterial LPS in theresistant and susceptible potato genotypes. Therefore, a transcriptome analysis duringthe microbial infection on the resistant S. commersonii and the susceptible S. tuberosumgenotypes has been undertaken. For this purpose, a differential expression approachhas been chosen based on the cDNA AFLP-TP technique [6] that allows a single cDNArestriction fragment to be amplified for each mRNA expressed. Indeed, beside gene-expression analysis performed through the microarray approach [7], the cDNA-AFLPtechnique has been successfully used for gene identification of many plant – pathogenmolecular interactions [8– 10].

CHEMISTRY & BIODIVERSITY – Vol. 5 (2008) 2663

Here, we report the extraction of the LPS from R. solanacearum and the structuraldetermination of the LPS OPS. The results of inoculation studies on host and non-hostplants pre-treated with LPS are also discussed, as well as those concerning withtranscript differentially expressed in the molecular interaction between R. solanacea-rum, and susceptible or resistant potato species.

Results and Discussion. – Isolation and Chemical Characterization of LPS and OPS.The LPS was isolated from bacterial cell mass (11.2 g wet weight) by hot phenol/H2Oextraction [11], followed by dialysis of the aqueous phase and freeze-drying. The yieldof LPS was 210 mg (1.9% of bacterial wet weight). The Bradford protein determination[12] showed that the LPS preparation contained less than 3% protein, thus, it was usedfor hydrolysis without further purification.

A portion of the LPS (60.0 mg) was hydrolyzed with 2% aqueous AcOH, and thelipid A was precipitated by centrifugation (4.9 mg, 8% of the LPS). The carbohydrate-containing supernatant was fractionated by gel-permeation chromatography (GPC) ona Sephadex G-50 column to give a low-molecular-mass OPS (17.0 mg, 28% of the LPS).

Sugar analysis of the OPS identified 6-deoxymannose (rhamnose, Rha) and xylose(Xyl) in a molar ratio of ca. 1 :0.1, and 2-amino-2-deoxyglucose (GlcN). In linkage(methylation) analysis, 2-substituted 6-deoxyhexose (6d-Hex), 3,6-disubstituted 6d-Hex, 3-substituted HexN, and a small amount of terminal 6d-Hex were identified.Identification of the terminal 6d-Hex could be accounted for by the presence of shortchains in the OPS preparation.

The 13C-NMR spectrum (Fig. 1 and Table 1) of the OPS contained two series ofsignals in a ratio of intensities of ca. 3 : 1. Interpretation of the major series led to theidentification of structure 1, whereas the minor one corresponded to a polysaccharidewith the structure 2, which differed only in the type of substitution of rhamnose A.Also, a trace amount of branched repeating units arising from non-stoichiometricxylosylation of a linear structure was present; however, the structure of this compoundcould not be determined.

! 2)-a-l-Rhap-(1!2)-a-l-Rhap-(1!3)-a-l-Rhap-(1!3)-a-d-GlcpNAc-(1 ! 1

A B C D

! 3)-a-l-Rhap-(1!2)-a-l-Rhap-(1!3)-a-l-Rhap-(1!3)-a-d-GlcpNAc-(1 ! 2

A’ B’ C’ D’

The structure 1 had earlier been identified in the LPS of P. solanacearum M4S [13]and in LPS of a number of different B. solanacearum strains [14 – 16]. Remarkably,structure 1 has also been reported for the OPS of human pathogen Serratia marcescensO22 [17] and differs from the OPS of Shigella flexneri serotype Y only by the anomericconfiguration of the GlcNAc residue [18]. Structure 2 has been identified in LPS ofstrains B. solanacearum ICMP 767, 7864, 7942, and 7955 [15] [16] [19].

The structures 1 and 2 showed a rhamnan trisaccharide identical to that synthesizedand oligomerized to obtain hexa- and nonasaccharide by Bedini et al. [20] which wereeffective in suppressing the hypersensitivity response (HR) in Arabisopsis thaliana.



Fig. 1. 13C-NMR Spectrum of the OPS of Ralstonia solanacearum (the region of C¼O resonances is notshown). Arabic numerals refer to C-atoms in the sugar residues denoted by letters as given in Table 1.

Table 1. 13C-NMR Data of the OPS of Ralstonia solanacearum (d in ppm). The published data [15] aregiven in parentheses. Additional chemical shifts for the N-Ac groups were d(C) 22.4, 22.6 (both Me), and

174.4 (both C¼O).

Sugar residue C(1) C(2) C(3) C(4) C(5) C(6)

OPS 1 (major)! 2)-a-l-Rhap-(1 ! A 100.1

(100.7)76.7

(77.6)70.4

(70.9)72.6

(73.7)69.9b)

(70.7)17.1

(18.0)! 2)-a-l-Rhap-(1 ! B 101.2

(102.0)78.8

(79.6)70.8

(71.4)72.6

(73.7)69.6b)

(70.4)17.1

(18.0)! 3)-a-l-Rhap-(1 ! C 101.2a)

(102.2)71.0

(71.9)77.8

(79.0)72.1

(72.9)69.4b)

(70.4)16.9

(17.8)! 3)-a-d-GlcpNAc-(1 ! D 96.9

(97.5)53.6

(54.4)79.6

(80.6)68.6

(69.8)72.8

(73.7)60.8

(62.0)OPS 2 (minor)! 3)-a-l-Rhap-(1 ! A’ 102.6

(103.3)67.0

(68.0)75.5

(76.7)71.0

(71.9)69.4b)

(70.4)16.9

(17.8)! 2)-a-l-Rhap-(1 ! B’ 101.5a)

(102.0)78.8

(79.6)70.4

(71.4)72.8

(73.6)69.4b)

(70.4)17.1

(18.0)! 3)-a-l-Rhap-(1 ! C’ 101.7

(102.4)71.0

(71.8)77.7

(78.8)72.4

(73.0)69.4b)

(70.4)17.1

(18.0)! 3)-a-d-GlcpNAc-(1 ! D’ 94.7

(95.6)53.6

(54.4)79.9

(80.9)68.6

(69.8)72.6

(73.5)60.8

(61.9)Xyloseb-Xylp-(1 ! X 104.1

(105.3)73.7

(75.0)76.3

(77.3)69.4b)

(70.5)65.3

(66.5)

a)b) Assignments may be interchanged.

LPS Prevention of the HR and Defense-Related Induction in a Non-Host and HostPlants. The purified LPS of R. solanacearum were tested on host (potato) and non-host(tobacco) plants. Tobacco leaves pre-treated with a LPS solution and successivelyinoculated with a suspension of viable bacteria gave no HR, when compared to theleaves of the same plant treated only with the bacteria (Fig. 2).

In independent experiments, potato leaves pretreated with the same LPS and theninoculated with the pathogen suspension did not show the typical wilting symptoms ofthe disease which were observed on untreated leaves of the same plant (Fig. 3).

The prevention of the HR induction of R. solanacearum LPS observed in tobacconon-host plant was in agreement with published data [3]. Early observations showedthat heat-killed cells of R. solanacearum infiltrated into tobacco leaves influenced theresponse to subsequent inoculation with living bacteria. Such pretreatment couldprevent or delay the HR induced by avirulent bacteria, or disease symptoms induced byvirulent pathogens. Fractionation of bacteria showed that LPS was responsible for thesuppression (LPS is not affected by a heat-killing process). The source of LPS used wasnot critical: preparations from a number of bacteria, including non-pathogens, wereeffective [21 – 24]. This phenomenon can be observed with many challenging bacteriaand in a number of plant species, although solanaceous plants have been mostcommonly used. The response is also observed in Arabidopsis thaliana (Brassicaceae)in which recently a mechanistic connection between microbe-associated molecularpattern (MAMP)-induced basal resistance and HR triggered by type III-secretedeffectors has been made [25].

The prevention of HR by LPS was strictly localized to the area of the leaf into whichthe LPS was infiltrated; adjacent areas of the leaf behave normally when subsequentlychallenged. The effects were independent of light. LPS Treatment alone did not cause

Fig. 2. Localized induced response effect induced on tobacco (Nicotiana tabacum) leaves by Ralstoniasolanacearum LPS. a) Control; tobacco leaves not pretreated with LPS, but inoculated with bacteriumcell suspension. b) Tobacco leaves pretreated with LPS and inoculated with R. solanacearum cell

suspension after 20 h.


necrosis or other visible symptoms. The prevention of HR required several hours tobecome established and was temporary. For example, in pepper the interval betweenLPS administration and challenge inoculation needed to be between 10 and 30 h todemonstrate HR suppression. The concentration of LPS required for a localizedsuppression of HR induction was around 50 mg/ml. However, it was difficult to interpretthis value because of uncertainties about the physical state of LPS preparation, which,in aqueous preparations, was likely to be micellar. However, it cannot be ruled out thatsuch a LPS preparation contained small amounts of a more readily accessible andactive form [3].

The phenomenon described above has been termed �localized induced resistance�(LIR) [26] which is believed to operate by creating an antimicrobial environmentwithin the plant [27], although the induction of increased tolerance of plant tissue tobacteria has also been suggested [23]. The structural requirements of LPS for theprevention of HR have been studied by two methods: chemical degradation and the useof truncated LPS forms produced by mutant bacteria. These studies carried out ondifferent plants demonstrated that the lipid A-inner core region of LPS represented theactive moiety to induce LIR [21] [28]. The OPS and the outer-core regions were notnecessary for activity as was the lipid A alone due to its insolubility in H2O [3].

Regarding the induction of defense-related response observed in potato host plant,there are numerous studies of plant responses to pathogens in terms of gene expression,and synthesis of macromolecules and substances such as phytoalexins, although the roleof such accumulated molecules remains unclear [3]. Several reports have describeddefense-related plant response by LPS treatment, but no literature was found on anydefense induced by R. solanacearum LPS in potato plants. In fact, it was reported thatR. solanacearum LPS induced in tobacco a polypeptide of unknown function, togetherwith soluble pereoxidase [29] [30]. LPS from Xanthomonas campestris induced b-1,3-


Fig. 3. Defense-response induced on potato (Solanum tuberosum) by Ralstonia solanacearum LPS. a)Control; potato leaves not pretreated with LPS, but inoculated with bacterium cell suspension. b) Potato

leaves pretreated with LPS and inoculated with R. solanacearum cell suspension after 20 h.

glucanase in Brassica [31], but LPS from Escherichia coli did not, even atconcentrations 100-times higher than that required for X. campestris LPS. Otherchanges induced by LPS included the induction of antimicrobial activity [27], inductionof glyceollin synthesis in soybean [32], and changes in the plant cell wall ultrastructure[21].

A recent review extensively described the induction and modulation of plantdefense response by bacterial lipopolysaccharides [33].

Molecular Interaction between R. solanacearum, and Susceptible and ResistantPotato Species. The pathogenic test performed on susceptible and resistant genotypesevidenced that, 24– 48 h after the infection, symptoms appeared on the inoculatedcultivar Blondy but not on the inoculated S. commersonii. This occurred when thebacteria were inoculated directly in the vascular system of the plant. When theinoculum was directly in the soil, symptoms appeared later, but 10 days after theinfection all the cv. Blondy plants showed severe symptoms or were dead, whereas S.commersonii did not show symptoms (Fig. 4). These results were confirmed by threedifferent repetitions of the pathogenic test.

This material was then analyzed by the cDNA-AFLP technique, for which fourdifferent samples were considered: S. commersonii infected and not infected, and S.tuberosum cv. Blondy infected and not infected. For each sample, plant material wascollected at different times after inoculation and processed for the differential analysis.When the acrylamide gels were analyzed, it was evident that no clear difference amongthe four samples could be observed until 48 h after the infection on the roots (Fig. 5).By contrast, after 72 h after the infection, many polymorphic fragments could beevidenced; therefore, molecular analyses were continued on plant material collected 72and 96 h after the infection. As a total, out of 32 different primer combinations tested,

Fig. 4. Symptoms revealed on potato 96 h after Ralstonia solanacearum inoculation. a) Control, S.tuberosum cv. Blondy not inoculated plant. b) S. tuberosum cv. Blondy inoculated plant. c) S.

commersonii inoculated plant.


2558 fragments ranging from 50 to 500 bp were analyzed, with an average of 80fragments for each combination. Among the differentially expressed fragments, sixdifferent classes (Fig. 6) were identified, for a total of 120 TDFs (Transcript DerivedFragments) (Table 2), which correspond to 4.7% out of the total fragments analyzed.Most of these TDFs (classes A and E; 72.5%) were only present or highly expressed inthe resistant S. commersonii, whereas 17.5% (classes B and F) were only present orhighly expressed in the susceptible cv. Blondy of S. tuberosum. These TDFs couldrepresent species-specific genes expressed in the resistant and susceptible genotypes, or


Fig. 5. cDNA-AFLP Patterns obtained on S. tuberosum and S. commersonii inoculated and notinoculated plants at different times after inoculation using different primer combinations. Lane 1: S.tuberosum cv. Blondy not inoculated, Lane 2: S. tuberosum cv. Blondy inoculated, Lane 3: S.

commersonii not inoculated, Lane 4: S. commersonii inoculated.

constitutively expressed in both genotypes but whose expression increases during theresistant or susceptible reaction. The TDFs belonging to class C (5.8%) could representS. commersonii specific-genes that are repressed during the infection. Finally, TDFsbelonging to class D (4.2%) could be genes activated by the pathogen attack to roots.

The selected TDFs were cloned, and some of them (34 out of 120, ca. 30%) werethen sequenced. Bioinformatic analyses performed through various software programsallowed the TDFs to be classified in three groups: a) those with a functional annotation,b) those similar to sequences with unknown function, c) those with no similarity found.In the first class, four TDFs were included, for which a high similarity index (e-valueequal or lower to 10�7) was detected with putative genes or proteins with knownfunction (Table 3). These were all sequences differentially expressed only in theresistant genotype S. commersonii 72 h after the infection, and three of them (TDFs 12,45, and 116) are involved in widely-spread biochemical pathways, such as acetylation,photosynthesis, and ribosomal protein synthesis. The fourth TDF (No. 113) isparticularly interesting, since it seems part of one gene that controls the synthesis of

Table 2. Number of Gene Fragments Present in the Resistant (S. commersonii) and Susceptible (S.tuberosum) Genotypes Inoculated and Non-Inoculated with Ralstonia solanacearum

TDFa) Class Class description TDFa)[No.]

TDFa)[%]

A Present in inoculated cmmb) 55 2.15B Present in inoculated tbrc) 9 0.35C Present in not inoculated cmm 7 0.27D Present in all inoculated plants 5 0.19E Present in all plants, but highly expressed in inoculated cmm 32 1.25F Present in all plants, but highly expressed in inoculated tbr 12 0.47Total 120 4.69

a) TDF¼Transcript Derived Fragment. b) cmm¼S. commersonii. c) tbr¼S. tuberosum.


Fig. 6. Examples of the six classes of TDFs (Transcript Derived Fragments) identified through the cDNA-AFLP analysis performed on S. tuberosum and S. commersonii plants. a) Class A-TDF, b) class B-TDF,c) class C-TDF, d) class D-TDF, e) class E-TDF, f) class F-TDF. Lane 1: S. tuberosum cv. Blondy notinoculated, Lane 2: S. tuberosum cv. Blondy inoculated, Lane 3: S. commersonii not inoculated, Lane 4:

S. commersonii inoculated.

an ABC transporter protein. In some cases, this type of protein is generally involved intransporting endogen and exogen compounds through the cellular membrane, such assmall peptides, steroid antibiotics, fungicides [34] [35], organic acids, heavy metals, andsteroid hormones [36]. The ABC transporters can also recognize and remove toxiccompounds from the cell, thus reducing the risk of cell death. In plants, their function isnot clear, even though their role in defense factors release and in protection againstpathogen attack has been hypothesized [37]. In particular, in potato four genes codingfor ABC transporters have been reported, and one of these (StABC2) seems to beinvolved in plant defense mechanisms against biotic and abiotic stresses [38].

As for TDFs belonging to the second class, five of them were included in this class offragments similar to sequences with unknown function. In three cases, the similaritywas with EST (Expressed Sequence Tag) collections that are derived from differentorganisms, such as the plant Panicum virgatum, man, and one fungus. In other twocases, the identified TDF was similar to cDNAs obtained from interactions betweenplants (soybean and potato, resp.) and pathogens (Phytophthora spp.). Therefore,these two TDFs will be further investigated in order to obtain their full-lengthsequences and to follow their expression by RT-PCR experiments. This will allow theirrole in our Ralstonia –potato resistant or susceptible interactions to be verified.

Finally, most of the sequenced TDFs were included in the third class, to whichfragments showing no similarity with other sequences available in the different geneand EST-queried banks were ascribed. These TDFs constitute an EST collectionspecific of the Ralstonia – potato interaction, which could be particularly interesting toinvestigate, since it could include genes specific of this resistance response. These ESTswill be submitted to GenBank, making them available to the whole scientificcommunity, and each will be studied according the same strategy adopted for thetwo TDFs belonging to the second class. Furthermore, since a collection of more than700 ESTs derived from Ralstonia solanacearum – tomato interaction are available(GenBank accession Nos. DV104321– DV105076), our potato ESTs will also becompared with those from tomato, and a custom chip will be designed and used for bothSolanaceae species. This will allow information on the key-genes involved in theresistance to Ralstonia solanacearum to be enhanced for both species.

Conclusions. – The results accumulated in the present study expand our knowledgeon the chemical structure of LPS from phytopatogenic bacteria and, in particular, frompathogenic Ralstonia species. Among R. solanacearum OPS, there is a group of

Table 3. Functional Annotation of 4 TDFs through KAAS (KEGG Automatic Annotation Server). Foreach fragment, the size in bp, the score, and the similarity level are reported.

TDF Code Fragment size[bp]

Functional annotation Score Similarity level(e-value)

KEGG referencecode

12 176 Ribosomal protein S7 385 7.5 e�13 K0299345 377 N-acetyltransferase 280 7 e�20 K03823

113 233 ABC transporter 279 7 e�24 K02010116 109 Chlorophyl a Apoprotein A1 246 2.3 e�7 K02689


polysaccharides with similar structures, including 1 and 2. Their main chain is built up oflinear tetrasaccharide repeating units, constituted by three l-rhamnose and one N-acetyl-d-glucosamine residues, and differing in configuration of the glycosidic linkageof GlcNAc and the site of its attachment (position 2 or 3) to the neighboring Rha. Insome strains, the OPS main chain carries a side chain of one l-Rha or l-xylose residue.Other strains possess several OPS with different structures of the main chain, whichcould originate from different polymerization of the same biological repeating unit withGlcNAc at the reducing end.

Furthermore, the results obtained showed the role of LPS in the host and non-hostplant interaction. In particular, their action in the suppression of HR response in non-host plant as well as the prevention of the development of typical disease symptomsinduced by the bacterium on the host plant was evidenced. The prevention of LIRinduced by R. solanacearum LPS in non-host tobacco plant is already well-known,while the induction of defense-related response in the host potato plant prompts tofurther investigation on the metabolites involved in this mechanism with the aim offinding natural compounds useful to prevent the serious disease induced by thispathogen.

Finally, the results of molecular investigation allowed the identification of putativegenes involved in the resistant response to Ralstonia solanacearum in potato species. Inparticular, one S. commersonii-specific TDF, which is part of one gene controlling thesynthesis of an ABC transporter, was identified. This type of protein is generallyinvolved in transporting endogenous and exogenous compounds through the cellularmembrane, thus probably being involved in cellular detoxification processes. Furtherinvestigation is required to better determine the role of these molecules identified andto underline their strict interaction in the defense gene activation mechanism.

Experimental Part

Bacterial Strain, Isolation, and Degradation of the LPS. Strain SA 93 of R. solanacearum R3 biovar 2,a voucher of which was deposited with the Collection of Dipartimento di Arboricoltura, Botanica ePatologia Vegetale dell�Universita di Napoli Fedrico II, was grown at 288 under shaking (150 rpm) in 500-ml Erlenmeyer flasks filled with 200 ml of liquid medium (1 g of NH4H2PO4, 0.2 g of KCl, 0.2 g of MgSO4 ·7 H2O, 1 g of peptone, 10 g of glucose, in 1 l of dist. H2O) inoculated with 500 ml of a bacterial suspensioncontaining 108 cfu/ml. After 48 h incubation, cultures were centrifuged (8,000 g at 48 for 30 min). The wetcells (11.2 g) were suspended in ultrapure H2O (130 ml), extracted with hot phenol/H2O [11], and theH2O phase of which was dialyzed (molecular cut-off 3500 Da) and lyophilized (210 mg). Proteins werequantified according to Bradford [12], and measurements were performed on a Helios Beta UV/VISspectrophotometer at 190–380 nm on sample solns. of 0.10, 0.20, and 0.30 mg/ml. A LPS sample (60 mg)was hydrolyzed with aq. 2% AcOH at 1008 for 10 h, and the lipid precipitate (lipid A) was removed bycentrifugation (13,000 g, 1 h), washed with H2O, and lyophilized (4.88 mg, 8% of the LPS). Thesupernatant was concentrated and fractionated by GPC on a column of Sephadex G-50 (80 cm�2.5 cm)in 0.05m pyridinium acetate buffer, pH 4.5, with monitoring by a Knauer differential refractometer. Theyield of the OPS was 16.98 mg (28% of the LPS).

Chemical Characterization of the OPS. For sugar analysis, the OPS was hydrolyzed with 2mCF3CO2H (1208, 2 h), and the resulting monosaccharides were analyzed by GLC as alditol acetateswith a Hewlett-Packard 5880 instrument, equipped with a cap. column (30 m�0.25 mm, 0.25-mm filmthickness) of HP-5MS and applying a temp. gradient of 1508 (3 min) to 3208 at 38/min. Methylation of theOPS was carried out according to Ciucanu and Kerek [28]. The methylated sample was extracted from


DMSO with CHCl3, then hydrolyzed (2m CF3CO2H, 1208, 2 h), reduced with NaBH4, and acetylated,and products were analyzed by GLC/MS using a Hewlett-Packard 5989A instrument equipped with a HP-5MS cap. column (30 m�0.25 mm, 0.25-mm film thickness) and applying a temp. gradient of 1508 (3 min)to 3208 at 58/min. NMR Spectra were recorded on solns. in D2O with a Bruker DRX-600 AVANCEspectrometer (operating frequency 600 MHz for 1H-NMR) and Bruker DPX-360 spectrometer(operating frequency 90.6 MHz for 13C-NMR) at 278. Chemical shifts were reported relative to internalacetone (d(H) 2.225; d(C) 31.45).

Bioassay of LPS on Potato and Tobacco Plants. The plants used in these assays were the cultivatedpotato species Solanum tuberosum cv. Blondy and tobacco (Nicotiana tabacum cv. White Burley), andcorresponding vouchers were deposited with the collection of Dipartimento di Scienze del Suolo, dellaPianta, dell�Ambiente e delle Produzioni Animali), Universita di Napoli Federico II, Napoli, Italy. Thepotato plants were obtained from not fractionated tubers, while those of tobacco were from seeds. Theywere grown in a greenhouse under controlled conditions (light/dark: 12 h/12 h; temp.: 218 in the dark and27/288 in the light; humidity 85%). The bioassay of LPS was carried out under the conditions describedbelow. The LPS, purified as reported above, were dissolved in H2O up to a final concentration of 50 mg/mlaccording to a conventional procedure [39]. Solns. (ca. 1 ml) were injected into the vessels of potato andthe mesophyll of tobacco leaves. In the experiments, three plants of each species were used, and threedifferent leaves of the plants were challenged with the test soln. Control experiments were conductedutilizing the same number of the two species identically treated but with dist. H2O. After 20 h, a R.solanacearum suspension (ca. 1 ml) was inoculated into the same points of the test plants at aconcentration of 2.5�107 cfu, according to the same method.

In vitro and in vivo Propagation of Plant Material. One genotype of S. commersonii and one of thecultivar Blondy of S. tuberosum, available at the germplasm collection of the Dipartimento di Scienze delSuolo, della Pianta, dell�Ambiente e delle Produzioni Animali, Universita di Napoli Federico II, were invitro propagated on the sterilized Murashige and Skoog medium [40] added with vitamins. In vitro plantswere grown in growth chamber at constant temp. (248) 18 h (light) and 6 h (dark). Several copies of eachgenotype were obtained for both pathogenic tests and molecular analyses. When plantlets reached 5 cmof height, they were transplanted in 6-cm pots with sterilized soil, and after an acclimation period atrelative humidity around 100%, they were grown in the greenhouse.

In vivo Pathogenicity Tests. When plantlets reached 10-cm height, they were inoculated with 2 ml of abacterium suspension at 2.5�107 cfu concentration, and roots were cut in order to favor the bacteriapenetration. This pathogenicity test was performed in a conditioned cell with standardized light (12 hlight and 12 h dark), temp. (218 during night and 278 during day), and humidity (85%). Followingbacteria inoculation, symptoms were revealed after 12, 24, 48, 72, and 96 h. Contemporarily, plantmaterial (leaves and collar) was also collected, freezed with liquid N2, and stored at �808. Symptoms oninfected plants were monitored 6, 9, and 12 days after inoculation.

Molecular and Bioinformatics Analyses. mRNA was extracted using the Dynabeads RNADIRECTTM kit (Invitrogen) from 100 mg of infected and non-infected S. commersonii and S. tuberosumcv. Blondy, according to the procedure described by the manufacturer. After spectrophotometricquantification of mRNA and its quality control on agarose gel, mRNA was retro-transcribed andprocessed for the cDNA-AFLP-TP, according to the protocols described by Breyne et al. [6], details ofwhich are also reported on-line (www.psb.rug.ac.be/papers/pebre/pnas.htm). The restriction enzymesused were BstYI and MseI, and, for the pre-amplification reaction, a MseI primer without selectivenucleotide was combined with a BstYI primer containing either a T or a C at the 3’-extremity. Forselective amplification, one selective nucleotide was added at the 3’-extremity of both MseI and BstYIprimers, the latter in each pair being labelled with FAM fluorochrome modification at 5’. As a total, 32primer combinations were used for transcript profiling. AFLP Fragments were separated on 6%acrylamide gel run at 1600 constant V, and, after running, the gel was scanned by Typhoon scanner(Amersham), then the image analysis was carried out with the ImageQuant software, and the TDFs wereidentified. The selected TDFs were cut from the gel, eluted DNA was re-amplified using the sameconditions of selective amplification, and the purified fragment was cloned using the Qiagen PCR cloningkit. Plasmid DNA was extracted by the NucleoSpin Plasmid Mini Kit (Macherey-Nagel). Afterquantification of DNA extracted on 1% agarose gel, the cloned TDFs were sequenced using the BigDye


Terminator Cycle Sequencing Kit and the ABI PRISM 3100 Genetic Analyzer. The sequences obtainedwere compared by BLAST alignment to nucleotide and protein databases publicly available. To find afunctional annotation, the software KAAS (KEGG Automatic Annotation Server: http://www.geno-me.jp/kegg/kaas/) was used. When no function was found by KEGG, sequences were analyzed byquerying four databases: CAB UNINA (http://biosrv.unina.it), NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), TIGR (http://www.tigr.org/tdb/potato/disease.db/disease_blast.shtml), and SGN (http://www.sgn.cornell.edu). TDFs showing no similarity at BLAST analysis with sequences with unknownfunction (e-value>10�7) were grouped to constitute an EST collection.

The authors thank Dr. A. Cimmino (Universita di Napoli Federico II) for helping with LPSextraction, H. Moll and R. Engel (Research Center Borstel) for help with GC/MS, H. Kaessner(Research Center Borstel) for recording the NMR spectra, and Raffaele Garramone for helping withpathogenic tests. This research was supported by the project AGRONANOTECH funded by MiPAF.Contribution DISSPAPA N: 173.

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Received May 28, 2008


host and non-host plant response to bacterial wilt in potato: role of the lipopolysaccharide...

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