Involvement of bacterial TonB-dependent signaling in the generation of an oligogalacturonide damage-associated molecular pattern from plant cell walls exposed to Xanthomonas campestris pv. campestris pectate lyases
© Vorhölter et al.; licensee BioMed Central Ltd. 2012
Received: 10 April 2012
Accepted: 25 September 2012
Published: 19 October 2012
Efficient perception of attacking pathogens is essential for plants. Plant defense is evoked by molecules termed elicitors. Endogenous elicitors or damage-associated molecular patterns (DAMPs) originate from plant materials upon injury or pathogen activity. While there are comparably well-characterized examples for DAMPs, often oligogalacturonides (OGAs), generated by the activity of fungal pathogens, endogenous elicitors evoked by bacterial pathogens have been rarely described. In particular, the signal perception and transduction processes involved in DAMP generation are poorly characterized.
A mutant strain of the phytopathogenic bacterium Xanthomonas campestris pv. campestris deficient in exbD2, which encodes a component of its unusual elaborate TonB system, had impaired pectate lyase activity and caused no visible symptoms for defense on the non-host plant pepper (Capsicum annuum). A co-incubation of X. campestris pv. campestris with isolated cell wall material from C. annuum led to the release of compounds which induced an oxidative burst in cell suspension cultures of the non-host plant. Lipopolysaccharides and proteins were ruled out as elicitors by polymyxin B and heat treatment, respectively. After hydrolysis with trifluoroacetic acid and subsequent HPAE chromatography, the elicitor preparation contained galacturonic acid, the monosaccharide constituent of pectate. OGAs were isolated from this crude elicitor preparation by HPAEC and tested for their biological activity. While small OGAs were unable to induce an oxidative burst, the elicitor activity in cell suspension cultures of the non-host plants tobacco and pepper increased with the degree of polymerization (DP). Maximal elicitor activity was observed for DPs exceeding 8. In contrast to the X. campestris pv. campestris wild type B100, the exbD2 mutant was unable to generate elicitor activity from plant cell wall material or from pectin.
To our knowledge, this is the second report on a DAMP generated by bacterial features. The generation of the OGA elicitor is embedded in a complex exchange of signals within the framework of the plant-microbe interaction of C. annuum and X. campestris pv. campestris. The bacterial TonB-system is essential for the substrate-induced generation of extracellular pectate lyase activity. This is the first demonstration that a TonB-system is involved in bacterial trans-envelope signaling in the context of a pathogenic interaction with a plant.
KeywordsTonB system Damage-associate molecular pattern DAMP Oligogalacturonide Trans-envelope signaling Molecular plant-microbe interaction Pathogen Xanthomonas campestris
Plant cells are permanently monitoring their immediate environment to identify attacking pathogens and subsequently initiate defense. Highly conserved molecular structures termed microbe-associated molecular patterns (MAMPs) or pathogen-associated molecular patterns (PAMPs) are obvious targets for this recognition process. The term PAMP-triggered immunity (PTI) is increasingly used for this innate immunity . Recognition by the plant employs transmembrane pattern recognition receptors (PRRs). Unfortunately, so far there are only a few detailed model systems that describe MAMP, PRR, and perception-induced signaling . An example for such a well-characterized PTI is the recognition of bacterial flagellin in Arabidopsis thaliana. In older literature, molecules which evoke defense-related plant reactions and which hence are assumed to be involved in the recognition process of non-host plants were termed elicitors . Plant defense upon pathogen recognition typically includes the induction of a so-called hypersensitive response (HR), which leads to the resistance of the non-host plants and which includes a rapid local generation of superoxide, the so-called oxidative burst, and a programmed cell death . Examples for MAMPs are the harpin proteins from Erwinia[5, 6], Xanthomonas[7, 8], or Pseudomonas, syringolides from Pseudomonas syringae or lipopolysaccharides (LPSs), characteristic glycoconjugate cell envelope constituents of Gram-negative bacteria .
In addition to monitoring for pathogen-derived MAMPs, plants recognize endogenous molecules that are released upon injury or infection as alarm signals. Such molecules are termed damage-associated molecular patterns (DAMPs) . Often DAMPs are generated by lytic enzymes of attacking pathogens when they breach structural barriers of plant tissues, in particular plant cell walls. DAMPs include oligosaccharide fragments, peptides resulting from protein degradation , and reactive oxygen species (ROS) . Plants can amplify the response to DAMPs by inducing specific enzymes that generate additional similar DAMP molecules . Examples for DAMPs known for a long time are oligogalacturonides (OGAs) that are released by fungal pectate lyases [16–18] from plant cell walls. Among the plant pathogenic bacteria, so far only Erwinia carotovora has been reported to induce the generation of a DAMP , which also turned out to be an OGA . Upon the discovery of the egg box conformation of OGA dimers , the A. thaliana wall-associated kinase 1 (WAK1) was identified as a candidate for a PRR that specifically recognizes OGAs. While the receptor-like kinase WAK2 was shown to be involved in pectin-dependent signaling , a recent domain-swap experiment confirmed the identification of WAK1 as OGA receptor , thereby turning the plant side of OGA perception into a comparably complete model of DAMP recognition.
Xanthomonas species are members of the γ subdivision of the Gram-negative Proteobacteria, which have adopted a plant-associated and usually plant pathogenic lifestyle [24, 25]. Xanthomonas campestris pv. campestris is a pathogen of Brassicacea including A. thaliana. Upon infection of cabbage plants it causes the black rot disease. In non-host plants like pepper (Capsicum annuum) and tobacco (Nicotiana tabacum), however, it induces an HR. For X. campestris pv. campestris, LPSs [26–29], as well as muropeptides , fragments of the bacterial cell wall material peptidoglycan, have been characterized as MAMPs. Non-host resistance of plants towards X. campestris pv. campestris seems to be a very complex situation, where multiple elicitors are active in parallel [26, 31]. The genetic analyses performed during the last years identified several gene loci that are linked to the pathogenicity of X. campestris pv. campestris in host plants and to the induction of a resistance response in non-host plants. Protein secretion systems, in particular the type III secretion system, have an important role in the pathogenic interactions with plants [32–35]. Further virulence factors are exported by type II secretion systems [32, 36]. They are involved in the secretion of extracellular enzymes including plant cell wall degrading enzymes like pectate lyases (EC 126.96.36.199), also known as polygalacturonate lyases [37–40], or polygalacturonases (EC 188.8.131.52) [40, 41]. Pectate lyases catalyze the cleavage of α1,4 glycosidic bonds between galacturonic acid residues of homogalacturonans. Likewise, polygalacturonases catalyze the cleavage of the glycosidic bonds between adjacent galacturonic acid residues, but the hydrolysis of the glycosidic linkage results in the addition of a water molecule from the environment. Genome data which are now available for several strains have further added to our understanding of pathogenicity loci in X. campestris[42–47]. More information can be derived from closely related pathogens like Xylella fastidiosa, where a polygalacturonase has been characterized that is similar to the pglA2 gene product of X. campestris pv. campestris B100 . Rapid progress is currently achieved in identifying and analyzing regulation in X. campestris[49–52]. Concerning signal transduction, there has been substantial advancement of science related to two complex systems of cell-cell communication that employ a diffusible signal factor (DSF)  and a diffusible factor (DF) , respectively. In addition, more and more X. campestris two-component systems signal-transduction systems are characterized experimentally [55–58].
In previous analyses, the X. campestris pv. campestris tonB gene cluster showed some very interesting characteristics. TonB systems of Gram-negative bacteria are multi-component transport systems that perform the specific active uptake of various compounds across the outer membrane . These systems consist of the core components TonB, ExbB, and ExbD, which are located at or within the inner membrane, and variable so-called TonB-dependent receptors, which are located in the outer membrane, and which are specific for the imported substrate . TonB is assumed to transfer energy, which is gathered by the inner membrane components ExbB and ExbD from the inner membrane’s proton motive force, to the TonB-dependent receptors in the outer membrane. The TonB system is particular known for the uptake of iron . For X. campestris pv. campestris, an unusual high number of diverse TonB-dependent receptors has been identified in a profound analysis . Functional data revealed, besides iron, carbohydrates as substrates imported by specific TonB-dependent receptors of X. campestris pv. campestris . A gene of a TonB-dependent receptor that was co-located wtih genes for two putative pectin/polygalacturonate degrading enzymes was induced by polygalacturonate . TonB-dependent receptors are part of a regulon involved in utilization of N-acetylglucosamine, but their specific role remained unclear . The contiguous X. campestris pv. campestris genes tonB, exbB, and exbD1, which code for the TonB system core components, are essential for iron uptake . They are also required to induce the black rot disease in Brassica oleracea, to induce an HR in the interaction with the non-host plant C. annuum, and they are involved in the infection of X. campestris pv. campestris by the lytic bacteriophage ΦL7 . Differing from other Gram-negative bacteria, in X. campestris pv. campestris there is a similar second exbD gene, termed exbD2, which is located in the same gene cluster in tandem directly downstream of exbD1. This gene is not essential for iron-uptake, not necessary to induce the black rot symptoms on host plants, and not essential for penetration by phage ΦL7, but it is required to induce an HR in non-host plants . A similar but not identical genetic organization with two exbD genes located in tandem has only been described for the fish-pathogenic Flavobacterium psychrophilum, where again the exbD2 gene, which was also not required for iron uptake, was involved in pathogenicity . Although the role of the X. campestris pv. campestris exbD2 gene is not well understood in detail, there are hints that the gene product is involved in the export of X. campestris pv. campestris exoenzymes.
In this study, we have analyzed the exbD2 gene in more detail. In the course of the analyses, we discovered that exbD2 is involved in the induction of bacterial pectate lyase activity, which then releases OGAs from plant-derived pectate that are subsequently recognized as a DAMP by the plant.
The structure of the tonB gene cluster of X. campestris pv. campestris is unusual, and the role of the second exbD gene located in this cluster is still puzzling. Differing from the genes tonB, exbB, and exbD1, exbD2 is not required for iron uptake , but it is essential to induce an HR on C. annuum. Hence, further analyses were performed to obtain a better understanding of this enigmatic pathogenicity-related gene.
Genomic analysis of X. campestrispv. campestris TonB core genes
The exbD2gene is involved in pectate lyase activity
These first results were checked in a more elaborate approach. The strains B100-5.05 (tonB1), B100-7.03 (exbB1), B100-9.01 (exbD1), B100-11.03 (exbD2), and the wild-type were grown in liquid medium under inducing conditions. The pectate lyase activity was determined in a photometric assay . In contrast to the wild-type, all mutant samples showed no pectate lyase activity, see Additional file 3: Table S1. As no structural genes coding for pectate lyase enzymes were affected by the X. campestris pv. campestris mutations analyzed, it seemed likely that the mutations in the genes tonB1, exbB1, exbD1, and exbD2 affected the induction of pectate lyase genes.
Pectate lyase activity is required for HR on C. annuum
Elicitor-activity upon co-incubation of X. campestris pv. campestris with C. annuumcell wall material
The successful complementation of an exbD2 mutant with a pectate lyase gene indicated an important role of this gene in the recognition of X. campestris pv. campestris pathogens by non-host plants. However, the molecular characteristics of the elicitor that caused the HR were still unknown. The pectate lyase itself could act as a MAMP. Alternatively, products of the pectate lyase reaction like depolymerized pectate fragments derived from the plant cell wall could be recognized by the non-host plants. So, also a DAMP could not be ruled out as a possible cause of the HR.
Evidence that the newly formed elicitor is an oligogalacturonide DAMP
Elicitor activity of pectate fragments in N. tabacum and C. annuumcell suspension cultures
To further verify the role of the TonB system core genes and particular exbD2 in generating the OGA DAMP, we resumed analyzing the mutants deficient in these genes [64, 66]. Cell-free supernatants of X. campestris pv. campestris cultivations that had been co-incubated with C. annuum cell wall material had been shown to induce oxidative burst reactions in suspension cell cultures of non-host plants (Figure 4), while the supernatant of an analogously cultivated mutant strain deficient in exbD2 evoked no oxidative burst in a non-host suspension cell culture (Figure 5). Now we tested the effect of cell-free supernatants obtained from co-incubating X. campestris pv. campestris strains with pectin on non-host cell suspension cultures concerning their ability to induce oxidative burst reactions. Mutants deficient in all genes of the X. campestris pv. campestris TonB core system including exbD2 were tested in this approach, and turned out to be clearly affected in evoking oxidative burst reactions. The oxidative burst reactions in non-host suspension cell cultures were recovered when the disrupted genes were complemented specifically with complete copies of the respective genes (Additional file 4). The hydrogen peroxide concentrations measured in response to aliquots of cell-free supernatants from cultivations of the complemented mutants in the presence of pectin was at least at wild-type level. This clearly underlines that the genes of the bacterial TonB core system including exbD2 are involved in linking the bacterial response to the presence of pectin with a specific defense reaction of non-host plants.
Most bacterial pathogens produce a wide variety of cell wall degrading enzymes like endoglucanases, cellulases, pectinases, hemicellulases and lyases. In case of X. campestris pv. campestris, genes coding for almost 30 enzymes probably involved in cell-wall degradation were identified based on genome data [43, 45, 46]. Among them were several genes involved in degrading polygalacturonic acid (Additional file 5: Table S2). In consequence, cell wall degradation by X. campestris pv. campestris is assumed to result in the release of a complex mixture of poly- and oligosaccharides to the surrounding medium. It is in the best advantage of plants to recognize such signals of microbial pathogenicity as DAMPs in order to initiate suitable defense reactions. Plants are able to perceive diverse signal molecules such as the yeast elicitor in tobacco , bacterial flagellin [71, 72], harpin proteins [5–9], Hrp proteins from X. campestris, fungal proteins in parsley  and fungal exoenzymes in tobacco . Rouet-Mayer et al. were also able to show that fungal lyase represents a different chemical stimulus than the OGAs produced from the cell walls by this enzyme’s activity and that both these elicitors despite their common origin activated at least partially differing signal transduction pathways. The fact that tobacco is not only able to perceive the products of enzymatic digestion, but also the enzyme itself, shows how crucial it is for the plant to recognize the pathogenic fungus.
Here we report on the release of elicitor-active compounds obtained from the co-incubation of C. annuum cell walls with X. campestris pv. campestris. The co-incubation was carried out using a crude cell wall extract from pepper leafs and the X. campestris pv. campestris strain Bac2. The use of crude cell wall extracts instead of complete plants or leafs has the advantage that all products resulting from the incubation can originate only from the plant cell wall material or the bacteria. Orientation experiments indicated that cell wall-derived oligosaccharides were responsible for the elicitor activity. To identify the elicitor-active compound, HPAE chromatography  was employed. First hints on the origin of the elicitor-active molecules were obtained by analyzing the composition of neutral sugars and uronic acids. In comparison to the controls, an increased abundance of typical cell wall sugars was observed when X. campestris pv. campestris and cell-free pepper cell wall material were co-incubated. In the subsequent characterization of the oligosaccharide composition using HPAEC , UV absorption was measured in addition to the PAD signal in order to detect double-bonds in the newly formed oligosaccharides. This resulted in identifying the elicitor-active compounds as pectin fragments with a varying degree of polymerization (DP) by comparing the elution profile to a standard derived from pectin digested by a pectate lyase from a commercially supplier. MALDI-TOF MS was used as a valuable tool to obtain further structural information on the isolated oligosaccharides. These fragments with different DPs were then isolated with preparative HPAEC and tested for their elicitor activities.
The highest elicitor activity was found for OGAs with a DP exceeding 8. This result corresponds well with data from Svalheim & Robertson , who showed that OGAs released by fungal enzymes with DPs ranging from 9 to12 are able to elicit oxidative burst reactions in cucumber hypocotyl segments. It also fits well with other data summarized by Ryan , showing that different oligosaccharides induce a vast variety of plant defense responses. For example, oligomeric fragments of chitosan with DPs ranging from 6 to 11 are able to induce defensive mechanisms in tissues of several plants. OGAs with a DP below 9 are unable to induce phytoalexin production in soybean cotyledons , which corresponds well with the X. campestris pv. campestris – pepper system, where most of the elicitor activity resides in OGAs of a DP exceeding 8.
Interestingly, OGAs can have different roles in other plant-pathogen interactions. In wheat plants, small oligomers of galacturonic acid (dimers and trimers) have a completely different function as they act as suppressors of the plant pathogen defense and thereby promote the growth of pathogenic fungi . In A. thaliana, where WAK1 was recently identified as OGA receptor [21, 23], only small cell wall-derived OGAs with DPs of 2 to 6 have been reported to induce genes involved in the plant response to cell wall-degrading enzymes from the pathogen E. carotovora.
Plants need to permanently monitor whether there are indications for pathogen attack, a task that is not trivial as it requires to efficiently filter pathogen-related signals from others, like those generated by commensal or symbiotic microorganism. For each plant it is of fundamental importance to decide correctly whether to initiate defense or not, as defense includes expensive measures like sacrificing plant tissue by intentional cell death at the assumed infection site, while mistakenly omitted defense can be lethal . Analyzing the interaction of pathogens with non-host plants is an approach to identify the molecular nature of plant-pathogen interactions. Beside the highly specific recognition of avr gene products interactions with host plants , lipopolysaccharides [26, 27], muropeptides , hrp gene products , secreted proteins  and the pectate-derived DAMP described in this study contribute to the reaction of non-host cells in response to Xanthomonas. Obviously, all these MAMPs and DAMPs are part of the very complex and specific damage- and microbe-associated molecular signal, where individual elicitors contribute in a complex manner  to obtain an optimal decision of the plant whether to initiate defense with all its costly consequences or not.
While the A. thaliana OGA receptor WAK1 was recently identified [21, 23], it is now fascinating to see that the generation of a DAMP similar to that perceived by WAK1 is related to bacterial trans-envelope signaling. Recently, trans-envelope signaling has been shown to be a second important function of TonB systems [61, 84] besides the uptake of nutrients. A model describing this signaling mechanism assumes that members of a specific subgroup of the TonB-dependent receptors, which share a common N-terminal extension and which were termed TonB-dependent transducers, perceive an environmental signal in the outer membrane . Such TonB-dependent transducers are energized via the TonB-ExbB-ExbD core complex, while their N-terminal extension permits contacting periplasmic structures of anti-sigma factors that are localized in the inner membrane. The anti-sigma factors can then interact with ECF family sigma factors [84, 85], which can modulate bacterial gene expression at the transcriptional level. Probably the best understood paradigm for TonB-dependent trans-envelope signaling is the Fec signaling pathway of E. coli. The exbD2 gene product of X. campestris pv. campestris B100 seems involved in trans-envelope signaling via the TonB system, while the exbD1 gene is also required to import substances like ferric iron . However the situation could be more complex, as exbD2 might also be involved in uptake of cell wall degradation products, and as exbD1 might be involved in further so far unidentified signaling processes. Currently there is no evidence that the products of both genes are involved in both functions, transportation and signaling. But likewise, so far there is no reason to assume strict task sharing, where the exbD1 gene product is exclusively required for transport, while ExbD2 is specialized on signaling.
Further research could shed more light on the processes involved in bacterial reaction to the presence of pectin. Obviously, extracellular pectin-degrading enzymes are induced. But it is completely unclear which mechanisms are involved, and what kind of role the TonB core system plays. It could be just involved in importing polygalacturonic acid or derivatives of it. Imported galacturonic acid compounds could be perceived by an intracellular factor like a transcriptional regulator. Alternatively, the TonB system could be directly involved in signaling via an anti-sigma factor as described by Koebnik . Further more, there is no reason to exclude regulatory processes at post-transcriptional levels. Likewise, the specific roles of the enzymes involved in pectin degradation are unclear. The genome of X. campestris pv. campestris B100 includes six genes of enzymes that cleave the glycosidic bonds between adjacent glucuronic acid residues (Additional file 5: Table S2). The product of the polygalacturonase gene pglA2 is similar to a recently characterized X. fastidiosa enzyme , and the truncated pectate lyase encoded by pel4 is partially similar to an enzyme from Pseudomonas cellulosa, but seemed to lack the carbohydrate-binding module (CBM)  of the P. cellulosa enzyme. A polygalacturonate-induced gene for an X. campestris pv. campestris TonB-dependent receptor like two neighboring genes encoding degrading enzymes  are promising candidates for being involved in the response to the presence of polygalacturonate. However, like for other Xanthomonas enzymes that degrade plant cell-wall constituents, the kinetic properties of the pectin-degrading enzymes are not known, nor is there evidence for the regulation and expression of their genes or for regulatory processes that directly address the enzymes.
As far as we know, we report here for the first time on a DAMP that is produced by Xanthomonas exoenzymes from non-host plant cell walls. With the characterization of a DAMP produced by X. campestris pv. campestris, which was identified as an OGA, we were able to identify a further component of the complex network of signals that determines whether a plant is a host for X. campestris pv. campestris or whether it is resistant to this pathogen. So far, DAMPs were mainly known to be generated by fungal pathogens [17–20], and so far there are rather few examples where the signaling mechanisms have been analyzed profoundly at a molecular level. Due to the reduced complexity of prokaryotes, spending more effort on analyzing bacteria-generated DAMPs may also be a promising complement to studying fungi-based systems for pragmatic reasons, as experiments may be simpler in design, with the additional perspective of utilizing results provided by high-throughput approaches in the genomics and post-genomics disciplines for many bacteria.
Against the emerging background of TonB-related signal transduction  it is not too surprising to see an isoform of ExbD being involved in signaling. Nevertheless, the experimental results that indicate the involvement of ExbD2 in transducing a plant cell-wall-derived signal raise the question whether the E. coli paradigm on tonB functionality needs to be adapted or extended for X. campestris pv. campestris, as in E. coli ExbD (like ExbB) is supposed to be involved in signaling exclusively by contributing to energizing the outer membrane TonB-dependent transducer via TonB. The specific involvement of ExbD2 in signaling may indicate a more direct role of this ExbD isoform in signal transduction.
Cultivation of Xanthomonas campestrispv. campestris
Bacterial strains and plasmids used in this study
Strain or plasmid
Relevant genotype and/or description
Source or reference
X. campestrispv. campestris strains
Control strain, carrying ΩKm(cat) in intergenic region flanked by tonB1 and exbB1, Smr, Kmr
tonB1-deficient mutant, Smr, Kmr
exbB1-deficient mutant, Smr, Kmr
exbD1-deficient mutant, Smr, Kmr
exbD2-deficient mutant, Smr, Kmr
Bacitracin-resistant spontaneous mutant of B100, unable to produce polysaccharides, Smr
D. Steinmann, CeBiTec culture collection
E. coli strain
recA1, thi, supE44, lac, [F’proAB lacIq, lacZΔM15, Tn10(Tcr)]
pUC19, lacZ, Cmr
pUC19, lacZ, Cmr
pSVB30, aacC1, Gmr
pHIP, aacC1ΔBglII, Gmr
pHGW31, tonB1, Gmr
pHGW31, exbB1, Gmr
pHGW31, exbD1, Gmr
pHGW31, exbD2, Gmr
pLAFRI-based cosmid carrying 27.9 kb chromosomal BamHI fragment of X. campestris pv. campestris 8004 with pglI, Tcr
pHGW31, 11.1 kb chromosomal BamHI fragment of X. campestris pv. campestris 8004 with pglI, Gmr
pBCKS+, 3.8 kb BamHI-ClaI subfragment with pglI from pHGW260, Cmr
pBCSK+, 3.8 kb BamHI-ClaI subfragment with pglI from pHGW260, Cmr
pUC6S, 3.8 kb BamHI-ClaI subfragment with pglI from pHGW260 expressed from aacC1 Pout promoter, Apr, Gmr
Cultivation of C. annuumplants
C. annuum (cultivar California Wonder) plants derived from seedlings were grown in the greenhouse at 21°C with 12/12 day/night hours. Cell wall material was isolated from 6 weeks old plants.
Analysis of enzyme activity
Extracellular pectate lyase activity was monitored by an agar plate test and quantified in a photometric assay . For the pectate lyase assay, X. campestris pv. campestris cultures were grown for 24 h in M9 medium supplemented with pectate and FeSO4. The resulting values were calibrated to the activity of glucose-6-phosphate dehydrogenase. For the tests on agar plates , X. campestris pv. campestris strains were cultivated for 2 days on M9 medium supplemented with pectate and FeSO4 as described elsewhere .
Genome analysis and recombinant DNA procedures
Genome data were analyzed and visualized by means of the GenDB annotation system . The EDGAR software  was employed to compare complete Xanthomonas genomes that were available from public databases [42, 43, 45, 46, 96–99]. For the analysis of genes encoding polysaccharide-degrading enzymes, information provided by the CAZy database (http://www.cazy.org/) has been considered .
All cloning was performed applying standard methods  and as described previously [64, 66]. An 11.1 kb chromosomal BamHI fragment of X. campestris pv. campestris 8004 carrying the pglI gene in cosmid pIJ3051  was inserted into the plasmid vector pHGW31 to obtain plasmid pHGW260. A 3.8 kb BamHI-ClaI sub-fragment with the pglI gene was then transferred to the cloning vectors pBCKS+ and pBCSK+, resulting in the plasmids pHGW261 and pHGW262, respectively. In pHGW262, pglI was constitutively expressed in E. coli from the lac promoter of the pBCSK+ multiple cloning site. To express pglI also in X. campestris pv. campestris, pHGW267 was constructed by cloning the 3.8 kb BamHI-ClaI sub-fragment with the X. campestris pv. campestris 8004 pglI gene into the multiple cloning site of pUC6S (Apr) , where it was under the control of the constitutive Pout promoter of the aacC1 gene from pMS246 , which was cloned as a 1 kb BamHI fragment into the BamHI site upstream of pglI.
Isolation of plant cell wall material
Leafs of C. annuum were employed to obtain cell wall material. Leafs (30 g) were homogenized in 150 ml sodium acetate (50mM, pH 5) for 3 min and filtered with a fluted filter. After the filtration, the cell wall material was washed with 1 l sodium acetate (4°C), 1 l ethanol (4°C) and with 1 l acetone (−20°C). The washed material was then air dried at room temperature and stored under inert atmosphere at -20°C.
Co-incubation of X. campestris pv. campestris and C. annuumcell wall material
5 ml X. campestris pv. campestris over-night liquid culture was centrifuged. After removal of the supernatant, the sediment was re-suspended in 5 ml phosphate buffer (50 mM potassium phosphate, pH 7.9). 100 mg of the isolated cell wall material was then added to this solution and incubated over night at 28°C. The sample was then centrifuged and the pellet discarded. After heating (5 min; 100°C), centrifugation (10 min 10,000×g) and dialysis (molecular weight cut off 1000), the sample was freeze-dried. Resuspended lyophilized material was then used for further experiments.
Removing LPS from the samples via polymyxin B agarose
X. campestris pv. campestris lipopolysaccharides (LPSs) were removed from the elicitor preparation using a batch technique by adding an excess amount of polymyxin B agarose  as described in . Upon addition of polymyxin B agarose (Sigma-Aldrich), the samples were shaken and centrifuged. While the pellet probably containing LPS bound to polymyxin B agarose was discarded, the supernatant was used for further analyses.
Identification, isolation and characterization of oligosaccharides
The analyses of oligosaccharides was performed by HPAEC using a DIONEX GP-40 gradient pump; a Merck-Hitachi D-2000 Chromato Integrator; a DIONEX pulsed amperometric detector and a DIONEX UV detector.
Monosaccharide composition of isolated oligosaccharides was analyzed upon acid hydrolysis in trifluoroacetic acid (2 M; 120°C for 2 h). Neutral sugars were separated and identified using an isocratic elution (10 mM sodium hydroxide; flow 1 ml/min) with amperometric detection on a CarboPac® PA-100 column. For charged sugars a linear sodium acetate gradient ranging from 0.02 M to 0.5 M under alkaline conditions (0.1 M NaOH) with a flow rate of 1 ml/min was used .
Pectate fragments were separated using a sodium acetate gradient (ranging from 0.01 M to 1.0 M with a plateau of 10 min. at a concentration 0.7 M sodium acetate; 0.1 M NaOH; CarboPac® PA-100 column; flow 1 ml/min). For the identification of pectate fragments a pectate standard was generated by digestion of pectin (Pectin esterified, Sigma P-9561) by pectate lyase (Sigma P-7052). The isolation of pectate fragments was carried out under the conditions described above, but a semi-preparative column (CarboPac® PA-1; flow 2.5 ml/min) was used.
MALDI-TOF MS of isolated oligosaccharides
Crude extracts were analyzed on a Bruker ultraflex I MALDI-TOF mass spectrometer (Bruker-Daltonics, Bremen, Germany) in the negative–ion mode. Samples were analyzed in the linear and in the reflector TOF. Gentisic acid was used as matrix. For sample preparation, 1 μl saturated gentisic acid solution was mixed with 1 μl of 50 mg ml–1 crude extract lyophilisate dissolved in demineralized water. One microliter of this mixture was dropped onto the MALDI target.
Determination of the oxidative burst reaction in plant cell suspension cultures
The detection of the oxidative burst was performed using the H2O2-dependent chemiluminescence reaction described by Warm . Three to five days after sub-cultivation 2 g of cell material from the cell suspension cultures was diluted in 8 ml of pre-incubation medium (3% w/v sucrose in 0.04 × MS ) and incubated for 3 to 4 hours. For the measurement of the oxidative burst 200 μl aliquots of these suspensions were mixed with phosphate buffer (50 mM potassium phosphate, pH 7.9) and 1.2 mM luminol in the same phosphate buffer. The reaction was started by the addition of 100 μl of 14 mM potassium hexacyanate. The luminescence was measured with a Luminometer 1250 (BioOrbit, Turku, Finland). The intensity of luminescence was calibrated for hydrogen peroxide concentrations of 0.01 mM, to 0.05 mM.
Polygalacturonic acid (sodium salt), pectin and polymyxin B agarose was from Sigma-Aldrich, Taufkirchen, Germany. Unless otherwise specified, other chemicals were obtained from Merck, Darmstadt, Germany.
Damage-associated Molecular Pattern
Degree of Polymerization
Diffusible Signal Factor
High-performance Anion Exchange
High-performance Anion Exchange Chromatography
High-performance Liquid Chromatography
Matrix-assisted Laser Desorption/ionization time-of-flight
Microbe-associated Molecular pattern
Pathogen-associated Molecular Pattern
Pattern Recognition Receptor
Reactive Oxygen Species
We gratefully acknowledge Dorothee Steinmann for providing the X. campestris pv. campestris mutant strain B100-Bac2. Also, we want to thank Dr. Bruno Moerschbacher from the Institut für Biochemie und Biotechnologie in Münster, Germany for the kind permission to use his HPAEC system. At Bielefeld University, the project benefitted from work carried out by, Julia Voß, Sergej Wendler, Anna Köpfer, and Tim Steffens. Jannis Harfmann provided supportive transcriptomics data. Completing the project successfully benefited substantially from oxidative burst measurements carried out by Barbara Samenfeld.
This work was financially supported by the BMBF program “GenoMik Plus”. We acknowledge support of the publication fee by Deutsche Forschungsgemeinschaft and by the Open Access Publication Funds of Bielefeld University.
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