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 188.8.131.52), also known as polygalacturonate lyases [37–40], or polygalacturonases (EC 184.108.40.206) [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.