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Research

Receptor kinases and pathogen recognition

Plants and animals can sense microbial attack by detection systems for "pathogen-associated molecular patterns" (PAMPs). These molecular patterns are conserved among whole groups and classes of microorganisms and can serve as hall-marks for the detection of ‘non-self’ by the hosts. So far, a series of PAMPs originating from fungi, oomycetes and bacteria have been described that act as potent elicitors of defense responses in plants. However, the corresponding receptors of the plants remain unknown for most of them. By concentrating on detection mechanisms for bacteria we have characterized the two bacterial proteins flagellin (Felix et al., 1999) and the elongation factor Tu (EF-Tu, Kunze et al., 2004) as PAMPs active in the model plant Arabidopsis. Perception in both cases is specific for particular domains of the proteins and synthetic peptides representing these epitopes, termed flg22 and elf18, fully mimic the elicitor activity of the intact proteins (Fig. 1). Using genetic and reverse-genetic approaches the flagellin receptor FLS2 (Gómez-Gómez and Boller, 2000) and the EF-Tu receptor EFR (Zipfel et al. unpublished) of Arabidopsis have been identified. Both receptor proteins have an ectodomain thought to function as the binding site for the respective ligand, a single-pass transmembrane domain and a cytoplasmic kinase domain. In plants, receptor kinases form a large family of proteins with >600 members in Arabidopsis. Based on genetic evidence it is clear that members of this family play crucial roles in aspects of the plant’s life ranging from growth and development, to fertilization, reproduction and interaction with symbionts and pathogens. However, most of these putative receptors remain poorly characterized with respect to the signals they perceive. Similarly, little is known about the molecular mechanism by which this important class of plant receptors converts extracellular signal inputs into intracellular signal outputs.

Current and future work will base on the well characterized ligand-receptor pairs flg22/FLS2 and elf18/ EFR as experimentally well suited models to study mechanism of receptor activation and transmembrane signaling (Fig. 2). Based on results with related receptors from animals one can anticipate that processes of homo- and/or hetero-dimerization with as yet unknown co-receptor elements relay extracellular signals to cytoplasmic signal output. For studying these protein-protein interactions we will make use of site-specific alterations and tagging of the receptors, affinity crosslinking, screens for interacting proteins and approaches with chimeric forms of FLS2 and EFR. These latter approaches will also help to identify the domains of the receptors responsible for interaction with the PAMPs.

Plants have perception systems for bacterial PAMPs in addition to flagellin and EF-Tu. For example, lipopolysacharide (LPS), a classical PAMP for the human innate immune system, has been reported to be detected by plants as well. Using the rapid and convenient cell-culture based bioassays that were successful to identify flagellin and EF-Tu, we will try to identify further bacterial structures for which plants have evolved perception systems. Knowing more about the repertoire of PAMPs recognized will allow studies on the interplay of the different perception systems in the defense response. Flagellin and EF-Tu activate a common signaling pathway and a congruent set of defense responses, but without a clear additive or synergistic effect. Thus, the apparent redundancy of perception systems might serve to ensure and enhance the detection for microbes that evolved to camouflage one or the other of the PAMPs. PAMPs usually represent highly conserved structures which carry essential functions and, consequently, are difficult to change or mutate. Nevertheless, some of the bacterial species pathogenic to plants have conspicuous changes in flagellin and EF-Tu which renders them ‘invisible’ to the detection systems of the plants. To test whether these changes in the PAMPs are due to selective pressure imposed by the defense system of the plant hosts, it will be important to study virulence of these pathogens when these camouflaged forms of the PAMPs are exchanged with forms that can be detected by the plant defense system.

Fig.1 Schematic view of EF-Tu and flagellin, two bacterial-derived PAMPs detected by Arabidopsis cells. The N-terminus of EF-Tu (elf18), recognized by the receptor termed EFR, and the epitope flg22 of flagellin, recognized by the flagellin receptor FLS2, are highlighted in red. Apart from flagellin and EF-Tu, plant cells can detect further bacterial PAMPs, including lipopolysacharides (LPS) and other, as yet unidentified, molecular patterns. Similarly, the receptors for these additional PAMPs are currently unknown (shaded, question marks). Based on the common set of responses induced by all PAMPs one can speculate that these receptors might resemble the receptor kinases EFR and FLS2.

Fig.2 Model for activation of the flagellin receptor FLS2. FLS2 is a receptor kinase with an ectodomain composed of leucine rich repeats (LRR). LRR-domains are involved also in PAMP detection by the Toll-like receptors of animal innate immunity and have been shown to adopt a horse-shoe type of configuration. Interaction of flg22 with FLS2 is a two-step process that leads to non-reversible binding of the ligand (steps I and II). Connected to this process one can imagine a structural change in the ectodomain that then allows an oligomerisation process (either homo- or hetero-dimerization, III) that leads to a corresponding change on the cytoplasmic side and activation of intracellular signaling. FLSx and FLS-adaptor are hypothetical elements of the flagellin receptor.

Publications

Albert, M., and Felix, G. (2010). Chimeric receptors of the Arabidopsis thaliana pattern recognition receptors EFR and FLS2. Plant Signal Behav 5, 108-110.

Albert, M., Jehle, A.K., Lipschis, M., Mueller, K., Zeng, Y., and Felix, G. (2010a). Regulation of cell behaviour by plant receptor kinases: Pattern recognition receptors as prototypical models. Eur J Cell Biol 89, 200-207.

Albert, M., Jehle, A.K., Mueller, K., Eisele, C., Lipschis, M., and Felix, G. (2010b). Arabidopsis thaliana pattern recognition receptors for bacterial elongation factor Tu and flagellin can be combined to form functional chimeric receptors. J Biol Chem 285, 19035-19042.

Schulze, B., Mentzel, T., Jehle, A.K., Mueller, K., Beeler, S., Boller, T., Felix, G., and Chinchilla, D. (2010). Rapid heteromerization and phosphorylation of ligand-activated plant transmembrane receptors and their associated kinase BAK1. J Biol Chem 285, 9444-9451.

Jeworutzki, E., Roelfsema, M.R., Anschutz, U., Krol, E., Elzenga, J.T., Felix, G., Boller, T., Hedrich, R., and Becker, D. (2010). Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca-associated opening of plasma membrane anion channels. Plant J 62, 367-378.

Krol, E., Mentzel, T., Chinchilla, D., Boller, T., Felix, G., Kemmerling, B., Postel, S., Arents, M., Jeworutzki, E., Al-Rasheid, K.A., Becker, D., and Hedrich, R. (2010). Perception of the Arabidopsis danger signal peptide 1 involves the pattern recognition receptor AtPEPR1 and its close homologue AtPEPR2. J Biol Chem 285, 13471-13479.

Boller, T., and Felix, G. (2009). A Renaissance of Elicitors: Perception of Microbe-Associated Molecular Patterns and Danger Signals by Pattern-Recognition Receptors. Annual Review of Plant Biology 60, 379-406.

Drissner, D., Kunze, G., Callewaert, N., Gehrig, P., Tamasloukht, M., Boller, T., Felix, G., Amrhein, N., and Bucher, M. (2007). Lyso-phosphatidylcholine is a signal in the arbuscular mycorrhizal symbiosis. Science 318, 265-268.

Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nurnberger, T., Jones, J.D., Felix, G., and Boller, T. (2007). A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497-500.

Robatzek, S., Bittel, P., Chinchilla, D., Kochner, P., Felix, G., Shiu, S.H., and Boller, T. (2007). Molecular identification and characterization of the tomato flagellin receptor LeFLS2, an orthologue of Arabidopsis FLS2 exhibiting characteristically different perception specificities. Plant Mol Biol 64, 539-547.

Gust, A.A., Biswas, R., Lenz, H.D., Rauhut, T., Ranf, S., Kemmerling, B., Gotz, F., Glawischnig, E., Lee, J., Felix, G., and Nurnberger, T. (2007). Bacteria-derived peptidoglycans constitute pathogen-associated molecular patterns triggering innate immunity in Arabidopsis. J Biol Chem 282, 32338-32348.

Chinchilla, D., Bauer, Z., Regenass, M., Boller, T., and Felix, G. (2006). The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell 18, 465-476.

Thuerig, B., Felix, G., Binder, A., Boller, T., and Tamm, L. (2006). An extract of Penicillium chrysogenum elicits early defense-related responses and induces resistance in Arabidopsis thaliana independently of known signalling pathways. Physiological and Molecular Plant Pathology 67, 180-193.

Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J.D., Boller, T., and Felix, G. (2006). Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125, 749-760.

Ludwig, A.A., Saitoh, H., Felix, G., Freymark, G., Miersch, O., Wasternack, C., Boller, T., Jones, J.D., and Romeis, T. (2005). Ethylene-mediated cross-talk between calcium-dependent protein kinase and MAPK signaling controls stress responses in plants. Proc Natl Acad Sci U S A 102, 10736-10741.

Zipfel, C., and Felix, G. (2005). Plants and animals: a different taste for microbes? Curr Opin Plant Biol 8, 353-360.

Danon, A., Miersch, O., Felix, G., Camp, R.G., and Apel, K. (2005). Concurrent activation of cell death-regulating signaling pathways by singlet oxygen in Arabidopsis thaliana. Plant J 41, 68-80.

Kunze, G., Zipfel, C., Robatzek, S., Niehaus, K., Boller, T., and Felix, G. (2004). The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16, 3496-3507.

Zipfel, C., Robatzek, S., Navarro, L., Oakeley, E.J., Jones, J.D., Felix, G., and Boller, T. (2004). Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428, 764-767.

Felix, G., and Boller, T. (2003). Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco. J Biol Chem 278, 6201-6208.