Characterization of flagellin perception in "Arabidopsis thaliana"

At the time when this work was started, a highly sensitive perception system for bacterial flagellin in plant cells had been described (Felix et al, 1999), and FLS1 and FLS2, had been identified in A. thaliana as essential loci for flagellin perception (Gomez-Gomez et al, 1999; Gomez-Gomez and Boller, 2000). In addition, radioactive binding studies had been established for tomato to characterize the flagellin binding site (Meindl et al, 2000). It became necessary to establish binding studies for A. thaliana as well, in order to assess if mutations in FLS2, causing insensitivity to flagellin, correlated with impairment in binding. Beyond the biochemical characterization of the flagellin binding site in A. thaliana, the goal of this work was to find out if the putative flagellin receptor FLS2 is the flagellin binding site. The A. thaliana flagellin binding site was found to exhibit the biochemical characteristics of a bona fida flagellin receptor: Binding was saturable and exhibited high affinity. It exhibited specificity for flagellin-derived peptides with biological activity as agonists or antagonists of the elicitor responses. Activation of flagellin receptor in A. thaliana appeared to occur as a two step process according to the addressmessage concept with the N-terminal part required for binding (address) and the Cterminal part for activation (message), as proposed for tomato before (Meindl et al, 2000). Additionally, sensitivity to salt and pH were determined, and reversibility was found to increase during cell fractionation, indicating that disassembly of a receptor complex or loss of cofactors take place. Furthermore, it was concluded that the flagellin binding site is localized at the plasma membrane. Comparison between the characteristics of flagellin binding in A. thaliana and tomato revealed that they show clear overall similarity but exhibit characteristic differences in detail, for instance in specificity, reversibility and sensitivity to pH and salts. The elution pattern from the ion exchange column indicated that two subclasses of the binding site occur. Concanavalin A chromatography showed, that the binding site is glycosylated, and optimization of ligand affinity chromatography paved the way for the identification of the binding site in future. Binding assays in extracts of different ecotypes and La-er FLS2 mutants showed a tight correlation between the presence of the binding site and sensitivity to flagellin, providing further evidence that the characterized binding site acts as the physiological receptor. Moreover, these results showed the pivotal role of FLS2 for flagellin binding. One mutation causing impairment of flg22 binding was localized in the LRR domain, indicating that this site might be involved in direct flagellin binding. Surprisingly, four mutations that disrupted binding, affected the cytoplasmic kinase domain. The significance of this finding is not understood yet. We speculated that the kinase activity might be important for proper targeting of FLS2 or for formation of a functional receptor complex. In order to prove that FLS2 is the flagellin binding site, epitope-tagged FLS2 was introduced into A. thaliana and tomato cell cultures and plants. When introduced into fls2 mutants, this construct complemented the mutation. However, properties of FLS2:myc protein, detected by immuno blot techniques, clearly differed from the properties of the flagellin binding site. Nevertheless, specificity of FLS2:myc transgenic tomato cell cultures for flagellin-derived peptides carried characteristic traits of A. thaliana binding and elicitor-response. This finding suggests that FLS2 determines specificity of flagellin perception. Another interesting aspect of this finding is that FLS2 seems compatible with tomato signal transduction components. It will be interesting to find out which part of FLS2 is responsible for the differences of flagellin perception, and which part is conserved in the two species. Although the results presented in this work clearly demonstrate an essential role of FLS2 for flagellin binding, direct evidence that FLS2 is the flagellin binding site is still lacking. Binding studies with heterologously expressed FLS2 could contribute to clearing this point. Also, optimization of the experimental conditions in order to prove flagellin binding by solubilized FLS2 could be reasonable. Alternatively, purification of the flagellin binding site would represent an independent approach. This method, like partial purification of FLS2, could provide further information’s about additional components of the receptor complex. It is likely that several components are needed for flagellin perception, as found for the CLAVATA and the self-incompatibility perception systems (for review see introduction).