Innate immunity mediated by the flagellin receptor FLS2 in Arabidopsis and tomato : a molecular approach to characterize ligand binding and function, using receptor chimeras

Flagellin, the major subunit of the bacterial motility organ flagellum is an archetypical elicitor molecule perceived by a variety of plant species (Felix et al., 1999). Flg22, a synthetic peptide comprising the highly conserved amino acid residues of the flagellin N-terminus, has been shown to be the active epitope of flagellin which is recognized by plants and sufficient to activate plant defense responses (Felix et al., 1999). Flagellin/flg22 recognition has been attributed to a single protein, FLS2. FLS2 is a leucine rich repeat (LRR) receptor like kinase (RLK), consisting of 28 extracellular LRRs, a single transmembrane domain and an intracellular ser/thr kinase domain, was first identified in the model plant Arabidopsis thaliana (Gómez-Gómez and Boller, 2000) and shown to directly bind flg22 (Chinchilla et al., 2006). Meanwhile, orthologues of FLS2 have been identified in a variety of species from different families, among them tomato (Lycopersicon esculentum), Nicotiana benthamiana, Ricinus communis and Populus trichocarpa, to name just a few (Robatzek et al., 2007b). Although all these plants recognize flg22 as an elicitor, distinct species specific differences were identified. In this work, the molecular differences between the flagellin recognition systems of Arabidopsis (AtFLS2) and tomato (LeFLS2) are analyzed in depth. It was shown that full length flg22 is required for activity in Arabidopsis while tomato requires only the 15 aa peptide flg15 for full stimulation of defense responses (Meindl et al., 2000). Receptor activation of FLS2 by flg22 occurs according to the address-message concept with binding of the address as a first step, and message-induced receptor activation as a second step (Meindl et al., 2000). By using a variety of flg22-derivatives, we analyze how Arabidopsis and tomato flagellin receptors discriminate between different variants of flg22 in terms of binding and receptor activation. By using the species specific differences of Arabidopsis and tomato flagellin perception, we identify areas within the LRR domain of the respective flagellin receptors which are responsible for interaction with the ligand. To achieve this, we constructed a series of chimeric receptors by swapping different parts of the LRR domain from LeFLS into the AtFLS2 sequence. These chimeric receptors were transformed into Nicotiana benthamiana and Arabidopsis thaliana and the transformed plants were tested for receptor function using various bioassays such as ethylene production and growth inhibition and we performed binding assays using immunoprecipitated receptors. Based on these experiments we show that the LeLRR 1 to 10 are sufficient to bind the minimum peptide flg15-Δ7, the shortest flg22-derivative perceived by tomato consisting only of the central 8 amino acids of flg22. We show that the initial ten N-terminal LRRs between the amino acids 32-337 and within this area, especially the amino acids 236-337 are import for the higher affinity of LeFLS2 to flg22 and N-terminally truncated flg22-derivatives. We further show that an additional region between the LRR 19 to 24 of LeFLS2 is involved in the recognition of the C-terminus of flg22. Because the C-terminus of flg22 has been shown to be part of the “message”, which activates receptor signaling (Meindl et al., 2000; Chinchilla et al., 2006), we propose the region of LRR 19 to 24 to be play an important role for activation of FLS2. Additionally a
chimeric receptor between AtFLS2 and LeFLS2 is presented which shows the characteristics of a constitutive active FLS2 allele when transformed into Arabidopsis. Interestingly, the constitutive signaling of this chimeric receptor can only be triggered via the artificial extracellular LRR domain, since the complete intracellular receptor part, e.g. transmembrane- juxtamembrane- and kinasedomain is not affected from the LRR domain swapping. Together, this study provides new insight towards the understanding of FLS2-ligand interaction and an interesting tool to further study receptor activation.