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Sensitivity of Different Ecotypes and Mutants ofArabidopsis thaliana toward the Bacterial Elicitor Flagellin Correlates with the Presence of Receptor-binding Sites

2001; Elsevier BV; Volume: 276; Issue: 49 Linguagem: Inglês

10.1074/jbc.m102390200

1083-351X

Zsuzsa Bauer, Lourdes Gómez‐Gómez, Thomas Boller, Georg Felix,

Plant and animal studies

Flagellin, the main building block of the bacterial flagellum, acts as potent elicitor of defense responses in different plant species. Genetic analysis in Arabidopsis thaliana identified two distinct loci, termed FLS1and FLS2, that are essential for perception of flagellin-derived elicitors. FLS2 was found to encode a leucine-rich repeat transmembrane receptor-like kinase with similarities to Toll-like receptors involved in the innate immune system of mammals and insects. Here we used a radiolabeled derivative of flg22, a synthetic peptide representing the elicitor-active domain of flagellin, to probe the interaction of flagellin with its receptor in A. thaliana. The high affinity binding site detected in intact cells and membrane preparations exhibited specificity for flagellin-derived peptides with biological activity as agonists or antagonists of the elicitor responses. Specific binding activity was measurable in all ecotypes ofA. thaliana that show sensitivity to flagellin but was barely detectable in the flagellin-insensitive ecotype Ws-0 affected inFLS1. A strongly impaired binding of flagellin was observed also in several independent flagellin-insensitive mutants isolated from the flagellin-sensitive ecotype La-er. In particular, no binding was found in plants carrying a mutation in the LRR domain ofFLS2. These data indicate that the formation of functional receptor-binding sites depends on genes encoded by both loci,FLS1 and FLS2. The tight correlation between the presence of the binding site and elicitor response provides strong evidence that this binding site acts as the physiological receptor of flagellin. Flagellin, the main building block of the bacterial flagellum, acts as potent elicitor of defense responses in different plant species. Genetic analysis in Arabidopsis thaliana identified two distinct loci, termed FLS1and FLS2, that are essential for perception of flagellin-derived elicitors. FLS2 was found to encode a leucine-rich repeat transmembrane receptor-like kinase with similarities to Toll-like receptors involved in the innate immune system of mammals and insects. Here we used a radiolabeled derivative of flg22, a synthetic peptide representing the elicitor-active domain of flagellin, to probe the interaction of flagellin with its receptor in A. thaliana. The high affinity binding site detected in intact cells and membrane preparations exhibited specificity for flagellin-derived peptides with biological activity as agonists or antagonists of the elicitor responses. Specific binding activity was measurable in all ecotypes ofA. thaliana that show sensitivity to flagellin but was barely detectable in the flagellin-insensitive ecotype Ws-0 affected inFLS1. A strongly impaired binding of flagellin was observed also in several independent flagellin-insensitive mutants isolated from the flagellin-sensitive ecotype La-er. In particular, no binding was found in plants carrying a mutation in the LRR domain ofFLS2. These data indicate that the formation of functional receptor-binding sites depends on genes encoded by both loci,FLS1 and FLS2. The tight correlation between the presence of the binding site and elicitor response provides strong evidence that this binding site acts as the physiological receptor of flagellin. leucine-rich repeat 4-morpholineethanesulfonic acid Induction of active defense responses by plants depends on the detection of the invading pathogen or detection of the stress condition. A variety of chemically different substances, either originating from microorganisms or released from the plant cells in the course of injury, have been shown to act as potent elicitors of active defense responses in plants. Perception of these elicitors is thought to occur via specific receptors present in the plant hosts (1Boller T. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 189-214Crossref Scopus (420) Google Scholar). Microbial elicitors have been classified into two groups: The first group, the “general elicitors,” are characteristic for whole groups or classes of microorganisms. Perception for these general elicitors is thought to occur via specific receptors and to signal the presence of “nonself” in general, i.e. the mere presence of fungi or bacteria (1Boller T. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 189-214Crossref Scopus (420) Google Scholar, 2Heath M.C. Curr. Opin. Plant Biol. 2000; 3: 315-319Crossref PubMed Scopus (450) Google Scholar). The second group comprises the race-specific elicitors encoded by Avr (avirulence) genes present in particular races of pathogens that elicit defense responses in plant hosts carrying the corresponding resistance genes. The interaction of these specific elicitors and the gene products underlies the classic gene-for-gene interaction (3Flor H.H. Annu. Rev. Phytopathol. 1971; 9: 275-296Crossref Google Scholar, 4Keen N.T. Plant Mol. Biol. 1992; 19: 109-122Crossref PubMed Scopus (144) Google Scholar), and it has been postulated that the products of the resistance genes function as receptors for the race-specific elicitors (5Hammond-Kosack K.E. Jones J.D.G. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 575-607Crossref PubMed Scopus (896) Google Scholar). Flagellin, the subunit building up the filament of bacterial flagella, has been identified as a potent general elicitor, active inArabidopsis thaliana, tomato, and other plant species (6Felix G. Duran J.D. Volko S. Boller T. Plant J. 1999; 18: 265-276Crossref PubMed Scopus (1137) Google Scholar). Elicitor activity could be attributed to the most conserved domain within the N-terminal part of flagellin. flg22, a synthetic peptide comprising the core 22 amino acids, exhibited full elicitor activity and induced responses in tomato and A. thaliana at subnanomolar concentrations. Interestingly, flagellins of the plant-associated species Agrobacterium andRhizobium exhibit exceptional divergence of this domain, and synthetic peptides representing these divergent sequences did not induce responses in tomato and A. thaliana (6Felix G. Duran J.D. Volko S. Boller T. Plant J. 1999; 18: 265-276Crossref PubMed Scopus (1137) Google Scholar, 7Gómez-Gómez L. Felix G. Boller T. Plant J. 1999; 18: 227-284Crossref Scopus (508) Google Scholar). In A. thaliana seedlings, flg22 elicits rapid general defense responses like ethylene production and oxidative burst but leads also to a striking growth retardation on prolonged treatment (7Gómez-Gómez L. Felix G. Boller T. Plant J. 1999; 18: 227-284Crossref Scopus (508) Google Scholar). Among different ecotypes tested, only ecotype Ws-0 proved insensitive to flagellin. Crosses of Ws-0 with the sensitive ecotypes Col-0 and La-er demonstrated that a single dominant locus on chromosome 5, termedFLS1, determines sensitivity to flagellin (7Gómez-Gómez L. Felix G. Boller T. Plant J. 1999; 18: 227-284Crossref Scopus (508) Google Scholar). In addition, several mutants that are not affected in growth by the flg22 peptide were isolated by screening a mutagenized population of the sensitive ecotype La-er. At least two independent point mutations mapped to a single gene encoding a putative membrane receptor-kinase with an extraplasmatic leucine-rich repeat (LRR)1 domain (8Gómez-Gómez L. Boller T. Mol. Cell. 2000; 5: 1003-1011Abstract Full Text Full Text PDF PubMed Google Scholar). This gene mapped to a locus genetically closely linked but different from FLS1 and was consequently termed FLS2. Complementation of these two mutants with the FLS2 gene of the wild type fully restored responsiveness to flagellin (8Gómez-Gómez L. Boller T. Mol. Cell. 2000; 5: 1003-1011Abstract Full Text Full Text PDF PubMed Google Scholar). The product of FLS2 encodes a receptor kinase with high homology to the resistance gene Xa21 from rice responsible for resistance toXanthomonas oryzae (9Song W.-Y. Pi L.-Y. Wang G.-L. Gardner J. Holsten T. Ronald P.C. Plant Cell. 1997; 9: 1279-1287PubMed Google Scholar), and its LRR domain is similar to that of the Cf gene family from tomato, providing resistance to various strains of Cladosporium fulvum (5Hammond-Kosack K.E. Jones J.D.G. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 575-607Crossref PubMed Scopus (896) Google Scholar, 10Parniske M. Hammond-Kosack K.E. Golstein C. Thomas C.M. Jones D.A. Harrison K. Wulff B.B.H. Jones J.D.G. Cell. 1997; 91: 821-832Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). The present work aimed at defining the physiological role ofFLS1 and FLS2 for flagellin perception inA. thaliana. Similar to the situation with most resistance genes, FLS1 and FLS2 are decisive for the formation of a functional perception system, but it remains to be demonstrated whether one or both encode the receptor site that physically interacts with the elicitor ligand. To study elicitor binding, we adapted the binding assay established previously for detection and characterization of flagellin receptor-binding sites in tomato (11Meindl T. Boller T. Felix G. Plant Cell. 2000; 12: 1783-1794Crossref PubMed Scopus (99) Google Scholar) to A. thaliana. Although similar in many aspects to tomato, perception of flagellin by A. thaliana showed characteristic differences with respect to the structural determinants of peptides recognized as agonists or antagonists. Applying the modified binding assay, we studied the presence of the flagellin receptor-binding site in A. thaliana differing in their sensitivity to flagellin because of changes in FLS1 andFLS2. The presence of binding sites and sensitivity to flagellin were closely linked in all plants tested, demonstrating that both FLS1 and FLS2 are important for the formation of functional binding sites acting as receptors for the flagellin elicitor. The flagellin-derived peptides were synthesized according to the consensus sequence for the most highly conserved region in the N terminus of eubacterial flagellin (6Felix G. Duran J.D. Volko S. Boller T. Plant J. 1999; 18: 265-276Crossref PubMed Scopus (1137) Google Scholar). flg22, Tyr-flg22, flg15, flg13, flg22-Δ2, flg15E.coli, flg15R.mel, and flg22A.tum were synthesized and purified on reversed phase high pressure liquid chromatography by F. Fischer (Friedrich Miescher Institute). Peptides were dissolved in H2O (stock solutions of 1–10 mm) and diluted in a solution containing 0.1% bovine serum albumin and 0.1 m NaCl. Tyr-flg22 was iodinated using chloramine T to I-Tyr-flg22 (11Meindl T. Boller T. Felix G. Plant Cell. 2000; 12: 1783-1794Crossref PubMed Scopus (99) Google Scholar) or labeled with [125I]iodine to yield 3-[125I]iodotyrosine-flg22 (125I-Tyr-flg22) with a specific radioactivity of >2000 Ci/mmol by Anawa Trading SA (Wangen, Switzerland). Flagellin protein was purified as described before (6Felix G. Duran J.D. Volko S. Boller T. Plant J. 1999; 18: 265-276Crossref PubMed Scopus (1137) Google Scholar). A. thaliana seeds of ecotypes La-er, Zürich and Ws-0 were obtained from J. Paszkowski (Friedrich Miescher Institute). Seeds of ecotypes Col-0, Mühlen, Estland, Cri, AUA-Rhon, No-0, Col-PRL, and Kandavill were obtained from Lehle Seeds (Round Rock, TX). Mutants insensitive to treatment with flg22 were selected from ethyl methanesulfonate-mutagenized La-er seedlings (Lehle Seeds) as described before (8Gómez-Gómez L. Boller T. Mol. Cell. 2000; 5: 1003-1011Abstract Full Text Full Text PDF PubMed Google Scholar). The seeds were grown in soil in growth chambers programmed for cycles of 12 h of light of 60 μE m−2s−1 (Biolux lamps; Osram, Munich, Germany) at 20 °C and 12 h of dark at 16 °C with 70% relative humidity. Cell cultures of A. thaliana, originally derived from plant tissue of ecotype Landsberg erecta, were grown as described (12May M.J. Leaver C.J. Plant Physiol. 1993; 103: 621-627Crossref PubMed Scopus (348) Google Scholar). The cells were subcultured in weekly intervals and used for assays 6–8 days after subculture, containing ∼80 mg cells/ml (fresh weight). Aliquots of theA. thaliana cell suspension were incubated in open flasks on a rotary shaker at 150 cycles/min (7Gómez-Gómez L. Felix G. Boller T. Plant J. 1999; 18: 227-284Crossref Scopus (508) Google Scholar). Extracellular pH was measured with a small combined glass pH electrode (Metrohm, Herisau, Switzerland) and either recorded continuously using a pen recorder or measured 20 min after start of the experimental treatment. For preparation of microsomal membranes, 100 g of cells were transferred to 200 ml of binding buffer (25 mmMES/KOH, pH 6.0, 3 mm MgCl2, 10 mmNaCl) supplemented with 4 mm dithiothreitol and broken in a Parr cell disruption bomb (Parr Instrument Co., Moline, IL) as described before for tomato cells (11Meindl T. Boller T. Felix G. Plant Cell. 2000; 12: 1783-1794Crossref PubMed Scopus (99) Google Scholar). The homogenate was sequentially centrifuged at 10,000 × g for 20 min to yield pellet 1 (P1) and at 100,000 × g for 45 min to yield pellet 2 (P2) containing microsomal membranes. The pellets were resuspended in binding buffer, and protein concentrations were determined by the Micro BCA protein assay kit from Pierce. Individual A. thaliana plants, weighing 0.1–0.5 g fresh weight, were homogenized in 1–5 ml ice-cold binding buffer (10 ml of buffer/g of tissue) with a Polytron mixer (Kinematica AG, Littau-Luzern, Switzerland). Big fragments of tissues were removed by passing the homogenate through one layer of Miracloth (Calbiochem). Aliquots of cells, microsomes, or plant homogenates were incubated in binding buffer in a total volume of 100 μl with 125I-Tyr-flg22 (60 fmol in standard assays; >2000 Ci/mmol) for 25 min either alone (total binding) or with 10 μm of competing flg22 (nonspecific binding). Cells, microsomes, or crude extracts were collected by vacuum filtration on glass fiber filters (Macherey-Nagel MN GF-2, 2.5-cm diameter, preincubated with 1% bovine serum albumin, 1% bactotrypton, and 1% bactopepton in binding buffer) and washed for 10 s with 15 ml of ice-cold binding buffer. The radioactivity retained on the filters was determined by γ-counting. Specific binding was calculated by subtracting nonspecific binding from total binding. For equilibrium binding assays, aliquots of plant homogenates containing 500 μg of protein were incubated with radioligand and competing unlabeled flg22 as described above. After incubation for 20 min on ice, free label was separated from label bound to P1 by centrifugation (10,000 × g for 5 min). Leaves ofA. thaliana plants were cut in 1–3-mm slices (∼30 mg fresh weight/assay) and floated overnight on H2O. The leaf slices were transferred to 6-ml glass tubes containing 1 ml of H2O. After addition of elicitor preparations to be tested, vials were closed with rubber septa and placed horizontally on an orbital shaker (100 rpm). Ethylene accumulating in the free air space was measured by gas chromatography after 2 h of incubation. Flagellin and synthetic peptides corresponding to the highly conserved N-terminal domain spanned by flg22 act as potent elicitors in tomato and A. thaliana (6Felix G. Duran J.D. Volko S. Boller T. Plant J. 1999; 18: 265-276Crossref PubMed Scopus (1137) Google Scholar). Suspension-cultured plant cells react to elicitors within minutes and have thus been widely used to study elicitor perception and elicitor responses (1Boller T. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 189-214Crossref Scopus (420) Google Scholar). Changes in protein phosphorylation, activation of MAP kinases, and altered ion fluxes across the plasma membrane, including increased efflux of K+ and Cl− and increased influx of H+ and Ca2+ are among the earliest responses observed (13Felix G. Grosskopf D.G. Regenass M. Boller T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8831-8834Crossref PubMed Scopus (217) Google Scholar, 14Mathieu Y. Kurkdjian A. Xia H. Guern J. Koller A. Spiro M.D. O'Neill M. Albersheim P. Darvill A. Plant J. 1991; 1: 333-343PubMed Google Scholar, 15Nürnberger T. Nennstiel D. Jabs T. Sacks W.R. Hahlbrock K. Scheel D. Cell. 1994; 78: 449-460Abstract Full Text PDF PubMed Scopus (471) Google Scholar, 16Suzuki K. Shinshi H. Plant Cell. 1995; 7: 639-647Crossref PubMed Scopus (192) Google Scholar). These alterations in ion fluxes precede the actual defense responses and are believed to be involved in elicitor signaling. We used medium alkalinization, an easily measurable consequence of the changed ion fluxes, as a rapid, sensitive, and quantitative bioassay to assess structural requirements important for elicitor activity of flagellin-derived elicitors. flg15, a peptide lacking the 7 amino acid residues at the N terminus of flg22, was nearly as active as flg22 in tomato but showed ∼100-fold lower activity than flg22 in A. thaliana (6Felix G. Duran J.D. Volko S. Boller T. Plant J. 1999; 18: 265-276Crossref PubMed Scopus (1137) Google Scholar). Peptides shortened at the C-terminal end exhibited an even more drastic difference in their activity in cells of tomato and A. thaliana. flg22-Δ2, a peptide shortened by 2 amino acid residues at the C terminus, triggers medium alkalinization in tomato cells nearly as efficiently as flg22 (EC50 of ∼0.03 nm; data not shown) but failed to induce significant medium alkalinization in cells of A. thaliana when added in concentration up to 30 μm (Fig. 1 A). When added concomitantly with 3 nm flg22, an excess of 30 μm flg22-Δ2 nearly completely suppressed alkalinization induced by 3 nm flg22 alone (Fig. 1 A). The suppressive effect of flg22-Δ2 was competitive and could be overcome by increasing concentrations of flg22. In the presence of 10 μm flg22-Δ2 the dose of flg22 required to induce a half-maximal response (EC50) increased from ∼0.3 to ∼10 nm (Fig. 1 B). In further experiments with different concentrations of flg22-Δ2 and flg22, aK i of ∼100 nm was estimated for flg22-Δ2 (data not shown). The antagonistic activity depended on the intact peptide flg22-Δ2 and was rapidly degraded by pretreatment of the peptide with trypsin. Similarly, the shorter peptide flg15-Δ2 exhibited no significant activity as antagonist of flg22 when tested in concentrations up to 30 μm (data not shown). In contrast, a small, transient acidification of the extracellular medium similar to the one observed after treatment with flg22-Δ2 (Fig. 1 A) was also found with flg15-Δ2 and with flg22-Δ2 after treatment with trypsin (data not shown), indicating that the slight acidification was due to smaller peptide fragments or due to as yet unidentified contaminants of the peptide preparations. In summary, flagellin perception by A. thaliana and tomato exhibit characteristic differences, although both species recognize essentially the same conserved domain in the flagellin protein. In particular, both the N-terminal as well as the C-terminal parts of the domain spanned by flg22 appear to be of greater importance for biological activity in A. thaliana than in tomato cells. In previous experiments we used 125I-Tyr-flg22, a radioactive derivative of flg22, to establish a binding assay for flagellin elicitors in tomato (11Meindl T. Boller T. Felix G. Plant Cell. 2000; 12: 1783-1794Crossref PubMed Scopus (99) Google Scholar). When we attempted to apply this assay toA. thaliana, we initially failed to detect specific binding sites in cells and membrane preparations. Variation of the experimental parameters showed that binding in A. thaliana had a pH optimum between pH 5 and 6, and buffering to pH > 7, as used for assays with tomato, reduced binding by >90% (data not shown). Similarly, binding was sensitive to concentrations of >100 mm NaCl, KCl, or MgCl2 (data not shown). Under appropriately modified and optimized assay conditions, buffering at pH 6.0 and lowering the concentration of NaCl to 10 mm, specific binding of 125I-Tyr-flg22 to A. thaliana cells and membrane preparations could readily be detected (Fig. 2). In accordance with the rapid onset of physiological responses like medium alkalinization, intact cells showed rapid binding of the radioligand (Fig. 2 A). Even at 4 °C, maximal binding was reached within 20 min, and label associated with cells remained essentially stable for at least 90 min. Nonspecific binding, assayed in the presence of an excess of 10 μm unlabeled Tyr-flg22, remained low throughout the experiment (Fig. 2 A). Binding to intact cells appeared nonreversible because the addition of excess unlabeled flg22 in midcourse did not result in a significant decrease of label associated with the cells (Fig. 2 A). This irreversibility of binding was found in repetitions with different batches of intact cells (n>4). Binding appeared to be irreversible also in cells preincubated in 10 mm NaN3 or 10 mm NaF (data not shown), indicating that nonreversibility of binding was not due to internalization or other processes dependent on energy or membrane flow. Nonreversibility of binding was peculiar for binding of flagellin both to intact cells and membrane preparations of tomato (11Meindl T. Boller T. Felix G. Plant Cell. 2000; 12: 1783-1794Crossref PubMed Scopus (99) Google Scholar). In contrast, binding of 125I-Tyr-flg22 to microsomal membranes ofA. thaliana proved to be reversible, and 60 ± 10% of label was replaced within 20 min in six independent membrane preparations. One example is shown in Fig. 2 B. To test saturability, and to estimate the affinity of the binding site, we incubated increasing concentrations of125I-Tyr-flg22 with intact cells and microsomes (Fig.3). Fitting the data of specific binding to rectangular hyperbola (solid lines in Fig. 3) resulted in an apparent K d of 1.3 nm for intact cells and a K d of 1.7 nm for microsomal membranes, respectively. In the experiments shown in Fig. 3 the number of binding sites (B max) corresponded to 1.6 pmol of binding sites/g of cells (fresh weight) and 1.2 pmol/mg of microsomal protein. In three repetitions of the saturation experiments with microsomes and in two repetitions with intact cells,K d values reproducibly ranged between 1 and 3 nm, and the values for B max varied 2–3-fold in different batches of cells and membranes (data not shown). The A. thaliana cells used in these assays contain ∼4 × 104 cells/mg fresh weight; thus there are 2–6 × 104 receptor sites/cell. 70–95% of total binding activity observed in cell homogenates was recovered in the rapidly sedimenting pellet P1 (10,000 × g pellet), 5–30% was recovered in the microsomal fraction P2 (100,000 × g pellet), and no measurable binding was detected in the soluble fraction (10,000 × g supernatant). In terms of specific activity (binding per mg protein), the microsomal fraction P2 showed a 3–5-fold higher binding activity than P1 (data not shown). Apart from the apparent change in reversibility, the binding characteristics in cells, crude extracts, P1, and microsomal membrane fraction P2 were indistinguishable with respect to affinity, measured with saturation kinetics, and specificity, tested in competition assays with different flagellin-derived peptides (data not shown). This indicates that binding activity detected in these different fractions all represent the same binding site. The specificity of binding was tested in competitive binding assays with increasing concentrations of flagellin protein or various flagellin-derived peptides as competitors of 125I-Tyr-flg22. Examples for competition by Tyr-flg22, flg22, flg15, flg22-Δ2, flg22A.tum, and flg13 in binding assays with microsomal preparations are shown in Fig.4. In binding competition assays with intact cells, the peptides tested exhibited the same relative order with similar IC50 values (data not shown). In Fig.5, the IC50 values for flagellin protein and various peptides, deduced from dose-competition curves such as the ones shown in Fig. 4, were plotted against their respective activity for induction of a half-maximal alkalinization response (EC50 values). The most efficient competition and the highest biological activity were observed for Tyr-flg22 and its iodinated form I-Tyr-flg22 (values for IC50 of 4 nm and for EC50 of 0.2 nm, respectively). flg22 was ∼3–5-fold less efficient in both assays, whereas intact flagellin protein was ∼20-fold less active. Peptides shortened at the N terminus, flg15 and flg13, showed decreasing binding affinity in parallel to dropping elicitor activity. flg22-Δ2, acting as antagonist for biological activity (Fig. 1), strongly competed for binding with an IC50 only 10-fold lower than that of flg22. Weak competition of binding was observed for flg15-Δ2 when added in millimolar concentrations. Peptides corresponding to the homologues of flg15 from Agrobacterium tumefaciens andRhizobium meliloti (flg15A.tum and flg15R.mel) were previously reported to be inactive as inducers of alkalinization (6Felix G. Duran J.D. Volko S. Boller T. Plant J. 1999; 18: 265-276Crossref PubMed Scopus (1137) Google Scholar), and flg15R.mel also did not show measurable activity in binding competition. In contrast, the homologue of flg22 from A. tumefaciens,flg22A.tum, competed for binding with an IC50of 20 μm but did not induce alkalinization in concentrations up to 30 μm. Structurally unrelated peptide such as the 18-amino acid systemin did not compete binding at all concentrations tested. In summary, the binding site detected exhibited clear specificity for flagellin-derived peptides with biological activity as agonists or antagonists.Figure 5Correlation of biological activities and binding affinities for flagellin and flagellin-derived peptides.Relative activities of flagellin and flagellin-derived peptides for induction of the alkalinization response (EC50 values) inA. thaliana cells are plotted against their activities in binding competition assays (IC50 values) with microsomal membranes. flg22-Δ2, flg15-Δ2, and flg22A.tum showed activity in binding competition but no agonistic activity in the alkalinization response up to concentrations of 30 μm. flg15R.mel and the unrelated peptide systemin showed no biological activity and did not compete in binding assays. At thebottom are peptide sequences with amino acid residues differing from the sequence in P. fluorescens. Each value represents the average of at least two determinations of IC50 and EC50 in independent dose response curves. Mean ± S.E. for IC50 values were 3.8 ± 1.6 nm for Tyr-flg22 (n = 4), 12.6 ± 3.3 nm for flg22 (n = 5), and 4400 ± 960 nm for flg15 (n = 5).View Large Image Figure ViewerDownload Hi-res image Download (PPT) For studying flagellin-binding sites in tissues of soil-grown A. thaliana plants, we homogenized leaves in binding buffer and assayed the crude homogenates for binding of125I-Tyr-flg22 as described for extracts from cultured cells. Specific binding, defined as the difference between total binding and nonspecific binding, was clearly detectable in homogenates of the flagellin-sensitive ecotype La-er (Fig.6 A). Specific binding of radioligand increased linearly with the amount of homogenate applied and homogenates containing 100–200 μg of protein, corresponding to ∼10 mg of plant tissue, were sufficient to detect significant binding. Thus, the assay was sensitive enough to measure binding activity in homogenates of individual plants. In contrast to ecotype La-er, seedlings of ecotype Ws-0 exhibit no sensitivity to treatment with flagellin. This insensitivity was previously attributed to a single locus termed FLS1 (7Gómez-Gómez L. Felix G. Boller T. Plant J. 1999; 18: 227-284Crossref Scopus (508) Google Scholar). When assayed for flagellin binding, homogenates of Ws-0 showed greatly reduced specific binding compared with the flagellin-sensitive ecotype La-er (Fig. 6 A). Although the difference between total and nonspecific binding was close to the detection limit in Ws-0 (Fig.6 A), a very low specific binding activity was detectable in most repetitions with independent homogenates of Ws-0 (n > 6; data not shown). Mixtures of homogenates from La-er and Ws-0 plants exhibited binding corresponding to the arithmetic of the mixtures, indicating that no soluble factors inhibit or enhance binding in the two homogenates (data not shown). Reduced binding, as observed in homogenates of Ws-0, could indicate a reduced number of binding sites, or it could reflect a reduced affinity of these sites. A reduced affinity could lead to the loss of bound radioligand during the washing step used to remove unbound ligand in the binding assays. To test this possibility we performed equilibrium binding assays with separation of bound and unbound ligand by centrifugation (Fig. 6 B). Although background in assays with excess unlabeled flg22 was higher than in standard assays with washing on filters, it clearly demonstrated significant specific binding in La-er and a strongly reduced number of binding sites in ecotype Ws-0. Several additional ecotypes of A. thaliana were assayed for the presence of flagellin-binding sites and their response to treatment with flg22. In Fig. 7, binding activity in homogenates was compared with the flg22-dependent induction of ethylene biosynthesis in leaf tissues of these ecotypes. With the exception of Ws-0, all ecotypes showed clear induction of ethylene biosynthesis and significant specific binding of125I-Tyr-flg22. We assessed total binding versus nonspecific binding in individual plants of wild type and several mutant lines selected for insensitivity to flagellin (8Gómez-Gómez L. Boller T. Mol. Cell. 2000; 5: 1003-1011Abstract Full Text Full Text PDF PubMed Google Scholar). The two mutants fls2-24 andfls2-17 carry two different point mutations in theFLS2 gene encoding a putative receptor kinase (8Gómez-Gómez L. Boller T. Mol. Cell. 2000; 5: 1003-1011Abstract Full Text Full Text PDF PubMed Google Scholar). Confirming previous results (17Gómez-Góm