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*,

Georg FelixDagger and Thomas Boller

From the Friedrich Miescher-Institute, P. O. Box 2543, CH-4002 Basel, Switzerland

Received for publication, September 26, 2002, and in revised form, November 21, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

To detect microbial infection multicellular organisms have evolved sensing systems for pathogen-associated molecular patterns (PAMPs). Here, we identify bacterial cold shock protein (CSP) as a new such PAMP that acts as a highly active elicitor of defense responses in tobacco. Tobacco cells perceive a conserved domain of CSP and synthetic peptides representing 15 amino acids of this domain-induced responses at subnanomolar concentrations. Central to the elicitor-active domain is the RNP-1 motif KGFGFITP, a motif conserved also in many RNA- and DNA-binding proteins of eukaryotes. Csp15-Nsyl, a peptide representing the domain with highest homology to csp15 in a protein of Nicotiana sylvestris exhibited only weak activity in tobacco cells. Crystallographic and genetic data from the literature show that the RNP-1 domain of bacterial CSPs resides on a protruding loop and exposes a series of aromatic and basic side chains to the surface that are essential for the nucleotide-binding activity of CSPs. Similarly, these side chains were also essential for elicitor activity and replacement of single residues in csp15 with Ala strongly reduced or abolished activity. Most strikingly, csp15-Ala10, a peptide with the RNP-1 motif modified to KGAGFITP, lacked elicitor activity but acted as a competitive antagonist for CSP-related elicitors. Bacteria commonly have a small family of CSP-like proteins including both cold-inducible and noninducible members, and Csp-related elicitor activity was detected in extracts from all bacteria tested. Thus, the CSP domain containing the RNP-1 motif provides a structure characteristic for bacteria in general, and tobacco plants have evolved a highly sensitive chemoperception system to detect this bacterial PAMP.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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A key aspect of active defense against invading microbial pathogens is the ability to discriminate between self and infectious nonself (1). In plants, recognition-dependent disease resistance has been studied most thoroughly and most successfully in cases that depend on the presence of specific resistance genes that confer immunity to particular races of plant pathogens. Several of these resistance genes were shown to be involved in the chemoperception of factors specifically attributed with particular strains of pathogens (2-4). In addition, plants have a broader, more basal, surveillance involving sensitive perception systems for patterns characteristic for entire groups or classes of microorganisms, and they respond to these general elicitors with activation of signaling pathways that initiate defense mechanisms (5). This is highly reminiscent of innate immunity in animals and humans. Among the elicitors that represent patterns characteristic for fungi are cell wall components like glucans, chitin and chitosan oligosaccharides, peptides and proteins with fungal-specific N-glycosylation, and the membrane component ergosterol (6, 7). Similarly, cells of many plant species have a perception system for the common bacterial surface protein flagellin, the building block of the flagella (8). Perception of flagellin by Arabidopsis thaliana was shown to depend on FLS2, a membrane-bound receptor kinase protein with an extracellular leucine-rich repeat (9). Bacterial flagellin has recently also been identified as one of the "pathogen-associated molecular patterns" (PAMPs)1 that activate the innate immune system of humans and animals (10) via the Toll-like receptor 5 (11, 12). Thus, perception of general elicitors in plants resembles perception of PAMPs in the innate immune system of animals with respect to the type of molecules perceived, the characteristics of pattern recognition receptors involved, as well as some of the signaling mechanisms and defense responses induced (13).

Flagellin was the predominant if not only elicitor present in crude bacterial extracts that activated elicitor responses in the tomato cells used in our previous experiments. Extracts from bacteria without flagella or with flagellins that are strongly divergent in the elicitor-active domain represented by the oligopeptide flg22 proved inactive in the tomato cells (8). These observations with one particular cell line, grown in vitro for several years, do not exclude the existence of chemoperception systems for other bacterial PAMPs in tomato or other plant species. Perception of several different PAMPs, indicative for the same class of microbial pathogens, appears characteristic for the innate immune system of animals. Similarly, redundancy of chemoperception systems for a variety of molecular patterns characteristic for fungi has also been observed in plants (6). Therefore, we set out to search for additional chemoperception systems of plants sensing molecular patterns characteristic for bacteria. Suspension cultured tobacco cells have long been known to respond with a rapid K+ efflux, a concomitant medium alkalinization and an oxidative burst when treated with bacterial preparations containing either living or heat-killed bacteria (14) but the bacterial factors eliciting these responses have not been identified. In initial experiments we tested commercial preparations containing peptidoglycan from Micrococcus lysodeikticus (Staphylococcus aureus) for induction of responses in cultured tobacco cells. Peptidoglycan has long been known as a PAMP signaling presence of Gram-positive bacteria in the innate immune systems of animals (10). The peptidoglycan preparation indeed induced significant and rapid responses in tobacco but, surprisingly, a preparation of total lyophilized M. lysodeikticus bacteria proved to be a far more potent source of elicitor activity. We concentrated on the purification and characterization of this latter activity and, in the present work, identified it as a small protein belonging to the family of so-called cold shock proteins.

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Materials-- Peptides were synthesized by F. Fischer (Friedrich Miescher-Institute, Basel) or by Bio-Synthesis Inc. (Lewisville, TX). Peptides were dissolved in H2O (stock solutions of 1 to 10 mM) and diluted in a solution containing 0.1% bovine serum albumin and 0.1 M NaCl. Agrobacterium tumefaciens (strain C58 T), Rhizobium meliloti, and Xanthomonas campestris were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM GmbH, Braunschweig, BRD) and grown in King's B broth at 26 °C on a rotary shaker. Bacteria were harvested by centrifugation, washed once with H2O, and resuspended in H2O (10% of original volume). Crude bacterial elicitors were prepared by boiling the bacterial suspensions for 5-10 min and removing of bacterial debris by centrifugation. Lyophilized bacteria of M. lysodeikticus (Sigma) and the peptidoglycan fraction from M. lysodeikticus (Fluka, Buchs, Switzerland) were applied as suspensions in H2O. The bacterial preparation "messenger" was obtained from EDEN Bioscience (Bothell, WA).

Purification of Elicitor from M. lysodeikticus-- Elicitor activity was purified from the lyophilized preparation of M. lysodeikticus (Sigma). Ten g of the lyophilisate was suspended in 100 ml of H2O and heated for 10 min at 95 °C. After centrifugation (30 min at 10,000 × g) the supernatant was mixed with 1 volume of acetone and the precipitate formed after overnight incubation at -20 °C was removed by centrifugation. The acetone concentration was brought to 80% (v/v) and the precipitate formed after 4 h at -20 °C was collected by centrifugation. This precipitate was dissolved in 20 mM Tris-HCl, pH 7.5, and passed over an anion-exchange column with diethylaminoethyl-cellulose (DE-cellulose, Whatman) equilibrated with 20 mM Tris-HCl, pH 7.5. Activity eluting in the flow-through was concentrated by acetone precipitation (80% acetone) and separated on a Sephacel C8 reversed phase column (Amersham Biosciences AB) at pH 6.5 (10 mM phosphate buffer, pH 6.5, as solvent A and 80% acetonitrile, 20% phosphate buffer as solvent B). The two fractions containing the highest elicitor activity were pooled, pH adjusted to 3.5, and rerun on a Sephacel C8 reversed phase column at pH 3.5 (0.1% trifluoroacetic acid in H2O at pH 3.5 as solvent A and 80% acetonitrile, 20% H2O with 0.1% trifluoroacetic acid as solvent B).

Plant Cell Cultures-- The tobacco (Nicotiana tabacum L.) cell culture line 275N, originally derived from pith tissue of Havanna 425 plants, was maintained and subcultured as described before (15) in a Murashige-Skoog based medium. Cells were maintained as suspension cultures and used 4 to 10 days after subculture for experiments. Cell cultures of tomato ("line Msk8" (16)), potato (17), Lycopersicon peruvianum (18), and A. thaliana (19) were cultured as described elsewhere.

Alkalinization Response-- To measure alkalinization of the growth medium (the alkalinization response), 3-ml aliquots of the cell suspensions were placed in open, 20-ml vials on a rotary shaker at 120 to 150 cycles per min. Using small combined glass electrodes (Metrohm, Herisau, Switzerland) extracellular pH values were either recorded continuously with a pen recorder or measured after 15 or 20 min of treatment.

Oxidative Burst and Ethylene Biosynthesis in Leaf Tissue-- Fully expanded leaves of different plant species were cut in 2-mm slices and floated on H2O overnight. For measuring the oxidative burst, active oxygen species released by the leaf tissue were measured by a luminol-dependent assay (20). Slices were transferred to assay tubes (2-4 slices corresponding to ~20 mg of fresh weight) containing 0.1 ml of H2O supplied with 20 µM luminol and 1 µg of horseradish peroxidase (Fluka). Luminescence was measured in a LKB 1250 luminometer (LKB Wallac, Turku, Finland) for 20 min after the addition of the test solution.

For assaying ethylene production, leaf slices (~50 mg of fresh per assay) were transferred to 6-ml glass tubes containing 1 ml of an aqueous solution of the peptide being tested. Vials were closed with rubber septa and ethylene accumulating in the free air space was measured by gas chromatography after 2 to 2.5 h of incubation.

Reproducibility-- The results shown in the figures represent single experiments that are representative for several independent repetitions.

    RESULTS
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Extracellular Alkalinization in Cultured Tobacco Cells Treated with Preparations from M. lysodeikticus-- Peptidoglycan, an essential cell wall component of all bacteria, acts as one of the PAMPs signaling the presence of Gram-positive bacteria to the innate immune system in animals (1, 10). In initial experiments we tested preparations containing peptidoglycan for induction of extracellular alkalinization in plant cells cultured in liquid medium. Medium alkalinization, occurring as a consequence of altered ion fluxes across the plasma membrane, can serve as a convenient, rapid, sensitive and quantitative bioassay to study elicitor perception by plant cells (16). As a source of peptidoglycan we used preparations from M. lysodeikticus (S. aureus) because lyophilized bacteria and a peptidoglycan fraction are commercially available. Also, as deduced from the genomes of the three fully sequenced strains of S. aureus (M. lysodeikticus) that do not encode proteins resembling flagellin, these preparations should be free of elicitor-active flagellin that could interfere in the assays. No alkalinization was observed in the tomato cells of the line Msk8 after treatment with lyophilized M. lysodeikticus bacteria or the peptidoglycan fraction derived from these bacteria (data not shown). Whereas these negative results confirmed the absence of elicitor-active flagellin they did not provide evidence for a chemoperception system responding to peptidoglycan in the tomato cells. When tested on tobacco cells, however, both preparations of M. lysodeikticus caused rapid and strong medium alkalinization (Fig. 1). As shown in the examples in Fig. 1A, extracellular pH started to increase after a lag of ~3 to 5 min and reached a maximum after ~10 to 15 min. Depending on the cell density and the initial pH of different batches of the cell culture the amplitude of the alkalinization response (Delta pHmax) varied from 1.2 to 2 pH units for lyophilized bacteria and from 0.6 to 1.4 pH units for the peptidoglycan fraction, respectively. In aliquots from a given batch of cells, however, Delta pHmax was highly reproducible and consistently showed a bigger response for the preparation of total bacteria than for the peptidoglycan fraction. The responses of the cells to both preparations of M. lysodeikticus were dose-dependent and lower, nonsaturating doses led to prolonged lag phases, smaller maximal pH increases, and shortened durations of medium alkalinization. The pH change occurring within 15 min (Delta pH15min) of treatment was a steady function of the dose applied and was used as a parameter to compare the relative strength of the two preparations of M. lysodeikticus (Fig. 1B). Half-maximal stimulation was observed with 30 µg/ml of the peptidoglycan (EC50) and <1 µg/ml with the lyophilized bacteria, respectively. Treatment with protease K strongly affected the activity of the bacterial preparation resulting in a 200-fold higher EC50 value (200 µg/ml) but led only to a 3-fold increase for the EC50 value of the peptidoglycan fraction (Fig. 1B). These results provided preliminary evidence for the presence of two distinct elicitor activities in M. lysodeikticus: a nonproteinaceous elicitor in the peptidoglycan fraction and a second, potent, proteinaceous elicitor predominating in the total bacteria preparation. On a per weight basis the proteinaceous factor was more than 100-fold more active than the peptidoglycan factor and further work focused on the characterization of this new protein elicitor.


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Fig. 1.   Extracellular alkalinization of tobacco cells in response to treatment with preparations from M. lysodeikticus. A, alkalinization in response to treatment with 10 µg/ml lyophilized M. lysodeikticus cells or to 100 µg/ml of the peptidoglycan fraction derived from M. lysodeikticus (peptidoglycan). B, effect of protease K treatment on alkalinization-inducing activity of lyophilized M. lysodeikticus bacteria and the peptidoglycan preparation. Different doses of lyophilized M. lysodeikticus bacteria (closed circles), bacteria after pretreatment with protease K (overnight incubation with 1 mg/ml protease K, open circles), peptidoglycan (closed triangles), and peptidoglycan after pretreatment with protease K (open triangles) were added to aliquots of the cell culture and the pH change measured after 15 min (initial pH 4.8).

Purification of an Elicitor-active Protein from M. lysodeikticus and Its Identification as Bacterial Cold Shock Protein-- The elicitor activity, extracted from the crude preparation of M. lysodeikticus (S. aureus), was heat-stable (5 min, 95 °C), passed ultrafilters with a molecular weight cut-off of 10,000, and was inactivated by treatment with trypsin (data not shown), indicating that the elicitor activity was attributable to a peptide or small protein. Activity was purified on a Sephacal C8 reversed phase column (Fig. 2A). In the first chromatography at pH 6.5 activity eluted as a single peak (Fig. 2B). The two fractions containing most of the activity were pooled and rerun on the C8 column at pH 3.5. The peak of activity eluting from this second run correlated with a single peak of A214. Separation by SDS-PAGE (14% (w/v) acrylamide) showed a band migrating with an apparent molecular weight of 7,000 to 9,000 and elicitor activity, detected in eluates of the sliced gel pieces, was found to co-migrate with this band (data not shown). N-terminal sequencing of the protein and sequence information obtained from some of the peptides after tryptic digestion identified the protein as a cold shock protein (CSP). In Fig. 3 the sequence information from the purified protein was aligned with the sequence of the major cold shock protein from M. luteus and a consensus sequence obtained from >150 bacterial cold shock proteins present in the data bank.


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Fig. 2.   Purification of the alkalinization-inducing activity by reversed phase chromatography. An extract from lyophilized M. lysodeikticus bacteria, pre-purified by ion-exchange chromatography as described under "Experimental Procedures," was fractionated on a C8 reversed phase column at pH 6.5. Fractions with highest activity, eluting between 24 and 28 min, were re-chromatographed on the C8 column at pH 3.5. Upper panel shows elution profile (OD280) of the first run at pH 6.5 (10 mM phosphate buffer) and the second run at pH 3.5 (0.1% trifluoroacetic acid). Lower panel shows extracellular alkalinization in tobacco cells (Delta pH15min) induced by aliquots of the fractions eluting in the first (open bars) and second (open bars) runs.


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Fig. 3.   Alignment of bacterial cold shock proteins. Sequence alignment of some CSPs, representative for different bacteria species. Letters indicate positions that differ from the consensus sequence. Consensus sequence and percentage of conservation for the amino acid residues were calculated from >150 bacterial CSP sequences present in the SwissProt data base. Partial sequence for M. lysodeikticus represents information obtained from the purified protein after tryptic digest and Edman degradation of some of the peptides. Csp22 and csp15 denote peptides synthesized according to the consensus sequence.

Identification of the "Cold Shock Domain" (CSD) as the Elicitor-active Epitope-- In attempts to localize the elicitor activity to a particular domain of the protein the purified CSP was subjected to peptide cleavage. Digestion with trypsin, Lys-C, or Glu-C (V8 protease) abolished the activity and did not result in smaller fragments with elicitor activity (data not shown). As in previous work with bacterial flagellin (8) we speculated that plant cells might have a perception system for the most characteristic and most conserved domain of the CSPs. Although these small bacterial proteins show a high overall homology they are particularly conserved in a domain close to the N terminus. Based on the consensus sequence of bacterial CSPs, a 22-amino acid peptide spanning this domain was synthesized (Fig. 3, underlined sequence) and tested for induction of alkalinization in tobacco cells. This peptide, termed csp22, proved even more active than the intact CSP purified from M. lysodeikticus and induced medium alkalinization with an EC50 of ~0.1 nM (Fig. 4).


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Fig. 4.   Elicitor activity of the purified Csp and synthetic peptides spanning the conserved N-terminal domain of bacterial CSPs. Dose-response curves for alkalinization induced by intact Csp (Csp7.4kDa) and synthetic peptides representing 22 (csp22) or 15 (csp15) amino acid residues of the conserved region from bacterial CSPs as indicated in the legend to Fig. 3.

To further delineate the epitope that activates responses in the plant cells, peptides lacking varying numbers of amino acid residues from the N- or C-terminal end were synthesized and assayed for activity in dose-response curves as described above for csp22 and CSP. The amino acid sequences and the EC50 values are summarized in (Fig. 5). Omitting 5 amino acid residues from the N terminus of csp22 reduced activity only slightly (EC50 of ~1.2 nM) but removal of the Lys residue at position 6 showed a much stronger effect (EC50 of ~220 nM) and further trimming by 4 amino acid residues resulted in an inactive peptide. The peptide termed csp15, comprising the 15 amino acid residues central to csp22, was nearly as active as csp22 (EC50 of 0.3 nM) and served as a core peptide for testing structural analogues with replacements of single amino acid residues with alanine. Csp15-Ala3, csp15-Ala4, csp15-Ala8, and csp15-Ala12 all exhibited at least 1000-fold reduced activity compared with csp15. Csp15-Ala10 was inactive even at the highest concentration of 100 µM tested (Fig. 5). In contrast, substitution of Phe at position 10 with a Tyr residue resulted in a peptide with full activity in the tobacco cells (Fig. 5). Among the peptides with single substitutions with Ala only csp15-Ala7 showed no significant decrease in activity. Interestingly, the Glu at this position also shows the least conservation in the different sequences of bacterial CSPs (Fig. 3).


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Fig. 5.   Alkalinization-inducing activity of csp-related peptides. EC50 values were determined from dose-response curves obtained for the different peptides. Specific activity relative to the activity of the most active peptide csp22 (hatched bar, EC50 of 0.1 nM). Logarithmic scaling was used to indicate residual activity in some of the peptides. No activity could be detected with peptides denoted with asterisks (relative activity <10-5).

The three-dimensional structure has been determined for the major bacterial cold shock proteins CspB from Bacillus subtilis (Molecular Modeling Database number 3622; Protein Data Bank number 1CSP) (21) and CspA (CS7.4) from E. coli (Molecular Modeling Database number 1677; Protein Data Bank number 1MJC) (22). CspB forms a dimer whereas CspA occurs as monomer. Besides this difference of dimerization the structures of both proteins are very similar, forming compact beta -barrel structures built up from five antiparallel beta -strands with connecting turns and loops. Fig. 6 shows models (secondary structure and a three-dimensional ribbon model) of the molecular structure of CspB, highlighting the domain spanned by the csp15 peptide. Clearly, elicitor activity can be attributed to the domain formed by antiparallel strands beta 1 and beta 2 and the loop L1. This domain includes a RNA-binding motif known as RNP-1 (also termed RNP-CS) and exposes a cluster of aromatic and basic side chains to the surface of the protein. An analysis using site-directed mutagenesis of CspB from B. subtilis has demonstrated that these conserved residues are essential for the interaction of the protein with nucleic acids (23). In Table I, these single amino acid replacements and their effects on nucleic acid binding were compared with the corresponding amino acid changes in csp15 and their effects on elicitor activity in tobacco. All the substitutions in csp15 that correspond to substitutions leading to strong or complete reduction in affinity of CspB for nucleic acids exhibited strongly reduced elicitor activity in tobacco cells (higher EC50 values). The substitution of Phe by Tyr at the position that corresponds to residue 10 in csp15 did not affect affinity of CspB for nucleic acids and also did not alter elicitor activity.


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Fig. 6.   Structure of bacterial CSPs. A, schematic scheme for secondary structure of CspB from B. subtilis from Schindelin et al. (21) with domains spanned by csp15 (hatched part) and the RNA-binding motifs RNP-1 (KGFGFITP) and RNP-2 (VFVHF) indicated. B, structure of CspB monomer (Molecular Modeling Database number 3622; Protein Data Bank number 1CSP (21)) drawn with WebLab ViewerLite (Molecular Simulations Inc., Cambridge, UK) with side groups exposed in the domain spanned by csp15.

                              
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Table I
Comparison of mutations in cspB affecting DNA-binding and single amino acid changes in csp15 on elicitor activity

Activity of Peptides Representing Homologous Domains Occurring of Proteins from Plants and Animals-- Csp-related proteins are common to all eubacteria and they usually form a small family of proteins that include both cold-inducible and noninducible members (24). The domain containing the RNP-1 motif is conserved also in many eukaryotic proteins that bind to RNA or DNA. Examples for proteins with this so-called CSD include human and animal transcription factors recognizing the Y-box sequence and glycine-rich RNA-binding proteins occurring in plants (see Supplementary Materials for gene structure and alignment with bacterial CSPs). Peptides corresponding to the homologues of a human Y-box protein and a Gly-rich protein from Nicotiana sylvestris were synthesized and tested for activity. The csp15 homologue from the human Y-box protein was inactive whereas the peptide representing N. sylvestris sequence induced responses with an EC50 of 300 nM and was thus ~1000-fold less active than the csp15 representing the bacterial sequence (Fig. 5).

Responses Induced by CSP in Different Plant Species-- We examined cell cultures derived from other plant species for alkalinization in response to CSP-related elicitors. Responses with characteristics similar to the ones of the tobacco line 275N were observed also with a second line of tobacco, originating from a plant of the variety SR1, with a cell line derived from potato and a cell culture from L. peruvianum (data not shown). In contrast, no responses could be detected in the cell culture line msk8, originally derived from a cross of Lycopersicon esculentum with L. peruvianum, and in cell lines from A. thaliana and rice (data not shown). Negative results with particular lines of cell cultures do not allow concluding on the absence of a perception system in the corresponding plant species because this perception system might be not expressed or might have been lost during the years of growth in vitro.

Induced release of active oxygen species, an oxidative burst, and increased biosynthesis of the stress hormone ethylene are responses characteristic for plants under attack by pathogens or treated by elicitor preparations (6, 25). We used these responses to monitor responsiveness toward CSP-derived elicitors in leaf tissues from different plant species. As exemplified in Fig. 7 for leaf tissue from tomato, a rapid, significant increase in ethylene biosynthesis and in active oxygen species was observed after treatment with csp15 but not after treatment with the same dose of csp15-Nsyl. Similarly, clear CSP-dependent induction of ethylene biosynthesis and oxidative burst was observed in tobacco and several other solanaceous plants including potato (Solanum tuberosum), Solanum dulcamara, Scopolia carniolica, and Mandragora officinarum. In contrast, no response could be detected from leaf tissue and cell cultures of A. thaliana, cucumber, and rice. Also, no signs of a hypersensitivity response could be detected after injection of CSP peptides to leaves of tobacco or tomato (data not shown). In summary, a perception system for CSP-related elicitors is common to solanaceous plants but has not yet been found outside of this plant family.


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Fig. 7.   Induction of ethylene biosynthesis and oxidative burst in tomato leaf tissue. A, ethylene biosynthesis in tomato leaf slices treated for 2 h with 1 µM csp15 or 1 µM csp15-Nsyl as indicated. Bars and error bars show mean ± S.D. of n = 4 replicates. B, luminescence of leaf slices in a solution with luminol and peroxidase after treatment with 1 µM concentrations of csp15 or csp15-Nsyl as indicated. Light emission at the very beginning of the experiments is caused by phosphorescence of the green tissue.

The Inactive Peptide csp15-Ala10 Antagonizes Elicitor Activity of CSP-- Peptides lacking either 4 amino acids from the C-terminal part (csp11) or 6 amino acids from the N-terminal part spanned by csp15 lacked activity even when applied in micromolar concentrations (data not shown). No response was observed also by application of these two peptides in combination (data not shown). Truncated forms of the biologically active peptides systemin and flg22 were previously found that showed characteristics of competitive antagonists for the respective nontruncated agonistic peptides (26-28). No antagonistic activity could be observed for the two truncated CSP peptides described above (data not shown). In contrast, csp15-Ala10, also inactive as agonist (Fig. 5), did exhibit antagonistic activity and suppressed responses induced by csp15 (Fig. 8). When added concomitantly with 3 nM csp15, a concentration of 3 µM strongly inhibited induction of the alkalinization response (Fig. 8A, "0 min"). Complete inhibition was observed when csp15-Ala10 was added 30 s before the agonist but progressively weaker effects were observed when the antagonist was added after the agonist and an addition after 3.5 min remained without apparent effect on the ongoing response. Inhibition by csp15-Ala10 was specific for CSP-derived elicitors and was not observed with unrelated elicitors like flagellin and chitin fragments (data not shown). Inhibition of CSP-related activity by csp15-Ala10 was competitive and could be overcome by increasing concentrations of active peptide or intact CSP. As shown in the example in Fig. 8B, this resulted in an increase of the EC50 for the CSP containing preparation of M. lysodeikticus bacteria from 1 µg/ml in the absence of the antagonist, to 20 µg/ml in the presence of 3 µM csp15-Ala10, respectively. In contrast, no shift in dose response was observed with the peptidoglycan fraction (Fig. 8B). These results confirm predominance of the CSP-related elicitor in the crude bacterial preparation and the presence of an activity unrelated to CSP in the peptidoglycan preparation.


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Fig. 8.   The peptide csp15-Ala10 acts as competitive suppressor of csp-related elicitors. A, extracellular alkalinization in suspension-cultured tobacco cells treated with 3 nM csp15 and 3 µM csp15-Ala10 at the time points indicated (slanted arrows). Extracellular pH of untreated cells was 4.9 and addition of 3 µM csp15-Ala10 alone did not cause significant pH changes. B, alkalinization induced by different doses of M. lysodeikticus (circles) and peptidoglucan preparation (triangles) in cells without pretreatment (closed symbols) or cells pretreated for 3 min with 10 µM csp15-Ala10 (open symbols). C and D, alkalinization induced by the harpin-containing preparation messenger (1 µg/ml, C) and by a crude extract from A. tumefaciens (1 µl/ml, D) in cells without pretreatment or cells pretreated for 3 min with 3 µM csp15-Ala10 as indicated.

Tobacco cells were found to respond to crude extracts from all bacterial species tested (n > 20). The antagonist csp15-Ala10 could serve as a diagnostic tool to test for the presence of csp-related activity. For example, cells responded with strong alkalinization when treated with messenger, an extract from E. coli expressing transgenic harpin from Erwinia amylovora (Fig. 8C). Interestingly, at least at limiting doses of messenger applied, activity was fully antagonized by csp15-Ala10. This indicated that a CSP-related stimulus and not harpinEa, previously reported to act as an inducer of alkalinization in tobacco (29), was the activity predominating in this preparation. As shown for the example of an extract from A. tumefaciens in Fig. 8D, csp15-Ala10 antagonized also the alkalinization inducing activity of crude extracts from the plant-associated species A. tumefaciens, R. meliloti, and X. campestris, extracts that were previously found to be devoid of elicitor-active flagellin (8). In summary, these results demonstrate the common occurrence of CSP-related elicitor activity in extracts from different, if not all, bacteria.

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Various types of living bacteria as well as preparations of heat-killed bacteria can trigger rapid responses in plant cell cultures and defense responses in intact plant tissues (20, 30, 31). Flagellin (8) and lipopolysaccharides (32) have been identified as common bacterial determinants or PAMPs that act as elicitors of defense responses in plant cells. In this report we add CSPs as a further bacterial PAMP that acts as an elicitor of defense responses in plants.

Cold shock proteins were named based on the original observation that rapid cooling with a Delta T of >-10 °C (cold shock) induces accumulation of specific proteins in many bacterial species. The major CSPs are small, ~7.4 kDa proteins that belong to a family of highly conserved proteins commonly occurring in all bacteria. At least some members of this family are (also) constitutively expressed or are induced under stress conditions different from cold shock (24). For example, the family of CspA-like proteins in E. coli consists of eight members (CspA through CspH) and only CspA, CspB, and CspG are cold-inducible. Thus, despite their name, members of the CSP family occur also in bacteria not subjected to a cold shock treatment. CSPs are implicated in various cellular processes, including cellular growth and adaptation to low temperatures, nutrient stress, and stationary phase. CSPs bind to nucleic acids and appear to function as RNA chaperones and anti-terminators of translation (33).

A shift to low temperature induces also a set of specific proteins in plants (34). Some of these cold-regulated proteins are small hydrophilic proteins of 6.6 kDa (35), like the major bacterial CSP but they are nonhomologous in sequence, and their physiological function in the cold acclimation process remains unknown. However, many eukaryotes including plants and animals have proteins with a nucleic acid-binding domain that shows a strikingly high homology and similar RNA-binding properties to bacterial CSPs (36). It is this universally conserved domain, also termed CSD, that contains the RNP-1 motif and the epitope found to act as elicitor of tobacco cells.

The elicitor activity of bacterial CSPs could be localized to a stretch of ~15 amino acid residues that forms a loop with two antiparallel beta -strands and exposes a series of aromatic and basic amino side chains to the surface of the protein. It is this epitope that exhibits highest conservation between the different bacterial CSPs and, as has been demonstrated by site-directed mutagenesis of CspB from B. subtilis, is essential for the interaction of the protein with nucleic acids (23). Most notably, synthetic peptides with amino acid sequences reflecting the changes leading to reduced or abolished binding to nucleic acids of CspB were also strongly affected in elicitor activity (Table I). This strong correlation raises the question of whether some sort of nucleic acid might be involved in the perception process by the plant cell. However, intact CSPs and the csp-derived peptide elicitors have characteristics of molecules that are not permeable for membranes and the first responses to subnanomolar concentrations of csp-derived elicitors occur after a lag phase of less than 2 min. These characteristics rather suggest a chemoperception system with a specific, high affinity primary interaction site in the apoplast, most likely the plasma membrane, of the plant cells. The strong correlation of nucleic acid binding and elicitor activity might thus reflect evolution of a chemoperception system directed at a particular surface epitope of the bacterial CSPs that is under a high selective pressure for retaining functionality of the protein.

Perception of csp-related elicitors resembles the chemoperception system for flagellin-derived elicitors studied before (8, 28). In both cases, elicitor activity could be attributed to an epitope of ~15 amino acids representing the most conserved part of the respective protein. Both elicitors are active at subnanomolar doses and activity is highly dependent on the genuine amino acid sequence of the conserved domain. "Mutational" analysis using structural analogues of the elicitors allowed identification of peptides lacking elicitor activity but exhibiting properties of competitive antagonists. Perception of flagellin was shown to involve a specific, high affinity binding site and the membrane-bound receptor kinase FLS2 (9, 37). A model involving a two-step process for receptor activation was proposed to explain the effects of agonistic and antagonistic peptides (28). At present, experiments that directly demonstrate a receptor site for the csp elicitors are lacking. Nevertheless, CSP- and flagellin-derived elicitors induce the same set of responses with similar kinetics, indicating a similar, receptor-mediated process for both elicitors. Thus, we hypothesize that csp perception occurs via a csp receptor that functions in a manner similar to the receptor for flagellin. Activation of this putative CSP receptor might also involve two consecutive steps with binding of the elicitor as a first step and activation of the receptor as a second step. An aromatic side chain on residue 10 of csp15, Phe in csp15, or Tyr in csp15-Tyr10, appears necessary for this second step to activate the receptor. The antagonist csp15-Ala10, apparently, does not undergo the second, locking step and interacts with the receptor site in a more readily reversible manner. This could explain the high excess of antagonist csp15-Ala10 over csp15 required to block elicitor action completely and the apparent inefficiency of the antagonist when applied subsequent to the csp agonists (Fig. 8).

Proteins with a cold shock domain comprising the RNP-1 motif are conserved also in eukaryotes and have been identified also in genes of A. thaliana and N. sylvestris. Although clearly homologous, the sequences corresponding to the elicitor-active epitope show some differences in comparison to the bacterial consensus. The synthetic peptide csp15-Nsyl, representing the least divergent form of this domain in genes known from N. sylvestris, indeed did show some activity in the bioassay with tobacco cell. However, the specific activity of this peptide was ~1000-fold lower than that of csp15 representing the bacterial epitope. A lower specific activity could be counterbalanced by the presence of high local concentrations of the stimulus. In initial attempts with extracts of tobacco plants or cells from tissue culture we failed to detect factors with CSP-like activity in bioassays (data not shown). Thus, at present, we do not have evidence for endogenous factors stimulating tobacco via the CSP perception system described in this report. Endogenous factors of tobacco, capable of stimulating medium alkalinization in cultured cells, have recently been described (38) but these peptidic factors show no apparent homology to CSPs.

Bacterial CSPs are molecules highly characteristic for bacteria in general and could thus serve as PAMPs signaling the presence of bacteria to the plant cells. An obvious problem with this hypothesis that is the localization of these proteins, which are generally assumed to function in the cytoplasm of the bacteria. So far, CSPs have not been reported to be exported or exposed to the surface by intact bacteria and, consequently, CSPs are probably not directly detectable by a chemoperception system assumed to reside on the surface of the plant cells. Further studies will be required to test whether CSPs are released from bacteria during invasion of their plant hosts. A release could be based on a bacterial export system activated in the course of the infection process, or it could result from bacterial or plant processes causing a general leakiness of the bacteria. As demonstrated for bacteria under mild osmotic shock (39), leakiness of bacteria leading to release of small cytoplasmic proteins might be more common than suggested by studies under optimal media conditions used to grow bacterial cells in the laboratory. Precedence for cytoplasmic components of bacteria that act as PAMPs and stimulate the innate immune responses via Toll-like receptors in animals include "nonsecreted" components such as the heat shock protein HSP60 (40) and bacterial DNA (12). Bacterial DNA is recognized via its content of nonmethylated CpG oligonucleotides and this PAMP was successfully applied as a potent immunostimulatory factor (41, 42) but the process leading to release of the DNA from the bacteria has not been elucidated. Similarly, no process that secretes HSP60 from intact bacterial cells has been described. HSP60 is well conserved from microbes to humans and HSP60 from both mammalian and microbial sources can trigger inflammatory responses via Toll-like receptor 4 (40), suggesting that Toll-like receptor 4 may detect both endogenous and exogenous ligands as alarm signals. Exposure of endogenous HSP60 to the Toll-like 4 receptor could be envisaged to occur via release from wounded or injured cells and perception by the receptor on different, intact cells. As discussed above, it remains to be seen whether the chemoperception system for CSPs described in this report might similarly react to both endogenous and exogenous ligands.

Peptidoglycan consists of a glycan backbone with alternating beta 1-4-linked residues of N-acetyl-D-glucosamine and muramic acid and forms the major component of the cell wall in Gram-positive bacteria. Peptidoglycans, sensed by a family of peptidoglycan recognition proteins that are conserved from insects to humans (43), are important PAMPs for the innate immunity of animals. The peptidoglycan preparation of M. lysodeikticus also triggered elicitor responses in tobacco cells. We have not yet characterized this activity in detail and cannot exclude that it is because of a minor component or a "contaminant" of the peptidoglycan fraction. Nevertheless, this nonproteinaceous factor is perceived as a quality of stimulus distinct from CSPs and provides evidence for a further bacterial PAMP with elicitor activity in tobacco cells. Thus, similar to the chemoperception systems for a variety of fungal-derived PAMPs, perception of bacteria by plant cells appears not to depend on a single bacterial factor but rather involves several different factors, including at least flagellin (8), lipopolysaccharides (32), peptidoglycan, and CSP. This redundancy, characteristic also for the recognition mechanisms in the innate immune system of animals, points at possible difficulties with approaches to demonstrate a direct physiological role for any particular of these chemoperception systems for plant defense. Inhibition or knockout of only one of the systems might be without a strong effect on the overall recognition system. In contrast to bacterial "avirulence factors," which act as elicitors that are specific and unique for a particular pathogen, the structures recognized as PAMPs are essential or "vital factors" for the functioning of the bacterial organisms in general and cannot easily be changed, removed, or mutated for probing their role in plant defense.

In summary, our results provide evidence for novel bacterial elicitors, cold shock protein, for which tobacco and other Solanaceae have evolved specific and sensitive chemoperception systems. The accuracy and sensitivity of the perception system for the CSP domain comprising the RNP-1 motif detailed in this report indicate a receptor mechanism involving a high affinity binding site on the surface of the plant and should provide the basis for further work to identify the protein acting as pattern recognition receptor for CSP.

    ACKNOWLEDGEMENTS

We thank Franz Fischer (Friedrich Miescher-Institute, Basel) for the synthesis of various peptides, Renate Matthies, Daniel Hess, and Jan Hofsteenge (Friedrich Miescher-Institute) for protein sequencing services and mass spectrometry, and Martin Regenass for maintaining the cell cultures and technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplementary figure.

Dagger To whom correspondence should be addressed. Fax: 41-61-697-45-27; E-mail: Felix@fmi.ch.

Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M209880200

    ABBREVIATIONS

The abbreviations used are: PAMP, pathogen-associated molecular pattern; CSP, cold shock protein; CSD, cold shock domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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