Felix et al. - 1

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 Felix1 and Thomas Boller Friedrich Miescher-Institute, P.O. B. 2543, CH-4002 Basel, Switzerland

Running title: Bacterial cold-shock proteins as elicitors in tobacco

Keywords : Cold shock protein, Cold shock domain, peptidoglycan, elicitor activity, PAMP,

Pattern recognition receptor (PRR)

1 To whom correspondence should be addressed.

E-mail: Felix@fmi.ch; fax: +41 61 697 45 27

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 5, 2002 as Manuscript M209880200 by guest on February 18, 2020

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Summary

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 aro-

matic 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 non-inducible 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.

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Introduction

A key aspect of active defense against invading microbial pathogens is the ability to discrimi-

nate 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 which confer immunity to particular races of plant pathogens. Several

of these resistance genes were shown to be involved in the chemoperception of factors specifi-

cally 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 reminis-

cent 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 oligosaccha-

rides, 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 com-

mon 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 (LRR) (9). Bacterial flagellin has

recently also been identified as one of the ‘pathogen associated molecular patterns’ (PAMPs’)

that activate the innate immune system of humans and animals (10) via the toll-like receptor 5

(TLR5) (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 per-

ceived, the characteristics of pattern recognition receptors (PRRs) 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 acti-

vated elicitor responses in the tomato cells used in our previous experiments. Extracts from

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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, redun-

dancy 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 chemoper-

ception 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 con-

taining 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 (Staphilococcus 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 lyophilized M. lysodeikticus bacteria proved far more potent

as 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.

Experimental Procedures

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Materials

Peptides were synthesised by F. Fischer (Friedrich Miescher-Institute, Basel) or by Bio-

Synthesis Inc. (Lewisville, Tx, USA). Peptides were dissolved in H2O (stock solutions of 1 to

10 mM) and diluted in a solution containing 0.1% BSA 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, Braun-

schweig, 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 to

10 min and removing of bacterial debris by centrifugation. Lyophilized bacteria of M.

lysodeikticus (Sigma, St Louis, MO, USA) and the peptidoglycan fraction from M.

lysodeikticus (Fluka, Buchs, Switzerland) applied as suspensions in H2O. The bacterial

preparation ‘messenger’ was obtained from EDEN Bioscience (Bothell, WA, USA).

Purification of elicitor from Micrococcus lysodeikticus

Elicitor activity was purified from lyophilized preparation of M. lysodeikticus (Sigma). Ten g

of the lyophilizate were suspended in 100 ml H2O and heated for 10 min at 95° C. After

centrifugation (30 min 10’000 x 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, Maidstone, England) 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

Sephasil C8 reversed phase column (Pharmacia, Uppsala, Sweden) at pH 6.5 (10 mM phos-

phate buffer pH 6.5 as solvent A and 80 % acetonitrile / 20 % phosphate buffer as solvent B).

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The two fractions containing highest elicitor activity were pooled, pH adjusted to 3.5 and rerun

on Sephasil C8 reversed phase column at pH 3.5 ( 0.1 % TFA in H2O at pH 3.5 as solvent A

and 80 % acetonitrile / 20 % H2O with 0.1 % TFA as solvent B).

Plant cell cultures

The tobacco (Nicotiana tabacum L.) cell culture line 275N, originally derived from pith tissue

of a Havanna 425 plants, was maintained and subcultured as described before (15) in a

Murashige-Skoog based medium. Cells were maintained as suspension cultures and were used

4- to 10-days after subculture for experiments. Cell cultures of tomato (“line Msk8“, (16)),

potato (17), Lycopersicon peruvianum (18) and Arabidopsis 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 fresh weight) containing 0.1 ml of H2O supplied with 20 µM

luminol and 1 µg 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.

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For assaying ethylene production, leaf slices (~50mg 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 which are representative

for several independent repetitions.

Results

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 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 (Staphylococcus

aureus) since lyophilized bacteria and a peptidoglycan fraction are commercially available.

Also, as deduced from the genomes of the three fully sequenced strains of Staphylococcus

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. lysodeik-

ticus bacteria or the peptidoglycan fraction derived from these bacteria (data not shown). While

these negative results confirmed the absence of elicitor-active flagellin they did not provide

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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 (Figure 1). As shown in the examples in Figure 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 (∆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, ∆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,

non-saturating doses led to prolonged lag phases, smaller maximal pH-increases and shortened

durations of medium alkalinization. The pH-change occurring within 15 min (∆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 (Figure 1B). Half-maximal stimu-

lation was observed with 30 µg/ml of the peptidoglycan (EC50) and <1 µg/ml with the lyophi-

lized bacteria, respectively. Treatment with protease K strongly affected the activity of the bac-

terial 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 (Figure 1B). These results provided

preliminary evidence for the presence of two distinct elicitor-activities in M. lysodeikticus: a

non-proteinaceous 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 focussed on the characterization of this new protein elicitor.

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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 (Staphylo-

coccus aureus), was heat-stable (5 min, 95°C), passed ultra-filters with a molecular weight cut-

off of 10 kDa 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

Sephasil C8 reversed phase column (Figure 2A). In the first chromatography at pH 6.5 activity

eluted as a single peak (Figure 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 OD214. Separation by SDS-PAGE (14 % (w/v) acrylamide)

showed a band migrating with an apparent molecular weight of 7 to 9 kDa 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

Figure 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.

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 puri-

fied CSP was subjected to peptide cleavage. Digestion with trypsin, Lys-C or Glu-C (V8 prote-

ase) 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 (Figure 3, underlined

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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 (Figure 4).

To further delineate the epitope that activates responses in the plant cells, peptides lacking

varying numbers of amino acid residues from the N-terminal or C-terminal end were synthe-

sized 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 (Figure 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 to csp15. Csp15-

Ala10 was inactive even at the highest concentration of 100 µM tested (Figure 5). In contrast,

substitution of Phe at position 10 with a Tyr residue resulted in a peptide with full activity in

the tobacco cells (Figure 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 least conservation in the different sequences of bacterial CSPs’ (Figure 3).

The three dimensional structure has been determined for the major bacterial cold shock

proteins CspB from B. subtilis (MMDB Id: 3622 PDB Id: 1CSP , (21) and CspA (CS7.4) from

E. coli (MMDB Id: 1677 PDB Id: 1MJC (22). CspB forms a dimer while CspA occurs as

monomer. Besides this difference of dimerization the structures of both proteins are very

similar, forming compact β-barrel structures built up from five antiparallel β-strands with

connecting turns and loops. Figure 6 shows models (secondary structure and a 3-D ribbon

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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 the antiparallel

strands β1 and β2 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 1, these single amino acid replacements and their effects on nucleic

acid binding were compared to 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.

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 non-inducible members (24). The domain con-

taining the RNP-1 motif is conserved also in many eukaryotic proteins that bind to RNA or

DNA. Examples for proteins with this so-called cold shock domain (CSD) include human and

animal transcription factors recognizing the Y-box sequence and glycine rich RNA-binding

proteins occurring in plants (see supplementary data for gene structure and alignment with bac-

terial CSPs’). Peptides corresponding to the homologues of a human Y-box protein and a Gly-

rich protein from N. sylvestris were synthesized and tested for activity. The csp15 homologue

from the human Y-box protein was inactive whereas the peptide representing N. sylvestris

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sequence induced responses with an EC50 of 300 nM and was thus ~1000-fold less active than

the csp15 representing the bacterial sequence (Figure 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 Lycopersicon peruvianum

(data not shown). In contrast, no responses could be detected in the cell culture line msk8,

originally derived from a cross Lycopersicon esculentum with L. peruvianum, and in cell lines

from A. thaliana and rice (data not shown). Negative results with particular lines of cell cul-

tures do not allow concluding on the absence of a perception system in the corresponding plant

species since 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

towards CSP-derived elicitors in leaf tissues from different plant species. As exemplified in

Figure 7 for leaf tissue from tomato, 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 offici-

narum. In contrast, no response could be detected leaf tissue and cell cultures of A. thaliana,

cucumber and rice. Also, no signs of a hypersensitivity response (HR) could be detected after

injection of CSP-peptides to leaves of tobacco or tomato (data not shown).

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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.

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 concen-

trations (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 non-truncated 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 (Figure 5), did exhibit antagonistic activity and

suppressed responses induced by csp15 (Figure 8). When added concomitantly with 3 nM

csp15, a concentration of 3 µM strongly inhibited induction of alkalinization response (Figure

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 Figure 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 (Figure 8B). These results confirm predominance of the CSP-related

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elicitor in the crude bacterial preparation and the presence of an activity unrelated to CSP in the

peptidoglycan preparation.

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

(Figure 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 pre-

dominating in this preparation. As shown for the example of an extract from Agrobacterium

tumefaciens in Figure 8D, csp15-Ala10 antagonized also the alkalinization-inducing activity of

crude extracts from the plant-associated species Agrobacterium tumefaciens, Rhizobium

meliloti and Xanthomonas 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.

Discussion

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 determi-

nants or PAMPs’ that act as elicitors of defense responses in plant cells. In this report we add

CSPs’ as further bacterial PAMP which acts as an elicitor of defense responses in plants.

Cold shock proteins were named based on the original observation that rapid cooling with

a ∆T of >-10 °C (cold shock) induces accumulation of specific proteins in many bacterial

species. The major CSPs’ are small, ~7.4 kD, proteins that belong to a family of highly con-

served proteins commonly occurring in all bacteria. At least some members of this family are

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(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 kD (35), like the major bac-

terial CSP but they are non-homologous in sequence, and their physiological function in cold

acclimation process remains uknown. 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 cold-shock domain (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 β-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 con-

servation 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 1). This strong correlation raises the question 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-de-

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rived 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 plasmamembrane, 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-de-

rived elicitors studied before (8;28). In both cases, elicitor activity could be attributed to an

epitope spanning 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 de-

pendent on the genuine amino acid sequence of the conserved domain. ‘Mutational’ analysis

using structural analogs of the elicitors allowed identification of peptides lacking elicitor

activity but exhibiting properties of competitive antagonists. Perception of flagellin was could

be 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

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block elicitor action completely and the apparent inefficiency of the antagonist when applied

subsequent to the csp agonists (Figure 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, repre-

senting 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 pep-

tide was ~1000-fold lower than that of csp15 representing the bacterial epitope. A lower spe-

cific 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 alka-

linization in cultured cells, have recently been described (38) but these peptidic factors show no

apparent homology to CSPs’.

Bacterial CSPs’ can be regarded as molecules that are 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 the hypothesis that bacterial CSPs’ serve as PAMP signaling the

presence of bacteria to the plant cells 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

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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’s for cytoplasmic components of bacteria that act

as PAMPs’ and stimulate the innate immune responses via toll-like receptors in animals

include ‘non-secreted’ components such as the heat shock protein HSP60 (40) and bacterial

DNA (12). Bacterial DNA is recognized via its content of non-methylated CpG

oligonucleotides and this PAMP was successfully applied as a potent immuno-stimulatory

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 TLR4 (40),

suggesting that TLR4 may detect both endogenous and exogenous ligands as alarm signals.

Exposure of endogenous HSP60 to the TLR4 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 β-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 (PGRPs’) 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 due to a minor component or a ‘contaminant’ of the

peptidoglycan fraction. Nevertheless, this non-proteinaceous factor is perceived as a quality of

stimulus distinct from CSPs’ and provides evidence for a further bacterial PAMP with elicitor-

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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 knock-out of only one of the systems

might be without 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 fora novel bacterial elicitors, cold shock

protein, for which tobacco and other Solanaceae have evolved specific and sensitive chemo-

perception 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 pep-tides, 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.

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Table 1. comparison of mutations in cspB affecting DNA-bindinga and single amino acid changes in csp15b on elicitor activity

substitution in CspB substitution in

csp15b fold-reduction of affinity for oligonucleotidea

fold-increase for EC50 b

CspB (wt) csp15 1 1 K7Q csp15-Ala2 3 1000 W8A csp15-Ala3 5 1000 F15A csp15-Ala10 inactive >10000 F17A csp15-Ala12 inactive 2000 F15Y csp15-Tyr10 1 1 F17Y 6 not tested a effect of site-directed point mutations in CspB on the binding of single stranded DNA

oligonucleotides containing Y-box motif. Data from Table 1 in Schröder et al. (23). b csp15 comprises amino acids 5 to 20 in CspB. EC50-values for the induction of the

alkalinization response from Fig. 5.

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Figure 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 pH-change measured after 15 min (initial pH 4.8).

M. lysodeikticus, µg/ml10-2 10-1 100 101 102 103

∆pH

at 1

5 m

in

0.0

0.4

0.8

1.2

1.6

2.0 bacteria

" protease digest

" protease digest

peptidoglycan

control

M. lysodeikticus

Time, min0 10 20 30 40 50 60

6.4

6.2

6.0

5.8

5.6

5.4

5.2

5.0

4.8

peptidoglycan

Extra

cellu

lar p

H

A

B

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Figure 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 in methods, 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 first run at pH 6.5 (10 mM phosphate buffer) and of second run at pH 3.5 (0.1 % TFA).

Lower panel shows extracellular alkalinization in tobacco cells (∆pH15min) induced by

aliquots of the fractions eluting in the first run (open bars) and second run (open bars)

Sephasil C8 reversed phase

Time, min0 10 20 30 40 50 60

OD

280n

m

0.00

0.05

0.10

0.15

0.20

60% B

100% B1st run, pH 6.5

2nd run, pH 3.5

∆pH

max

0.0

0.5

1.0 1st run, pH 6.5 2nd run, pH 3.5

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Figure 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 database. Partial sequence for M.

lysodeikticus represents information obtained from the purified after tryptic digest and

Edman degradation of some of the peptides. Csp22 and csp15 denote peptides

synthesized according to the consensus sequence.

mekgt*****na******c****ge*i*a*y*t**m**yrt*ka**s*q*dvh**pk*nh*svivpveveaava meigi*****na******sa**v*a*i*a*y*v*em**yr**ka****q**vlhsdk*sh*tkiipi*dtqe matgt*************aqd**gp*****y***nat**r****n*v*n*dvth*e.****e**spa masgt*****s*******aqd**gp***a*y*n*naq*yre*q***a*t*d*t**qk****e*i*pa skikgn*****es******t**d*s***********tn***t*a***r*e***tn*ak**s****ia* akikgq*****es******t*ad*s************n***t*a***n*e***qd*qk**a*v***ai sgkmtgi******d******t*dd*s***********n**y***d*******t*es*ak**a*g***s* sgkmtgi******d******t*dd*s***********n**y***d*******t*es*ak**a*****s* msnkmtgl******d******s*vd*s***********n*nyrt*f*****t*s*es*ak**a****iitd maqgt*************t*dds*g*****y*e**tg***t*d*nar*q***g**ak****tg**lv meqgt**************r*n**.**********s******d*******dve**a**a*****q*a mnmeqgt**************r*n**.*****************d***a*t*dvee*q********q*a mqgr**************r*d**.**********q**y*******q*e*d*vd*a********v** mqrgk*****n***y****v***s.******t****e***t*****e*****v***********v** mqrgk*****n***y****v***s.******t****e*********e*****v***********v** mqngk*****n********v****.******t**e***y*******e*****ve*******s**v** mlegk*****s********v**q*.***********e***t*****a*****ve************e* i****s********v**q*.***********e***c*****a*****ve********* *****s*****l**v**q*.***********e***t***s*a*****ve********* mqtgk*****g********v***e.***************t*****e*****vd************n mngk*****n********m**se.**********s**y*a*****e***d*te**********a** mavgt*********y***a**dnsa***********n***e*q*ndrve**t*dgpk*l******** avgtv wfnaek elqendrvefetqdgpk

E. coli cspDH. influenzae cspD

Str. clavuligerus csp7Str. coelicolor cspF

E. coli cspEE. coli cspCE. coli cspA

S. typhimurium cspAE. coli cspB

Arthr. globiformisB. subtilis cspC

L. monocytogenes csp1B. cereus cspC

B. caldolyticus cspBB. stearothermophilus cspB

B. subtilis cspDB. subtilis cspB

B. globisporus cspBB. globigii cspBB. cereus cspDB. cereus cspBM. luteus cspA

M. lysodeikticus (purified protein)

CSP consensus ---TGTVKWFNAEKGFGFITPDGGDKDVFVHFSAIQGDGFKSLEEGQKVSFEI-QGNRGPQAANVTKLA ______________________

csp22 AVGTVKWFNAEKGFGFITPDGG identity : x 60-90%, x >90% csp15 VKWFNAEKGFGFITP

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Figure 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 Fig. 3 .

Concentration, nM10-2 10-1 100 101 102 103

∆pH

at 1

5 m

in

0.0

0.2

0.4

0.6

0.8

1.0

1.2

csp15

Csp7.4kDa

csp22

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Figure 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).

N-term

relative activity, log scale10

-3 -2 -1 010 10 1010

-410

-5

VKWFNAEKGFG

VKWFNAEKGFGF

VKWFNAEKGFGFI

VKWFNAEKGFGFITP

csp22 AVGTVKWFNAEKGFGFITPDDG

KWFNAEKGFGFITPDDG

WFNAEKGFGFITPDDG

EKGFGFITPDDG

csp15 VKWFNAEKGFGFITP

VKAFNAEKGFGFITP

VKWANAEKGFGFITP

VKWFNAAKGFGFITP

VKWFNAEAGFGFITP

VKWFNAEKGAGFITP

VKWFNAEKGFGAITP

csp15Ybox VKWFNVRNGYGFITP

csp15Nsyl VKWFSDQKGFGFITP

VKWFNAEKGYGFITP

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N

C

2

46

54 57

65

6263

β1

β3

β4β5

V

KWFNADK

GFGFITP

β2

csp15

VFV

HF

RNP-2 : VFVHFRNP-1 : KGFGFITP

A B

Figure 6. Structure of bacterial CSPs’

(A) Schematic scheme for secondary structure of CspB from B. subtilis from Schindelin et al.

(21) with domain spanned by csp15 (hatched part) and the RNA-binding motifs RNP-1 and

RNP-2 indicated.

(B) Structure of CspB monomer (MMDB Id: 3622 PDB Id: 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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5 min

10 uV (RLU)

control

csp15-Nsyl

csp15

A

B

Figure 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 and standard deviation 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.

Ethylene, nmol / (g*h)0 1 2 3 4

csp15

csp15-Nsylv

control

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M. lysodeikticus, µg/ml10-2 10-1 100 101 102 103

∆ pH

at 1

5 m

in

0.0

0.4

0.8

1.2

1.6 +csp15-Alal10bacteria

peptidoglycan +csp15-Ala10

∆pH 0.2

2 min

at -0.5 min

at 0 min

at 1 min

at 3.5 min+ csp15-Ala10:

csp1

5

controlA

B

D

∆pH 0.2

10 min

∆pH 0.2

10 min

C

csp15-Ala10+ 'messenger'

'messenger'

csp15-Ala10 + A. tum.

A. tumefaciens

csp15-Ala10

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Figure 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.

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Supplementary data:

Comparison of proteins with cold shock domains from animals and plants with

bacterial CSP

Schematic representation of Y-box binding proteins with CSD (top) and glycine rich proteins

containing CSDs’ from plant (bottom). Alignment of CSD sequences with consensus

sequence of bacterial CSPs’ (middle, with * denoting residues conserved in Y-box proteins or

grp2 proteins, respectively. Shaded sequences indicate RNP-1 (left) and RNP-2 (right).

EKKIIASQVS GTVKWFNVKS GYGFINRDDT KEDVFVHQTA IVKNNPRKYL RSVGDGEKVE FDVVEGEKG NEAANVTGPE GSNVQGEKKVLATKVL GTVKWFNVRN GYGFINRNDT KEDVFVHQTA IKKNNPRKYL RSVGDGETVE FDVVEGEKG AEAANVTGPD GVPVEGDKKVIATKVL GTVKWFNVRN GYGFINRNDT KEDVFVHQTA IKKNNPRKYL RSVGDGETVE FDVVEGEKG EEAANVTGPG GVPVQGDKKVIATKVL GTVKWFNVRN GYGFINRNDT KEDVFVHQTA IKKNNPRKYL RSVGDGETVE FDVVEGEKG AEAANVTGPE GVPVQG

******* * *** * ***** * * * * * * * * *****

Aplysia californicaMus musculusHomo sapiens

Xenopus laevis

bacterial CSP (consensus)

Y box transcription factors

N-term C-termcold shock domain (CSD) ++++ ---- ++++ ---- ++++ ---- ++++

MSNMXT GTVKWFNAEK GFGFIEPEDG SKDVFVHFSA IQGDG....F KSLEEGQKVS FEIEQGARGP QAANVTKL ****** * ***** * ** * *** * * * * ** * ** * * *

MAEESGQRAK GTVKWFSDQK GFGFITPDDG GEDLFVHQSG IRSEG....F RSLAEGETVE FEVESGGDGR TKAVDVTGPD GAAVQGVNMSGGDRRK GTVKWFDTQK GFGFITPSDG GDDLFVHQSS IRSEG....F RSLAAEESVE FDVEVDNSGR PKAIEVSGPD GAPVQGGDNGGGERRK GSVKWFDTQK GFGFITPDDG GDDLFVHQSS IRSEG....F RSLAAEEAVE FEVEIDNNNR PKAIDVSGPD GAPVQG

N. sylvestris (grp2)A. thaliana (grp2b)A. thaliana (grp2)

MSGGGDMS

glycine rich proteins (grp2) in plants

alternating regions with positive and negative charges

N-term C-termcold shock domain (CSD) glycine rich domain

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Georg Felix and Thomas BollerRNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobaccoMolecular sensing of bacteria in plants: The highly conserved RNA-binding motif

published online December 5, 2002J. Biol. Chem. 

  10.1074/jbc.M209880200Access the most updated version of this article at doi:

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