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
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ABSTRACT |
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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.
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.
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 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.
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 ( 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.
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).
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).
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 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.
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.
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.
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 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
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
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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, nonsaturating 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 (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).
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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
( 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.
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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.
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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).
-barrel structures built up from five antiparallel
-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
1 and
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.
Comparison of mutations in cspB affecting DNA-binding and single amino
acid changes in csp15 on elicitor activity
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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.
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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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
-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.
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.
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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.
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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.
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
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ABBREVIATIONS |
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The abbreviations used are: PAMP, pathogen-associated molecular pattern; CSP, cold shock protein; CSD, cold shock domain.
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