Damage on plants activates Ca2+-dependent metacaspases for release of immunomodulatory peptides.

Physical damage to cells leads to the release of immunomodulatory peptides to elicit a wound defense response in the surrounding tissue. In Arabidopsis thaliana, the plant elicitor peptide 1 (Pep1) is processed from its protein precursor, PRECURSOR OF PEP1 (PROPEP1). We demonstrate that upon damage, both at the tissue and single-cell levels, the cysteine protease METACASPASE4 (MC4) is instantly and spatiotemporally activated by binding high levels of Ca2+ and is necessary and sufficient for Pep1 maturation. Cytosol-localized PROPEP1 and MC4 react only after loss of plasma membrane integrity and prolonged extracellular Ca2+ entry. Our results reveal that a robust mechanism consisting of conserved molecular components links the intracellular and Ca2+-dependent activation of a specific cysteine protease with the maturation of damage-induced wound defense signals.

Differential Suppression of Nicotiana benthamiana Innate Immune Responses by Transiently Expressed Pseudomonas syringae Type III Effectors.

The plant pathogen Pseudomonas syringae injects about 30 different virulence proteins, so-called effectors, via a type III secretion system into plant cells to promote disease. Although some of these effectors are known to suppress either pattern-triggered immunity (PTI) or effector-triggered immunity (ETI), the mode of action of most of them remains unknown. Here, we used transient expression in Nicotiana benthamiana, to test the abilities of type III effectors of Pseudomonas syringae pv. tomato (Pto) DC3000 and Pseudomonas syringae pv. tabaci (Pta) 11528 to interfere with plant immunity. We monitored the sequential and rapid bursts of cytoplasmic Ca2+ and reactive oxygen species (ROS), the subsequent induction of defense gene expression, and promotion of cell death. We found that several effector proteins caused cell death, but independently of the known plant immune regulator NbSGT1, a gene essential for ETI. Furthermore, many effectors delayed or blocked the cell death-promoting activity of other effectors, thereby potentially contributing to pathogenesis. Secondly, a large number of effectors were able to suppress PAMP-induced defense responses. In the majority of cases, this resulted in suppression of all studied PAMP responses, suggesting that these effectors target common elements of PTI. However, effectors also targeted different steps within defense pathways and could be divided into three major groups based on their suppressive activities. Finally, the abilities of effectors of both Pto DC3000 and Pta 11528 to suppress plant immunity was conserved in most but not all cases. Overall, our data present a comprehensive picture of the mode of action of these effectors and indicate that most of them suppress plant defenses in various ways.

Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases.

Sensing of potential pathogenic bacteria is of critical importance for immunity. In plants, this involves plasma membrane-resident pattern recognition receptors, one of which is the FLAGELLIN SENSING 2 (FLS2) receptor kinase. Ligand-activated FLS2 receptors are internalized into endosomes. However, the extent to which these spatiotemporal dynamics are generally present among pattern recognition receptors (PRRs) and their regulation remain elusive. Using live-cell imaging, we show that at least three other receptor kinases associated with plant immunity, PEP RECEPTOR 1/2 (PEPR1/2) and EF-TU RECEPTOR (EFR), internalize in a ligand-specific manner. In all cases, endocytosis requires the coreceptor BRI1-ASSOCIATED KINASE 1 (BAK1), and thus depends on receptor activation status. We also show the internalization of liganded FLS2, suggesting the transport of signaling competent receptors. Trafficking of activated PRRs requires clathrin and converges onto the same endosomal vesicles that are also shared with the hormone receptor BRASSINOSTERIOD INSENSITIVE 1 (BRI1). Importantly, clathrin-dependent endocytosis participates in plant defense against bacterial infection involving FLS2-mediated stomatal closure and callose deposition, but is uncoupled from activation of the flagellin-induced oxidative burst and MAP kinase signaling. In conclusion, immunity mediated by pattern recognition receptors depends on clathrin, a critical component for the endocytosis of signaling competent receptors into a common endosomal pathway.

Evolutionary divergence of the plant elicitor peptides (Peps) and their receptors: interfamily incompatibility of perception but compatibility of downstream signalling.

Plant elicitor peptides (Peps) are potent inducers of pattern-triggered immunity and amplify the immune response against diverse pathogens. Peps have been discovered and studied extensively in Arabidopsis and only recently orthologues in maize were also identified and characterized in more detail.Here, the presence of PROPEPs, the Pep precursors, and PEPRs, the Pep receptors, was investigated within the plant kingdom. PROPEPs and PEPRs were identified in most sequenced species of the angiosperms. The conservation and compatibility of the Pep-PEPR-system was analysed by using plants of two distantly related dicot families, Brassicaceae and Solanaceae, and a representative family of monocot plants, the Poaceae. All three plant families contain important crop plants, including maize, rice, tomato, potato, and canola. Peps were not recognized by species outside of their plant family of origin, apparently because of a divergence of the Pep sequences. Three family-specific Pep motifs were defined and the integration of such a motif into the Pep sequence of an unrelated Pep enabled its perception. Transient transformation of Nicotiana benthamiana with the coding sequences of the AtPEPR1 and ZmPEPR1a led to the recognition of Pep peptides of Brassicaceae or Poaceae origin, respectively, and to the proper activation of downstream signalling. It was concluded that signalling machinery downstream of the PEPRs is highly conserved whereas the leucine-rich repeat domains of the PEPRs co-evolved with the Peps, leading to distinct motifs and, with it, interfamily incompatibility.

Quo vadis, Pep? Plant elicitor peptides at the crossroads of immunity, stress, and development.

The first line of inducible plant defence, pattern-triggered immunity (PTI), is activated by the recognition of exogenous as well as endogenous elicitors. Exogenous elicitors, also called microbe-associated molecular patterns, signal the presence of microbes. In contrast, endogenous elicitors seem to be generated and recognized under more diverse circumstances, making the evaluation of their biological relevance much more complex. Plant elicitor peptides (Peps) are one class of such endogenous elicitors, which contribute to immunity against attack by bacteria, fungi, as well as herbivores. Recent studies indicate that the Pep-triggered signalling pathways also operate during the response to a more diverse set of stresses including starvation stress. In addition, in silico data point to an involvement in the regulation of plant development, and a study on Pep-mediated inhibition of root growth supports this indication. Importantly, Peps are neither limited to the model plant Arabidopsis nor to a specific plant family like the previously intensively studied systemin peptides. On the contrary, they are present and active in angiosperms all across the phylogenetic tree, including many important crop plants. Here we summarize the progress made in research on Peps from their discovery in 2006 until now. We discuss the two main models which describe their likely function in plant immunity, highlight the studies supporting additional roles of Pep-triggered signalling and identify urgent research tasks to further uncover their biological relevance.

The family of Peps and their precursors in Arabidopsis: differential expression and localization but similar induction of pattern-triggered immune responses.

In Arabidopsis thaliana, the endogenous danger peptides, AtPeps, have been associated with plant defences reminiscent of those induced in pattern-triggered immunity. AtPeps are perceived by two homologous receptor kinases, PEPR1 and PEPR2, and are encoded in the C termini of the PROPEP precursors. Here, we report that, contrary to the seemingly redundant AtPeps, the PROPEPs fall at least into two distinct groups. As revealed by promoter-ß-glucuronidase studies, expression patterns of PROPEP1-3, -5, and -8 partially overlapped and correlated with those of the PEPR1 and -2 receptors, whereas those of PROPEP4 and -7 did not share any similarities with the former. Moreover, bi-clustering analysis indicated an association of PROPEP1, -2, and -3 with plant defence, whereas PROPEP5 expression was related to patterns of plant reproduction. In addition, at the protein level, PROPEPs appeared to be distinct. PROPEP3::YFP (fused to yellow fluorescent protein) was present in the cytosol, but, in contrast to previous predictions, PROPEP1::YFP and PROPEP6::YFP localized to the tonoplast. Together with the expression patterns, this could point to potentially non-redundant roles among the members of the PROPEP family. By contrast, their derived AtPeps, including the newly reported AtPep8, when applied exogenously, provoked activation of defence-related responses in a similar manner, suggesting a high level of functional redundancy between the AtPeps. Taken together, our findings reveal an apparent antagonism between AtPep redundancy and PROPEP variability, and indicate new roles for PROPEPs besides plant immunity.

The nodulation factor hydrolase of Medicago truncatula: characterization of an enzyme specifically cleaving rhizobial nodulation signals.

Nodule formation induced by nitrogen-fixing rhizobia depends on bacterial nodulation factors (NFs), modified chitin oligosaccharides with a fatty acid moiety. Certain NFs can be cleaved and inactivated by plant chitinases. However, the most abundant NF of Sinorhizobium meliloti, an O-acetylated and sulfated tetramer, is resistant to hydrolysis by all plant chitinases tested so far. Nevertheless, this NF is rapidly degraded in the host rhizosphere. Here, we identify and characterize MtNFH1 (for Medicago truncatula Nod factor hydrolase 1), a legume enzyme structurally related to defense-related class V chitinases (glycoside hydrolase family 18). MtNFH1 lacks chitinase activity but efficiently hydrolyzes all tested NFs of S. meliloti. The enzyme shows a high cleavage preference, releasing exclusively lipodisaccharides from NFs. Substrate specificity and kinetic properties of MtNFH1 were compared with those of class V chitinases from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), which cannot hydrolyze tetrameric NFs of S. meliloti. The Michaelis-Menten constants of MtNFH1 for NFs are in the micromolar concentration range, whereas nonmodified chitin oligosaccharides represent neither substrates nor inhibitors for MtNFH1. The three-dimensional structure of MtNFH1 was modeled on the basis of the known structure of class V chitinases. Docking simulation of NFs to MtNFH1 predicted a distinct binding cleft for the fatty acid moiety, which is absent in the class V chitinases. Point mutation analysis confirmed the modeled NF-MtNFH1 interaction. Silencing of MtNFH1 by RNA interference resulted in reduced NF degradation in the rhizosphere of M. truncatula. In conclusion, we have found a novel legume hydrolase that specifically inactivates NFs.

Several MAMPs, including chitin fragments, enhance AtPep-triggered oxidative burst independently of wounding.

AtPeps are a family of small peptides in Arabidopsis that are believed to act as endogenous amplifiers of the plant's innate immune response. In our recent study, (10) we showed that in Arabidopsis leaf disks, bacterial MAMPs (microbe-associated molecular patterns) such as the flagellin derived elicitor flg22, greatly enhanced the release of reactive oxygen species (ROS) triggered by a subsequent AtPep-perception. This enhanced ROS production could be a hallmark either of improved local defense or of the initiation of ROS-based systemic signaling. Here, we established a superior ROS detection system based on a new derivative of luminol (L-012). With this sensitive system we were able to show that chitin, too, acts as an enhancer of AtPep-triggered ROS, linking this specific defense response amplification also to the recognition of fungal pathogens. In addition we used the more sensitive ROS assay to transfer the experimental setup from cut leaf disks to unwounded seedlings. Thereby we revealed that wounding is not a prerequisite to enable the MAMP-induced enhancement of the AtPep-triggered ROS response.

Contamination risks in work with synthetic peptides: flg22 as an example of a pirate in commercial peptide preparations.

The pattern recognition receptor FLAGELLIN SENSING2 (FLS2) renders plant cells responsive to subnanomolar concentrations of flg22, the active epitope of bacterial flagellin. We recently observed that a preparation of the peptide IDL1, a signal known to regulate abscission processes via the receptor kinases HAESA and HAESA-like2, apparently triggered Arabidopsis thaliana cells in an FLS2-dependent manner. However, closer investigation revealed that this activity was due to contamination by a flg22-type peptide, and newly synthesized IDL1 peptide was completely inactive in FLS2 signaling. This raised alert over contamination events occurring in the process of synthesis or handling of peptides. Two recent reports have suggested that FLS2 has further specificities for structurally unrelated peptides derived from CLV3 and from Ax21. We thus scrutinized these peptides for activity in Arabidopsis cells as well. While responding to <1 nM flg22, Arabidopsis cells proved blind even to 100 µM concentrations of CLV3p and axY(s)22. Our results confirm the exquisite sensitivity and selectivity of FLS2 for flg22. They also show that inadvertent contaminations with flg22-type peptides do occur and can be detected even in trace amounts by FLS2.

Functional analysis of NopM, a novel E3 ubiquitin ligase (NEL) domain effector of Rhizobium sp. strain NGR234.

Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS) are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia) and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M) of Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL) domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP) kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant's defense response, as shown by a reduced production of reactive oxygen species (ROS) in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.

Microbe-associated molecular pattern (MAMP) signatures, synergy, size and charge: influences on perception or mobility and host defence responses.

Triggering of defences by microbes has mainly been investigated using single elicitors or microbe-associated molecular patterns (MAMPs), but MAMPs are released in planta as complex mixtures together with endogenous oligogalacturonan (OGA) elicitor. We investigated the early responses in Arabidopsis of calcium influx and oxidative burst induced by non-saturating concentrations of bacterial MAMPs, used singly and in combination: flagellin peptide (flg22), elongation factor peptide (elf18), peptidoglycan (PGN) and component muropeptides, lipo-oligosaccharide (LOS) and core oligosaccharides. This revealed that some MAMPs have additive (e.g. flg22 with elf18) and even synergistic (flg22 and LOS) effects, whereas others mutually interfere (flg22 with OGA). OGA suppression of flg22-induced defences was not a result of the interference with the binding of flg22 to its receptor flagellin-sensitive 2 (FLS2). MAMPs induce different calcium influx signatures, but these are concentration dependent and unlikely to explain the differential induction of defence genes [pathogenesis-related gene 1 (PR1), plant defensin gene 1.2 (PDF1.2) and phenylalanine ammonia lyase gene 1 (PAL1)] by flg22, elf18 and OGA. The peptide MAMPs are potent elicitors at subnanomolar levels, whereas PGN and LOS at high concentrations induce low and late host responses. This difference might be a result of the restricted access by plant cell walls of MAMPs to their putative cellular receptors. flg22 is restricted by ionic effects, yet rapidly permeates a cell wall matrix, whereas LOS, which forms supramolecular aggregates, is severely constrained, presumably by molecular sieving. Thus, MAMPs can interact with each other, whether directly or indirectly, and with the host wall matrix. These phenomena, which have not been considered in detail previously, are likely to influence the speed, magnitude, versatility and composition of plant defences.

Molecular identification and characterization of the tomato flagellin receptor LeFLS2, an orthologue of Arabidopsis FLS2 exhibiting characteristically different perception specificities.

Bacterial flagellin is known to stimulate host immune responses in mammals and plants. In Arabidopsis thaliana, the receptor kinase FLS2 mediates flagellin perception through physical interaction with a highly conserved epitope in the N-terminus of flagellin, represented by the peptide flg22 derived from Pseudomonas syringae. The peptide flg22 is highly active as an elicitor in many plant species. In contrast, a shortened version of the same epitope derived from Escherichia coli, flg15(E coli), is highly active as an elicitor in tomato but not in A. thaliana or Nicotiana benthamiana. Here, we make use of these species-specific differences in flagellin perception abilities to identify LeFLS2 as the flagellin receptor in tomato. LeFLS2 is most closely related to AtFLS2, indicating that it may represent the flagellin receptor of tomato. Expression of the LeFLS2 gene in Arabidopsis did not result in accumulation of its corresponding gene product, as indicated by experiments with LeFLS2-GFP fusions. In contrast, expression of LeFLS2-GFP fusions in N. benthamiana, a species that, like tomato, belongs to the Solanaceae, was obviously functional. N. benthamiana plants transiently expressing a LeFLS2-GFP fusion acquired responsiveness to flg15(E coli) to which they are normally unresponsive. Thus, LeFLS2 encodes a functional, specific flagellin receptor, the first to be identified in a plant family other than the Brassicaceae.

Developing fructan-synthesizing capability in a plant invertase via mutations in the sucrose-binding box.

Fructans are fructose polymers that are synthesized from sucrose by fructosyltransferases. Fructosyltransferases are present in unrelated plant families suggesting a polyphyletic origin for their transglycosylation activity. Based on sequence comparisons and enzymatic properties, fructosyltransferases are proposed to have evolved from vacuolar invertases. Between 1% and 5% of the total activity of vacuolar invertase is transglycosylating activity. We investigated the nature of the changes that can convert a hydrolysing invertase into a transglycosylating enzyme. Remarkably, replacing 33 amino acids (amino acids 143-175) corresponding to the N-terminus of the mature onion vacuolar invertase with the corresponding region of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) led to a shift in activity from hydrolysis of sucrose towards transglycosylation between two sucrose molecules. The substituted N-terminal region contains the sucrose-binding box that harbours the nucleophile involved in sucrose hydrolysis (Asp164). Subsequent research into the individual amino acids responsible for the enhanced transglycosylation activity revealed that mutations in amino acids Trp161 and Asn166, can give rise to a shift towards polymerase activity. Changing the amino acid at either of these positions in the sucrose-binding box increases the transglycosylation capacity of invertases two- to threefold compared to wild type. Combining the two mutations had an additive effect on transglycosylation ability, resulting in an approximately fourfold enhancement. The mutations generated correspond with natural variation present in the sucrose-binding boxes of vacuolar invertases and fructosyltransferases. These relatively small changes that increase the transglycosylation capacity of invertases might explain the polyphyletic origin of the fructan accumulation trait.

Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation.

Higher eukaryotes sense microbes through the perception of pathogen-associated molecular patterns (PAMPs). Arabidopsis plants detect a variety of PAMPs including conserved domains of bacterial flagellin and of bacterial EF-Tu. Here, we show that flagellin and EF-Tu activate a common set of signaling events and defense responses but without clear synergistic effects. Treatment with either PAMP results in increased binding sites for both PAMPs. We used this finding in a targeted reverse-genetic approach to identify a receptor kinase essential for EF-Tu perception, which we called EFR. Nicotiana benthamiana, a plant unable to perceive EF-Tu, acquires EF-Tu binding sites and responsiveness upon transient expression of EFR. Arabidopsis efr mutants show enhanced susceptibility to the bacterium Agrobacterium tumefaciens, as revealed by a higher efficiency of T-DNA transformation. These results demonstrate that EFR is the EF-Tu receptor and that plant defense responses induced by PAMPs such as EF-Tu reduce transformation by Agrobacterium.

Ethylene-mediated cross-talk between calcium-dependent protein kinase and MAPK signaling controls stress responses in plants.

Plants are constantly exposed to environmental changes and need to integrate multiple external stress cues. Calcium-dependent protein kinases (CDPKs) are implicated as major primary Ca2+ sensors in plants. CDPK activation, like activation of mitogen-activated protein kinases (MAPKs), is triggered by biotic and abiotic stresses, although distinct stimulus-specific stress responses are induced. To investigate whether CDPKs are part of an underlying mechanism to guarantee response specificity, we identified CDPK-controlled signaling pathways. A truncated form of Nicotiana tabacum CDPK2 lacking its regulatory autoinhibitor and calcium-binding domains was ectopically expressed in Nicotiana benthamiana. Infiltrated leaves responded to an abiotic stress stimulus with the activation of biotic stress reactions. These responses included synthesis of reactive oxygen species, defense gene induction, and SGT1-dependent cell death. Furthermore, N-terminal CDPK2 signaling triggered enhanced levels of the phytohormones jasmonic acid, 12-oxo-phytodienoic acid, and ethylene but not salicylic acid. These responses, commonly only observed after challenge with a strong biotic stimulus, were prevented when the CDPK's intrinsic autoinhibitory peptide was coexpressed. Remarkably, elevated CDPK signaling compromised stress-induced MAPK activation, and this inhibition required ethylene synthesis and perception. These data indicate that CDPK and MAPK pathways do not function independently and that a concerted activation of both pathways controls response specificity to biotic and abiotic stress.

Peptide signalling in plant development and self/non-self perception.

Plant genomes contain very large families of genes encoding receptor-like kinases (RLKs). In recent years, several of these RLKs have been shown by biochemical and mutational analysis to represent receptors for peptides, and the emerging picture shows that peptide signalling in development and self/non-self perception is based on a similar repertoire of receptors and signalling cascades. The need to recognize multiple peptide signals in self/non-self recognition may have led to the surprising radiation and diversification of RLKs in the plant kingdom.

The transcriptional innate immune response to flg22. Interplay and overlap with Avr gene-dependent defense responses and bacterial pathogenesis.

Animals and plants carry recognition systems to sense bacterial flagellin. Flagellin perception in Arabidopsis involves FLS2, a Leu-rich-repeat receptor kinase. We surveyed the early transcriptional response of Arabidopsis cell cultures and seedlings within 60 min of treatment with flg22, a peptide corresponding to the most conserved domain of flagellin. Using Affymetrix microarrays, approximately 3.0% of 8,200 genes displayed transcript level changes in flg22 elicited suspension cultures and seedlings. FLARE (Flagellin Rapidly Elicited) genes mostly encode signaling components, such as transcription factors, protein kinases/phosphatases, and proteins that regulate protein turnover. Approximately 80% of flg22-induced genes were also up-regulated in Arabidopsis seedlings treated with cycloheximide. This suggests that many FLARE genes are negatively regulated by rapidly turned-over repressor proteins. Twenty-one tobacco Avr9/Cf-9 rapidly elicited (ACRE) cDNA full-length sequences were used to search for their Arabidopsis orthologs (AtACRE). We identified either single or multiple putative orthologs for 17 ACRE genes. For 13 of these ACRE genes, at least one Arabidopsis ortholog was induced in flg22-elicited Arabidopsis suspension cells and seedlings. This result revealed a substantial overlap between the Arabidopsis flg22 response and the tobacco Avr9 race-specific defense response. We also compared FLARE gene sets and genes induced in basal or gene-for-gene interactions upon different Pseudomonas syringae treatments, and infer that Pseudomonas syringae pv tomato represses the flagellin-initiated defense response.

A plasma membrane syntaxin is phosphorylated in response to the bacterial elicitor flagellin.

In vivo pulse labeling of suspension-cultured Arabidopsis cells with [32P]orthophosphate allows a systematic analysis of dynamic changes in protein phosphorylation. Here, we use this technique to investigate signal transduction events at the plant plasma membrane triggered upon perception of microbial elicitors of defense responses, using as a model elicitor flg22, a peptide corresponding to the most conserved domain of bacterial flagellin. We demonstrate that two-dimensional gel electrophoresis in conjunction with mass spectrometry is a suitable tool for the identification of intrinsic membrane proteins, and we show that among them a syntaxin, AtSyp122, is phosphorylated rapidly in response to flg22. Although incorporation of radioactive phosphate into the protein only occurs significantly after elicitation, immunoblot analysis after two-dimensional gel separation indicates that the protein is also phosphorylated prior to elicitation. These results indicate that flg22 elicits either an increase in the rate of turnover of phosphate or an additional de novo phosphorylation event. In vitro, phosphorylation of AtSyp122 is calcium-dependent. In vitro phosphorylated peptides separated by two-dimensional thin layer chromatography comigrate with two of the three in vivo phosphopeptides, indicating that this calcium-dependent phosphorylation is biologically relevant. These results indicate a regulatory link between elicitor-induced calcium fluxes and the rapid phosphorylation of a syntaxin. Because syntaxins are known to be important in membrane fusion and exocytosis, we hypothesize that one of the functions of the calcium signal is to stimulate exocytosis of defense-related proteins and compounds.

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.

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.