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non-self perception
Peptide signalling in plant development and self/non-self perception Thomas Boller 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. Addresses Botanisches Institut, Universita¨t Basel, Zurich-Basel Plant Science Center, Hebelstrasse 1, CH-4056 Basel, Switzerland Corresponding author: Boller, Thomas ([email protected])
larities and differences between the perception of endogenous and exogenous signals. Peptide signalling and receptor-like kinases (RLKs) in plants have been reviewed previously in this journal [3] and elsewhere [4–6]. The present review is focussed on the cases where RLKs have been implicated most clearly in peptide signalling. Of particular interest are the RLKs with an extracellular leucine rich repeat (LRR) domain, the LRR-RLKs. The Arabidopsis genome contains >210 LRR-LRK genes [1], and the rice genome >370 [2]. LRR domains are known to be involved in protein– protein or protein–peptide interactions, and LRR-RLKs comprise putative receptors of both non-self and hormonal peptide signals in plants (Figure 1).
Peptide hormones in plants Current Opinion in Cell Biology 2005, 17:116–122 This review comes from a themed issue on Cell regulation Edited by Brian Hemmings and Peter Parker
0955-0674/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2005.02.007
Introduction In animal biology, there is a clear conceptual dichotomy in peptide signalling. On the one hand, peptide hormones are key players in development: peptide hormones are evolutionarily conserved, and perceived by evolutionarily conserved receptors. On the other hand, peptides allow the discrimination of self and non-self in the immune system, and ultimately enable an effective defence against pathogens. Almost any arbitrary peptide can be an antigen, and the receptors recognizing them — the antibodies — are characterized by a maximal lack of evolutionary conservation, as their genes are freshly assembled in each individual. In plants, like in animals, peptides are signals in development as well as in self and non-self perception, but the distinction between the corresponding receptors is less clear. Plants have large families of receptor-like kinases (RLKs) [1,2], which appear to be involved in both types of signalling. Even the downstream pathways appear to be similar in several instances. This raises intriguing questions about evolution, and about the simiCurrent Opinion in Cell Biology 2005, 17:116–122
The time-honoured five classes of phytohormones (auxins, cytokinins, gibberellins, ethylene and abscisic acid) are micromolecules of various types but are not peptides. Several similar compounds have been elevated to phytohormone status in the last decades, including brassinosteroids, jasmonate and salicylic acid, but peptides were only recently added to the list. Systemin and the systemin receptor SR160
The first peptide that acquired the status of an acknowledged plant hormone was systemin [7]. As its name implies, this 18-amino-acid peptide was believed to be a systemic signal of wounding in tomato. However, elegant grafting experiments indicate that systemin is required only locally, and that the systemic signal is in fact jasmonate, a prototypic phytohormone, or a related compound of the octadecanoid pathway [8,9]. A high-affinity binding site for systemin was found in cell cultures of Lycopersicon peruvianum, a close relative of tomato (L. esculentum). This binding site was identified as a 160 kDa protein, called ‘SR160’, by labelling with a radioactive photoaffinity analogue of systemin, and purified by preparative gel electrophoresis, concanavalin A sepharose chromatography and analytical gel electrophoresis [10]. Surprisingly, tryptic peptides generated from the radioactive band showed homology to BRI1, an Arabidopsis LRR-RLK thought to be involved in the perception of brassinolides (Figure 1b). On the basis of this finding, a L. peruvianum cDNA library was screened with an Arabidopsis BRI1 fragment and a fragment of a tomato EST encoding a BRI1 homolog. The resulting cDNA encoded a LRR-RLK with high homology to BRI1 and clearly represented the gene encoding the major www.sciencedirect.com
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Figure 1
(a) Flagellin receptor FLS2
flg22
(b) Systemin receptor
(d) Clavata system
BRI1
SR160
Systemin
(c) Phytosulfokine receptor
120 kDa receptor
(?) =
(??)
brl psk
(??)
CLV1 CLV2
CLV3
MAPKKK
(??)
MAPKK
(??)
M APK
WRKY
(WUS)
Current Opinion in Cell Biology
LRR-RLKs (receptor-like kinases with leucine-rich repeats) involved in peptide signalling. (a) Flagellin receptor in Arabidopsis thaliana. The gene FLS2 encodes a LRR-LRK, which is essential for flg22 binding and signalling. Signal perception activates a MAP kinase cascade and culminates in the activation of WRKY-type transcription factors. (b) Systemin receptor in Lycopersicon peruvianum. Systemin binds to a receptor of 160 kDa (SR160). Perception of systemin activates a MAP kinase cascade and leads to a response that resembles the flg22 response. According to current evidence, the gene encoding SR160 is the tomato ortholog of the Arabidopsis gene BRI1, encoding the putative receptor of brassinolides (brl). Since the responses to systemin and brassinolides are completely different, there remains a question mark about the identity of SR160 and BRI1. (c) Phytosulfokine receptor in Daucus carota. Phytosulfokine (psk) binds to a LRR-RLK of 120 kDa. Little is known about phytosulfokine signal transduction. (d) The clavata system in Arabidopsis thaliana. The 5 kDa peptide CLV3 is expressed in the apical meristem and serves to limit the size of the meristem by negatively regulating expression of WUSCHEL, the gene encoding the transcription factor WUS. According to earlier publications, CLV3 interacts with a receptor consisting of CLV1, a LRR-RLK, and CLV2, a LRR-RLP (receptor-like protein with leucine-rich repeats). However, since clv1 null mutations have a much weaker phenotype than clv3 mutations, it is unlikely that CLV3 acts through CLV1. Possibly, CLV3 binds to a different, unknown RLK and RLP (depicted in grey, with question marks). The CLV1/CLV2 and CLV3 pathways both regulate WUS expression but their interplay remains unknown.
protein in the radioactive band of SR160, as shown by lysyl endopeptidase fingerprinting [10]. The same gene was independently found to be mutated in the tomato mutant cu-3, which is dwarf and does not respond to brassinosteroids, exactly like the Arabidopsis bri1 mutants [11]. This led to the notion that the same LRR-RLK may be the receptor for both brassinosteroids and systemin [11]. There is a caveat, however. The Cu-3/SR160 gene clearly represents the tomato orthologue of BRI1 [11], and the major protein in the radioactive band isolated in the search for the systemin receptor [10], but not necessarily the photoaffinity-labelled systemin receptor itself, since purification was based primarily on size in gel electrophoresis, and not on specific properties of the systemin receptor. www.sciencedirect.com
More direct evidence that Cu-3/SR160 indeed encodes the systemin receptor came from an experiment with tobacco, a plant that does not respond to systemin (see below). A transgenic tobacco plant expressing the Cu-3/ SR160 gene was generated [12]. This plant produced seeds that segregated when grown on kanamycin. However, surprisingly, these transgenic seedlings were not further analysed. Instead, a suspension cell culture was generated from the transgenic tobacco plant, and this culture was shown to express SR160 and to respond to systemin with an alkalinization response, as would be the case with a L. peruvianum cell culture [12]. Confirmation of this result with transgenic plants is eagerly awaited. Unequivocal evidence for or against the identity of the Cu-3/SR160 gene and the gene encoding the systemin Current Opinion in Cell Biology 2005, 17:116–122
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receptor would come from complementation experiments similar to the ones performed with the cotton BRI1 ortholog GhBRI1, which complemented the Arabidopsis bri1 mutation [13]. If Cu-3/SR160 encoded the systemin receptor, then the Arabidopsis bri1 mutant complemented with tomato Cu-3/SR160 should show systemin binding or a systemin response, whereas the tomato cu-3 mutant complemented with Arabidopsis BRI1 should fail to respond to systemin. The presence of the prosystemin gene (encoding the systemin precursor), and the capability to perceive systemin, is restricted to tomato and its closest relatives (Lycopersicon peruvianum, potato, pepper and nightshade) [7], and even among these plants, the 18-amino-acid sequence of systemin differs in at least two positions. Both tobacco (a closely related solanaceous plant) and Arabidopsis lack a prosystemin gene and do not respond to systemin [7]. This is in stark contrast to the situation with peptide hormones in animals, where sequence and function are conserved even across taxonomic orders and classes, and it indicates that systemin-type signalling may be subject to diversifying selection. In tomato, the gene encoding prosystemin appears to be expressed exclusively in phloem parenchyma cells, and the gene product appears to reside in the cytoplasm [14]. Expression of prosystemin in phloem parenchyma cells is strongly induced by wounding as well as by jasmonate [14]. These results indicate that systemin is an ‘endogenous elicitor’ rather than a hormone in the usual sense. When it is released upon wounding or death of phloem parenchyma cells, it is recognized by systemin receptors residing in the plasma membrane of adjacent mesophyll cells, and it thereby elicits the herbivore defence program. In contrast to other endogenous elicitors, it seems to be subject to diversifying selection, perhaps in response to herbivores evolving to counteract systeminmediated signalling.
80-amino-acid precursor protein and promotes dedifferentiation and cell division in plant cells at nanomolar concentrations [5]. A binding site for phytosulfokine has been identified by ligand-based affinity chromatography and shown to be an LRR-type receptor-like kinase [17]. The corresponding gene was cloned, and its overexpression in carrot cells was shown to lead to a considerable increase in phytosulfokine binding sites and to altered growth responses, strongly indicating that this gene encodes the phytosulfokine receptor [17] (Figure 1c). Although phytosulfokine promotes cell proliferation in tissue culture, and can be used for ‘chemical nursing’ (i.e. to promote cell division of plant cells grown at high dilutions) [18], its actual role in the growth and development of plants remains to be determined. The clavata system
The CLAVATA (CLV) signalling pathway has become a paradigm of stem cell maintenance in plant meristems [19]. The CLV pathway negatively regulates expression of WUSCHEL (WUS), a gene encoding a homeodomain transcription factor essential for maintaining stem cell identity, and thereby limits meristem size. According to current reviews, the CLV signalling pathway comprises the extracellular peptide CLV3 (encoded by the CLV3 gene), thought to act as a peptide signal, and a receptor complex consisting of CLV1 (an LRR-RLK) and CLV2 (a receptorlike protein with leucine-rich repeats [LRR-RLP]), thought to interact with CLV3 [5,6,20] (Figure 1d).
Phytosulfokine and the phytosulfokine receptor
However, no one has been able to show that CLV3 actually interacts with CLV1/CLV2; earlier evidence to this effect [21] has been retracted [22]. What is the current status of this model? Studies with a CLV3– GFP fusion protein expressed under the CLV3 promoter indicate that CLV3 spreads extracellularly from the location of its synthesis; this spread appears to be restrained in the region where CLV1 is expressed, with the result that CLV3 does not enter the organizing centre where WUS is expressed [19]. Although these studies are tantalizingly suggestive of a function for CLV3 as a short-distance morphogen, it remains to be seen how CLV3 is perceived, and how CLV1 is involved. Null mutants of CLV3 have a very strong phenotype, whereas the recently isolated null mutants of CLV1 have very weak phenotypes [23]. This supports the importance of CLV3 in meristem regulation, but is in contradiction to a model in which CLV3 exerts its activity through interaction with CLV1. The previously isolated ‘strong’ alleles of CLV1 appear to have a dominant-negative effect on CLV3 signalling [23]. This may be explained by a new model in which the CLV3 signal is mediated by another, unknown RLK, and in which the dominant-negative CLV1 mutants interfere with the function of this other RLK [23].
Another plant peptide hormone is phytosulfokine, a sulfated pentapeptide [Tyr(SO3H)-Ile-Tyr(SO3H)-ThrGln]. This molecule is generated from a ubiquitous
Interestingly, there are at least 25 homologues of the CLV3 gene in Arabidopsis, the CLE genes (genes that
Using bioassays designed for the identification of elicitors, two other groups of peptides have been isolated that may function as endogenous elicitors, namely RALF [15] and ‘tobacco systemin’, both of which are glycopeptides unrelated to tomato systemin; see [7]. These peptides are evolutionarily more conserved than systemin and also occur in other plants, including tomato [15,16]. They may be released upon wounding and induce the typical defence responses also seen in response to exogenous ‘general elicitors’ and PAMPS (pathogen-associated molecular patterns; see below). Their receptors are currently unknown.
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share a conserved C-terminal domain with CLV3 and have putative homologues in maize called ESR) [24]. They all encode small secreted peptides. Modulation of the expression of some of these CLE genes can cause interesting developmental aberrations. For example, loss of CLE40 causes a root-waving phenotype [25], and overexpression of CLV19 causes consumption of the root meristem [26,27]. All of these putative peptide signals, including CLV3, may act as morphogens [28], but their receptors are currently unknown. On the other hand, a CLV1-type RLK has appeared in an unexpected context. Long-distance signalling from roots via leaves to roots is known to restrict nodulation in legumes, and loss of this autoregulation of nodules (AON) causes hypernodulation and increased numbers of lateral roots. The genes responsible for AON in Lotus japonicus (HAR-1, [29,30]) and in soybean (NTS-1, [31]) have been cloned; they encode LRR-RLKs highly homologous to CLV1. It will be interesting to find out whether a CLV3-like peptide signal is produced in nodulated roots and transferred to the shoots to activate HAR1/NTS1. Other peptides potentially serving as hormonal signals
Two gain-of-function screens for genes that influence development in Arabidopsis have independently yielded a small gene family encoding small peptides of 50 amino acids, named DVL (devil) because of their horned-fruit phenotype [32] and ROTUNDIFOLIA-FOUR-LIKE because of their leaf phenotype [33]. This gene family consists of 20 genes encoding similar small peptides, all lacking an apparent signal sequence, and all with a highly similar C-terminal end. Overexpression of the conserved C terminus of these peptides is sufficient to cause morphological aberrations [33]. Although it remains to be demonstrated that these peptides are actually involved in development at natural expression levels, and although their potential receptors are completely unknown, the data hint at the existence of multiple peptide signals acting as morphogens [28] to determine organ development.
Peptide-mediated self perception in plants A key innovation in plant evolution has been the formation of a gynoecium (pistil) in angiosperms, allowing rejection of self-pollen and thereby the enforcement of outbreeding. In the Brassicaceae, ‘sporophytic selfincompatibility’ is based on the specific interaction of two matching components encoded at the S-locus, namely the SCR (the S-locus cysteine-rich peptide produced by pollen) and the SRK (the S-locus-specific RLK expressed in the pistil) [34]. A given plant species may have as many as 100 S haplotypes, and for each of these the SRK appears to interact only with the cognate SCR and not with any of the other 99. How did this remarkable specificity evolve? As demonstrated in a case study, four contiguous amino acids are sufficient to create specificity of SCR function, and SCRs have a high degree of evoluwww.sciencedirect.com
tionary flexibility [35]. This has lead to a hypothesis explaining how new self-incompatibility specificities can be generated by gradual modification of SRK-SCR affinities without invoking a self-compatible transition state [35]. How is the signal provided by SCR-SRK interaction transduced? An analysis of a recessive modifier mutation which eliminates the self-incompatibility response in the stigma yielded a downstream signalling element, namely the M locus protein kinase (MLPK) [36]. Thus, two membrane-bound kinases, SRK with its extracellular recognition domain and MLPK with its myristoyl membrane anchor (but without an extracellular domain), are both required for the self-incompatibility response. It is tempting to speculate that the two kinases interact physically in the receptor complex, as is the case for receptor tyrosine kinases and membrane-anchored Src family kinases in animal hormone signalling. Interestingly, Arabidopsis thaliana, a self-compatible species, possesses all the elements of a functional selfincompatibility response except for functional SCR-SRK alleles, as shown by the establishment of self-incompatibility by the expression of an SCR-SRK pair from the self-incompatible Arabidopsis lyrata [37,38]. An analysis of the pseudogenes present in the non-functional S-loci of 21 A. thaliana ecotypes indicates that self-compatibility has arisen recently and may be associated with the post-Pleistocene expansion of A. thaliana from glacial refugia [39]. A similar interaction between peptide signals and RLKs may also be important in self-acceptance, in other words the acceptance of conspecific pollen by a pistil. It has been shown in tomato that a small cysteine-rich peptide produced by the stigma specifically binds to a LRR-LRK specifically expressed in pollen tubes, and that the stigma-produced peptide promotes pollen tube growth in vitro [40].
Peptide-mediated non-self perception in plants Non-self perception is a key element in the defence against potential pathogens. Plants have two different non-self perception systems for microbial molecules: a basal non-self recognition system with similarities to the innate immunity in animals, which is described in more detail below, and a more specialized gene-for-gene recognition system where a specific AVR (avirulence) gene product of a given pathogen is recognized by a cognate resistance gene product of a host plant. Conceptually the gene-for-gene system is based on peptide recognition as well. However, the most common resistance gene products are LRR-containing proteins without protein kinases, and they appear to interact in an indirect way with the AVR products; this subject is thus outside the Current Opinion in Cell Biology 2005, 17:116–122
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scope of the present review, but has been covered by excellent recent reviews elsewhere [41,42]. Flagellin and the flagellin receptor FLS2
In animal and human innate immunity, TLRs (toll-like receptors) perceive PAMPs (molecular patterns that are universally conserved in a whole class of microbes). Similarly, plants recognize ‘general elicitors’ derived from pathogens. These general elicitors may be peptides such as flg22, which represents the most highly conserved domain of bacterial flagellin [43]. Perception of flg22 in Arabidopsis depends on FLS2, a gene encoding a LRRRLK [44] (Figure 1a). Subsequently, mammalian perception of bacterial flagellin was shown to depend on TLR5 [45], highlighting similarities between the plant and animal innate immunity systems. Interestingly, the flagellin domain recognized by TLR5 differs from the flg22 domain, indicating that the two perception systems have arisen independently by convergent evolution [46]. A global gene expression study indicated that flg22 stimulation of wild-type Arabidopsis plants caused the induction of almost 1000 genes (3% of the annotated genes) within 30 min, whereas flg22 did not induce a single gene in plantlets carrying the fls2 mutation [47]. RLKs were massively over-represented among the induced genes: 110 RLKs were induced, corresponding to 16% of all RLKs in Arabidopsis. When fls2-mutant plants were spray-inoculated with Pseudomonas syringae DC3000, they displayed much stronger disease symptoms and strongly enhanced bacterial growth compared to the corresponding wild-type, Landsberg-erecta. The ecotype Wassiljewskaja (Ws-0), long known anecdotally to be particularly sensitive to bacterial colonization, is a natural fls2 mutant. In spray-inoculation experiments with Pseudomonas syringae DC3000, Ws-0 indeed displayed enhanced disease susceptibility, and normal disease resistance was reestablished in the transgenic plants expressing an FLS2 construct [47]. Taken together, these results indicate that the PAMP receptor FLS2 contributes to basal disease resistance. Other peptides as PAMP signals
The flagellin-derived peptide is active as a PAMP in many different plant species [48]. In other cases, bacterial molecules are recognized in only a subset of plant species. For example, bacterial cold shock protein, CSP, and csp16, a peptide derived from CSP, acts as a PAMP in tobacco and many other Solanaceae at subnanomolar concentrations, but it does not induce any response in Arabidopsis [49]. Similarly, pep13, a peptide derived from the extracellular trans-glutaminase of oomycete pathogens, is active as a potent elicitor in parsley and potato but not in Arabidopsis [50]. Vice versa, bacterial elongation factor Tu (Ef-TU) and elf26, a peptide derived from its N terminus, is recognized by Arabidopsis and many other Brassicaceae, again at subnanomolar concentrations, but does not have any effect in Solanaceae [48]. In all these Current Opinion in Cell Biology 2005, 17:116–122
cases, the proteins recognized by the plants are highly abundant in a whole class of microorganisms, and the plant recognition system is directed against a particularly highly conserved domain of these microbial proteins. Thus these proteins fit the definition of a PAMP. It is likely that in evolution specific lineages of plants have acquired distinct, diverse PAMP recognition capabilities, making it difficult for a given microbe to avoid recognition in all plant species and thereby to become a ‘generalist’ plant pathogen. A similar principle appears to govern the diversification of secondary metabolites in plants, such as phytoalexins or alkaloids. Each family, or even each genus or species, has its own characteristic mix of secondary metabolites, and its own guild of specialist pathogens and herbivores that can cope with this specific mix. This may also provide an explanation for the large-scale diversification of RLKs in plants [1,2].
Conclusions Peptide signalling in plants is important in development as well as in self/non-self perception. Plants appear to have diversified their RLK families, and the corresponding signalling cascades, to accommodate both functions. In evolutionary terms, peptides acting as hormones and morphogens, and consequently their receptors, are expected to be conserved, whereas peptides acting in self/non-self discrimination are expected to be subject to diversifying selection. However, there is a grey zone: PAMP receptors involved in non-self recognition perceive universally conserved molecules of potential pathogens, and ‘successful’ PAMP receptors, such as the flagellin receptor, may be subject to stabilizing selection.
Acknowledgements Work in the author’s laboratory is supported by the Swiss National Science Foundation.
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40. Tang WH, Kelley D, Ezcurra I, Cotter R, McCormick S: LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro. Plant J 2004, 39:343-353. 41. Jones DA, Takemoto D: Plant innate immunity — direct and indirect recognition of general and specific pathogenassociated molecules. Curr Opin Immunol 2004, 16:48-62. 42. Belkhadir Y, Subramaniam R, Dangl JL: Plant disease resistance protein signaling: NBS-LRR proteins and their partners. Curr Opin Plant Biol 2004, 7:391-399. 43. Gomez-Gomez L, Boller T: Flagellin perception: a paradigm for innate immunity. Trends Plant Sci 2002, 7:251-256. 44. Gomez-Gomez L, Boller T: FLS2: An LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 2000, 5:1003-1011. 45. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A: The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001, 410:1099-1103. 46. Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA, Barrett SLR, Cookson BT, Aderem A: Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat Immunol 2003, 4:1247-1253. The authors identify the epitope of bacterial flagellin required to activate mammalian TLR5. The epitope recognized by TLR5 is found to be highly
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conserved but different from the flg22 epitope recognized by plants, indicating that the capability to recognize flagellin arose independently in plants and animals by convergent evolution. 47. Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JDG, Felix G, Boller T: Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 2004, 428:764-767. This publication demonstrates the biological importance of PAMP perception and innate immunity by showing that Arabidopsis thaliana mutants lacking the flagellin receptor FLS2 are more sensitive to bacterial disease than wild-type plants. It also shows induction of almost 1000 genes, including 112 RLK genes, in response to flg22 in wild-type plants. 48. Kunze G, Zipfel C, Robatzek S, Niehaus K, Boller T, Felix G: The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 2004, 16:3496-3507. The highly conserved N terminus of bacterial elongation factor Tu (the most abundant bacterial protein) is recognized as a PAMP in Arabidopsis thaliana. 49. Felix G, Boller T: 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. J Biol Chem 2003, 278:6201-6208. 50. Brunner F, Rosahl S, Lee J, Rudd JJ, Geiler C, Kauppinen S, Rasmussen G, Scheel D, Nu¨ rnberger T: Pep-13, a plant defenseinducing pathogen-associated pattern from Phytophthora transglutaminases. EMBO J 2002, 21:6681-6688.
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larities and differences between the perception of endogenous and exogenous signals. Peptide signalling and receptor-like kinases (RLKs) in plants have been reviewed previously in this journal [3] and elsewhere [4–6]. The present review is focussed on the cases where RLKs have been implicated most clearly in peptide signalling. Of particular interest are the RLKs with an extracellular leucine rich repeat (LRR) domain, the LRR-RLKs. The Arabidopsis genome contains >210 LRR-LRK genes [1], and the rice genome >370 [2]. LRR domains are known to be involved in protein– protein or protein–peptide interactions, and LRR-RLKs comprise putative receptors of both non-self and hormonal peptide signals in plants (Figure 1).
Peptide hormones in plants Current Opinion in Cell Biology 2005, 17:116–122 This review comes from a themed issue on Cell regulation Edited by Brian Hemmings and Peter Parker
0955-0674/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2005.02.007
Introduction In animal biology, there is a clear conceptual dichotomy in peptide signalling. On the one hand, peptide hormones are key players in development: peptide hormones are evolutionarily conserved, and perceived by evolutionarily conserved receptors. On the other hand, peptides allow the discrimination of self and non-self in the immune system, and ultimately enable an effective defence against pathogens. Almost any arbitrary peptide can be an antigen, and the receptors recognizing them — the antibodies — are characterized by a maximal lack of evolutionary conservation, as their genes are freshly assembled in each individual. In plants, like in animals, peptides are signals in development as well as in self and non-self perception, but the distinction between the corresponding receptors is less clear. Plants have large families of receptor-like kinases (RLKs) [1,2], which appear to be involved in both types of signalling. Even the downstream pathways appear to be similar in several instances. This raises intriguing questions about evolution, and about the simiCurrent Opinion in Cell Biology 2005, 17:116–122
The time-honoured five classes of phytohormones (auxins, cytokinins, gibberellins, ethylene and abscisic acid) are micromolecules of various types but are not peptides. Several similar compounds have been elevated to phytohormone status in the last decades, including brassinosteroids, jasmonate and salicylic acid, but peptides were only recently added to the list. Systemin and the systemin receptor SR160
The first peptide that acquired the status of an acknowledged plant hormone was systemin [7]. As its name implies, this 18-amino-acid peptide was believed to be a systemic signal of wounding in tomato. However, elegant grafting experiments indicate that systemin is required only locally, and that the systemic signal is in fact jasmonate, a prototypic phytohormone, or a related compound of the octadecanoid pathway [8,9]. A high-affinity binding site for systemin was found in cell cultures of Lycopersicon peruvianum, a close relative of tomato (L. esculentum). This binding site was identified as a 160 kDa protein, called ‘SR160’, by labelling with a radioactive photoaffinity analogue of systemin, and purified by preparative gel electrophoresis, concanavalin A sepharose chromatography and analytical gel electrophoresis [10]. Surprisingly, tryptic peptides generated from the radioactive band showed homology to BRI1, an Arabidopsis LRR-RLK thought to be involved in the perception of brassinolides (Figure 1b). On the basis of this finding, a L. peruvianum cDNA library was screened with an Arabidopsis BRI1 fragment and a fragment of a tomato EST encoding a BRI1 homolog. The resulting cDNA encoded a LRR-RLK with high homology to BRI1 and clearly represented the gene encoding the major www.sciencedirect.com
Peptide signalling in plants Boller 117
Figure 1
(a) Flagellin receptor FLS2
flg22
(b) Systemin receptor
(d) Clavata system
BRI1
SR160
Systemin
(c) Phytosulfokine receptor
120 kDa receptor
(?) =
(??)
brl psk
(??)
CLV1 CLV2
CLV3
MAPKKK
(??)
MAPKK
(??)
M APK
WRKY
(WUS)
Current Opinion in Cell Biology
LRR-RLKs (receptor-like kinases with leucine-rich repeats) involved in peptide signalling. (a) Flagellin receptor in Arabidopsis thaliana. The gene FLS2 encodes a LRR-LRK, which is essential for flg22 binding and signalling. Signal perception activates a MAP kinase cascade and culminates in the activation of WRKY-type transcription factors. (b) Systemin receptor in Lycopersicon peruvianum. Systemin binds to a receptor of 160 kDa (SR160). Perception of systemin activates a MAP kinase cascade and leads to a response that resembles the flg22 response. According to current evidence, the gene encoding SR160 is the tomato ortholog of the Arabidopsis gene BRI1, encoding the putative receptor of brassinolides (brl). Since the responses to systemin and brassinolides are completely different, there remains a question mark about the identity of SR160 and BRI1. (c) Phytosulfokine receptor in Daucus carota. Phytosulfokine (psk) binds to a LRR-RLK of 120 kDa. Little is known about phytosulfokine signal transduction. (d) The clavata system in Arabidopsis thaliana. The 5 kDa peptide CLV3 is expressed in the apical meristem and serves to limit the size of the meristem by negatively regulating expression of WUSCHEL, the gene encoding the transcription factor WUS. According to earlier publications, CLV3 interacts with a receptor consisting of CLV1, a LRR-RLK, and CLV2, a LRR-RLP (receptor-like protein with leucine-rich repeats). However, since clv1 null mutations have a much weaker phenotype than clv3 mutations, it is unlikely that CLV3 acts through CLV1. Possibly, CLV3 binds to a different, unknown RLK and RLP (depicted in grey, with question marks). The CLV1/CLV2 and CLV3 pathways both regulate WUS expression but their interplay remains unknown.
protein in the radioactive band of SR160, as shown by lysyl endopeptidase fingerprinting [10]. The same gene was independently found to be mutated in the tomato mutant cu-3, which is dwarf and does not respond to brassinosteroids, exactly like the Arabidopsis bri1 mutants [11]. This led to the notion that the same LRR-RLK may be the receptor for both brassinosteroids and systemin [11]. There is a caveat, however. The Cu-3/SR160 gene clearly represents the tomato orthologue of BRI1 [11], and the major protein in the radioactive band isolated in the search for the systemin receptor [10], but not necessarily the photoaffinity-labelled systemin receptor itself, since purification was based primarily on size in gel electrophoresis, and not on specific properties of the systemin receptor. www.sciencedirect.com
More direct evidence that Cu-3/SR160 indeed encodes the systemin receptor came from an experiment with tobacco, a plant that does not respond to systemin (see below). A transgenic tobacco plant expressing the Cu-3/ SR160 gene was generated [12]. This plant produced seeds that segregated when grown on kanamycin. However, surprisingly, these transgenic seedlings were not further analysed. Instead, a suspension cell culture was generated from the transgenic tobacco plant, and this culture was shown to express SR160 and to respond to systemin with an alkalinization response, as would be the case with a L. peruvianum cell culture [12]. Confirmation of this result with transgenic plants is eagerly awaited. Unequivocal evidence for or against the identity of the Cu-3/SR160 gene and the gene encoding the systemin Current Opinion in Cell Biology 2005, 17:116–122
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receptor would come from complementation experiments similar to the ones performed with the cotton BRI1 ortholog GhBRI1, which complemented the Arabidopsis bri1 mutation [13]. If Cu-3/SR160 encoded the systemin receptor, then the Arabidopsis bri1 mutant complemented with tomato Cu-3/SR160 should show systemin binding or a systemin response, whereas the tomato cu-3 mutant complemented with Arabidopsis BRI1 should fail to respond to systemin. The presence of the prosystemin gene (encoding the systemin precursor), and the capability to perceive systemin, is restricted to tomato and its closest relatives (Lycopersicon peruvianum, potato, pepper and nightshade) [7], and even among these plants, the 18-amino-acid sequence of systemin differs in at least two positions. Both tobacco (a closely related solanaceous plant) and Arabidopsis lack a prosystemin gene and do not respond to systemin [7]. This is in stark contrast to the situation with peptide hormones in animals, where sequence and function are conserved even across taxonomic orders and classes, and it indicates that systemin-type signalling may be subject to diversifying selection. In tomato, the gene encoding prosystemin appears to be expressed exclusively in phloem parenchyma cells, and the gene product appears to reside in the cytoplasm [14]. Expression of prosystemin in phloem parenchyma cells is strongly induced by wounding as well as by jasmonate [14]. These results indicate that systemin is an ‘endogenous elicitor’ rather than a hormone in the usual sense. When it is released upon wounding or death of phloem parenchyma cells, it is recognized by systemin receptors residing in the plasma membrane of adjacent mesophyll cells, and it thereby elicits the herbivore defence program. In contrast to other endogenous elicitors, it seems to be subject to diversifying selection, perhaps in response to herbivores evolving to counteract systeminmediated signalling.
80-amino-acid precursor protein and promotes dedifferentiation and cell division in plant cells at nanomolar concentrations [5]. A binding site for phytosulfokine has been identified by ligand-based affinity chromatography and shown to be an LRR-type receptor-like kinase [17]. The corresponding gene was cloned, and its overexpression in carrot cells was shown to lead to a considerable increase in phytosulfokine binding sites and to altered growth responses, strongly indicating that this gene encodes the phytosulfokine receptor [17] (Figure 1c). Although phytosulfokine promotes cell proliferation in tissue culture, and can be used for ‘chemical nursing’ (i.e. to promote cell division of plant cells grown at high dilutions) [18], its actual role in the growth and development of plants remains to be determined. The clavata system
The CLAVATA (CLV) signalling pathway has become a paradigm of stem cell maintenance in plant meristems [19]. The CLV pathway negatively regulates expression of WUSCHEL (WUS), a gene encoding a homeodomain transcription factor essential for maintaining stem cell identity, and thereby limits meristem size. According to current reviews, the CLV signalling pathway comprises the extracellular peptide CLV3 (encoded by the CLV3 gene), thought to act as a peptide signal, and a receptor complex consisting of CLV1 (an LRR-RLK) and CLV2 (a receptorlike protein with leucine-rich repeats [LRR-RLP]), thought to interact with CLV3 [5,6,20] (Figure 1d).
Phytosulfokine and the phytosulfokine receptor
However, no one has been able to show that CLV3 actually interacts with CLV1/CLV2; earlier evidence to this effect [21] has been retracted [22]. What is the current status of this model? Studies with a CLV3– GFP fusion protein expressed under the CLV3 promoter indicate that CLV3 spreads extracellularly from the location of its synthesis; this spread appears to be restrained in the region where CLV1 is expressed, with the result that CLV3 does not enter the organizing centre where WUS is expressed [19]. Although these studies are tantalizingly suggestive of a function for CLV3 as a short-distance morphogen, it remains to be seen how CLV3 is perceived, and how CLV1 is involved. Null mutants of CLV3 have a very strong phenotype, whereas the recently isolated null mutants of CLV1 have very weak phenotypes [23]. This supports the importance of CLV3 in meristem regulation, but is in contradiction to a model in which CLV3 exerts its activity through interaction with CLV1. The previously isolated ‘strong’ alleles of CLV1 appear to have a dominant-negative effect on CLV3 signalling [23]. This may be explained by a new model in which the CLV3 signal is mediated by another, unknown RLK, and in which the dominant-negative CLV1 mutants interfere with the function of this other RLK [23].
Another plant peptide hormone is phytosulfokine, a sulfated pentapeptide [Tyr(SO3H)-Ile-Tyr(SO3H)-ThrGln]. This molecule is generated from a ubiquitous
Interestingly, there are at least 25 homologues of the CLV3 gene in Arabidopsis, the CLE genes (genes that
Using bioassays designed for the identification of elicitors, two other groups of peptides have been isolated that may function as endogenous elicitors, namely RALF [15] and ‘tobacco systemin’, both of which are glycopeptides unrelated to tomato systemin; see [7]. These peptides are evolutionarily more conserved than systemin and also occur in other plants, including tomato [15,16]. They may be released upon wounding and induce the typical defence responses also seen in response to exogenous ‘general elicitors’ and PAMPS (pathogen-associated molecular patterns; see below). Their receptors are currently unknown.
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Peptide signalling in plants Boller 119
share a conserved C-terminal domain with CLV3 and have putative homologues in maize called ESR) [24]. They all encode small secreted peptides. Modulation of the expression of some of these CLE genes can cause interesting developmental aberrations. For example, loss of CLE40 causes a root-waving phenotype [25], and overexpression of CLV19 causes consumption of the root meristem [26,27]. All of these putative peptide signals, including CLV3, may act as morphogens [28], but their receptors are currently unknown. On the other hand, a CLV1-type RLK has appeared in an unexpected context. Long-distance signalling from roots via leaves to roots is known to restrict nodulation in legumes, and loss of this autoregulation of nodules (AON) causes hypernodulation and increased numbers of lateral roots. The genes responsible for AON in Lotus japonicus (HAR-1, [29,30]) and in soybean (NTS-1, [31]) have been cloned; they encode LRR-RLKs highly homologous to CLV1. It will be interesting to find out whether a CLV3-like peptide signal is produced in nodulated roots and transferred to the shoots to activate HAR1/NTS1. Other peptides potentially serving as hormonal signals
Two gain-of-function screens for genes that influence development in Arabidopsis have independently yielded a small gene family encoding small peptides of 50 amino acids, named DVL (devil) because of their horned-fruit phenotype [32] and ROTUNDIFOLIA-FOUR-LIKE because of their leaf phenotype [33]. This gene family consists of 20 genes encoding similar small peptides, all lacking an apparent signal sequence, and all with a highly similar C-terminal end. Overexpression of the conserved C terminus of these peptides is sufficient to cause morphological aberrations [33]. Although it remains to be demonstrated that these peptides are actually involved in development at natural expression levels, and although their potential receptors are completely unknown, the data hint at the existence of multiple peptide signals acting as morphogens [28] to determine organ development.
Peptide-mediated self perception in plants A key innovation in plant evolution has been the formation of a gynoecium (pistil) in angiosperms, allowing rejection of self-pollen and thereby the enforcement of outbreeding. In the Brassicaceae, ‘sporophytic selfincompatibility’ is based on the specific interaction of two matching components encoded at the S-locus, namely the SCR (the S-locus cysteine-rich peptide produced by pollen) and the SRK (the S-locus-specific RLK expressed in the pistil) [34]. A given plant species may have as many as 100 S haplotypes, and for each of these the SRK appears to interact only with the cognate SCR and not with any of the other 99. How did this remarkable specificity evolve? As demonstrated in a case study, four contiguous amino acids are sufficient to create specificity of SCR function, and SCRs have a high degree of evoluwww.sciencedirect.com
tionary flexibility [35]. This has lead to a hypothesis explaining how new self-incompatibility specificities can be generated by gradual modification of SRK-SCR affinities without invoking a self-compatible transition state [35]. How is the signal provided by SCR-SRK interaction transduced? An analysis of a recessive modifier mutation which eliminates the self-incompatibility response in the stigma yielded a downstream signalling element, namely the M locus protein kinase (MLPK) [36]. Thus, two membrane-bound kinases, SRK with its extracellular recognition domain and MLPK with its myristoyl membrane anchor (but without an extracellular domain), are both required for the self-incompatibility response. It is tempting to speculate that the two kinases interact physically in the receptor complex, as is the case for receptor tyrosine kinases and membrane-anchored Src family kinases in animal hormone signalling. Interestingly, Arabidopsis thaliana, a self-compatible species, possesses all the elements of a functional selfincompatibility response except for functional SCR-SRK alleles, as shown by the establishment of self-incompatibility by the expression of an SCR-SRK pair from the self-incompatible Arabidopsis lyrata [37,38]. An analysis of the pseudogenes present in the non-functional S-loci of 21 A. thaliana ecotypes indicates that self-compatibility has arisen recently and may be associated with the post-Pleistocene expansion of A. thaliana from glacial refugia [39]. A similar interaction between peptide signals and RLKs may also be important in self-acceptance, in other words the acceptance of conspecific pollen by a pistil. It has been shown in tomato that a small cysteine-rich peptide produced by the stigma specifically binds to a LRR-LRK specifically expressed in pollen tubes, and that the stigma-produced peptide promotes pollen tube growth in vitro [40].
Peptide-mediated non-self perception in plants Non-self perception is a key element in the defence against potential pathogens. Plants have two different non-self perception systems for microbial molecules: a basal non-self recognition system with similarities to the innate immunity in animals, which is described in more detail below, and a more specialized gene-for-gene recognition system where a specific AVR (avirulence) gene product of a given pathogen is recognized by a cognate resistance gene product of a host plant. Conceptually the gene-for-gene system is based on peptide recognition as well. However, the most common resistance gene products are LRR-containing proteins without protein kinases, and they appear to interact in an indirect way with the AVR products; this subject is thus outside the Current Opinion in Cell Biology 2005, 17:116–122
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scope of the present review, but has been covered by excellent recent reviews elsewhere [41,42]. Flagellin and the flagellin receptor FLS2
In animal and human innate immunity, TLRs (toll-like receptors) perceive PAMPs (molecular patterns that are universally conserved in a whole class of microbes). Similarly, plants recognize ‘general elicitors’ derived from pathogens. These general elicitors may be peptides such as flg22, which represents the most highly conserved domain of bacterial flagellin [43]. Perception of flg22 in Arabidopsis depends on FLS2, a gene encoding a LRRRLK [44] (Figure 1a). Subsequently, mammalian perception of bacterial flagellin was shown to depend on TLR5 [45], highlighting similarities between the plant and animal innate immunity systems. Interestingly, the flagellin domain recognized by TLR5 differs from the flg22 domain, indicating that the two perception systems have arisen independently by convergent evolution [46]. A global gene expression study indicated that flg22 stimulation of wild-type Arabidopsis plants caused the induction of almost 1000 genes (3% of the annotated genes) within 30 min, whereas flg22 did not induce a single gene in plantlets carrying the fls2 mutation [47]. RLKs were massively over-represented among the induced genes: 110 RLKs were induced, corresponding to 16% of all RLKs in Arabidopsis. When fls2-mutant plants were spray-inoculated with Pseudomonas syringae DC3000, they displayed much stronger disease symptoms and strongly enhanced bacterial growth compared to the corresponding wild-type, Landsberg-erecta. The ecotype Wassiljewskaja (Ws-0), long known anecdotally to be particularly sensitive to bacterial colonization, is a natural fls2 mutant. In spray-inoculation experiments with Pseudomonas syringae DC3000, Ws-0 indeed displayed enhanced disease susceptibility, and normal disease resistance was reestablished in the transgenic plants expressing an FLS2 construct [47]. Taken together, these results indicate that the PAMP receptor FLS2 contributes to basal disease resistance. Other peptides as PAMP signals
The flagellin-derived peptide is active as a PAMP in many different plant species [48]. In other cases, bacterial molecules are recognized in only a subset of plant species. For example, bacterial cold shock protein, CSP, and csp16, a peptide derived from CSP, acts as a PAMP in tobacco and many other Solanaceae at subnanomolar concentrations, but it does not induce any response in Arabidopsis [49]. Similarly, pep13, a peptide derived from the extracellular trans-glutaminase of oomycete pathogens, is active as a potent elicitor in parsley and potato but not in Arabidopsis [50]. Vice versa, bacterial elongation factor Tu (Ef-TU) and elf26, a peptide derived from its N terminus, is recognized by Arabidopsis and many other Brassicaceae, again at subnanomolar concentrations, but does not have any effect in Solanaceae [48]. In all these Current Opinion in Cell Biology 2005, 17:116–122
cases, the proteins recognized by the plants are highly abundant in a whole class of microorganisms, and the plant recognition system is directed against a particularly highly conserved domain of these microbial proteins. Thus these proteins fit the definition of a PAMP. It is likely that in evolution specific lineages of plants have acquired distinct, diverse PAMP recognition capabilities, making it difficult for a given microbe to avoid recognition in all plant species and thereby to become a ‘generalist’ plant pathogen. A similar principle appears to govern the diversification of secondary metabolites in plants, such as phytoalexins or alkaloids. Each family, or even each genus or species, has its own characteristic mix of secondary metabolites, and its own guild of specialist pathogens and herbivores that can cope with this specific mix. This may also provide an explanation for the large-scale diversification of RLKs in plants [1,2].
Conclusions Peptide signalling in plants is important in development as well as in self/non-self perception. Plants appear to have diversified their RLK families, and the corresponding signalling cascades, to accommodate both functions. In evolutionary terms, peptides acting as hormones and morphogens, and consequently their receptors, are expected to be conserved, whereas peptides acting in self/non-self discrimination are expected to be subject to diversifying selection. However, there is a grey zone: PAMP receptors involved in non-self recognition perceive universally conserved molecules of potential pathogens, and ‘successful’ PAMP receptors, such as the flagellin receptor, may be subject to stabilizing selection.
Acknowledgements Work in the author’s laboratory is supported by the Swiss National Science Foundation.
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