PERSPECTIVE. Are innate immune signaling pathways in plants and animals conserved? Frederick M Ausubel

Transcription

1 Are innate immune signaling pathways in plants and animals conserved? Frederick M Ausubel Although adaptive immunity is unique to vertebrates, the innate immune response seems to have ancient origins. Common features of innate immunity in vertebrates, invertebrate animals and plants include defined receptors for microbe-associated molecules, conserved mitogen-associated protein kinase signaling cascades and the production of antimicrobial peptides. It is commonly reported that these similarities in innate immunity represent a process of divergent evolution from an ancient unicellular eukaryote that pre-dated the divergence of the plant and animal kingdoms. However, at present, data suggest that the seemingly analogous regulatory modules used in plant and animal innate immunity are a consequence of convergent evolution and reflect inherent constraints on how an innate immune system can be constructed. Both plants and animals have complex innate mechanisms to recognize and respond to attack by pathogenic microorganisms. Innate immunity relies on a defense strategy that involves a set of defined receptors referred to as pathogen- or pattern-recognition receptors (PRRs) that recognize microbe-associated molecules. There is a wealth of evidence that the mammalian innate immune response has ancient origins in arthropods 1,2 and perhaps nematodes as well 3 7. In vertebrates, the innate immune response not only serves as a first line of defense in the response to pathogenic microbes but also is key in the production of costimulatory molecules involved in T cell activation and chemokines and cytokines. Protists, fungi and other unicellular eukaryotes may also have evolved defensive mechanisms that confer resistance to microbial pathogens. However, that is not addressed here and neither is the involvement of highly conserved RNA interference related antiviral defenses. Instead, the generally accepted view that PRRs, signal transduction pathways and downstream effectors are evolutionarily conserved and are used by plants, insects and vertebrates is examined. Pathogen-associated versus microbe-associated molecules Pathogen-associated molecular pattern (PAMP) is the term generally used when referring to the molecules that elicit innate immune responses. As classically defined, PAMPs are evolutionarily conserved Frederick M. Ausubel is in the Department of Genetics, Harvard Medical School, and Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA. ausubel@molbio.mgh.harvard.edu Published online 21 September 2005; doi: /ni1253 pathogen-derived molecules that distinguish hosts from pathogens. PAMPs include lipopolysaccharide, peptidoglycan, bacterial flagellin and mannans of yeast. However, because nonpathogens also synthesize these molecules, the term pathogen-associated is a misnomer and a more precise term would seem to be microbe-associated molecular pattern. Thus, it makes sense that hosts would also have defined receptors for molecules that are truly pathogen specific, but only in plants is there definitive evidence for immune receptors that recognize pathogen-encoded virulence-related molecules such as type III effectors. To avoid confusion here, the term microbe-associated molecule(s) is used instead of PAMP. Toll-like receptors in insects and mammals In insects and mammals, a family of conserved transmembrane Toll-like receptors (TLRs) functions directly or indirectly as PRRs for microbeassociated molecules Study of conserved TLR signaling pathways has served as a paradigm for insights that can be gained due to evolutionary conservation of innate immune signaling components 14,15. Initially, the Toll pathway was identified because of its involvement in pattern formation in early drosophila embryo development 16. Later, the cytoplasmic domain of Toll (the Toll interleukin 1 (IL-1) receptor (TIR) domain) was noted to have homology with the cytoplasmic domain of human IL-1 (ref. 17), and analysis of the promoters of genes encoding antimicrobial peptides in drosophila suggested that they were regulated by NF-κB-like transcription factors that also function in the Toll pathway 18. This led to experiments demonstrating that drosophila Toll pathway mutants are immunocompromised 19. Meanwhile, a human homolog of Toll was shown to activate expression of NF-κBcontrolled genes 20, and positional cloning of mouse Lps, which confers resistance to lipopolysaccharide-induced endotoxin shock, identified TLR4 as the lipopolysaccharide sensor 21. Thus, a core component of the mammalian innate immune system involved in mediating the cellular response to lipopolysaccharide and the proinflammatory cytokine IL-1 was shown to have features in common with the signaling pathways of the insect immune response. TLRs are characterized by an extracellular leucine-rich repeat (LRR) domain and an intracellular TIR protein-protein interaction domain. LRRs are found in a variety of receptors in both plants and animals. TLRs are coupled to signaling adaptors such as MyD88, which also have TIR domains. Activation of the TLR signaling cascade results in the nuclear translocation of NF-κB-like transcription factors, leading to the production of antimicrobial peptides in both insects and vertebrates and signaling molecules such as cytokines in vertebrates. In drosophila, the TLR pathway responds to fungi and Gram-positive bacteria 10,19,22, NATURE IMMUNOLOGY VOLUME 6 NUMBER 10 OCTOBER

2 Figure 1 Extracellular and intracellular PRRs in plants and animals. Animal TLRs and plant receptorlike kinases are similar in overall structure in that all are transmembrane receptors that have C-terminal LRRs. However, the cytoplasmic domains are not conserved. In both plants and animals, extracellular receptors respond to highly conserved microbe-associated molecules such as bacterial flagellin. Like the extracellular receptors, the cytoplasmic animal CLR proteins and plant NBS-LRR pathogen-resistance proteins have the same overall tripartite structure with C-terminal LRR and central nucleotide-binding site (NBS) domains. As with TLRs and receptor-like kinases, however, the N-terminal domains are not conserved between plants and animals. In animals, the N-terminal domain can be, for example, one or two caspase activation and recruitment domains in Nod1 and Nod2, respectively, and pyrin and NACHT domains in NALP proteins. In plants, the N-terminal domains are usually TIR or coiled-coiled domains. Nod1, Nod2 and NALP3 respond to peptidoglycan degradation products, whereas the plant NBS-LRR proteins respond to pathogen-specific virulence factors. Although the presence of the TIR domain in some NBS-LRR plant disease-resistance proteins suggests a common evolutionary origin of plant and animal PRRs, the downstream signaling pathways have nothing in common. The overall conservation of the tripartite structure of animal CLR and plant NBS-LRR proteins also suggests evolutionary conservation, but these proteins are not present in nematodes or arthropods, suggesting independent evolutionary origins in plants and animals. whereas a second immune signaling pathway, the immune-deficient mutant (Imd) pathway, responds to Gram-negative bacteria via a peptidoglycan-recognition protein Like the TLR pathway, the Imd pathway leads to the activation of NF-κB-like transcription factors. In comparing the drosophila and mammalian innate immune signaling pathways, the mammalian TLR pathway is made up of components analogous to those found in the proximal and distal portions of the drosophila TLR and Imd pathways, respectively 26. Mammalian CLR (Nod) receptors In addition to the transmembrane TLRs, mammals have a family of cytosolic PRRs that belong to a family of proteins variously referred to as CATERPILLER (CLR) or Nod proteins that are involved in apoptotic and inflammatory responses 13, CLR (Nod) proteins, hereafter referred to CLR proteins, are characterized by a tripartite domain architecture consisting of a variable N-terminal domain, a central nucleotide-binding domain and C-terminal LRRs and are structurally similar to the TIR-NBS-LRR and CC-NBS-LRR pathogen-resistance proteins in plants (discussed below) that function in the plant immune response (Fig. 1). CLR proteins include NALPs, Nods and NAIPs. The two Nod proteins Nod1 and Nod2 mediate intracellular responsiveness to peptidoglycan degradation products, leading to the activation of NF-κB. Nod1 responds to γ-d-iso-glutamyl diaminopimelic acid, and Nod2 responds to muramyl dipeptide The two Nod proteins differ mainly in that Nod1 has one and Nod2 has two N-terminal caspase activation and recruitment domains. There are 14 NALP proteins in humans 31,35. Like Nod2, NALP3, which is a key component of the inflammasome, is activated by the muramyl dipeptide component of peptidoglycan 36. Muramyl dipeptide mediated activation of the inflammasome leads in turn to activation of caspase 1 and maturation of pro-il-1b. It is not known whether the peptidoglycan degradation products interact directly with the LRRs of Nod1, Nod2 or NALP3 or whether there is a linker protein involved. In any case, peptidoglycan must translocate into the cytoplasm, and at least some cell types such as macrophages have intracellular hydrolyases that can digest peptidoglycan 36. Plant response to microbial molecules Like insects and vertebrates, plants have receptors for microbe-associated molecules and respond to many of the same molecules that animals respond to, including lipopolysaccharide and flagellin (Fig. 1). Plants also respond to a wide variety of molecules associated with fungi or oomycetes, including cell wall components such as chitin and ergosterol 37. Has the comparison of innate immune signaling pathways in plants and animals led to new insights analogous to the comparison of TLR signaling in mammals and flies? The response of the reference plant Arabidopsis thaliana to eubacterial flagellin has been a focus of interest Arabidopsis responds both to flagellin and to a highly conserved 22 amino acid fragment of the flagellin protein called Flg22 (ref. 39). Flg22 activates a signal transduction cascade that includes a transmembrane LRR receptor kinase (FLS2), a MAP kinase cascade, so-called WRKY transcription factors and downstream effector proteins 38,40,44 (Fig. 2). The structure of the FLS2 receptor is reminiscent of that of TLRs in that FLS2 has an extracellular LRR domain. In addition, both the FLS2 and TLR signaling pathways involve a conserved family of cytoplasmic serine-threonine kinases (cytoplasmic kinase domain of FLS2 and IRAK or PELLE kinases in mammals and drosophila, respectively) 45,46. However, the similarity between the FLS2 and the TLR signaling pathways does not extend beyond those features. FLS2 corresponds functionally to TLR5 in mammals, which is also a flagellin receptor, but TLR5 and FLS2 respond to different epitopes in the flagellin protein 47. The LRR domains of FLS2 and TLR5 are very divergent. The FLS2 receptor does not have an intracellular TIR domain like the TLRs, and no downstream signaling components have been identified in plants homologous to the MyD88 family of TIR domain containing adaptor proteins. If a functional flagellin receptor had evolved in an ancestral eukaryote, it is difficult to explain why both the flagellin ligand specificity and the intracellular signaling domain are different in the plant and animal receptors. Finally, plants do not have transcription factors homologous to the Rel-like family of transcription factors that includes Dif and NF-κB in flies and mammals, respectively, and the WRKY family of transcription factors activated downstream of FLS2 in arabidopsis is not found in animals. Thus, although the overall structure of microbe-associated molecule signaling pathways in plants and animals is similar in that both involve transmembrane LRR receptors, mitogen-associated protein kinase 974 VOLUME 6 NUMBER 10 OCTOBER 2005 NATURE IMMUNOLOGY

3 (MAPK) signaling cascades, production of active oxygen and nitrogen species, calcium fluxes, activation of transcription factors and the inducible expression of immune effectors, there is little evidence that this overall similarity reflects evolutionary conservation of an ancient signaling pathway. Indeed, the apparent conservation that is typically cited seems to be equally well explained as being a reflection of the overall conservation of the components of canonical MAPK signaling cascades that form a bridge between diverse signal sensors and/or receptors and target genes in eukaryotes. These MAPK signaling pathways, which often include transmembrane receptors, MAPKKKs, MAPKKs, MAPKs and downstream transcription factors, are present in yeast, plants, invertebrates and vertebrates and presumably appeared very early in evolution before the emergence of multicellularity. Intracellular CLR receptors in plants The best-characterized plant immune receptors are a large class of intracellular receptors referred to as NBS-LRR pathogen-resistance proteins, which have an overall tripartite structure similar to that of the mammalian CLR proteins 48,49 (Fig. 1). In general, plants have large families of these NBS-LRR proteins; arabidopsis has 140 (ref. 50) and rice has over 500 (ref. 51). Most of the NBS-LRR pathogen-resistance proteins have either a TIR or a coiled-coil N-terminal domain. In contrast to the Nod1, Nod2 and NALP3 proteins, which respond to peptidoglycan degradation products, the plant NBS-LRR pathogen-resistance proteins that have been studied respond to pathogen-specific signals, including pathogen-encoded virulence-related factors. In the best-studied cases, NBS-LRR proteins function indirectly as receptors for bacterial effector proteins that are translocated directly into host cells by the type III secretion system, in that they recognize the host cell proteins targeted by the type III effector proteins 48,49. These targets include components of the host defense response, including innate immune response pathways or other critical cellular processes If a plant host can recognize a particular effector protein directly, or indirectly by virtue of the action of the effector protein on a target, a rapid and potent defense response ensues that often involves localized programmed cell death of infected cells. An important issue that has not been fully resolved is whether the defense responses activated downstream of NBS-LRR pathogen-resistance proteins are qualitatively or quantitatively different from those activated downstream of receptors such as FLS2 that respond to generic highly conserved microbe-associated molecules. Some data (S. Ferrari, C. Denoux, J. Dewdney, G. de Lorenzo and F.M.A., unpublished data and ref. 43), however, suggest that Flg22 rapidly activates a set of genes independently of the signaling pathways known to be involved in NBS-LRR signaling. Notably, activation of programmed cell death is a feature shared by both plant NBS- LRR proteins and at least some animal CLR proteins. But there is no evidence that the programmed cell death signaling pathways downstream of the plant and animal CLR proteins share any common components, in that caspase-dependent apoptosis is restricted to animals. Thus, although the conserved structure of CLR proteins and the fact that the CLR receptors activate programmed cell death in both plants and animals seem to provide evidence for an ancient origin for innate immune signaling pathways, it is not possible to distinguish a common evolutionary origin from convergent evolution. One line of evidence that may favor the convergent evolution model is that CLR proteins as a class do not seem to be encoded in the Caenorhabditis elegans and drosophila genomes and that the vertebrate CLR family did not evolve until the teleost lineage 31. Thus, it seems likely that the plant NBS-LRR and vertebrate CLR proteins have evolved independently as immune signaling components. In mammals, both the extracellular TLRs and intracellular CLR PRRs can lead to NF-κB activation. Analogously in plants, both transmembrane LRR-containing receptors and intracellular NBS-LRR receptors can activate defense responses involving programmed cell death of infected cells. It is not known whether any intracellular plant NBS-LRR proteins respond to highly conserved microbe-associated molecules and activate a relatively weak response like the FLS2 flagellin receptor. It is also not known whether any CLR proteins in mammals function as receptors for microbe-associated molecules in addition to peptidoglycan or for pathogen-specific virulence factors as they do in plants. Evolutionary conservation of TIR domain receptors The presence of a TIR domain in some of the plant NBS-LRR pathogenresistance proteins has fueled speculation about a common evolutionary origin between the plant and animal innate immunity systems. Figure 2 Signaling pathways downstream of PRRs in mammals, insects, nematodes and plants. In insects and mammals, a family of TLRs mediates the recognition of highly conserved microbeassociated molecules, and there is considerable correspondence between the downstream signaling components. The C. elegans genome encodes a single Toll-like protein that does not seem to function in immune signaling and does not encode Rel-like transcription factors such as mammalian NF-κB or drosophila Dif and Relish. However, C. elegans, drosophila and mammals share a conserved p38 MAPK signaling module. Moreover, C. elegans has a TIR domain containing protein, TIR-1, that functions in innate immune signaling upstream of p38 (refs. 61,64), controls the expression of antimicrobial peptides (D. Kim and F.M.A., unpublished data; N. Pujol and J. Ewbank, personal communication) and is homologous to the mammalian SARM protein. Plants have a family of receptorlike kinases such as the flagellin receptor FLS2. Although the overall structure of the FLS2 signaling pathway seems similar to that of the PRR signaling pathways in animals, there is no conservation of any individual components and the similarity most likely reflects the ubiquity of eukaryotic MAPK stress-response cassettes that respond to environmental signals. NATURE IMMUNOLOGY VOLUME 6 NUMBER 10 OCTOBER

4 In animals, TIR domains are found only in immune-related proteins, but this does not seem to be the case in plants. Moreover, there are no similarities in the signaling pathways downstream of TLRs in animals and TIR-NBS-LRR pathogen-resistance proteins in plants, obscuring any potential evolutionary relationship. Investigation of innate immune signaling pathways in C. elegans has supported the conclusion that the presence of TIR domains in plant NBS-LRR pathogen-resistance proteins and in animal TLRs and TIR domain adaptor proteins is not indicative of evolutionary conservation. Like plants, the nematode C. elegans does not have Rellike transcription factors, and although a single TLR-like protein is encoded in the C. elegans genome, mutation of this TLR does not confer an immunocompromised phenotype 59. Nevertheless, transcriptional profiling analysis has shown that C. elegans respond to bacterial pathogens by activating a variety of genes, homologs of which are involved in antimicrobial responses in insects and mammals 60,61. Moreover, other results have demonstrated a key requirement for an ancient, highly conserved p38 MAPK signaling cassette in C. elegans immunity 62,63 that also is an important in insect and mammalian immunity. Despite the apparent lack of involvement of TLR signaling in C. elegans immunity, a TIR domain protein has been identified that functions upstream of p38 MAPK 61,64 that is highly homologous to the human protein SARM. Although a clear function for human SARM in innate immune signaling has not been established, it is not likely that SARM functions upstream of NF-κB 64. It can argued from these data that the C. elegans TIR-1 (p38) MAPK pathway may be an ancient conserved immune pathway and that the evolutionary recruitment of TLRs and NF-κB into innate immune signaling pathways occurred after the divergence of insects and nematodes 4,64. Work in mammalian cells and with knockout mice has also suggested that the p38 MAPK pathway is a more ancient component of innate immune signaling than is NF-κB 65,66. Additional plant immune response pathways Another reason to think that plant and animal immune signaling pathways have distinct evolutionary origins is that there is essentially no overlap between a large set of immune signaling components identified in plants and those found in animals. For example, a variety of arabidopsis genes involved in innate immune signaling have been identified, and a large body of genetic, physiological and biochemical work has shown that these immunity-related genes are important in conferring pathogen resistance 67. Confirming a variety of biochemical studies, this collective mutant analysis has also shown that several low-molecular-weight signaling molecules, including salicylic acid, jasmonic acid, ethylene and nitric oxide, are key in the regulation of plant innate immune pathways and that the plant innate immune response is complex, involving several parallel defense response signaling pathways that interact or intersect at key regulatory steps 56, However, either the genes identified in these signaling pathways have no mammalian homologs or there is no evidence that the mammalian homologs are involved in innate immune responses. Although the innate immune signaling pathways seem to have little in common in plants and animals, some of the defense reactions that are directly involved in attacking invading pathogens may indeed have very ancient origins. Both plants and animals, for example, synthesize a wide range of small antimicrobial peptides and both produce an oxidative burst via conserved gp91 phox NADPH oxidases. Arabidopsis mutants that contain disruptions of both AtrbohD and AtrbohF, which encode gp91 phox like NADPH oxidases, fail to generate a full oxidative burst in response to infection by bacterial and fungal pathogens However, plant gp91 phox NADPH oxidases also function in a variety of developmental and physiological processes, including root development and stomatal closure. Thus, the multiple functions of gp91 phox NADPH oxidases may reflect the reuse of a highly evolved process for diverse functions. Thus, it seems just as likely that the recruitment of NADPH oxidases for immune response pathways may have occurred independently in the plant and animal kingdoms. Why do plants have species-specific PRRs? An important distinction between the plant and animal innate immune systems is that plants seem to encode a much larger array of PRRs than do animals, including receptors for pathogen-specific virulence factors. The TLRs in flies and mammals, the Imd system in flies and the CLR proteins in mammals respond only to highly conserved microbe-associated molecules. For vertebrates, the evolution of the adaptive immune system and concomitant ability to somatically generate a vast array of pathogen-specific receptors may have obviated the need for the expansion of defined PRRs to include pathogen-specific ones or may have allowed their disappearance. From this perspective, the presence of pathogen-specific receptors in plants such as the NBS-LRR pathogen-resistance proteins suggests that they may function analogously to the adaptive immune system in mammals in providing pathogen-specific immunity. If it is true that plants have many more innate immune receptors than do invertebrate animals, this may be a consequence of the fact that plants are sessile and have relatively long lives compared with those of many invertebrate animals and they do not have any mobile cells that can become specialized in pathogen defense. Cells in plants are fixed in place by their cell walls and every cell in a plant is therefore both responsible for and capable of its own defense. As discussed above, plants have at least two categories of PRRs. The arabidopsis FLS2 flagellin receptor is an example of a plant PRR that functions analogously to the animal PRRs, responding to generic microbe-associated molecules. FLS2 is a member of a large family of receptor-like kinases in plants that have a domain configuration resembling that of transmembrane receptors 45,46. Other members of the family function in defense responses, and it seems likely that many receptor-like kinases function as receptors for microbe-associated molecules. The NBS-LRR pathogen-resistance proteins are a second class of PRRs in plants, responding to strain-specific pathogen-encoded virulence factors. A third potential class of plant PRRs is the rice Xa21 resistance protein 74. Xa21 is a receptor-like kinase that is highly homologous to FLS2, but it seems to respond to a speciesspecific secreted molecule from Xanthomonas oryzae rather than to a broadly conserved microbe-associated molecule like lipopolysaccharide Despite its structural similarity to FLS2, Xa21 functions similarly to NBS-LRR pathogen-resistance proteins in that it elicits a strong defense response that confers resistance to X. oryzae strains expressing the corresponding AvrXa21 elicitor. Because Xa21 responds to an extracellular signal that may not be a virulence factor itself, it blurs the distinction between receptor-like kinases that recognize microbe-associated molecules and NBS-LRR proteins that recognize pathogen-encoded virulence factors. How do the various categories of plant PRRs function together to provide a coordinated defense response? One possibility is that the different types of receptors mutually reinforce the immune response, thereby constituting a two-hit requirement to reduce the wasteful production of a full-blown response to nonpathogenic microbes. Extracellular non species-specific receptors such as FLS2 may serve as early warning sentinels to alert the plant to the presence of a potential pathogen, activating local transient responses and/or priming the plant defense response such that a more vigorous and systemic 976 VOLUME 6 NUMBER 10 OCTOBER 2005 NATURE IMMUNOLOGY

5 long-lasting response is mounted when a second cytosolic signal is detected by an NBS-LRR receptor. Alternatively, having receptors for both highly conserved microbe-associated molecules and pathogenspecific virulence factors may allow plants to directly distinguish pathogens from saprophytic and commensal microbes. In a long-term evolutionary process of pathogen-host warfare, it is possible to envisage that pathogens evolved type III effectors to abrogate the plant response pathways to microbe-associated molecules 56 and that plants in turn evolved receptors for the type III effectors. Because there are an almost unlimited number of potential type III effectors that plants would have to recognize, the NBS-LRR receptors evolved to guard the targets of the type III effectors rather than functioning as receptors for the type III effectors themselves 78. These targets may be individual signaling components or so-called functional modules, collections of proteins that carry out signal transduction or the synthesis of complex secondary products. Published data have provided compelling evidence for the latter scenario and have explained how plants can potentially recognize a diverse set of pathogens and pathogen-specific molecules using a relatively limited number of pathogenreceptors 56, Thus, the evolutionary solution in plants to identify pathogens involves self surveillance, whereas the evolutionary solution in the adaptive immune response in vertebrates for the same problem involves detection of foreign antigens. Conclusions Although it seems to be generally accepted that the innate immune responses of plants and animals share at least some common evolutionary origins, examination of the available data fails to support that conclusion, despite similarities in the overall logic of the innate immune response in diverse multicellular eukaryotes (Fig. 2). It is notable that different transcription factor families are used in plant and animal innate immune responses. Indeed, the WKRY family of transcription factors used in plants is absent from animals, the NF-κB family used in insects and vertebrates is not present in plants, and C. elegans does not use either family. Moreover, even though individual components of the plant and animal innate immune signaling pathways share some common protein motifs, including TIR, NBS and LRR domains, it seems just as likely that these motifs have been recruited independently in different evolutionary lineages to function in immune signaling as it is that they evolved from common ancestral innate immune signaling proteins. Phylogentic studies of gene families involved in innate immunity support that conclusion, suggesting that other components of innate immune signaling in insects and vertebrates, including TLRs and NF-κB, represent highly conserved generalized signaling proteins that were co-opted independently to function in innate immune signaling 82,83. This is not necessarily unexpected. LRRs, for example, are used as receptors for a variety of ligands in many different eukaryotic proteins. The idea of a process of convergent evolution is also supported by the recombinatorial immune system in lampreys (a jawless vertebrate), which involves highly diverse LRRs instead of the immunoglobulin gene segments found in jawed vertebrates 84. Although the conclusion that convergent evolution best explains the similarities in the plant and animal immune response pathways is expressed in some reviews 37,85, it is generally not stated explicitly. Similar conclusions have been reached about the evolution of multicellularity and developmental processes in plants and animals. Although the underlying logic of multicellular development in plants and animals is unexpectedly similar, it seems that multicellularity evolved independently in plants and animals and that the basic molecular mechanisms specifying pattern formation were independently derived 86. Given the compelling case for convergent evolution of innate immune pathways, an important issue is why evolution has chosen a limited number of apparently analogous regulatory modules in disparate evolutionary lineages. Does this reflect inherent biochemical constraints that result from a similar overall logic of how an effective immune system can be constructed? For example, TIR domain containing proteins are involved in three apparently disparate innate immune signaling pathways: TIR-NBS-LRR proteins in plants, TIR (p38) MAPK signaling in nematodes (and possibly other animal phyla) and TLR-mediated signaling in insects and vertebrates. Moreover, at least for animals, TIR domain proteins seem to be used exclusively in immune signaling pathways. It is conceivable that TIR domains, as well other domains such as LRRs and pelle-receptor-like kinases commonly found in immune pathways, have intrinsic biochemical properties that are particularly well suited for these pathways or are particularly resistant to disruption by pathogen-encoded counter-defense mechanisms. Assuming that the last common ancestor of plants and animals was a primitive unicellular eukaryote, it would be informative to determine how many of the components of innate immune signaling pathways are present in and have immune functions in these organisms. Despite many similarities, plants and animals do have one main strategic difference in responding to pathogen attack in that plants elaborate a variety of pathogen-specific PRRs, whereas PRRs in animals seem to be limited to the recognition of very highly conserved microbeassociated molecules. Although it can be argued that this difference in strategy may be an evolutionary consequence of plant architecture, this line of reasoning is not fully explanatory and raises the possibility that both invertebrate and vertebrate animals also have pathogen-specific receptors that have yet to be identified. Indeed, it has been suggested that mammals may express pathogen-specific factors that confer resistance to particular pathogens. Specific alleles of mouse Naip5 (also known as Birc1e), which encodes a member of the CLR protein family, is associated with resistance to Legionella pneumophila 87,88. In work that illuminates the molecular basis of the resistance of Old World monkeys to infection with human immunodeficiency virus, it was shown that such infection is blocked more efficiently in cells expressing rhesus monkey TRIM5α, a RING finger protein, than in those expressing human TRIM5α 89. In a final example, a mouse gene, Ipr1, has been identified that confers resistance to Mycobacterium tuberculosis and Listeria monocytogenes when expressed in macrophages 90 ; however, because Ipr1 confers resistance to two very different pathogens, this suggests that it may function analogously to a plant NBS-LRR guard protein rather than a PRR. Although preliminary, these examples may illustrate yet another common feature of the overall logic of the innate immune response in plants and animals. ACKNOWLEDGEMENTS I thank C. Dardick, W. Dietrich, J. Dangl, J. Ewbank, J. Jones, T. Nurnberger, P. Ronald, P. Schulze-Lefert and J. Sheen for comments. COMPETING INTERESTS STATEMENT The author declares that he has no competing financial interests. Published online at Reprints and permissions information is available online at reprintsandpermissions/ 1. Medzhitov, R. & Janeway, C., Jr. Innate immunity. N. Engl. J. Med. 343, (2000). 2. Medzhitov, R. & Janeway, C.A., Jr. An ancient system of host defense. Curr. Opin. Immunol. 10, (1998). 3. Gravato-Nobre, M.J. & Hodgkin, J. Caenorhabditis elegans as a model for innate immunity to pathogens. Cell. Microbiol. 7, (2005). 4. Kim, D.H. & Ausubel, F.M. Evolutionary perspectives on innate immunity from the study of Caenorhabditis elegans. Curr. Opin. Immunol. 17, 4 10 (2005). NATURE IMMUNOLOGY VOLUME 6 NUMBER 10 OCTOBER

6 5. Kurz, C.L. & Ewbank, J.J. Caenorhabditis elegans: an emerging genetic model for the study of innate immunity. Nat. Rev. Genet. 4, (2003). 6. Millet, A.C. & Ewbank, J.J. Immunity in Caenorhabditis elegans. Curr. Opin. Immunol. 16, 4 9 (2004). 7. Schulenburg, H., Kurz, C.L. & Ewbank, J.J. Evolution of the innate immune system: the worm perspective. Immunol. Rev. 198, (2004). 8. Beutler, B. & Rehli, M. Evolution of the TIR, tolls and TLRs: functional inferences from computational biology. Curr. Top. Microbiol. Immunol. 270, 1 21 (2002). 9. Brennan, C.A. & Anderson, K.V. Drosophila: the genetics of innate immune recognition and response. Annu. Rev. Immunol. 22, (2004). 10. Hoffmann, J.A. & Reichhart, J.M. Drosophila innate immunity: an evolutionary perspective. Nat. Immunol. 3, (2002). 11. Janeway, C.A., Jr. & Medzhitov, R. Innate immune recognition. Annu. Rev. Immunol. 20, (2002). 12. Medzhitov, R. & Janeway, C., Jr. The Toll receptor family and microbial recognition. Trends Microbiol. 8, (2000). 13. Royet, J. Infectious non-self recognition in invertebrates: lessons from Drosophila and other insect models. Mol. Immunol. 41, (2004). 14. Aderem, A. & Ulevitch, R.J. Toll-like receptors in the induction of the innate immune response. Nature 406, (2000). 15. Anderson, K.V. Toll signaling pathways in the innate immune response. Curr. Opin. Immunol. 12, (2000). 16. Belvin, M.P. & Anderson, K.V. A conserved signaling pathway: the Drosophila tolldorsal pathway. Annu. Rev. Cell Dev. Biol. 12, (1996). 17. Gay, N.J. & Keith, F.J. Drosophila Toll and IL-1 receptor. Nature 351, (1991). 18. Sun, S.C., Lindstrom, I., Lee, J.Y. & Faye, I. Structure and expression of the attacin genes in Hyalophora cecropia. Eur. J. Biochem. 196, (1991). 19. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J.M. & Hoffmann, J.A. The dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, (1996). 20. Medzhitov, R., Preston-Hurlburt, P. & Janeway, C.A., Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, (1997). 21. Poltorak, A. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, (1998). 22. Georgel, P. et al. Drosophila immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis. Dev. Cell 1, (2001). 23. Gottar, M. et al. The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 416, (2002). 24. Ramet, M., Manfruelli, P., Pearson, A., Mathey-Prevot, B. & Ezekowitz, R.A. Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416, (2002). 25. Steiner, H. Peptidoglycan recognition proteins: on and off switches for innate immunity. Immunol. Rev. 198, (2004). 26. Hoffmann, J.A. The immune response of Drosophila. Nature 426, (2003). 27. Athman, R. & Philpott, D. Innate immunity via Toll-like receptors and Nod proteins. Curr. Opin. Microbiol. 7, (2004). 28. Girardin, S.E. & Philpott, D.J. Mini-review: the role of peptidoglycan recognition in innate immunity. Eur. J. Immunol. 34, (2004). 29. Philpott, D.J. & Girardin, S.E. The role of Toll-like receptors and Nod proteins in bacterial infection. Mol. Immunol. 41, (2004). 30. Viala, J., Sansonetti, P. & Philpott, D.J. Nods and intracellular innate immunity. C. R. Biol. 327, (2004). 31. Ting, J.P. & Davis, B.K. CATERPILLER: a novel gene family important in immunity, cell death, and diseases. Annu. Rev. Immunol. 23, (2005). 32. Chamaillard, M. et al. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat. Immunol. 4, (2003). 33. Chamaillard, M., Girardin, S.E., Viala, J. & Philpott, D.J. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell. Microbiol. 5, (2003). 34. Girardin, S.E. et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278, (2003). 35. Tschopp, J., Martinon, F. & Burns, K. NALPs: a novel protein family involved in inflammation. Nat. Rev. Mol. Cell Biol. 4, (2003). 36. Martinon, F., Agostini, L., Meylan, E. & Tschopp, J. Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome. Curr. Biol. 14, (2004). 37. Nurnberger, T., Brunner, F., Kemmerling, B. & Piater, L. Innate immunity in plants and animals: striking similarities and obvious differences. Immunol. Rev. 198, (2004). 38. Asai, T. et al. MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415, (2002). 39. Felix, G., Duran, J.D., Volko, S. & Boller, T. Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J. 18, (1999). 40. Gomez-Gomez, L. & Boller, T. FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol. Cell 5, (2000). 41. Gomez-Gomez, L., Felix, G. & Boller, T. A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana. Plant J. 18, (1999). 42. Meindl, T., Boller, T. & Felix, G. The bacterial elicitor flagellin activates its receptor in tomato cells according to the address-message concept. Plant Cell 12, (2000). 43. Zipfel, C. et al. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428, (2004). 44. Gomez-Gomez, L. & Boller, T. Flagellin perception: a paradigm for innate immunity. Trends Plant Sci. 7, (2002). 45. Shiu, S.H. & Bleecker, A.B. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc. Natl. Acad. Sci. USA 98, (2001). 46. Shiu, S.H. & Bleecker, A.B. Expansion of the receptor-like kinase/pelle gene family and receptor-like proteins in Arabidopsis. Plant Physiol. 132, (2003). 47. Donnelly, M.A. & Steiner, T.S. Two nonadjacent regions in enteroaggregative Escherichia coli flagellin are required for activation of toll-like receptor 5. J. Biol. Chem. 277, (2002). 48. Holt, B.F., III, Hubert, D.A. & Dangl, J.L. Resistance gene signaling in plants complex similarities to animal innate immunity. Curr. Opin. Immunol. 15, (2003). 49. Nimchuk, Z., Eulgem, T., Holt, B.F., III & Dangl, J.L. Recognition and response in the plant immune system. Annu. Rev. Genet. 37, (2003). 50. Meyers, B.C., Kozik, A., Griego, A., Kuang, H. & Michelmore, R.W. Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell 15, (2003). 51. Zhou, T. et al. Genome-wide identification of NBS genes in japonica rice reveals significant expansion of divergent non-tir NBS-LRR genes. Mol. Genet. Genomics 271, (2004). 52. Bais, H.P., Prithiviraj, B., Jha, A.K., Ausubel, F.M. & Vivanco, J.M. Mediation of pathogen resistance by exudation of antimicrobials from roots. Nature 434, (2005). 53. Hauck, P., Thilmony, R. & He, S.Y. A Pseudomonas syringae type III effector suppresses cell wall-based extracellular defense in susceptible Arabidopsis plants. Proc. Natl. Acad. Sci. USA 100, (2003). 54. He, P. et al. Activation of a COI1-dependent pathway in Arabidopsis by Pseudomonas syringae type III effectors and coronatine. Plant J. 37, (2004). 55. Hotson, A., Chosed, R., Shu, H., Orth, K. & Mudgett, M.B. Xanthomonas type III effector XopD targets SUMO-conjugated proteins in planta. Mol. Microbiol. 50, (2003). 56. Kim, M.G. et al. Two Pseudomonas syringae type III effectors inhibit RIN4-regulated basal defense in Arabidopsis. Cell 121, (2005). 57. Orth, K. et al. Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease. Science 290, (2000). 58. Zhao, Y. et al. Virulence systems of Pseudomonas syringae pv. tomato promote bacterial speck disease in tomato by targeting the jasmonate signaling pathway. Plant J. 36, (2003). 59. Pujol, N. et al. A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans. Curr. Biol. 11, (2001). 60. Mallo, G.V. et al. Inducible antibacterial defense system in C. elegans. Curr. Biol. 12, (2002). 61. Couillault, C. et al. TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM. Nat. Immunol. 5, (2004). 62. Kim, D.H. et al. A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity. Science 297, (2002). 63. Kim, D.H. et al. Integration of Caenorhabditis elegans MAPK pathways mediating immunity and stress resistance by MEK-1 MAPK kinase and VHP-1 MAPK phosphatase. Proc. Natl. Acad. Sci. USA 101, (2004). 64. Liberati, N.T. et al. Requirement for a conserved Toll/interleukin-1 resistance domain protein in the Caenorhabditis elegans immune response. Proc. Natl. Acad. Sci. USA 101, (2004). 65. Matsuzawa, A. et al. ROS-dependent activation of the TRAF6 ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity. Nat. Immunol. 6, (2005). 66. Mochida, Y. et al. ASK1 inhibits interleukin-1-induced NF-kappa B activity through disruption of TRAF6 TAK1 interaction. J. Biol. Chem. 275, (2000). 67. Glazebrook, J. Genes controlling expression of defense responses in Arabidopsis 2001 status. Curr. Opin. Plant Biol. 4, (2001). 68. Chern, M., Canlas, P.E., Fitzgerald, H.A. & Ronald, P.C. Rice NRR, a negative regulator of disease resistance, interacts with Arabidopsis NPR1 and rice NH1. Plant J. 43, (2005). 69. Durrant, W.E. & Dong, X. Systemic acquired resistance. Annu. Rev. Phytopathol. 42, (2004). 70. Pieterse, C.M. & Van Loon, L.C. NPR1: the spider in the web of induced resistance signaling pathways. Curr. Opin. Plant Biol. 7, (2004). 71. Keller, T. et al. A plant homolog of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca 2+ binding motifs. Plant Cell 10, (1998). 72. Torres, M.A. et al. Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox). Plant J. 14, (1998). 73. Torres, M.A., Dangl, J.L. & Jones, J.D. Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc. Natl. Acad. Sci. USA 99, (2002). 74. Song, W.Y. et al. A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science 270, (1995). 75. Burdman, S., Shen, Y., Lee, S.W., Xue, Q. & Ronald, P. RaxH/RaxR: a two-component regulatory system in Xanthomonas oryzae pv. oryzae required for AvrXa21 activity. Mol. Plant Microbe Interact. 17, (2004). 978 VOLUME 6 NUMBER 10 OCTOBER 2005 NATURE IMMUNOLOGY

7 76. da Silva, F.G. et al. Bacterial genes involved in type I secretion and sulfation are required to elicit the rice Xa21-mediated innate immune response. Mol. Plant Microbe Interact. 17, (2004). 77. Shen, Y., Sharma, P., da Silva, F.G. & Ronald, P. The Xanthomonas oryzae pv. lozengeoryzae raxp and raxq genes encode an ATP sulphurylase and adenosine- 5 -phosphosulphate kinase that are required for AvrXa21 avirulence activity. Mol. Microbiol. 44, (2002). 78. Dangl, J.L. & Jones, J.D. Plant pathogens and integrated defence responses to infection. Nature 411, (2001). 79. Axtell, M.J. & Staskawicz, B.J. Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4. Cell 112, (2003). 80. Mackey, D., Belkhadir, Y., Alonso, J.M., Ecker, J.R. & Dangl, J.L. Arabidopsis RIN4 is a target of the type III virulence effector AvrRpt2 and modulates RPS2-mediated resistance. Cell 112, (2003). 81. Mackey, D., Holt, B.F., Wiig, A. & Dangl, J.L. RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 108, (2002). 82. Friedman, R. & Hughes, A.L. Molecular evolution of the NF-κB signaling system. Immunogenetics 53, (2002). 83. Luo, C. & Zheng, L. Independent evolution of Toll and related genes in insects and mammals. Immunogenetics 51, (2000). 84. Pancer, Z. et al. Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey. Nature 430, (2004). 85. Nurnberger, T. & Volker, L. Non-host resistance in plants: new insights into an old phenomenon. Mol. Plant Pathol. 6, (2005). 86. Meyerowitz, E.M. Plants compared to animals: the broadest comparative study of development. Science 295, (2002). 87. Diez, E. et al. Birc1e is the gene within the Lgn1 locus associated with resistance to Legionella pneumophila. Nat. Genet. 33, (2003). 88. Wright, E.K. et al. Naip5 affects host susceptibility to the intracellular pathogen Legionella pneumophila. Curr. Biol. 13, (2003). 89. Stremlau, M. et al. The cytoplasmic body component TRIM5α restricts HIV-1 infection in Old World monkeys. Nature 427, (2004). 90. Pan, H. et al. Ipr1 gene mediates innate immunity to tuberculosis. Nature 434, (2005). NATURE IMMUNOLOGY VOLUME 6 NUMBER 10 OCTOBER