Plants have the capacity to recognise and reject pathogens at various stages of their attempted colonisation of the plant. Non-specific rejection often arises as a consequence of the potential pathogen’s attempt to breach the first lines of plant defence.
Pathogens able to penetrate beyond this barrier of non-host resistance may seek a subtle and persuasive relationship with the plant. For some, this may be limited to molecular signals released outside the plant cell wall, but for others it includes penetration of the cell wall and the delivery of signal molecules to the plant cytosol. Direct or indirect recognition of these signals triggers specific resistance. Our understanding of host-specific resistance and its possible links to non-host-host-specific resistance has advanced significantly as more is discovered about the nature and function of the molecules underpinning both kinds of resistance.
Addresses
Plant Cell Biology, Research School of Biological Sciences, Australian National University, Canberra ACT 2601, Australia
1e-mail: [email protected]
Current Opinion in Immunology 2004, 16:48–62 This review comes from a themed issue on Innate immunity
Edited by Bruce Beutler and Jules Hoffmann 0952-7915/$ – see front matter
ß2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.coi.2003.11.016
Abbreviations
Avr avirulence
CC coiled-coil
CLV clavata
COI1 coronatine insensitive 1
COP9 constitutively photomorphogenic 9
CSN COP9 signalosome
CUL1 cullin 1
EDS1 enhanced disease susceptibility 1 flg22 22 amino acid domain of bacterial flagellin HR hypersensitive response
HSP heat-shock protein
IL interleukin
LRR leucine-rich repeat
MAPK mitogen-activated protein kinase MLA1 powdery mildew resistance locus a 1 NBS nucleotide-binding site
NDR1 non-race-specific disease resistance 1 NOD nucleotide-binding oligomerisation domain NPP1 necrosis-inducing Phytophthora protein 1 PAD4 phytoalexin deficient 4
PR pathogenesis-related
PVX potato virus X
R resistance
RAR1 required for barley Mla resistance 1
RIN4 RPM1 interacting 4
RLK receptor-like kinase RLP receptor-like protein
RPM1 resistance to Pseudomonas syringae pv. maculicola 1 RPP resistance to Peronospora parasitica
RPS resistance to P. syringae pv. tomato SGT1 suppressor of G2 allele of SKP1
SHD shepherd
SKP1 S-phase kinase-associated protein 1 TIR Toll and IL-1 receptor
TLR Toll-like receptor
TMV tobacco mosaic virus
WRKY domain tryptophan-arginine-lysine-tyrosine domain
Introduction
Plants and mammals have fundamental biological differ-ences that affect their capacity to defend themselves against potential pathogens. Plants are sessile, requiring pathogen mobility, whereas mammals are mobile and therefore able to spread infections by contact. Mammals have a circulatory system able to deliver somatically generated, adaptive immune responses to sites of infec-tion, but plants lack adaptive immunity. Plant cells have a cell wall that provides an effective barrier to many poten-tial pathogens, but mammalian cells lack a similar phys-ical barrier. Despite these and other differences, plant disease resistance and mammalian innate immunity share several remarkable similarities at the molecular level.
Innate immunity in mammals
In mammals, the major players in innate immunity are the leucine-rich repeat (LRR)-containing Toll-like receptors (TLRs; reviewed in [1,2]) and the nucleotide-binding oligomerisation domain (NOD)-LRR proteins (reviewed in[3,4]). Extracytosolic and cytosolic pathogen-associated molecular patterns (PAMPs) are perceived by TLRs and NOD-LRRS, respectively, and these receptors are involved in the subsequent activation of innate immune responses, including inflammation and production of antimicrobial proteins. In man, ten TLR genes have been identified, of which at least five are involved in PAMP perception, and 23 NOD-LRR genes have been identified, of which at least two (NOD1 and NOD2) are involved in PAMP perception[3]. The NOD-LRR proteins have a modular structure (see Figure 1) with a central, NOD regulatory domain, carboxy-terminal LRR recognition domain and, in the case of NOD1 and NOD2,
amino-CARD-related, amino-terminal, pyrin domains. Similarly, TLRs have a modular structure with an amino-terminal, extracytosolic LRR receptor domain and a carboxy-terminal cytosolic Toll and IL-1 receptor (TIR) hom-ology effector domain (Figure 1).
Innate immunity in plants
In plants, the most important molecules in disease resis-tance include the nucleotide-binding site (NBS)-LRR proteins and, to a lesser extent, LRR-receptor-like kinases (RLKs) and membrane-anchored LRR-recep-tor-like proteins (RLPs), which are analogous to the TLRs (reviewed in [5]). Similar to the NOD-LRR pro-teins, plant NBS–LRR proteins have a modular structure with a central NBS regulatory domain homologous to the NOD domain, a carboxy-terminal LRR recognition domain and amino-terminal TIR or coiled-coil (CC) effector domains (Figure 1). Similar to the TLRs, the LRR-RLKs and LRR-RLPs have amino-terminal extra-cytosolic LRR recognition domains, but differ by the
The NBS-LRR, LRR-RLK and LRR-RLP resistance proteins, similar to the NOD-LRRs and TLRs, are also involved in the activation of an array of resistance responses, including a form of programmed cell death called the hypersensitive response (HR), and the produc-tion of antimicrobial proteins [5].
NBS-LRR genes in Arabidopsis
In the model plant Arabidopsis, there are 149 NBS-LRR genes [6], indicating much greater elaboration of this gene family in plants compared to the NOD-LRR genes in mammals, and no member of the NBS-LRR gene family has yet been shown to have a role other than disease resistance. The larger number of NBS-LRR genes in plants compared to NOD-LRR genes in mam-mals may reflect adaptive elaboration and evolution of an ancestral innate immune system by gene duplication, gene divergence, sequence exchange and diversifying selection[7,8], analogous to that proposed for the major histocompatability complex. The somatically generated
Figure 1
Current Opinion in Immunology
Mammals Plants
FLS2 Xa21 CLV1
CLV2 Cf-9 Flagellin
TLR5
AvrXa21 CLV3 Avr9
TLR4 CD14
Peptidoglycan NOD1
NOD2
RPS4
RPS2
AvrRps4 AvrRpt2 Lipopolysaccharide
TLR2 Peptidoglycan
LRR TIR PK CARD CC NOD/NBS
Domains
Schematic representation of pathogen surveillance receptors in mammals and plants (adapted from[4,88]). The mammalian Toll-like receptors (TLRs) 2, 4 and 5 detect the bacterial pathogen-associated molecular patterns (PAMPs) peptidoglycan, lipopolysaccharide and flagellin, respectively [1,2]. CD14 is a co-receptor for lipopolysaccharide. The Arabidopsis flagellin-sensing (FLS2) receptor has structural similarity to the TLRs and performs a similar function although using a different signalling domain (reviewed in[10]). The rice Xa21 resistance protein has structural similarity to FLS2, but detects a specific bacterial (Xanthomonas oryzae pv. oryzae) avirulence determinant (AvrXa21;[11]). The tomato Cf-9 receptor detects a fungal (Cladosporium fulvum) avirulence determinant (Avr9) and has structural similarity to FLS2 and Xa21, but lacks a signalling domain[12]. Structurally, Cf-9 is also similar to CD14. The Arabidopsis CLAVATA complex has structural similarity to both FLS2 and Xa21 on the one hand (CLV1) and Cf-9 on the other (CLV2), but is involved in plant development rather than pathogen perception (reviewed in[84]). The nucleotide-binding oligomerisation domain (NOD) proteins 1 and 2 also detect peptidoglycan, but detect different constituents to one another[89]. The Arabidopsis RPS2 and RPS4 resistance proteins have structural similarity to the NOD proteins, but detect specific bacterial (Pseudomonas syringae) avirulence determinants and have different amino-terminal domains[5,6]. Abbreviations: CARD, caspase recruitment effector domain; CC, coiled coil; LRR, leucine-rich repeat; NBS, nucleotide binding site; PK, serine/threonine protein kinase; TIR, Toll and interleukin-1 receptor cytosolic domain homology.
system, allowing it to be adapted to other roles including potentiation of the adaptive immune system. As a con-sequence of this adaptive elaboration, NBS-LRR resis-tance (R) genes also show a greater degree of variation and individual specificity for the pathogen molecules they recognise and, in turn, selection pressure is imposed on the pathogen, leading to variation to avoid recognition but retaining effector function where possible.
LRR-RLK and LRR-RLP genes in Arabidopsis
Whereas the NBS-LRRs are involved in cytosolic percep-tion, the LRR-RLKs and LRR-RLPs are involved in extracytosolic perception of various ligands including pathogen molecules. In Arabidopsis, there are 233 LRR-RLK genes and 110 LRR-RLP genes [9], but it is clear that many have roles in plant development rather than disease resistance, so the number involved in plant disease resistance remains to be determined. Indeed, no LRR-RLK gene has yet been shown to play a disease resistance role in Arabidopsis, although FLS2, an Arabi-dopsis flagellin-sensing LRR-RLK, appears to play a role in plant innate immunity that is directly comparable to the role of TLR5 in mammalian innate immunity (Figure 1; reviewed in [10]). In fact, the only LRR-RLK gene with a role in disease resistance that has been described to date is the Xa21 gene from rice, which confers resistance to the bacterium Xanthomonas oryzae pv. oryzae [11]. Until recently, no LRR-RLP gene had been shown to play a disease resistance role in Arabidopsis, and the only LRR-RLP genes with roles in disease resistance were the tomato Cf and Ve genes, conferring resistance to the fungi Cladosporium fulvum [12] and Verticillium dahliae [13], respectively. However, reports from the recent Molecular Plant–Microbe Interaction meeting in St. Petersburg (11th International Congress on Molecular Plant–Microbe Interactions, July 18–27 2003, St Petersberg, Russia; URL: http://www.arriam.
spb.ru/mpmi/) indicate that that the Arabidopsis RPP27 gene, which confers resistance to the oomycete Peronos-pora parasitica, encodes an LRR-RLP.
Advances in understanding R-protein function
The past year has seen significant advances in two major areas of R-protein function. One is the perception of pathogen-effector or avirulence (Avr) proteins by R pro-teins and the other is the enigmatic role of chaperone and protein degradation complexes in R-protein signalling.
There has also been progress in determining the func-tional properties of R-protein domains. The poor relation in plant innate immunity has often been non-host resis-tance, mediated by pathogen-associated molecules anal-ogous to PAMPs in animals. However, some significant advances have been made in this area that hint at sig-nificant overlaps with host-specific resistance mediated by R proteins and the possible integration of the two
Non-host resistance — a form of plant innate immunity
A potential plant pathogen has to overcome many barriers to become an actual pathogen (reviewed in [14]). The majority of potential pathogens fail to overcome these barriers and are never able to colonise a potential host plant. This non-host resistance may depend on passive preformed barriers, but it often depends on active responses following recognition of the pathogen or its activities as it attempts to penetrate the plant. Induced non-host resistance in plants is comparable to animal innate immunity, which activates pathogen resistance following host recognition of general PAMPs, which are both indispensable for pathogenicity and unique to pathogens [15]. Surface-derived structural molecules from plant pathogens, such as fungal cell wall constituents (chitin, glucan, protein and glycoprotein), bacterial lipo-polysaccharide (LPS) and flagellin, elicit defence responses from a wide range of plant species[10,16,17], and these ‘elicitors’ are conceptually similar to PAMPs.
Cell-wall-degrading enzymes, including endopolygalac-turonase and xylanase, are ubiquitous as virulence effec-tors among plant pathogens [18,19,20], but can also function as elicitors. Their enzymatic products, such as plant-cell-wall-derived oligogalacturonides, are also known to induce plant defence responses (Figure 2;[21]).
The range and activity of elicitor molecules
The recently characterised NPP1 (necrosis-inducing Phy-tophthora protein 1), derived from PhyPhy-tophthora cell walls, is a member of a protein family that is widespread among oomycetes, fungi and bacteria, and has elicitor activity in dicots[22]. The surface-exposed Pep13 elicitor, recently identified as part of a cell wall transglutaminase, is highly conserved among the genus Phytophthora and activates resistance responses in solanaceous plants[23]. Elicitins, which are secreted sterol carrier proteins produced by Phytophthora and some Pythium species, have elicitor activity on most Nicotiana species and a few cultivars of Raphanus sativus and Brassica rapa[24]. These variations in the type and range of elicitor activities suggest that a much looser definition of PAMPs is appropriate for plants compared to animals.
Although these elicitor molecules can induce plant defences, the biological role of elicitor detection in host–pathogen interactions is not clear, because suscep-tible plants can support the growth of pathogens capable of producing the elicitor without triggering a defence response. For example, the 22 amino acid domain of bacterial flagellin (flg22) is conserved even among patho-genic bacteria able to colonise flg22-sensitive plants[10].
Similarly, Phytophthora Pep13 is recognised by potato, which is highly susceptible to Phytophthora infestans[23], and NPP1 is recognised by tobacco, which is susceptible
signalling induced by general elicitors or ways to avoid their detection by host plants (Figure 2).
Virulence function of elicitor molecules
Many pathogen Avr proteins (specific elicitors) are patho-genicity factors with virulence effector functions [25– 27]. Expression in Arabidopsis of AvrPto, a type III effector/Avr protein produced by the leaf speck bacter-ium Pseudomonas syringae, delivered into the plant cytosol by a type III secretion mechanism and recognised by the tomato Pto R protein, was found to suppress the expres-sion of a set of genes for putative secreted cell-wall defence proteins [25]. In AvrPto-expressing plants, the accumulation of defence-inducible callose was abolished,
ond type III effector detected by Pto[28]) was identified as a suppressor of HR (a form of programmed cell death thought to limit pathogen growth) triggered by Pto–
AvrPto or Cf-9–Avr9 R–Avr protein interactions [26].
These examples suggest active suppression of host defences, but passive intervention is also possible.
Avr4, an extracellular Avr protein produced by the leaf mould fungus C. fulvum and detected by the tomato Cf-4 LRR-RLP protein, has chitin-binding activity, and is proposed to protect the fungus against degradation by tomato chitinases, which might liberate elicitor-active chitin oligomers in addition to weakening the fungal cell wall [27]. These observations suggest that pathogens produce virulence effectors with many different
strate-Figure 2
Current Opinion in Immunology
Plant cell Conserved signal
transduction pathways
Cell-wall degrading enzymes Pathogens
Virulence/avirulence factors
Resistance reaction Attempted
penetration Cell-wall degradation products
Key factors
• MAP kinase cascades
• EDS1, PAD4, NDR1
• Transcription regulators Cell wall
abnormality
• PR-proteins
• Phytoalexins
Expression of resistance Signalling
Recognition
• R gene products
• PAMP receptors
• Programmed cell death (HR) PAMPs
Masking of PAMPs
A simplified model for plant signalling responses induced by various pathogen elicitors. The model illustrates the multifaceted nature of pathogen attack and the broad spectrum of elicitors produced as a consequence, ranging from non-specific elicitors (that may be loosely defined as plant PAMPs), through to highly specific elicitors (virulence effector/avirulence factors) with narrow specificity. One role of the latter may be to suppress plant mechanisms capable of responding to the former. Together with mechanisms that mask PAMPs, this may be one of the main strategies used by plant pathogens to avoid detection. Plants have evolved mechanisms to detect elicitors from both extremes of the spectrum, and whilst detection of non-specific elicitors may play a role in non-host resistance, detection of specific elicitors is required to resist pathogens that have evolved to overcome all the non-specific barriers and detection mechanisms. Nevertheless, non-host and host-specific resistance often result in the activation of similar responses and the model also illustrates the possible integration of non-host and host-specific resistance signalling pathways suggested by evidence for a number of shared signalling components. Red arrows indicate pathogen strategies for infection and black arrows indicate plant signalling for resistance. Although detection of PAMPs is shown at the cell surface, and the action of virulence effector proteins and their detection as avirulence factors is shown in the cytosol, these locations are not mutually exclusive. Abbreviations: EDS1, enhanced disease susceptibility 1; MAP, mitogen activated protein; NDR1, non-race-specific disease resistance 1; PAD4, phytoalexin deficient 4; PAMP, pathogen-associated molecular pattern; PR, pathogenesis related.
recognition of these specific (or semi-specific) virulence factors has therefore evolved as a critical determinant in many plant–microbe interactions (thus making these virulence effectors conditional avirulence factors). How-ever, the fact that plants maintain the capacity to recog-nise and respond to general elicitors suggests that this system remains effective against a wide range of potential pathogens and saprophytes.
Signal transduction pathways in resistance
It is possible that resistance mechanisms induced by recognition of general elicitors and R-protein-mediated recognition of specific elicitors share similar signal trans-duction pathways (Figure 2). The tobacco mitogen-acti-vated protein kinases (MAPKs), SIPK (salicylate-inducible protein kinase) and WIPK (wound-(salicylate-inducible protein kinase), are activated by the N–TMV (tobacco mosaic virus) and Cf-9–Avr9 R–Avr protein interactions.
These MAPKs, and their orthologues in other species, are also activated by flagellin, fungal cell-wall-derived elici-tors, elicitin, Pep13 and NPP1, suggesting that the MAPK cascade could be a convergence point for signal transduc-tion pathways leading to both host-specific and non-host resistance (reviewed in[29]). Recent reports also impli-cate signalling components from R-protein signalling pathways in non-host resistance and/or responses to general elicitors. For example, the silencing of SGT1 (covered in more detail later) compromised pathogen resistance mediated by N–TMV, Rx–PVX (potato virus X) and Pto–AvrPto R–Avr protein interactions, elicitin-mediated HR, and non-host resistance to some bacteria in Nicotiana benthamiana[30]. In addition, NPP1-mediated induction of pathogenesis-related (PR) proteins required both functional NDR1 (non-race-specific disease resis-tance 1) and PAD4 (phytoalexin deficient 4)[22], which are well-characterised signalling components involved in resistance mediated by CC-NBS-LRR and TIR-NBS-LRR R proteins, respectively[31]. Furthermore, PAD4 and EDS1 (enhanced disease susceptibility 1), signalling factors for resistance mediated by TIR-NBS-LRR R proteins are necessary for full Arabidopsis non-host resis-tance to the wheat powdery mildew fungus, Blumeria graminis [32]. EDS1 is also essential in Arabidopsis for broad-spectrum resistance to powdery mildew pathogens, conferred by RPW8 (resistance to powdery mildew 8), a novel membrane-anchored CC protein[33].
Involvement of the plant cell wall
Several recent reports suggest the possible involvement of the plant cell wall in sensing environmental stresses, including attempted penetration by pathogens and wounding. The Arabidopsis cev1 (constitutive expression of VSP1) and eli1 (ectopic lignin) mutants have mutations in a cellulose synthase gene CESA3, leading to constitu-tive activation of jasmonate- and ethylene-mediated
PMR4 (powdery mildew resistant 4) gene encodes a biosynthetic enzyme responsible for stress-related callose deposition, and the pmr4 mutant produces less callose in response to pathogen infection and wounding[36]. Sur-prisingly, the pmr4 mutant shows weak constitutive upre-gulation of salicylate- and pathogen-responsive genes, and resistance to several biotrophic plant pathogens.
These results suggest that plants are able to detect and respond rapidly to biotic and abiotic challenges by mon-itoring the integrity of their cell walls and cell-wall defences (Figure 2). As the pmr4 phenotype is PAD4-and NPR1 (non-expresser of PR genes 1)-dependent [36], cell-wall-mediated signalling also shares signal transduction components with resistance pathways in-duced by recognition of general elicitors and R-protein-mediated recognition of specific elicitors, although the recognition and early signal transduction components may be unique.
Thus, it seems likely that plants are able to detect a range of more or less specific elicitors, comprising pathogen cellular components or host components released or modified by pathogen effectors, using signalling systems that have various degrees of overlap with R-protein signalling. Plant innate immunity may, therefore, com-prise a continuum from non-host resistance involving the detection of general elicitors to host-specific resistance involving detection of specific elicitors by R proteins.
Flagellin perception — bridging the conceptual gap between non-host and host-specific resistance
The perception of general pathogen-associated mole-cules by the host is perhaps best exemplified by flagellin perception in Arabidopsis (reviewed in[10]). In Arabidop-sis, flagellin perception is controlled by the FLS2 gene, which encodes an LRR-RLK. A conserved 22 amino acid subdomain of flagellin, designated flg22, is necessary and
The perception of general pathogen-associated mole-cules by the host is perhaps best exemplified by flagellin perception in Arabidopsis (reviewed in[10]). In Arabidop-sis, flagellin perception is controlled by the FLS2 gene, which encodes an LRR-RLK. A conserved 22 amino acid subdomain of flagellin, designated flg22, is necessary and