Plant innate immunity – direct and indirect recognition of general and specific pathogen-associated molecules

Plant innate immunity – direct and indirect recognition of general and specific pathogen-associated molecules David A Jones1 and Daigo Takemoto Plants have the capacity to recognise and reject pathogens at various stages of their attempted colonisation of the plant. Nonspecific 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 host-specific resistance. Our understanding of hostspecific resistance and its possible links to non-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 1 e-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 ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2003.11.016

Abbreviations Avr CC CLV COI1 COP9 CSN CUL1 EDS1 flg22 HR HSP IL LRR MAPK MLA1 NBS NDR1 NOD NPP1 PAD4 PAMP PBS1 PMR4

PR PVX R RAR1 RIN4 RLK RLP RPM1 RPP RPS SGT1 SHD SKP1 TIR TLR TMV WRKY domain

pathogenesis-related potato virus X resistance required for barley Mla resistance 1 RPM1 interacting 4 receptor-like kinase receptor-like protein resistance to Pseudomonas syringae pv. maculicola 1 resistance to Peronospora parasitica resistance to P. syringae pv. tomato suppressor of G2 allele of SKP1 shepherd S-phase kinase-associated protein 1 Toll and IL-1 receptor Toll-like receptor tobacco mosaic virus tryptophan-arginine-lysine-tyrosine domain

Introduction Plants and mammals have fundamental biological differences 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 infection, but plants lack adaptive immunity. Plant cells have a cell wall that provides an effective barrier to many potential pathogens, but mammalian cells lack a similar physical 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

avirulence coiled-coil clavata coronatine insensitive 1 constitutively photomorphogenic 9 COP9 signalosome cullin 1 enhanced disease susceptibility 1 22 amino acid domain of bacterial flagellin hypersensitive response heat-shock protein interleukin leucine-rich repeat mitogen-activated protein kinase powdery mildew resistance locus a 1 nucleotide-binding site non-race-specific disease resistance 1 nucleotide-binding oligomerisation domain necrosis-inducing Phytophthora protein 1 phytoalexin deficient 4 pathogen-associated molecular pattern AvrPphB susceptible 1 powdery mildew resistant 4

Current Opinion in Immunology 2004, 16:48–62

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, aminoterminal caspase recruitment effector domains (CARDs); the majority of the remaining NOD–LRR proteins have www.sciencedirect.com

Plant innate immunity Jones and Takemoto 49

Figure 1

Mammals

Plants

Peptidoglycan Lipopolysaccharide

TLR2

CD14

TLR4

Flagellin

TLR5

AvrXa21

FLS2

CLV3

Xa21

Avr9

CLV1 CLV2

Cf-9

RPS4

NOD1

AvrRps4 Peptidoglycan AvrRpt2 RPS2

NOD2

Domains

LRR

TIR

PK

CARD

CC

NOD/NBS

Current Opinion in Immunology

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.

CARD-related, amino-terminal, pyrin domains. Similarly, TLRs have a modular structure with an amino-terminal, extracytosolic LRR receptor domain and a carboxyterminal cytosolic Toll and IL-1 receptor (TIR) homology effector domain (Figure 1).

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 production of antimicrobial proteins [5].

Innate immunity in plants

In plants, the most important molecules in disease resistance include the nucleotide-binding site (NBS)-LRR proteins and, to a lesser extent, LRR-receptor-like kinases (RLKs) and membrane-anchored LRR-receptor-like proteins (RLPs), which are analogous to the TLRs (reviewed in [5]). Similar to the NOD-LRR proteins, 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 extracytosolic LRR recognition domains, but differ by the presence or absence of carboxy-terminal cytosolic serine/threonine protein kinase effector domains (Figure 1). www.sciencedirect.com

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 mammals 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 adaptive immune system of mammals, however, may have largely superseded the ancestral innate immune Current Opinion in Immunology 2004, 16:48–62

50 Innate immunity

system, allowing it to be adapted to other roles including potentiation of the adaptive immune system. As a consequence of this adaptive elaboration, NBS-LRR resistance (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 perception, 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 Arabidopsis 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 LRRRLK 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 Peronospora 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 proteins 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 functional properties of R-protein domains. The poor relation in plant innate immunity has often been non-host resistance, mediated by pathogen-associated molecules analogous to PAMPs in animals. However, some significant advances have been made in this area that hint at significant overlaps with host-specific resistance mediated by R proteins and the possible integration of the two kinds of resistance. This review covers both extremes of plant innate immunity with this possibility in mind. Current Opinion in Immunology 2004, 16:48–62

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 lipopolysaccharide (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 endopolygalacturonase and xylanase, are ubiquitous as virulence effectors 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 Phytophthora protein 1), derived from Phytophthora 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 susceptible 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 pathogenic 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 to P. parasitica [22
]. These data suggest that pathogens must have mechanisms to suppress plant defence www.sciencedirect.com

Plant innate immunity Jones and Takemoto 51

Figure 2

Pathogens

Masking of PAMPs

Cell-wall degrading enzymes Attempted PAMPs Cell-wall degradation penetration products Key factors Cell wall abnormality

Recognition • R gene products • PAMP receptors

Virulence/avirulence factors

Signalling • EDS1, PAD4, NDR1

Conserved signal transduction pathways

• MAP kinase cascades • Transcription regulators Expression of resistance

Resistance reaction Plant cell

• PR-proteins • Phytoalexins • Programmed cell death (HR) Current Opinion in Immunology

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.

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 pathogenicity factors with virulence effector functions [25
– 27
]. Expression in Arabidopsis of AvrPto, a type III effector/Avr protein produced by the leaf speck bacterium 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 expression 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, and the growth of bacteria unable to secrete type III effectors was enhanced [25
]. Recently, AvrPtoB (a secwww.sciencedirect.com

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 strategies to prevent recognition of general elicitors and thereby assist infection (Figure 2). R-protein-dependent Current Opinion in Immunology 2004, 16:48–62

52 Innate immunity

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). However, the fact that plants maintain the capacity to recognise 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 transduction pathways (Figure 2). The tobacco mitogen-activated protein kinases (MAPKs), SIPK (salicylateinducible protein kinase) and WIPK (wound-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 elicitors, elicitin, Pep13 and NPP1, suggesting that the MAPK cascade could be a convergence point for signal transduction pathways leading to both host-specific and non-host resistance (reviewed in [29]). Recent reports also implicate 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, elicitinmediated 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 resistance 1) and PAD4 (phytoalexin deficient 4) [22
], which are well-characterised signalling components involved in resistance mediated by CC-NBS-LRR and TIR-NBSLRR 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 resistance 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 constitutive activation of jasmonate- and ethylene-mediated defence-gene expression and enhanced resistance to powdery mildew pathogens [34
,35
]. The Arabidopsis Current Opinion in Immunology 2004, 16:48–62

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]. Surprisingly, the pmr4 mutant shows weak constitutive upregulation 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 monitoring the integrity of their cell walls and cell-wall defences (Figure 2). As the pmr4 phenotype is PAD4and NPR1 (non-expresser of PR genes 1)-dependent [36], cell-wall-mediated signalling also shares signal transduction components with resistance pathways induced by recognition of general elicitors and R-proteinmediated 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, comprise 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 molecules by the host is perhaps best exemplified by flagellin perception in Arabidopsis (reviewed in [10]). In Arabidopsis, 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 sufficient to produce the flagellin response, including induction of PR proteins, but not an HR. Despite elegant molecular characterisation in Arabidopsis, a clear biological role for flagellin perception has only emerged in other plant pathosystems. Flagellin from a millet strain of the bacterium Acidovorax avenae (formerly Pseudomonas avenae), but not a rice strain, has been shown to induce a resistance response in rice cells, suggesting that flagellin may determine the host range of different strains of A. avenae [36]. More recent experiments indicate that flagellin from the millet strain of A. avenae can induce an HR and expression of PR proteins in rice, but the inability of a flagellin deletion mutant to overcome resistance indicates that flagellin perception is not the only determinant of resistance [37
]. By contrast, flagellin deletion mutants of P. syringae pv. tabaci become pathogenic on tomato, indicating that flagellin perception is the major determinant of non-host resistance in this interaction [38

]. Clearly, flagellin perception has a role in determining www.sciencedirect.com

Plant innate immunity Jones and Takemoto 53

the host range of both A. avenae and P. syringae and, although a role in host cultivar/pathogen strain-specific resistance has yet to be demonstrated, a role in host species/pathogen strain specific resistance goes some way towards bridging the gap, as does natural variation in Arabidopsis for the ability to detect flagellin [10]. The perception of P. syringae flagellins may differ to the perception of A. avenae flagellin, and may also differ from one plant species to another. The ability of flagellins from A. avenae to induce a resistance response in rice appears to correlate with sequence differences in the flagellin proteins of the two strains [36], whereas the ability of flagellins from P. syringae pv. glycinea, but not P. syringae pv. tabaci, to induce a resistance response in tobacco appears to correlate with the differential glycosylation of otherwise identical proteins [39

]. Moreover, the sequence differences in the A. avenae flagellins and the amino-glycosylation sites in the P. syringae flagellins lie outside the flg22 subregion, suggesting that different subregions may be perceived in rice and tobacco in comparison to Arabidopsis or to one another. Interestingly, the flagellin from the millet strain of A. avenae has two amino-glycosylation sites that are missing from the rice strain [36], suggesting that differential glycosylation may also play a role in flagellin perception or lack of it in this pathosystem. Perception of flagellins may also lead to different downstream responses in different pathosystems; for example, triggering an HR in rice [37
] and tobacco [39

] but not in Arabidopsis [10].

The ‘guard’ hypothesis Until recently, the only example of direct interaction between an NBS-LRR protein and a pathogen Avr determinant was the interaction described between the LRR domain of the rice Pi-ta CC-NBS-LRR protein and the Avr-Pita protein of the rice blast fungus Magnaporthe grisea [40]. However, the absence of evidence for direct interaction in many other systems examined led to the formulation of the ‘guard’ hypothesis, which proposes that the interaction between an R protein and its cognate Avr determinant is mediated by a host protein that is the target for the effector function of the Avr determinant on the one hand, and under R-protein surveillance for such interference on the other [41]. Observations supporting the guard hypothesis

Strong support for the ‘guard’ hypothesis has been shown recently in three papers [42

–44

] describing the protein RIN4 (RPM1 interacting 4) from Arabidopsis. RIN4 mediates interactions between the RPM1 (resistance to P. syringae pv. maculicola 1) CC-NBS-LRR protein and the AvrB and AvrRpm1 type III effector proteins from the leaf speck bacterium P. syringae on the one hand and between the RPS2 (resistance to P. syringae pv. tomato 2) CC-NBS-LRR protein and the AvrRpt2 type III effector protein also from P. syringae on the other. RPM1 and www.sciencedirect.com

RPS2 form a complex with RIN4 [42

–44

]. AvrB and AvrRpm1 cause the phosphorylation of RIN4 and the consequent activation of RPM1 [42

], and AvrRpt2 causes the degradation of RIN4, thereby interfering with RPM1 function, but activating RPS2 [43

,44

]. Further strong support for the ‘guard’ hypothesis has also been shown recently for another interaction between Arabidopsis and P. syringae involving the RPS5 CCNBS-LRR protein and the AvrPphB type III effector protein. Previously, the RPS5 protein was shown to require PBS1 (AvrPphB susceptible 1), a protein kinase, to function [45] and AvrPphB was shown to be a cysteine protease that self-cleaved to reveal a cryptic amino-terminal myristylation site [46
]. Subsequently, AvrPphB has been shown to cleave PBS1 at a site in the kinaseactivation region homologous to its own cleavage site (Figure 3; [47

]). Both cleavage and kinase activity of PBS1 were shown to be required for RPS5 activation, leading to the inference that cleaved, phosphorylated PBS1 is recognised by RPS5 [47

]. This observation raises the possibility that AvrRpt2, which is also processed to reveal a cryptic amino-terminal myristylation site [48], may be a protease that cleaves both RIN4 and itself. A comparison of the amino-acid sequence adjacent to the cleavage site identified in AvrRpt2 [48] reveals two regions of homology in RIN4 supporting this proposition (Figure 3). These observations also raise the possibility that Avr-Pita, which has been identified as a putative protease [49], may also be recognised through its proteolytic action. Significantly, a mutation in the putative catalytic site of AvrPita destroys its ability to trigger Pi-ta activation [49]. Taken together with the observation that Avr-Pita interacts poorly with a full-length Pi-ta bait construct in a yeast two-hybrid analysis (i.e. with a DNA-binding domain fused to the amino terminus of Pi-ta), but interacted well with an LRR-domain bait construct [40], it is possible that AvrPita might activate Pi-ta by binding to the LRR domain and effecting its removal.

Figure 3

AvrPphB PBS1

60 GDK↓GC 64 241 GDK SH 245

AvrRpt2 RIN4 RIN4

67 VPAFG↓GW 73 6 VPKFG NW 12 149 VPKFG DW 154 Current Opinion in Immunology

Homology between proteolytic cleavage sites in avirulence proteins AvrPphB [46
] and AvrRpt2 [48], and actual or potential cleavage sites in target host proteins PBS1 [47

] and RIN4 guarded by RPS5 and RPS2, respectively. Cleavage sites are indicated by arrows and amino-acid identities are underlined. Current Opinion in Immunology 2004, 16:48–62

54 Innate immunity

Interpretation of observations contradicting the guard hypothesis

The recently described Arabidopsis RRS1-R resistance gene [50
] provides a potential counter example to the guard hypothesis, because the RRS1-R protein shows direct interaction with PopP2, a type III effector protein from the wilt bacterium Ralstonia solanacearum [51

]. The RRS1-R protein is unusual because it has a carboxy-terminal WRKY (tryptophan-arginine-lysinetyrosine) domain. WRKY proteins are plant-specific transcription factors that bind W-box domains in the promoters of various genes, including some encoding PR proteins (reviewed in [52]). The juxtaposition of an R protein and a possible downstream function led to the conclusion that the WRKY domain provides a direct link to transcriptional activation of resistance effector proteins [50
,51

]. Another interpretation, however, is that the RRS1-R protein carries its own version of the protein(s) it guards as a bait. Thus, rather than arguing against the guard hypothesis, the direct interaction between RRS1-R and PopP2 may strengthen it. Other TIR-NBS-LRR proteins also possess carboxy-terminal extensions beyond those defined by the core TIR-NBSLRR domains [6
], and these could also be potential bait domains.

Resistance protein function Nucleotide binding and hydrolysis functions in NBS domains

Consistent with a long-held and generally accepted view, there is now evidence for a nucleotide binding and hydrolysis function in the NBS domain of at least two NBS-LRR proteins [53
], but there is no evidence as yet for an oligomerisation function similar to that of the NOD domain. However, evidence for intramolecular interaction has been shown for the potato Rx CC-NBS-LRR protein, which confers resistance to potato virus X (PVX; [54

]). Trans interactions between separated CC and NBS-LRR domains, and between separated CC-NBS and LRR domains, combined with deletion analyses, have been used to restore Rx function and demonstrate that the LRR domain of Rx interacts with the NBS domain, and the CC domain interacts with the NBS or LRR domains [54

]. Cis interaction was shown to occur preferentially over trans interaction, suggesting that Rx normally exists as a monomer [54

]. The cognate PVX Avr protein disrupted these interactions, suggesting a possible mechanism for activation of the Rx protein [54

]. The role of ATP binding and hydrolysis in this disruption/activation mechanism has not yet been determined, although interaction between the CC and NBS or LRR domains of Rx were shown to require a functional NBS region, whereas interaction between the LRR and NBS domains did not [54

]. One scenario might be that ATP binding is part of the assembly process, and ATP hydrolysis and NBS-mediated oligomerisation are part of the activation process. Current Opinion in Immunology 2004, 16:48–62

Multiple roles for LRR domains

The above-mentioned data suggest that the LRR domain of NBS-LRR proteins plays at least two roles, one in intramolecular interaction and the other in ligand recognition. Site-directed mutagenesis of the tomato Mi CC-NBS-LRR gene for nematode, aphid and thrip resistance, and a constitutively active domain swap with a close homologue suggest that these functions may be physically separated within the LRR domain, with negative regulation of Mi activation located upstream of nematode recognition [55
]. A constitutive-activation mutant, caused by substitution of a single residue from the homologue into the LRR domain of Mi, was suppressed in trans by the amino-terminal domain of the homologue, suggesting complementation of disrupted intramolecular interaction between the amino-terminal and LRR domains of Mi by co-adapted residues in the amino terminus of the homologue [55
]. Similarly, some TIR-NBS-LRR proteins encoded by alleles of the flax L resistance locus differ from one another by co-adapted polymorphic TIR and LRR regions, and disruption of these associations by domain swaps has been shown to either destroy or alter R-protein function [56]. This suggests that intramolecular interactions are not confined to CC-NBS-LRR proteins and, in this case, involve interaction between TIR and LRR domains. There is also evidence that the LRR domain may play a third role. The rps5-1 mutation in LRR3 of the RPS5 CC-NBS-LRR protein, which confers resistance to P. syringae, not only affects RPS5 but also partially suppresses the function of several other NBS-LRR genes that require the NDR1 downstream signalling protein [57]. This includes RPS2, RPM1 and RPP4, a TIR-NBS-LRR, which requires NDR1 for resistance to the downy mildew oomycete, P. parasitica in cotyledons but not in true leaves [57,58]. This negative interference is consistent with interaction between NDR1, or another component of the NDR1 signalling pathway, and the LRR domain. Domain-swapping experiments between the barley MLA1 (powdery mildew resistance locus a 1) and MLA6 CC-NBS-LRR proteins, which confer resistance to the powdery mildew fungus B. graminis, have shown that the dependence of MLA6 on RAR1 (required for barley Mla resistance 1) and SGT1 (suppressor of G2 allele of SKP1) and independence of MLA1 (discussed in the next section) was determined by LRRs 2–7, whereas recognitional specificity of MLA6 was determined by LRRs 8–10 and its carboxyl terminus [59

]. Similarly, a six amino acid polymorphism in the LRR domain of a natural variant of RPS2 leads to differing requirements for additional host factors in Arabidopsis [60]. Overall, these observations suggest that the LRR domain proximal to the NBS may be involved in multiple intra- and inter-molecular interactions separate from recognition. www.sciencedirect.com

Plant innate immunity Jones and Takemoto 55

Protein folding, activation and degradation — possible roles for RAR1 and SGT1 in plant resistance The role of SCF-type E3 ubiquitin ligases and the COP9 signalosome

In 2002 there was a flurry of research publications [30

,59

,61,62

,63–66] reporting the varied requirements of R proteins for RAR1 and SGT1 and suggesting that protein degradation is a part of resistance signalling (reviewed in [67,68,69

]). RAR1 and SGT1 interact with one another, and SGT1 interacts with SKP1 (S-phase kinase-associated protein 1), thereby providing a link to SCF (SKP1 cullin F-box)-type E3 ubiquitin ligases (SCFE3s), which are able to cause protein ubiquitination and ultimately protein degradation by the 26S proteasome. The exact role that protein degradation might play in resistance signalling is not clear, because no clear target for SCF-E3-mediated degradation has been identified. The various requirements for RAR1 and SGT1 by R proteins seem to reflect a role in close proximity to R proteins themselves rather than in shared downstream signalling pathways. Consistent with this idea, domain swapping experiments between the barley MLA6 CCNBS-LRR protein, which requires both RAR1 and SGT1 to function, and the closely-related MLA1 protein, which does not, show that the requirement for RAR1 and SGT1 is determined by LRRs 2–7 of MLA6 and recognitional specificity is determined by LRRs 8–10 and by the carboxyl terminus of MLA6 [59

]. An obvious role for protein degradation might be the removal of defective, and perhaps dangerous, R proteins capable of causing inappropriate cell death. An alternative, and highly favoured hypothesis, is that controlled degradation of R proteins or their negative regulators may be an integral part of resistance signalling. Knowledge of the roles of SGT1 and RAR1 has been extended by two key papers [62

,70

] examining the role of SCF-E3s and the COP9 (constitutively photomorphogenic 9) signalosome (CSN) in Mla6-mediated powdery mildew resistance in barley and in N-mediated TMV resistance in N. benthamiana. The SCF-E3 complex comprises SKP1 (suppressor of kinetochore protein), CUL1 (cullin), RBX1 (RING box) and an F-box protein, which provides target protein specificity for ubiquitination. In barley, SKP1 and CUL1 homologues were detected in SGT1 immunoprecipitates (which also contain RAR1), but not in RAR1 immunoprecipitates (which also contained SGT1), suggesting that SGT1 forms at least two distinct complexes, one containing RAR1 and the other SKP1 and CUL1 [62

]. In N. benthamiana, however, SKP1 and CUL1 homologues were detected in both RAR1 and SGT1 immunoprecipitates, suggesting a single complex [70

]. The CSN is a multiprotein complex with metalloproteinase activity that cleaves covalently attached RUB1 (related to ubiquitin) protein from the CUL1 component of SCF-E3s, thereby activating them. www.sciencedirect.com

Given that the CSN interacts with SCF-E3s, it is not surprising that CSN components were also detected in SGT1 and RAR1 immunoprecipitates from N. benthamiana [70

]. Surprisingly, CSN components were also detected in both SGT1 and RAR1 immunoprecipitates from barley [62

]. The presence of the CSN in the barley RAR1–SGT1 complex without the SCF-E3 is enigmatic and might suggest an alternative role for the CSN in the barley RAR1–SGT1 complex. However, N-mediated TMV resistance in N. benthamiana was shown by virusinduced gene silencing to depend on SKP1 and the CSN components CSN3 and CSN8, suggesting a direct role for both SCF-E3 and the CSN in resistance signalling [70

]. Activation versus degradation

If SCF-E3s and the CSN are recruited by SGT1 into complexes with NBS-LRR proteins, what might their role be in R-protein function? R-protein degradation is an obvious role, but R-protein activation via mono-ubiquitination before degradation is another possibility [71]. Degradation simply to remove dysfunctional R proteins would not explain the requirement for SKP1 and CSN components, as demonstrated for N-mediated TMV resistance in N. benthamiana. Intriguingly, the Arabidopsis RPM1 resistance protein is degraded coincident with perception of the cognate AvrRpm1 or AvrB proteins [72]. Perhaps the perception of Avr proteins triggers SCF-E3 activity, resulting in transient R-protein activation and subsequent degradation. The role of heat shock protein 90

In 2003 it became clear that another molecule, heat-shock protein 90 (HSP90), was also involved in R protein function, as suggested by at least one research publication [70

] and two reviews [68,69

]. No doubt a flurry of research publications reporting the requirement of various R proteins for HSP90 will soon appear. RAR1 and SGT1 both interact with HSP90 in yeast two-hybrid experiments [68,69

,70

], and portions of SGT1 are suggested to resemble the HSP90-interacting domains of the co-chaperones Hop (HSP70 and HSP90 organising protein) and p23 [69

]. RAR1, SGT1 and HSP90 are suggested to form a chaperone complex mediating the folding of R proteins and/or their incorporation into functional complexes [69

]. How would a chaperone complex fit with a protein degradation complex? Two scenarios are possible depending on whether SGT1 is a component shared by distinct RAR1 and SCF-E3 complexes, as suggested by the barley data [62

], or whether SGT1 physically links the two together, as suggested by the N. benthamiana data [70

]. F-box–LRR proteins

SGT1 plays multiple roles and multiple examples of its interaction with LRR proteins have been noted [69

]. Significantly, in yeast, SGT1 interacts with adenylate cyclase, a non-F-box–LRR protein, and mutational Current Opinion in Immunology 2004, 16:48–62

56 Innate immunity

analysis shows that the SGS (SGT-specific) domain of SGT1 interacts with the LRR domain of adenylate cyclase [73,74]. SGT1 may also chaperone SCF-E3s through interactions with the LRR domains of F-box-LRR proteins. In mouse, SGT1 and HSP90 interact with the SCFSKP2 F-box-LRR protein SKP2 (suppressor of kinetochore 2) [75], consistent with a role as a chaperone. The F-box gene family of Arabidopsis comprises 694 genes, of which 42 have LRR domains and 160 have variant LRR domains [76
]. Characterised examples of F-box–LRR proteins that form SCF-E3 complexes include COI1 (coronatine insensitive), which is involved in jasmonate signalling [77,78], TIR1 (transport inhibitor response), which is involved in auxin signalling [79,80
], and ORE9 (oresara meaning long lived in Korean)/MAX2 (more axillary growth), which is involved in leaf senescence [81] and the regulation of axillary shoot formation [82]. Of these, SCFTIR1 requires SGT1, and sgt1 mutants affect both auxin and jasmonate signalling, suggesting that SCFCOI may also require SGT1 [80
]. Moreover, sgt1 mutants enhance the effects of the tir1-1 mutant, but overexpression of SGT1 has a suppressive effect on tir1-1 [80
], consistent with a chaperone effect. SGT1 is not required for SCFTIR1 assembly [80
], which involves interaction between the F-box domain and SKP1, consistent with interaction between SGT1 and the LRR domain of TIR1. Interestingly, RAR1 does not seem to be required for normal auxin signalling [80
]. It is possible that SGT1 and HSP90, but probably not RAR1, play a role in SCF-E3 activity through the correct folding and incorporation of F-box–LRR proteins into SCF complexes (Figure 4a), so it will be interesting to determine the range of SGT1 and HSP90 requirements for other Fbox–LRRs, including COI1 and ORE9/MAX2. If SGT and HSP90 are involved in chaperoning the formation of functional SCF-E3s, then it could be argued that their association with SCF-E3s occurs independently of any association with R proteins. However, this does not explain the requirement for SCF-E3 and CSN components in N-mediated TMV resistance in N. benthamiana. It is conceivable that SGT1 and HSP90 might operate at two different steps in resistance signalling, one chaperoning R proteins (Figure 4b) and the other chaperoning F-box components of SCF-E3s that degrade downstream signalling components (Figure 4a). Some support for a role for SCF-E3s in downstream resistance signalling is provided by preliminary findings that mutations in Arabidopsis affecting core SCF subunits result in increased disease susceptibility [80
]. However, this idea does not explain the different R-protein requirements for SGT1, especially for very closely related R proteins, such as barley MLA1 and MLA6, which differ in their SGT1 requirements, but probably share a common signalling pathway [59

]. A simpler, more direct hypothesis is that the chaperone and degradation functions both act on R proteins or R-protein complexes (Figure 4c), but, this Current Opinion in Immunology 2004, 16:48–62

Figure 4

(a)

F-box-LRR SGS

RBX

CUL1

SKP1

CS

SGT1

TPR

ATP

EEDV HSP90

(b)

SGS CS

NBS-LRR

SGT1

CH2 RAR1

TPR CH1

ATP

EEDV HSP90

(c)

SGS CS

NBS-LRR

SGT1

CH2 RAR1

SKP1 CH1

CUL1

RBX

TPR

ATP

EEDV HSP90 Current Opinion in Immunology

Schematic diagram of possible SGT1 complexes formed in plants (adapted from [69

]). (a) SGT1-HSP90 chaperone complex formed with an F-box-LRR protein component of a SCF-E3. (b) RAR1-SGT1-HSP90 chaperone complex formed with an NBS-LRR protein. (c) Combined RAR1-SGT1-HSP90 chaperone and SCF-E3 (minus F-box protein) complex formed with an NBS-LRR protein. Abbreviations: ATP, ATPbinding domain; CH1, CHORD-I cysteine- and histidine-rich domain; CH2, CHORD-II; CS, motif present in metazoan CHORD and SGT1 proteins; EEDV, glutamate-glutamate-aspartate-valine amino acid motif that interacts with tetratricopeptide repeat domains; SGS, SGT1specific motif; TPR, tetratricopeptide repeat domain.

does not explain the occurrence of two distinct SGT1 complexes in barley; one containing RAR1 and the other SKP1 and CUL1. It is conceivable that SGT1 recruits chaperones and SCF-E3s as their activities are required in barley, but assembles a combined complex that enables a switch from one activity to the other, as required in N. benthamiana. One outstanding question in considering the role of SCFE3s in resistance signalling is the requirement for F-box www.sciencedirect.com

Plant innate immunity Jones and Takemoto 57

proteins. No mutation in an F-box gene has so far been identified in mutation screens for genes required for Rprotein function. Such mutations would seem recoverable, given the target specificity of F-box proteins and the observation that silencing of the SKP1 component of SCF-E3s abolishes N-gene-mediated resistance in N. benthamiana [70

]. This raises a question mark about the involvement of F-box proteins in resistance signalling. SGT1 interacts with SKP1 and the LRR domain of adenylate cyclase, so it is conceivable that SGT1 could link SCF-E3s to LRR domains of R proteins in an F-boxindependent fashion (Figure 4c). The question would then be whether the R protein plays a targeting role, analogous to the F-box protein, or if it is a target, in much the same way as F-box proteins are themselves degraded following ubiquitination; alternatively, it could be both. Regardless of the presence or absence of an F-box protein, an extension of the guard hypothesis might be that the protein being guarded acts as a negative regulator of R-protein signalling. In this instance, the role of the SCFE3 might be to degrade the protein being guarded to allow R-protein activation or to act directly on the R protein to activate it by monoubiquitination and, through its continued action, target it for degradation by polyubiquitination. Protein degradation is an important component of RPM1 and RPS2 activation, but does the pattern of degradation fit either scenario? RPM1 is degraded in the absence of either RAR1 or RIN4 [42

,64,66]. AvrB and AvrRpm1 cause RPM1-independent phosphorylation of RIN4, but not its degradation [42

]. Phosphorylated RIN4 activates RPM1 and causes its degradation [42

,72]. RPS2 requires RAR1 [64,66] and HSP90 (GenBank accession no. AAP87284), but its fate in their absence is unknown. AvrRpt2 causes RPS2independent degradation of RIN4 (and consequently RPM1), and this activates RPS2, but does not cause it to degrade [43

,44

]. Neither RPM1 nor RPS2, however, require SGT1 [61], so the protein degradation that does occur during RPM1 or RPS2 activation is not caused by SGT1-mediated recruitment of SCF-E3s. Moreover, the ability to separate resistance specificity from SGT1 requirement in barley MLA6 [59

] argues against this kind of role. Perhaps the answer to this complex puzzle lies in the multifunctionality of SGT1 and a degree of functional redundancy. SGT1 can potentially play at least three roles in resistance signalling, each of which can, in some cases, also be carried out in the absence of SGT1 or with assistance from another protein. In chaperoning NBSLRR proteins or F-box–LRRs, it may be that some require little help in their folding or are able to interact with HSP90 without assistance; however, if assistance is required to recruit HSP90, either RAR1 or SGT1 may be sufficient or both may be required. When linking SCFE3s to target R proteins or the proteins they guard, it may www.sciencedirect.com

be that some F-box proteins need little help in targeting the SCF-E3, but if assistance is required to recruit the SCF-E3, SGT1 may provide it through interaction with SKP1. Many pieces of the puzzle are still missing and the range of R-protein requirements for components of the SCF-E3, the CSN and HSP90 has not yet been ascertained. Are the SCF-E3 and the CSN universally required, or are they only necessary for R proteins that require SGT1? Similarly, is HSP90 universally required or is it only necessary for R proteins that require SGT1 or RAR1? The puzzle is further complicated by the observation that SGT1 also appears to be required for Cf-4–Avr4- and Cf9–Avr9-dependent HR, and for non-host resistance in N. benthamiana [30

]. Cf-4 and Cf-9 are LRR-RLPs, with the majority of the protein (including the LRR domain) in a different subcellular compartment to SGT1. This might support the idea that SGT1 is involved in a downstream step of resistance signalling shared with other kinds of R proteins. Similarly, the role of HSP90 as a chaperone of R proteins may not be confined to the cytosolic NBS-LRR proteins, but may also extend to the LRR-RLK and LRR-RLP resistance proteins with their extracytosolic LRRs. SHD (shepherd), an HSP90like protein localized in the endoplasmic reticulum, is responsible for the formation of a functional CLV (clavata) complex in Arabidopsis [83
]. The CLV complex, involved in shoot apical meristem development (reviewed in [84]), comprises CLV1, a LRR-RLK, and CLV2, a LRR-RLP (Figure 1), and it is likely that SHD chaperones the correct folding and complex formation of either or both LRR proteins. Interestingly, the shd mutation has pleiotropic effects beyond those of clv1 or clv2 mutations, suggesting that it plays a role in chaperoning other extracytosolic proteins or protein domains involved in development. It would be interesting to test the SHD requirement of LRR-RLKs or LRR-RLPs with known function; for example, the RPP27 resistance protein. However, SHD may not be required for all members of these two protein families. Presumably it is not required for the LRR-RLK BRI (brassinosteroid insensitive) because the shd mutation did not phenocopy the dwarf phenotype of the bri mutation. It is also possible that SHD chaperones non-LRR proteins. Interestingly, gp96, an HSP90-like protein localized in the endoplasmic reticulum, is responsible for the formation of functional TLRs in mice [85]. TLRs and integrins were not translocated to the cell surface in a gp96 mutant cell line, but instead remained in the cytoplasm, whereas CD14 (a glycosylphosphatidylinositol-anchored LRR protein that is a component of the TLR4 LPS-recognition complex; see Figure 1) was translocated normally, indicating that LRR association is not absolute, with some non-LRR proteins requiring gp96 and at least one LRR protein that does not. Current Opinion in Immunology 2004, 16:48–62

58 Innate immunity

It has been proposed that HSP90 acts as a buffer for phenotypic variation [86,87
]. On one level this may be an overly sophisticated explanation for the observation that loss of HSP90 function can cause phenotypic changes arising from the misfolding of proteins. On another level, however, it offers a degree of tolerance to the adaptive genetic variation arising between members of gene families undergoing diversifying selection. Thus, rather than LRR proteins being difficult to fold per se, it might be that the LRR domains of some R proteins (and perhaps Fbox–LRRs) need more assistance in their folding because they have undergone adaptive changes that might otherwise have caused them to fold and function poorly.

Conclusions In the past year, exciting progress has been made in several specific areas of R-protein function and in the recognition of general elicitors, or PAMPs, for which a looser definition seems appropriate in plant pathogens compared to animal pathogens. Significant overlaps between the protein components and signalling pathways involved suggest there may be both shared and unique features for both branches of plant innate immunity (nonhost and host-specific resistance). As a consequence, nonhost resistance is likely to attract more attention in the future than it currently receives. For host-specific resistance, it would appear that arguments in favour of the guard hypothesis have strengthened, and even examples of direct interaction between plant R proteins and pathogen Avr proteins may support it. The degradation of host target proteins by pathogen-encoded proteases may be a common theme in host-specific resistance, and recognition of the products of pathogen Avr protein effector function may be another. Chaperone-assisted folding and/or assembly of complexes appears to be important for some R proteins, but the extent to which it is required has yet to be ascertained. Similarly, SCF-E3 targeting of proteins for degradation appears to be important for some R proteins, but just where this degradation fits into resistance signalling is not yet clear. Although more has been learnt about the function of domains within resistance proteins, clear connections to downstream signalling components await discovery. Clearly, there is much to anticipate in the coming year.

Acknowledgements We thank Jeff Ellis and Adrienne Hardham for critical reading of the manuscript.

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Gaulin E, Esquerre´ -Tugaye´ M-T, Dumas B: Elicitor activity of a fungal endopolygalacturonase in tobacco required a functional catalytic site and cell wall localization. Plant Physiol 2003, 131:93-101. There is controversy about whether induction of plant resistance responses by endopolygalacturonase treatment depends on oligogalacturonides produced by their enzymatic activity or on the endopolygalacturonase proteins as elicitors in their own right (see [19
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effector suppresses cell wall-based extracellular defense in susceptible Arabidopsis plants. Proc Natl Acad Sci USA 2003, 100:8577-8582. This report demonstrates that AvrPto, an avirulence factor recognised by the tomato Pto R protein, has a type III virulence effector function. Expression of AvrPto in Arabidopsis suppressed expression of genes for putative secreted cell-wall defence proteins. Accumulation of defence-inducible callose was abolished in AvrPto plants, suggesting that AvrPto is a suppressor of cell-wall-based host defences. In contrast to HR inhibition by AvrPtoB [26
], induction of R/Avr-dependent HR was retained in AvrPto plants (RPS2/AvrRpt2 interaction). 26. Abramovitch RB, Kim Y-J, Chen S, Dickman MB, Matrin GB:
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Woestenenk E, Boeren S, de Wit PJGM, Joosten MHAJ, Vervoort J: Natural disulfide bond-disrupted mutants of AVR4 of the tomato pathogen Cladosporium fulvum are sensitive to proteolysis, circumvent Cf-4-mediated resistance, but retain their chitin binding ability. J Biol Chem 2003, 278:27340-27346. Avr4, an extracellular Avr protein produced by the leaf mould fungus, Cladosporium fulvum, and detected by the tomato Cf-4 LRR-RLP protein, has chitin-binding activity thought to protect chitin in the fungal cell wall from degradation by plant chitinases. Most natural C. fulvum isolates, virulent on Cf-4 tomato, possesses Avr4 alleles with cysteine to tyrosine substitutions disrupting disulphide bonds, resulting in greater sensitivity to proteolysis in the intercellular spaces of tomato plants. However, these natural isoforms of Avr4 still retain chitin-binding activity, which appears to protect them from degradation. These results indicate selection has been imposed for Avr4 variants that avoid recognition by Cf-4, but retain www.sciencedirect.com

chitin-binding ability, suggesting chitin protection may be an important function of Avr4. 28. Kim YJ, Lin N-C, Martin GB: Two distinct Pseudomonas effector proteins interact with the Pto kinase and activate plant immunity. Cell 2002, 109:589-598. 29. Zhang S, Klessig DF: MAPK cascades in plant defense signaling. Trends Plant Sci 2001, 6:520-527. 30. Peart JR, Lu R, Sadanandom A, Malcuit I, Moffett P, Brice DC,

Schauser L, Jaggard DAW, Xiao S, Coleman MJ et al.: Ubiquitin ligase-associated protein SGT1 is required for host and nonhost disease resistance in plants. Proc Natl Acad Sci USA 2002, 99:10865-10869. Using virus-induced gene silencing in Nicotiana benthamiana, this paper shows that silencing of SGT1 compromised N, Rx and Pto R genemediated pathogen resistance, HR induced by transient expression of Rx, Pto, Cf-4 and Cf-9 R genes and their cognate Avr genes, HR induced by transient expression of the RPW8 R gene, the AvrRpt2 Avr gene or the Inf1 elicitin gene, and non-host resistance to Pseudomonas syringae pv. maculicola and Xanthomonas axonopodis pv. vesicatoria, but not X. campestris pv. campestris or cauliflower mosaic virus. The broad requirement for SGT1 in N. benthamiana suggests it may be involved in a shared signalling pathway. 31. Yun B-W, Atkinson HA, Gaborit C, Greenland A, Read ND,
Pallas JA, Loake GJ: Loss of actin cytoskeletal function and EDS1 activity, in combination, severely compromises non-host resistance in Arabidopsis against wheat powdery mildew. Plant J 2003, 34:768-777. The authors of this paper report the synergistic roles of the actin cytoskeleton and the resistance signalling protein EDS1 on non-host resistance, showing that the combination of an actin-polymerisation inhibitor and the eds1 mutation allows a wheat pathogen to complete its lifecycle on Arabidopsis, a non-host plant. 32. Xiao S, Ellwood S, Calis O, Patrick E, Li T, Coleman M, Turner JG: Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8. Science 2001, 291:118-120. 33. Ellis C, Karafyllidis I, Wasternack C, Turner JG: The Arabidopsis
mutant cev1 links cell wall signaling to jasmonate and ethylene responses. Plant Cell 2002, 14:1557-1566. Arabidopsis cev1 [33
] and eli1-1 [34
] mutants have mutations in a conserved domain of the cellulose synthase gene CESA3, leading to aberrant deposition of lignin, reduced cell expansion and enhanced resistance to powdery mildew pathogens. In cev1 and eli1-1 mutants, jasmonic acid (JA)- and ethylene-mediated defence gene expression is activated, and treatment with both JA and 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor) reproduced their phenotypes in wildtype plants. These results indicate that reduced cellulose synthesis activates JA and ethylene signal transduction and leads to defence gene expression. 34. Cano˜ -Delgado A, Penfield S, Smith C, Catley M, Bevan M:
Reduced cellulose synthesis invokes lignification and defence responses in Arabidopsis thaliana. Plant J 2003, 34:351-362. See annotation to [33
]. 35. Nishialra MT, Stein M, Hou B-H, Vogel JP, Edwards H,
Somerville SC: Loss of a callose synthase results in salicylic acid-dependent disease resistance. Science 2003, 301:969-972. The Arabidopsis pmr4 (powdery mildew resistant) mutant shows resistance to various biotrophic pathogens, including Erysiphe cichoracearum, E. oronti and Peronospora parasitica, and produces less callose in response to pathogen infection and wounding. PMR4 encodes a biosynthetic enzyme that appears to be responsible for stress-related callose deposition. Salicylic acid (SA)- and pathogen-responsive genes were upregulated in pmr4 plants, but mutations in the SA signalling pathway abolished or reduced pmr4-induced resistance. 36. Che F-S, Nakajima Y, Tanaka N, Iwano M, Yoshida T, Takayama S, Kadota I, Isogai A: Flagellin from an incompatible strain of Pseudomonas avenae induces a resistance response in cultured rice cells. J Biol Chem 2000, 275:32347-32356. 37. Tanaka N, Che F-S, Watanabe N, Fujiwara S, Takayama S, Isogai A:
Flagellin from an incompatible strain of Acidovorax avenae mediates H2O2 generation accompanying hypersensitive cell death and expression of PAL, Cht-1, and PBZ1, but not of Lox in rice. Mol Plant Microbe Interact 2003, 16:422-428. Flagellin from a non-pathogenic strain, but not a pathogenic strain, of bacteria was able to induce an HR and some pathogen-response genes in rice, but despite the inability to induce an HR, a flagellin-deficient nonCurrent Opinion in Immunology 2004, 16:48–62

60 Innate immunity

pathogenic strain remained non pathogenic and retained the ability to induce pathogen-response genes. This indicates that flagellin detection contributes to pathogen recognition, but is not the only contributing factor. 38. Shimizu R, Taguchi F, Marutani M, Mukaihara T, Inagaki Y,

Toyoda K, Shiraishi T, Ichinose Y: The DfliD mutant of Pseudomonas syringae pv. tabaci, which secretes flagellin monomers, induces a strong hypersensitive reaction (HR) in non-host tomato cells. Mol Genet Genomics 2003, 269:21-30. This paper describes a role for detection of flagellin monomers in nonhost resistance by showing that bacterial mutants unable to produce flagellin became pathogenic on tomato, whereas those producing flagellin monomers but unable to assemble flagella caused an enhanced resistance response. 39. Taguchi F, Shimizu R, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y:

Post-translational modification of flagellin determines the specificity of HR induction. Plant Cell Physiol 2003, 44:342-349. This paper highlights the role of post-translational modification of otherwise sequence-identical flagellins from pathogenic and non-pathogenic bacteria, most likely by differential glycosylation, in avoiding or allowing their detection by tobacco plants. 40. Jia Y, McAdams SA, Bryan GT, Hershey HP, Valent B: Direct interaction of resistance gene and avirulence gene products confers rice blast resistance. EMBO J 2000, 19:4004-4014. 41. Van der Biezen EA, Jones JDG: Plant disease-resistance proteins and the gene-for-gene concept. Trends Biochem Sci 1998, 23:454-456. 42. Mackey D, Holt BF III, Wiig A, Dangl JL: RIN4 interacts with

Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 2002, 108:743-754. The authors identify RIN4 from a yeast two-hybrid screen using AvrB as bait and shows that RIN4 interacts with the amino terminus of RPM1 by yeast two-hybrid analysis and could be co-immunoprecipitated with RPM1, AvrB and AvrRpm1. They also show that AvrB and AvrRpm1 cause RPM1-independent phosphorylation of RIN4, and that a reduction in RIN4 expression causes reduction in RPM1 protein levels, but not vice versa. 43. Axtell MJ, Staskawicz BJ: Initiation of RPS2-specified disease

resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4. Cell 2003, 112:369-377. Together with [44

], this paper shows that RPS2 co-immunoprecipitates with RIN4 and that AvrRpt2 causes RPS2-independent degradation of RIN4, resulting in the activation of RPS2. It also shows that RPS2 levels do not change with RPS2 or RPM1 activation and that AvrRpt2-induced degradation of RIN4 is RAR1 independent. 44. Mackey D, Belkhadir Y, Alonso JM, Ecker JR, Dangl JL:

Arabidopsis RIN4 is a target of the type III virulence effector AvrRpt2 and modulates RPS2-mediated resistance. Cell 2003, 112:379-389. Together with [43

], this paper shows that RPS2 co-immunoprecipitates with RIN4 and that AvrRpt2 causes RPS2-independent degradation of RIN4 resulting in the activation of RPS2. It also shows that overexpression of RIN4 blocks RPS2 signalling but does not affect RPM1 signalling, and that elimination of RIN4 eliminates RPM1 signalling. 45. Swiderski MR, Innes RW: The Arabidopsis PBS1 resistance gene encodes a member of a novel protein kinase subfamily. Plant J 2001, 26:101-112. 46. Shao F, Merritt PM, Bao Z, Innes RW, Dixon JE: A Yersinia effector
and a Pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis. Cell 2002, 109:575-588. See annotation to [47

]. 47. Shao F, Golstein C, Ade J, Stoutemyer M, Dixon JE, Innes RW:

Cleavage of Arabidopsis PBS1 by a bacterial type III effector. Science 2003, 301:1230-1233. Shows that the cysteine protease activity of AvrPphB responsible for cleavage of its own amino terminus [46
] is also responsible for the RPS5and RAR1-independent cleavage of the protein kinase PBS1 in the activation segment of the kinase domain, leading to its kinase-activitydependent recognition by RPS5. 48. Mudgett MB, Staskawicz BJ: Characterization of the Pseudomonas syringae pv. tomato AvrRpt2 protein: Current Opinion in Immunology 2004, 16:48–62

demonstration of secretion and processing during bacterial pathogenesis. Mol Microbiol 1999, 32:927-941. 49. Orbach MJ, Farrall L, Sweigard JA, Chumley FG, Valent B: A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta. Plant Cell 2000, 12:2019-2032. 50. Deslandes L, Olivier J, Theulie`res F, Hirsch J, Feng DX,
Bittner-Eddy P, Beynon J, Marco Y: Resistance to Ralstonia solanacearum in Arabidopsis thaliana is conferred by the recessive RRS1-R gene, a member of a novel family of resistance genes. Proc Natl Acad Sci USA 2002, 99:2404-2409. This paper reports the identification of RRS1-R as a novel TIR-NBS-LRR protein containing a potential nuclear localisation signal (NLS) and a carboxy-terminal WRKY domain, which may function as a direct link to transcriptional activation of resistance effector genes. However, the functionality of either the NLS or the WRKY domain was not established. This may be an example of the principle that physical linkage of protein domains separated in other systems indicates physical interaction between the domains, however the combination of TIR-NBS-LRR and WRKY domains in RRS1 could also be a unique variation rather than a guiding principle. Resistance conferred by RRS-1R is also novel because it is shown to be dependent on NDR1, which hitherto has been associated almost exclusively with genes encoding CC-NBS-LRR proteins. 51. Deslandes L, Olivier J, Peeters N, Feng DX, Khounlotham M,

Boucher C, Somssich I, Genin S, Marco Y: Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus. Proc Natl Acad Sci USA 2003, 100:8024-8029. The authors of this paper have Identified the pathogen molecule recognised by the RRS1-R resistance protein [50
] as PopP2, a type III effector protein of the YopJ/AvrRxv protein family. Direct interaction between PopP2 and both RRS1-R and RRS1-S (which does not confer resistance) is shown using a yeast split-ubiquitin two-hybrid system. Tagging with fluorescent proteins revealed that PopP2 carries a functional nuclear localisation signal (NLS) and forms a nuclear localised complex with both RRS1-R and RRS1-S, suggesting that delivery of the WRKY domain to the nucleus where it might act as a transcriptional activator is not, by itself, sufficient for resistance. Neither fluorescent-protein-tagged RRS-1R nor RRS1-S could be detected alone, but could be detected in a cytosolic complex with a derivative of PopP2 lacking a NLS, suggesting that the potential NLS in RRS1 may not be functional. 52. Eulgem T, Rushton PJ, Robatzek S, Somssich IE: The WRKY superfamily of plant transcription factors. Trends Plant Sci 2000, 5:199-206. 53. Tameling WIL, Elzinga SDJ, Darmin PS, Vossen JH, Takken FLW,
Haring MA, Cornelissen BJC: The tomato R gene products I-2 and Mi-1 are functional ATP binding proteins with ATPase activity. Plant Cell 2002, 14:2929-2939. This paper confirms the long held, but previously unconfirmed, belief that the NBS domain of plant NBS-LRR proteins, like the NOD domains of mammals, really do function as nucleotide-binding domains. The NBS domain of the I-2 resistance protein is shown to bind ATP, dATP, ADP or dADP, but not GTP, in a Mg2þ-dependent fashion. The NBS domains of the Mi and I-2 resistance proteins are both shown to hydrolyse ATP. 54. Moffett P, Farnham G, Peart J, Baulcombe DC: Interaction

between domains of a plant NBS-LRR protein in disease resistance-related cell death. EMBO J 2002, 21:4511-4519. This paper reports the physical interaction and functional complementation of separated domains of the Rx CC-NBS-LRR protein transiently expressed in N. benthamiana. It also describes the clever use of virusinduced gene silencing of SGT1 to allow immunoprecipitation of epitopetagged Rx protein domains following co-expression of the cognate Avr protein, which would otherwise trigger destructive resistance responses. The data obtained suggest intramolecular interaction of the CC and LRR domains with different subregions of the NBS domain (or differential control of intramolecular interaction by the ATP-binding subregion) and that the cognate Avr protein disrupts these interactions leading to Rx activation. 55. Hwang CF, Williamson VM: Leucine-rich repeat-mediated
intramolecular interactions in nematode recognition and cell death signaling by the tomato resistance protein Mi. Plant J 2003, 34:585-593. Based on the observation that transient expression of a fusion of the amino terminus of Mi-1.1 to the carboxyl terminus of Mi-1.2 (the functional Mi R protein) causes necrosis in Nicotiana benthamiana, which suggests a mismatch between amino- and carboxy-terminal residues responsible for negative regulation of Mi activity, this paper uses site directedmutagenesis of polymorphic residues in the LRR domain to determine www.sciencedirect.com

Plant innate immunity Jones and Takemoto 61

which residues are involved. A single Mi-1.1 residue from an LRR proximal to the NBS domain caused lethality in a tomato root transformation assay when substituted into Mi-1.2, but substitution of three contiguous residues from a downstream LRR were found to affect nematode recognition in transformed tomato roots, suggesting separation of negative regulation from recognition. The lethal mutation was suppressed in trans by transient co-expression of the amino-terminal region of Mi-1.1, suggesting intramolecular interaction between the amino terminus and a region of the LRR domain proximal to the NBS domain. 56. Luck JE, Lawrence GJ, Dodds PN, Shepherd KW, Ellis JG: Regions outside of the leucine-rich repeats of flax rust resistance proteins play a role in specificity determination. Plant Cell 2000, 12:1367-1377. 57. Warren RF, Henk A, Mowery P, Holub E, Innes RW: A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes. Plant Cell 1998, 10:1439-1452. 58. van der Biezen EA, Freddie CT, Kahn K, Parker JE, Jones JDG: Arabidopsis RPP4 is a member of the RPP5 multigene family of TIR-NB-LRR genes and confers downy mildew resistance through multiple signalling components. Plant J 2002, 29:439-451. 59. Shen Q-H, Zhou F, Bieri S, Haizel T, Shirasu K, Schulze-Lefert P:

Recognition specificity and RAR1/SGT1 dependence in barley Mla disease resistance genes to the powdery mildew fungus. Plant Cell 2003, 15:732-744. RAR1 and SGT1 dependence of MLA6 can be separated from recognition specificity by domain swaps with MLA1, which does not depend on RAR1 and SGT1, resulting in a RAR1- and SGT1-independent version of MLA6. However, the reciprocal swap, rather than creating a RAR1- and SGT1dependent version of MLA1, produced a non-functional R protein. RAR1 and SGT1 dependence was found to be determined by LRRs 2–7 of MLA6, which could indicate a region of physical interaction, but could also indicate an unstable region requiring greater assistance in folding, given that RAR1, SGT1 and HSP90 may form a chaperone complex. 60. Banerjee D, Zhang X, Bent AF: The leucine-rich repeat domain can determine effective interaction between RPS2 and other host factors in Arabidopsis RPS2-mediated disease resistance. Genetics 2001, 158:439-450. 61. Austin MJ, Muskett P, Kahn K, Feys BJ, Jones JDG, Parker JE: Regulatory role of SGT1 in early R gene-mediated plant defenses. Science 2002, 295:2077-2080. 62. Azevedo C, Sadanandom A, Kitagawa K, Freialdenhoven A,

Shirasu K, Schulze-Lefert P: The RAR1 interactor SGT1, an essential component of R gene-triggered disease resistance. Science 2002, 295:2073-2076. These authors report the isolation of barley SGT1 as a RAR1 interacting protein in a yeast two-hybrid screen. They also use gene silencing and pathogen infection to demonstrate that MLA6 requires SGT1, but MLA1 does not, in line with their RAR1 requirements. Immunoprecipitation experiments show that RAR1 and SGT1 co-immunoprecipitate with one another and that SGT1 co-immunoprecipitates with SCF-E3 components SKP1 and CUL1, but RAR1 does not, whereas both co-immunoprecipitate with CSN components CSN4 and CSN5. These data suggest that there may be at least two different kinds of SGT1 complex in barley. 63. Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP: Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 2002, 30:415-429. 64. Muskett PR, Kahn K, Austin MJ, Moisan LJ, Sadanandom A, Shirasu K, Jones JDG, Parker JE: Arabidopsis RAR1 exerts ratelimiting control of R gene-mediated defenses against multiple pathogens. Plant Cell 2002, 14:979-992. 65. To¨ r M, Gordon P, Cuzick A, Eulgem T, Sinapidou E, Mert-Tu¨ rk F, Can C, Dangl JL, Holub EB: Arabidopsis SGT1b is required for defense signaling conferred by several downy mildew resistance genes. Plant Cell 2002, 14:993-1003. 66. Tornero P, Merritt P, Sadanandom A, Shirasu K, Innes RW, Dangl JL: RAR1 and NDR1 contribute quantitatively to disease resistance in Arabidopsis, and their relative contributions are dependent on the R gene assayed. Plant Cell 2002, 14:1005-1015. www.sciencedirect.com

67. Devoto A, Muskett PR, Shirasu K: Role of ubiquitination in the regulation of plant defence against pathogens. Curr Opin Plant Biol 2003, 6:307-311. 68. Holt BF III, Hubert DA, Dangl JL: Resistance gene signaling in plants -complex similarities to animal innate immunity. Curr Opin Immunol 2003, 15:20-25. 69. Shirasu K, Schulze-Lefert P: Complex formation, promiscuity

and multi-functionality: protein interactions in diseaseresistance pathways. Trends Plant Sci 2003, 8:252-258. A stimulating review that postulates a chaperone function for RAR1 and SGT1, and introduces HSP90 as a new player in resistance signalling. It also reports unpublished data indicating interactions between HSP90 and both RAR1 and SGT1 in yeast two-hybrid analyses. 70. Liu Y, Schiff M, Serino G, Deng X-W, Dinesh-Kumar SP: Role

of SCF ubiquitin-ligase and the COP9 signalosome in the N gene-mediated resistance response to tobacco mosaic virus. Plant Cell 2002, 14:1483-1496. These authors report the isolation of Nicotiana benthamiana SGT1 and HSP90 as RAR1-interacting proteins in a yeast two-hybrid screen, and show that SKP1 interacts with SGT1 in a yeast two-hybrid analysis. Interactions between SGT1 and both RAR1 and SKP1 were confirmed by in vitro binding assays. Immunoprecipitation experiments show that RAR1 and SGT1 co-immunoprecipitate with one another, with SCF-E3 components SKP1 and CUL1, and with CSN component CSN4. Virusinduced gene silencing of SGT1, SKP1 and CSN components CSN3 and CSN8 compromised N gene-mediated resistance to TMV in N. benthamiana, suggesting both SCF-E3 and CSN complexes are required for N protein function. These data suggest that RAR1, SGT1, SCFE3 and CSN may form a single complex in N. benthamiana. 71. Pickart CM: Ubiquitin enters the new millennium. Mol Cell 2001, 8:499-504. 72. Boyes DC, Nam J, Dangl JL: The Arabidopsis thaliana RPM1 disease resistance gene product is a peripheral plasma membrane protein that is degraded coincident with the hypersensitive response. Proc Natl Acad Sci USA 1998, 95:15849-15854. 73. Dubacq C, Guerois R, Courbeyrette R, Kitagawa K, Mann C: Sgt1p contributes to cyclic AMP pathway activity and physically interacts with the adenylyl cyclase Cyr1p/Cdc35p in budding yeast. Eukaryot Cell 2002, 1:568-582. 74. Schadick K, Fourcade HM, Boumenot P, Seitz JJ, Morrell JL, Chang L, Gould KL, Partridge JF, Allshire RC, Kitagawa K et al.: Schizosaccharomyces pombe Git7p, a member of the Saccharomyces cerevisiae Sgtlp family, is required for glucose and cyclic AMP signaling, cell wall integrity, and septation. Eukaryot Cell 2002, 1:558-567. 75. Lyapina S, Cope G, Shevchenko A, Serino G, Tsuge T, Zhou C, Wolf DA, Wei N, Shevchenko A, Deshaies RJ: Promotion of NEDD8-CUL1 conjugate cleavage by COP9 signalosome. Science 2001, 292:1382-1385. 76. Gagne JM, Downes BP, Shiu S-H, Durski AM, Vierstra RD: The
F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis. Proc Natl Acad Sci USA 2002, 99:11519-11524. Analysis of the F-box gene family of Arabidopsis shows that 42 of the 694 F-box genes found, including COI1, TIR1 and ORE9/MAX2, encode Fbox proteins with LRR domains, and another 160 encode F-box proteins with variant LRR domains. 77. Xie D-X, Feys BF, James S, Nieto-Rostro M, Turner JG: COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 1998, 280:1091-1094. 78. Devoto A, Nieto-Rostro M, Xie D, Ellis C, Harmston R, Patrick E, Davis J, Sherratt L, Coleman M, Turner JG: COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis. Plant J 2002, 32:457-466. 79. Ruegger M, Dewey E, Gray WM, Hobbie L, Turner J, Estelle M: The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast Grr1p. Genes Dev 1998, 12:198-207. 80. Gray WM, Muskett PR, Chuang H-W, Parker JE: Arabidopsis
SGT1b is required for SCFTIR1-mediated auxin response. Plant Cell 2003, 15:1310-1319. Current Opinion in Immunology 2004, 16:48–62

62 Innate immunity

The authors of this paper identified the eta3 mutation that enhanced the defective auxin response phenotype of the tir1-1 mutant (defective for the TIR1 F-box-LRR protein that targets SCF-E3 to transcriptional regulators involved in the auxin response). They also carried out map-based cloning of the ETA3 gene, which was found to correspond to SGT1. This paper also shows that plants mutant for RAR1 do not show altered auxin responses, suggesting that RAR1 is not required for TIR1 function. 81. Woo HR, Chung KM, Park J-H, Oh SA, Ahn T, Hong SH, Jang SK, Nam HG: ORE9, an F-box protein that regulates leaf senescence in Arabidopsis. Plant Cell 2001, 13:1779-1790. 82. Stirnberg P, van de Sande K, Leyser HMO: MAX1 and MAX2 control shoot lateral branching in Arabidopsis. Development 2002, 129:1131-1141. 83. Ishiguro S, Watanabe Y, Ito N, Nonaka H, Takeda N, Sakai T,
Kanaya H, Okada K: SHEPHERD is the Arabidopsis GRP94 responsible for the formation of functional CLAVATA proteins. EMBO J 2002, 21:898-908. This paper reports the identification of the shd (shepherd) mutation in Arabidopsis, which phenocopies defects in apical meristem formation caused by clv (clavata) mutations. Gene isolation and sequencing revealed SHD to encode an endoplasmic reticulum HSP90-like protein. Analysis of epistatic interactions between shd and genes involved in clavata signalling indicated that SHD is required for correct folding or incorporation of CLV proteins into a signalling complex. The additional

Current Opinion in Immunology 2004, 16:48–62

phenotypic effects of the shd mutation suggest other signalling proteins or complexes may also be chaperoned by SHD. 84. Clark SE: Cell signalling at the shoot meristem. Nat Rev Mol Cell Biol 2001, 2:276-284. 85. Randow F, Seed B: Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability. Nat Cell Biol 2001, 3:891-896. 86. Rutherford SL, Lindquist S: Hsp90 as a capacitor for morphological evolution. Nature 1998, 396:336-342. 87. Queitsch C, Sangster TA, Lindquist S: Hsp90 as a capacitor of
phenotypic variation. Nature 2002, 417:618-624. This paper further examines the concept proposed previously [86] that HSP90 buffers genetic variation by chaperoning protein variants that might be adaptive on the one hand but maladaptive in terms of their innate folding capacity. Reduction in HSP90 function in Arabidopsis revealed significant phenotypic variation between different ecotypes, supporting this idea. 88. Parker JE: Plant recognition of microbial patterns. Trends Plant Sci 2003, 8:245-247. 89. Girardin SE, Travassos LH, Herve´ M, Blanot D, Boneca IG, Philpott DJ, Sansonetti PJ, Mengin-Lecreulx D: Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2. J Biol Chem 2003, 278:41702-41708.

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