Plant Molecular Biology 50: 971–980, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Protein complexes mediate signalling in plant responses to hormones,light, sucrose and pathogens

Christine Ellis, John G. Turner and Alessandra Devoto∗School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK (∗author for correspondence;e-mail a.devoto@uea.ac.uk)

Received 26 July 2001; accepted in revised form 23 January 2002

Key words: proteasome, resistance and avirulence proteins, ubiquitin

Abstract

Living organisms use complex pathways of signal perception and transduction to respond to stimuli in their en-vironments. In plants, putative signal transduction components have been identified through mutant screens andcomparative analysis of genome sequences of model eukaryotes. Several pieces in a large series of puzzles havenow been identified and a current challenge is to determine how these pieces interconnect. Functional analysisof the encoded proteins has necessitated a change from genetic to biochemical approaches. In recent years, theapplication of techniques such as two-hybrid screening and epitope tagging has facilitated the study of protein-protein interactions and has increased our understanding of cellular signalling mechanisms. One focus of presentresearch is the ubiquitin/proteasome-mediated degradation of proteins. Increasing evidence suggests this is a con-trol common to many plant signalling pathways including development and responsiveness to hormones, light andsucrose. A central challenge in the study of plant disease resistance has been to identify protein complexes thatcontain host defence proteins and pathogenicity factors. In this review we summarize the latest developments inthese areas where the existence of protein complexes has been demonstrated to be of fundamental importance inplant signalling.

Introduction

A variety of environmental and developmental stim-uli regulate diverse and often overlapping processesin plants. These processes involve signal perception,transduction, and execution of the response. Signaltransduction is mediated through protein-protein in-teractions and growing evidence indicates that theseinteractions occur within protein complexes that werefer to here as signalling complexes. Several of thegenes in the signalling pathways that regulate responseto light, auxin, sucrose and pathogens have now beenidentified and characterised (for other reviews see delPozo and Estelle, 2000; McCarty and Chory, 2000;Dangl and Jones, 2001; Estelle, 2001; Glazebrook,2001; Nimchuk et al., 2001). Most of these genes weredefined by mutations that either suppressed or en-hanced response to the stimulus; others were isolatedbecause they determined natural phenotypic variation,

for example in response to pathogens. These isolatedgenes have been used as functional ‘anchor’ pointsfor that signal pathway. A current effort is to identifyproteins that bind to these ‘anchor’ proteins in orderto build up a picture of the signalling complex. Animportant corollary is to evaluate the function of theinteracting proteins.

Two of the main techniques used to identify in-teracting proteins are the yeast two-hybrid assay and‘pull-down’ assays, reviewed elsewhere in this issue.In the former, the ‘anchor’ protein may be expressedin yeast as a fusion protein and used to screen a cDNAexpression library for interacting proteins; in the latterantibodies that recognize the ‘anchor’ protein, or an-tibodies or ligands that bind epitopes translationallyfused to the ‘anchor’ protein, are used to precipi-tate this from whole cell extracts. The precipitatesare then characterized to identify interacting proteins.The genome sequences of model eukaryotes has al-

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lowed comparative analysis of signalling pathwaysbetween plants and animals and this has acceleratedthe identification of putative interacting proteins.

An unexpected finding of present research has beenthat the ubiquitin/proteasome forms complexes with‘anchor’ proteins from several signalling pathways,adding to evidence that targeted degradation of pro-teins has an important role in signal pathways. Analy-sis of the protein complexes that bind to plant diseaseresistance gene products has significantly increasedour understanding of the mechanisms that control re-sistance to pathogen infection. Here we summarizerecent advances in these two areas.

The ubiquitin/proteasome system: a platform forsignalling

Protein turnover via the ubiquitination/proteasomepathway is an important cellular control mechanismin many eukaryotes (Hershko and Ciechanover, 1998;Deschaies, 1999; Ciechanover et al., 2000). In thispathway, ubiquitin is attached to substrate proteinstargeted for degradation via the successive action ofthree enzymes, E1 (ubiquitin-activating enzyme), E2(ubiquitin-conjugating enzyme), and E3 (ubiquitinligase). The 26S proteasome is responsible for thedegradation of most ubiquitinated proteins. This is amultisubunit protease complex found in the nucleusand cytoplasm of cells, which consists of two largesubcomplexes, a 19S regulatory unit and a 20S cat-alytic unit (Vierstra, 1996; Hershko and Ciechanover,1998). It is speculated that the 19S subunit is responsi-ble for the binding and unfolding of ubiquitin-taggedproteins. The 20S subunit, which contains a numberof different proteolytic activities, is responsible for thesubsequent degradation of target proteins.

In Arabidopsis, only a small number of E1 geneshave been identified and these have no known func-tional specificity (Hatfield et al., 1997), whereas thereare at least 36 isoforms in the E2 family (Callisand Vierstra, 2000). E3 activity is exhibited by alarge number of proteins that can be divided into sixmain classes: HECT domain proteins, Ubr1p pro-teins, APC/C complexes, SCF complexes, pVHLcomplexes, and RING-finger proteins (Ciechanoveret al., 2000). The E3 proteins or E2/E3 combina-tions are responsible for the selection of target proteinsand thereby confer specificity on the ubiquitinationprocess.

The SCF ubiquitin ligase was originally identi-fied in yeast and was named for three of its subunits:

Skp1, Cdc53p (called cullin in other species), and F-box proteins (reviewed in Deschaies, 1999). A fourthsubunit has been more recently identified and is re-ferred to as Roc1/Rbx/Hrt1. E2 enzymes have alsobeen shown to interact directly with these enzymes(Patton et al., 1998; Seol et al., 1999). Cullin is oftencovalently modified by the attachment of a ubiquitin-related protein called RUB1, also known as NEDD8(Lammer et al., 1998; Deschaies, 1999). Attachmentof RUB1 is analogous to ubiquitination and requiresE1-like and E2-like activities. However, in RUB1 con-jugation, two proteins that share homology with theN-terminal and C-terminal halves of E1 carry out theE1-like activity. RUB1 modification has been pro-posed to function in the stabilization or assembly ofthe SCF complex (Lammer et al., 1998) and in thepromotion of ubiquitin-chain formation (Wu et al.,2000).

In Arabidopsis, there are at least 3 cullin proteins(del Pozo and Estelle, 2000), 19 Skp1-like proteins, orASKs (Farras et al., 2001), and 337 F-box proteins(Arabidopsis Genome Initiative, 2000). The F-boxproteins provide specificity to the E3 complex. PlantF-box proteins with known function include TIR1,involved in auxin response (Ruegger et al., 1998),UFO/FIM, required for floral development (Ingram,1997), COI1, required for jasmonate response (Xieet al., 1998), and FKF and ZTL, both involved inthe control of circadian rhythms (Nelson et al., 2000;Somers et al., 2000). Database searches have identi-fied a large number of F-box proteins in Arabidopsis,most of which have unknown functions (ArabidopsisGenome Initiative, 2000; Xiao and Jang, 2000).

In yeast two-hybrid screens, F-box proteins inter-act preferentially with certain SKP1 proteins (Grayet al., 1999; Samach et al., 1999). However, it is notknown whether F-box proteins interact with only oneSKP1 and cullin in vivo or with numerous SKP1 andcullin proteins to form a combinatorial variety of SCFcomplexes. Crystallographic data of complexes of hu-man SKP1 with the F-box protein SKP2 suggests thatdivergent C-terminal sequences of SKP1 proteins mayallow preferential binding of subsets of F-box proteins(Schulman et al., 2000). ASK1 has been shown to in-teract with TIR1 (Gray et al., 1999), UFO (Samachet al., 1999) and COI1 (Devoto, and Turner, unpub-lished) in yeast two-hybrid studies. Similarly, each ofthese proteins binds to multiple ASKs. Because of thehigh degree of homology amongst ASKs, experimentswith ask1 mutants (Gray et al., 1999; Zhao et al. 1999)and epitope-tagged ASK1 (Farras et al., 2001) were

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required to confirm the specificity of the ASK1 inter-actions. Apparently, ASK1 can interact with severalF-box proteins that regulate different plant processes,indicating the possibility of a higher level of cellularcontrol through the combinatorial assembly of SCFcomplexes.

The large number of potential SCF complexes andthe diversity of the pathways they affect suggest thatthe regulation of protein turnover is a control elementcommon to many plant processes. As yet, however,the involvement of ubiquitin-mediated proteolysis hasbeen substantiated in only a limited number of sys-tems. Progress has been particularly significant in theareas of auxin signalling, sucrose regulation, lightresponse and pathogen defence.

Involvement of ubiquitin-mediated proteolysis inauxin signalling

In plants, the role of ubiquitin-mediated degradation incell signalling has been established most extensivelyin the auxin response pathway (reviewed in del Pozoand Estelle, 1999a; Gray and Estelle, 2000). The tir1mutant was isolated in a screen for resistance to auxintransport inhibitors (Ruegger et al., 1998) and is defec-tive in several auxin-related growth responses such aslateral root formation and auxin-mediated root growthinhibition. TIR1 is an F-box protein that forms anSCF complex with ASK1 and AtCul1 (Gray et al.,1999). Mutations in the ASK1 gene have pleiotropicdefects, including male sterility (Yang et al., 1999)and reduced auxin responsiveness (Gray et al., 1999).TIR1 and ASK1 interact genetically, reinforcing thenotion that TIR1 and ASK1 function together in auxinsignalling.

Auxin responsiveness also requires the AXR1 gene,which bears homology to the N-terminal half of E1(Leyser et al., 1993). Database searches have iden-tified Arabidopsis genes ECR1, analogous to the E1C-terminal region (del Pozo et al., 1998), and RCE1(RUB-conjugating enzyme 1), analogous to the E2-like enzymes involved in RUB1/NEDD8 transfer inyeast and man (del Pozo and Estelle, 1999b). Invitro translated proteins have been used to demon-strate that the successive action of the AXR1-ECR1heterodimer and RCE1 results in the attachment ofRUB1 to AtCul1. RUB1-AtCul1 conjugates have alsobeen detected in plant extracts (del Pozo and Estelle,1999b).

Auxin treatment causes the rapid synthesis of afamily of related short-lived proteins called Aux/IAA

proteins (Abel and Theologis, 1996). A number ofauxin mutants contain alterations in domain II ofAux/IAA proteins (Rouse et al., 1998; Tian et al.,1999; Nagpal et al., 2000). Engineering two of thesemutations into domain II of a pea IAA6-luciferasefusion protein increased its longevity in vivo (Wor-ley et al., 2000) and plants that over-express theAux/IAA17 (AXR3) gene show a mutant phenotypesimilar to axr3-1 plants. This indicates that the degra-dation of auxin-regulated proteins is essential fornormal auxin signalling. Aux/IAA proteins form het-erodimers with auxin-response factors (ARFs), a fam-ily of DNA-binding proteins, and may thus regulateexpression of auxin-responsive genes (Kim et al.,1997; Ulmasov et al., 1999).

An elegant series of experiments have recentlylinked auxin signalling to the COP9 signalosome(Schwechheimer et al., 2001). The COP9 signalosomeis a multiprotein complex that acts as a negative regu-lator of photomorphogenic development in Arabidop-sis. It bears homology to the 19S lid sub-complex ofthe 26S proteasome and has been found in a range ofeukaryotes from yeast to man where it is involved indiverse processes such as cell cycle progression andthyroid hormone reception (Hardtke and Deng, 2000).Similarities between the 19S proteasome sub-complexand the COP9 signalosome have led to the speculationthat it may function as a lid for 26S-proteasomal-likecomplexes. However, electron microscopy data showthat the COP9 signalosome has several structural dif-ferences from the 19S sub-complex (Kapelari et al.,2000), suggesting that their functions may also differ.

Reduction of the COP9 signalosome subunit CSN5through antisense and co-suppression strategies leadsto a reduction in COP9 signalosome levels (Schwech-heimer et al., 2001). In addition to a photomor-phogenic phenotype when grown in the dark, CSN5transgenic plants have pleiotropic anomalies, a num-ber of which are similar to those found in axr1 andtir1 plants. Degradation of pea IAA6-luciferase fusionprotein is also decreased in the CSN5 background.

Immunoprecipitation experiments reveal that thecomponents of the SCFTIR1 ubiquitin ligase, TIR1,ASK1, and AtCul1, form a complex with the COP9signalosome. Yeast two-hybrid results show direct in-teractions between COP9 signalosome subunit CSN2and AtCul1, and that Rbx1 interacts with both CSN1and CSN6. The COP9 signalosome also appears tomediate RUB1 deconjugation from AtCul1. COP9 sig-nalosome mutants preferentially accumulate RUB1-conjugated AtCul1 whereas in wild-type plants, the

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Figure 1. Auxin signalling as a model for ubiquitin-mediated protein degradation in Arabidopsis. ASK1, TIR1, AtCUL1, and RBX1 forman SCF-type ubiquitin ligase complex that is associated with the COP9 signalosome. RUB1 is conjugated to AtCUL1 through the actionof the AXR1-ECR1 heterodimer and RCE1. The COP9 signalosome may function in the deconjugation of RUB1 and/or as a lid for a26S-proteasome-like complex. In response to auxin, repressors of auxin-mediated development become targets for ubiquitination via the actionof E1, E2, and SCFTIR1. Polyubiquinated proteins are ultimately degraded in the proteasome. The mechanism that targets these repressors fordegradation remains unclear, but may involve modification of target proteins by phosphorylation. Alternatively, auxin may control the activityof the SCFTIR1 complex through regulation of RUB1-conjugation. Abbreviations: UB, ubiquitin; RUB1, related to ubiquitin; REP, repressorsof auxin-mediated development.

distribution is shifted towards unconjugated AtCul1(Schwechheimer et al., 2001). A similar phenomenonhas been reported in yeast, where the COP9 signalo-some was also shown to have deconjugating activity(Lyapina et al., 2001).

A clear picture of auxin signalling is now emerging(Figure 1). Although no direct link has as yet beenmade between Aux/IAA proteins and the SCFTIR1

protein complex, it is plausible that these short-livedproteins are degraded via the ubiquitin/proteasomepathway, resulting in expression of the auxin re-sponse. CSN5 plants also show pleiotropic defectsnot accounted for by light and auxin responses. It istherefore possible that the function of ubiquitin ligasesfrom other signalling pathways also requires the COP9signalosome.

Involvement of an SCF complex in sucrose regulation

Mutations in the PRL1 (PLEIOTROPIC REGULA-TORY LOCUS 1) gene result in transcriptional de-repression of many sucrose-related genes as wellas hypersensitivity to a number of plant hormones(Nemeth et al., 1998). AMP-activated protein ki-nases, such as the yeast Snf1 (sucrose non-fermenting1), play an important role in metabolic and stressresponses in eukaryotes (Hardie et al., 1998). In Ara-

bidopsis, a number of Snf1-related protein kinases, orSnRKs, have been identified, two of which, AKIN10and AKIN11, show enhanced activation in the prl1background and have been shown to bind to PRL1(Bhalerao et al., 1999; Farras et al., 2001).

Co-immunoprecipitation experiments and purifica-tion of 20S proteasomal complexes demonstrate thatthe proteasome associates with SnRK and componentsof the SCF complex (Farras et al., 2001). In yeast two-hybrid experiments and in vitro, AKIN10 interacts di-rectly with ASK1, PRL1, and the α4/PAD1 subunit ofthe 20S proteasome. α4/PAD1 promotes binding be-tween AKIN10 and ASK1 and the three proteins forma ternary complex. However, PRL1 competitively in-hibits ASK1 binding to AKIN10. PRL1, ASK1, andSnRK complexes were not detected in plant extracts.Immunolocalization experiments reveal that ASK1,AtCul1, and the 26 proteasome co-localize with themitotic spindles and phragmoplasts during mitosisleading to speculation that components of the mitoticspindle may serve as substrates for the proteasomalcomplex (Farras et al., 2001).

These results illustrate the association betweenSCF components and the 26S proteasome. In addition,they suggest that SnRKs and PRL1 may modulate theactivity of the SCF/proteasome complex by altering

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the phosphorylation states of target proteins or thebinding affinities amongst components of the com-plex. Further work is needed to clarify their exactroles. As this study did not examine the F-box compo-nent of the SCF complexes, it has yet to be determinedwhich specific signalling pathways are regulated bySnRKs and PRL1. However, given the pleiotropic phe-notype of the prl1 mutant, it is possible that severalpathways are affected.

Involvement of ubiquitin-mediated proteolysis in lightresponses

HY5 and COP1 are key proteins in the control of pho-tomorphogenesis (Hardtke and Deng, 2000). HY5, apositive effector of photomorphogenic development,is a bZIP transcription factor that binds to the promot-ers of light-inducible genes and accumulates in light-grown plants (Chattopadhyay et al., 1998; Oyamaet al., 1997). The stability and activity of HY5 isdecreased by phosphorylation (Hardtke et al., 2000).COP1 negatively regulates HY5 activity. It contains aRING-finger motif such as those found in many E3ubiquitin ligases (Joazeiro and Weissman, 2000) andWD40 repeats, through which it interacts with HY5(Holm et al., 2001).

In the light, COP1 is excluded from the nucleus(von Arnim and Deng, 1994), HY5 accumulates inits active unphosphorylated form, and light-inducedgenes are expressed. In the dark, COP1 localizes to thenucleus where it can interact with HY5, and triggerits degradation (Osterlund et al., 2000). Some HY5may be inactivated by phosphorylation. Phosphory-lated HY5 has decreased affinity for COP1 and maytherefore persist in the nucleus and serve to providea rapid response to light. All COP/DET/FUS loci, in-cluding those that make up the COP9 signalosome, arerequired for HY5 degradation. Furthermore, protea-some inhibitors diminish HY5 degradation (Osterlundet al., 2000), suggesting that the ubiquitin/proteasomepathway is essential for COP1-mediated removal ofHY5. Based on these results and on the presence ofa RING finger motif, it appears that COP1 is an E3ubiquitin ligase or part of an E3 complex. Characteri-zation of other COP1-interacting proteins identified byyeast two-hybrid experiments (Yamamoto et al., 1998;Torii et al., 1999; Yamamoto et al., 2001) may providecorroboration for this hypothesis.

Signalling complexes in the regulation of diseaseresistance

There is a growing body of evidence suggesting that insome situations, disease resistance is mediated by pro-teasomal degradation. The cloning of the gene Rar1from barley has unveiled a potential link betweenubiquitin mediated proteolysis and resistance. Rar1represents a convergence point in the signalling of re-sistance to powdery mildew, and in higher metazoan,shows colinearity with a protein domain homologousto the yeast SGT1 (Kitagawa et al. 1999) which regu-lates delivery of targets to the SCF complex (Shirasuet al., 1999). The degradation of the resistance geneproduct RPM1 coincides with the hypersensitive re-sponse (Boyes et al., 1998), although whether thisoccurs via the proteasome has yet to be demonstrated.

Much effort has also been put into identifyingsignalling complexes mediating pathogen recogni-tion. Plant resistance to pathogens requires productsof resistance (R) genes that recognize pathogen-specific avirulence (Avr) proteins (Hammond-Kosackand Jones, 1997). Five main classes of plant disease-resistance proteins have been identified so far (Dangland Jones, 2001; Glazebrook, 2001). At least 150 Ara-bidopsis R protein homologues contain an N-terminalnucleotide-binding site (NBS) and leucine-rich repeats(LRRs) (http://pgfsun.ucdavis.edu/niblrrs/At_RGenes/)although some R genes are notably different (Xiaoet al. 2001.). The presence in most cases, of threeor more distinct domains with the potential to inter-act with multiple proteins, render R proteins attractivecandidates for components of signal complexes. Afavoured model based on genetic data postulates thatR proteins act as receptors for pathogen-encoded Avrproteins to initiate a signal which activates plant de-fence responses and the arrest of pathogen ingress.A central challenge therefore has been to determinewhether R proteins interact physically with Avr pro-teins, and to identify those proteins through which thedefence response signal is transduced.

Interaction of R proteins with Avr proteins

Evidence for direct interaction between an R proteingene product and an Avr protein came from yeast two-hybrid analysis which demonstrated binding of thetomato bacterial speck resistance gene product, Pto,to the corresponding AvrPto avirulence gene prod-uct of Pseudomonas syringae pv. tomato (Scofieldet al. 1996; Tang et al., 1996). In addition, the

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genetic evidence for an interaction between the NBS-LRR-containing rice R protein Pita and the corre-sponding Avr protein from the rice fungal pathogenMagnaporthe grisea was confirmed in yeast and byfar-western analysis. Mutations in the Pita leucine-rich repeat domain or in the Avr-Pita protease motifresulted in loss of resistance and disruption of thephysical interaction, both in yeast and in vitro (Jiaet al., 2000; Bogdanove, this issue).

Genetic evidence indicates that Arabidopsis plantscarrying either of the R genes RPS2 or RPM1 are resis-tant to strains of P. syringae carrying either of the Avrgenes AvrRpt2 or AvrB respectively. The identifica-tion of RPS2-AvrRpt2 and RPS2-AvrB complexes inco-immunoprecipitation assays (Leister et al., 2000)might suggest that these R proteins can bind differentAvr products in vivo, indicating that the interactionsare not simple receptor-ligand combinations (see Fig-ure 2A). Significantly, these interactions could not bereproduced with in vitro translated proteins, emphasiz-ing the importance of obtaining in vivo data. Possiblythe interaction may require additional plant factorsand/or the in vitro translated protein may not assumeproper conformation or additional post-translationalmodifications.

The tomato Cf-9 gene confers race-specific resis-tance to the fungal pathogen Cladosporium fulvumexpressing the corresponding avirulence gene Avr9.The Cf gene contains an extracytoplasmic, membrane-anchored domain predominantly comprosed of LRRs(Jones et al., 1994; Thomas et al., 1997). However,Cf-9 does not appear to bind Avr9 directly, suggestingthat other proteins may be required for recognition ofAvr9 by Cf-9 (Luderer et al., 2001 and Figure 2B).Searches have therefore begun for other proteins thatbind to Avr proteins, or to R proteins.

Multi-component defence complexes

Genetic evidence indicates that the highly vari-able LRR regions of R proteins are responsible forpathogen recognition (Ellis et al., 1999, 2000) but sup-porting biochemical evidence has not been forthcom-ing. Luck et al. (2000) have shown genetically thatnon-LRR regions of flax L alleles can also determinespecificity differences of flax rust resistance.

It may be significant therefore that RPS2 proteinco-immunoprecipitates in vitro with a host protein of75 kDa (Figure 2B). The 75 kDa protein interacts withthe N-terminal portion of RPS2, which contains anNBS and a leucine zipper, suggesting these motifs may

function in signal transduction. Transient expressionassay results indicate that the N-terminal domain ofRPS2 product can mediate cell death signalling (Taoet al., 2000).

The Cf proteins are highly glycosylated proteinswith apparent molecular mass of ca. 145 kDa (Cf-4) or165 kDa (Cf-9). Lack of a signalling domain in the Cf-proteins is suggestive of a requirement for at least onefurther component mediating ligand binding, similarto that observed in the CLAVATA system regulatingboth meristem and organ development in Arabidopsis(Trotochaud et al., 2000). Preliminary results indicatethat epitope-tagged functional Cf-4 and Cf-9 (Piedraset al., 2000) might be part of a higher-molecular-weight complex (S. Rivas and J.D.G. Jones, personalcommunication). This would be the first indicationthat these R proteins may be part of signalling com-plexes. Van der Biezen and Jones (1998) anticipatedthis idea with the ‘guard hypothesis’ that R proteinsare not merely direct receptors for Avr proteins, but‘guard’ host defence proteins that are targeted bypathogenicity factors. The elicitor/receptor model maystill hold true for some systems but for many othersdirect interaction of R and Avr products has not beendetected (Dangl and Jones, 2001).

Signalling complexes could be inferred whenmolecules have more than one conspicuous bindingdomain and in addition to protein-protein interactions,recognition between protein and DNA may occur.AvrXa7 is a member of the avrBs3 avirulence genefamily, which encodes proteins and acts as a virulencefactor in Xanthomonas oryzae pv. oryzae. Yang et al.(2000) demonstrated through domain-swapping analy-sis that the nuclear localization signal and the acidictranscriptional activation domains of the AvrXa7 arerequired for its virulence and avirulence. Gel shift as-says indicate that AvrXa7 can interact with host DNA.These results suggest that AvrXa7 may interact bothwith host transcriptional machinery as well as DNAin the formation of a signalling complex. A specificinteraction with a host transcription cofactor might bealso required for virulence.

A first step towards the identification of host pro-teins interacting with R proteins only in the presenceof Avr proteins and their distinction from those bind-ing directly to Avr proteins, has been taken by uti-lizing the AvrPto-Pto system (see also Bogdanove,this issue). To pursue the hypothesis that tertiaryinteractions are involved between Pto and AvrPto,Bogdanove and Martin (2000) modified the yeasttwo-hybrid protein interaction trap and conducted

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Figure 2. Different models for the interaction of plant R proteins with pathogen Avr proteins. The situations described are not mutuallyexclusive. A. Direct binding between resistance (R) and avirulence (Avr) proteins is consistent with the interaction between tomato Pto andP. syringae Avr-Pto as well as for rice Pita and M. grisea Avr-Pita. The model also considers the possibility that the same R protein couldrecognize more than one Avr peptide as shown for the Arabidopsis RPS2 and P. syringae AvrRPT2 and AvrB. B. Host protein mediated R-Avrbinding. The R protein binds the Avr protein only in the presence of one or more host protein (HP). RPS2-p75 binding and Cf9 recognition ofAvr9, may be consistent with this model. B1. Binding to the Avr protein does not require modification of the R protein and the formation of astable tertiary complex may occur only if all three components are present. B2. The host protein forms a complex with the R protein, which ismodified prior to Avr binding. C. Avr-mediated R-HP binding. In the absence of the Avr peptide, no interaction of the R protein with the hostprotein occurs. The host protein(s) form a complex with the Avr protein, resulting in a modification or conformational change, which allowsbinding of the R protein. R, resistance protein; Avr, avirulence protein; HP, host protein; ∗protein modification.

a search for tomato proteins that interact with Ptoonly in the presence of AvrPto, allowing differentia-tion between AvrPto-interacting proteins and AvrPto-dependent Pto-interacting partners (Figure 2C). Tothe latter category belong clones corresponding to acatalase, putative serine-threonine protein kinases andPti2, a truncated proteasome α-subunit, previouslyisolated by Zhou et al. (1998). Homologues of a stressrelated protein and Ras-related small, putative GTP-binding protein have been identified as AvrPto directinteractors. Direct physical interaction between Prf,

an NBS-LRR protein required in the AvrPto-Pto re-sistance response pathway (Salmeron et al., 1996),and Pto and/or AvrPto is yet to be demonstrated.Techniques such as this may prove useful in identi-fying protein complexes, which require more than twoproteins for stable interactions.

Conclusions and future directions

The genetic analysis of plant signalling pathways isrelatively advanced in plants but biochemical con-

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firmation of the interactions between the identifiedcomponents is only beginning. Although several sig-nalling pathways can be linked to the proteasome, fewtargets for ubiquitination have been confirmed. Sim-ilarly, the functions of proteins that interact with Rproteins are largely unknown. Additional proteins arelikely to participate in Avr perception and subsequentsignal transduction. The genetic approaches utilisedso far to isolate and characterise the signalling genesneed to be complemented by biochemical studies onthe protein to understand function.

The relative ease of yeast two hybrid screens (re-viewed in this issue) has made it a popular toolto identify proteins capable of forming complexeswith these signalling components. Results obtainedwith this system are indicative but insufficient to es-tablish the existence of protein-protein interactionsin planta. In the yeast system, proteins are over-expressed in the nucleus of a heterologous host irre-spective of the native localization. Protein interactionsin planta may also require post-translational mod-ifications not accomplished by the host yeast cellshence many interactions would not be detected bythis system. Interactions must be confirmed by tech-niques such as co-immunoprecipitation and localiza-tion experiments where proteins can be expressed andpost-translationally modified in their original envi-ronment. A limitation of co-immunoprecipitation asan investigative tool is that the interacting protein isoften detected by western analysis. In this case previ-ous knowledge of potential interactors and antibodiesraised against them are required. The improvementof mass spectrometric and other analytical techniquesfor the direct identification of components of im-munoprecipitates will remove the present limitationof prior hypothesis. Although the potential of thesetechnologies has yet to be fully exploited, they willundoubtedly accelerate the identification of protein-protein interactions and further our understanding ofprotein complexes in plant signalling.

Acknowledgements

We are grateful to C. Koncz, A. Ferrando, H. Ma,J.D.J. Jones and S. Rivas who made available un-published information to assist the preparation of thisreview. Many thanks also to M. Coleman, C. Thomas,S. Xiao and T. Lahaye for critical comments. Researchwork in J.G.T.’s laboratory is supported by grants from

the Biotechnology and Biological Sciences ResearchCouncil.

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