The role of proteolysis in R gene mediated defence in plants

transcript

MOLECULAR PLANT PATHOLOGY

(2003)

(4 ) , 287–296 DOI : 10 .1046/ J .1364-3703.2003.00169.X

Blackwell Publishing Ltd.

Review

The role of proteolysis in

gene mediated defence in plants

MAHMUT TÖR*, ANTONY YEMM AND ER IC HOLUB

Sustainable Disease Resistance Team, Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK

SUMMARY

Within the last 10 years, numerous

genes have been clonedfrom natural genetic variation in model as well as crop plants,and these have been classified according to their motifs. Some ofthe downstream signalling components have also been identifiedby artificial mutagenesis. Recently, cloning of three of these sig-nalling genes (

) from

Arabidopsis

, barleyand tobacco have helped uncover the physiological link betweendefence signalling and ubiquitin-mediated protein degradation.The physical association of COI1 and SGT1b with the componentsof ubiquitin–ligase complexes has been shown. In addition, post-transcriptional silencing of some of the subunits of the ubiquitin-ligase complex has led to a loss of resistance, indicating thatprotein degradation may also act as a regulatory mechanism inplant defence. Over the next few years, we should expect to seemore examples of the interplay between the defence response

and protein degradation in plants.

INTRODUCTION

In their natural environments, plants are routinely challenged byparasites including viruses, bacteria, fungi, nematodes and insects,all of which have the molecular capability to manipulate hostplants for their own sustenance. As discussed below, plants haveevolved an innate disease resistance involving a complex array ofconstitutively expressed

genes to detect foreign invaders anddefend themselves (Dangl and Jones, 2001; Holub, 2001).

When a pathogen attempts to infect a host plant, pathogenassociated molecules (

genes, effector molecules) are recog-nized at the infection site by receptors in the host plasma mem-brane, or within the cytosol. This initiates a multicomponent defenceresponse at the local and systemic level. Physiological studieshave shown a directed movement of nucleus and organellestowards the site of pathogen attack (Gross

., 1993; Heath

., 1997). Papilla formation (Bolwell

., 2001) or callose

deposition at the penetration sites or around the haustoria offungal pathogens (Mellersh and Heath, 2001) has often beenobserved. In the initial stage of the innate defence response, asmall burst of reactive oxygen species (ROS) has been reportedfor both compatible and incompatible interactions (Lamb andDixon, 1997). In incompatible interactions, the permeability ofthe plasma membrane is altered and ion channels are opened,leading to a second but much stronger burst of ROS (Grant

.,2000) which amplifies the signalling process further (McDowelland Dangl, 2000). These responses are usually accompanied bycellular collapse from irreversible damage of membranes and thesynthesis and accumulation of antimicrobial compounds (referredto as programmed cell death, PCD or hypersensitive response,HR) (Dangl and Jones, 1998). These local defence responsesare then amplified throughout the plant with the help of wellknown signal molecules such as salicylic and jasmonic acid,ethylene and MAP kinases (Dong, 1988; Glazebrook, 2001; Zhangand Klessig, 2001).

Physiological studies have been complemented by the mole-cular characterization of several

genes (see below). Some of thedownstream signalling components, including kinases, putativelipases, phosphatases and transcription factors have been iden-tified. Recently, a new picture has emerged showing that proteindegradation may also be an essential component of the

genemediated defence response. The aim of this review is to describerecent advances in the knowledge of protein degradation inplants and our present understanding of how this process may beinvolved in disease resistance by acting as a regulatory mechanismof defence in plants.

GENES AND DEFENCE SIGNALLING

Over the last few years, several

genes have been cloned froma wide range of plants including

Arabidopsis

, tomato, rice andmaize. Their structural and functional comparisons have beenwell documented (see reviews by Ellis and Jones, 1998;Hammond-Kosack and Jones, 1997) revealing at least five differentclasses (Table 1) distinguished according to structural motifs andpotential functions of the proteins. The first group contains thecytoplasmic Ser/Thr kinase and has been represented by

Correspondence

: E-mail: Mahmut.tor@hri.ac.uk

M. TÖR

et al.

(2003)

(Martin

., 1993), which confers resistance to the bacterialpathogen

Pseudomonas syringae

tomato

. The second groupis represented by tomato

genes conferring resistance to thefungal pathogen

Cladosporium fulvum

(Dixon

., 1996; Jones

., 1994). The characteristic features of these genes are anextracellular leucine rich repeat domain (LRR) with a single mem-brane spanning region and a short cytoplasmic carboxyl domain.Several genes in this class, including

have been cloned (Dixon

., 1996; Jones

., 1994). Thethird group of

genes is similar to the

genes, in that theyhave the extracellular LRR and the membrane-spanning domain.However, they also have a cytoplasmic kinase domain. This groupis represented by the rice

gene (Song

., 1995) that con-fers resistance to the bacterial pathogen

Xanthomonas oryzae

oryzae

. A further interesting example is

gene in

Arabidopsis

(Gömez-Gömez and Boller, 2000) that recognizes bacterial flag-ellin. The fourth group is represented by the

gene of

Ara-bidopsis

for powdery mildew resistance (Xiao

., 2001). Thisgene product has a putative signal anchor at the N-terminus, acentral coiled-coil domain (CC) and a carboxyl LRR domain. Thefifth and the largest group is comprised of genes that contain acentral nucleotide binding site (NB) and carboxyl LRR domain(referred to henceforth as NB-LRR genes). This group can be fur-ther subdivided into two subclasses: one that has an aminoterminal CC domain (CC-NB-LRR) and others that have a TIRdomain resembling the cytoplasmic signalling domain of the Tolland Interleukin I transmembrane receptors (TIR-NB-LRR). The CC-NB-LRR subclass includes examples such as the

Arabidopsis RPS2

Table 1 Classification of R genes according to their structural motifs and potential functions.

R-gene classes

Characteristic features of protein Members

Plant origin

Pathogen/ Avr gene Reference

1 Cytoplasmic Ser/Thr PTO Tomato Pseudomonas syrningae Martin et al. (1993)protein kinase pv. tomato (avrPto)

2 Extracellular LRR, a single membrane spanning region, a short cytoplasmic carboxyl domain

Cf-2 Cf-4 Cf-5 Cf-9

Tomato Cladospsorium fulvum (Avr2, Avr4, Avr5, Avr9)

Jones et al. (1994) Dixon et al. (1996)

3 Extracellular LRR, a single membrane spanning region, a cytoplasmic kinase domain

Xa-21 Rice Xanthomosa oryzae pv. oryzae (all races)

Song et al. (1995)

FLS2 Arabidopsis Bacterial flagellin Gömez-Gömez

and Boller (2000)4 A putative signal anchor RPW8 Arabidopsis Erysiphe cruciferarum Xiao et al. (2001)

at the N-terminus, a UEA1central (CC) domain and Erysiphe cichoracearuma carboxyl LRR domain UCSC1

5a An amino terminal CC RPS2 Arabidopsis Pseudomonas syringae Mindrinos et al. (1994)domain, a central NB, and (AvrRpt2 )a carboxyl LRR domain RPM1 Arabidopsis P. syringae Grant et al. (1995)(CC-NB-LRR) (AvrRpm1, AvrB )

RPP8 Arabidopsis Peronospora parasitica McDowell et al. (1998)(ATR8)

RPP13 Arabidopsis P. parasitica (ATR13) Bittner-Eddy et al. (2000)5b An amino terminal TIR N Tobacco mosaic virus Whitham et al. (1994)

domain, a central NB and L6 Flax Melampsora lini Lawrence et al. (1995)a carboxyl LRR domain (AL6)(TIR-NB-LRR) RPP4 Arabidopsis P. parasitica van der Biezen et al. (2002)

(ATR4) RPP5 Arabidopsis P. parasitica Parker et al. (1997)

(ATR5)

Abbreviations: Avr, avirulence; Ser, Serine; Thr, Threonine, LRR, Leucine Rich Repeat; CC, Coiled-Coil; NB, Nucleotide Binding site; ATR, Arabidopsis thaliana recognized avirulence; TIR, Toll and Interleukin I receptor.

Proteolysis in defence signalling

(2003)

(4 ) , 287–296

(Mindrinos

., 1994) and

(Grant

., 1995) for bac-terial resistance and

(McDowell

., 1998) and

(Bittner-Eddy

., 2000) for downy mildew resistance. The TIR-NB-LRR subclass includes genes such as the tobacco

(Whitham

., 1994) for viral resistance, the flax

(Lawrence

.,1995) for rust resistance

Arabidopsis RPP4

(van der Biezen

., 2002) and

(Parker

., 1997) for downy mildew resist-ance. Sequencing of the complete genome of

Arabidopsis

hasrevealed more than 150 NB-LRR genes <

http://mips.gsf.de/cgi-bin/proj/thal/displayrgenes.pl/

http://niblrrs.ucdavis.edu/

>.However, the function of most of these genes has yet to bediscovered.

The products of

genes recognize the effector molecules eitherdirectly (Bryan

., 2000) or through an interactor (Mackey

., 2002) and trigger the downstream signalling pathways. Inorder to gain insight into signalling components, mutational dis-section of signalling pathways and high-throughput screeningsof gene expression involving DNA microarray analysis (Kazan

., 2001; Maleck

., 2000) have been carried out.A genetic approach involving the selection of ‘enhanced sus-

ceptibility’ mutants has identified numerous genes that play arole in plant–pathogen interactions (Glazebrook, 1999; Shapiro,2000). For example,

(enhanced disease susceptibility) and

(nonspecific disease resistance) suggest that at least twodistinct pathways exist in

Arabidopsis

encodes a solubleprotein with a homology to eukaryotic lipases (Falk

., 1999;Parker

., 1996) and is required for the function of several

Ara-bidopsis RPP

(Resistance to

Peronospora

parasitica

) genes and atleast one

(Resistance to

Pseudomonas

syringae) gene (Aartset al., 1998). Similarly, NDR1 (Century et al., 1997) is required forresistance to different isolates of both pathogens and encodes apotentially membrane-associated protein of unknown function.Interestingly, eds1 affects the TIR-NB-LRR class of genes, whilstndr1 affects most of the CC-NB-LRR class of genes indicating thatresistance gene structure plays a more important role than thepathogen type in defining the signalling pathway. Similar to EDS1and NDR1, the RAR1 (required for Mla resistance) gene of barleyfor powdery mildew resistance (Freialdenhoven et al., 1994) alsodiscriminates between highly related resistance genes. The RAR1homologue in Arabidopsis (previously designated as PBS2; Warrenet al., 1999) has recently been isolated (Muskett et al., 2002;Tornero et al., 2002) and in contrast to EDS1 and NDR1, RAR1 isrequired by some members of R genes belonging to both CC-NB-LRR and TIR-NB-LRR class. While both EDS1 and NDR1 arerestricted to the plant species, domains of the RAR1 proteinhave been conserved across the kingdoms (Shirasu et al., 1999).RAR1 encodes a protein with two novel zinc finger motifs calledCHORD-I and CHORD II (Cys- and His-rich domain) that is presentin all eukaryotes except yeast. In addition, RAR1 lacks a domainthat is found in metazoan CHORD proteins. This domain, calledCS (CHORD containing proteins and Sgt1), has homology to yeast

SGT1 (suppressor of G two allele of skp1-4; Kitagawa et al.,1999) protein, which has been shown to be involved in proteindegradation. These findings have led Shirasu et al. (1999) topostulate that plant SGT1-like proteins with a CS domain mayprovide a link between RAR1 and the protein degradationpathway.

INTRACELLULAR PROTEOLYSIS

Protein molecules are continuously synthesized and degraded inall living organisms as directed by physiological need. A balancebetween the rates of synthesis and degradation has to be main-tained and the level of important structural proteins, enzymes,and regulatory proteins like transcription factors has to be keptunder control. Too much or too little of these regulatory proteinsmay have catastrophic consequences for the cell. The only waythat cells can reduce the steady state level of a particular proteinis by proteolytic degradation (Vierstra, 1996). An increased rateof proteolysis has been observed in cells which are subjected tostresses such as starvation and heat-shock (Gottesman, 1999).Challenges by pathogens cannot be an exception to this rule. Forexample, product of the Arabidopsis disease resistance geneRPM1 degrades rapidly in infected cells (Boyes et al., 1998).Although it has yet to be documented, it is reasonable to assumethat all the pathogen-associated molecules (effector molecules),will be subjected to proteolysis within the host cell as foreign,abnormal or misfolded proteins are removed by the degradationmachinery of the cell (Kirshner, 1999).

Intracellular proteolysis is selective, fast, and requires energyto maintain the proteolytic enzymes and to deliver the substratefor degradation (Vierstra, 1996). In addition, it is irreversible anda failure in the process has been reported to lead to diseasessuch as Huntington’s and Parkinson’s in humans (Kirshner, 1999).The degradation machinery of the cell has lysosomal and non-lysosomal components. Although the interrelationship betweenthese components has been reported, it is the non-lysosomal,ubiquitin/26S proteosome-dependent proteolytic pathway whichhas emerged as a powerful regulatory mechanism in a widerange of cellular processes (Ciechanover, 1998; Hochstrasser,1996). Ubiquitin-mediated proteolysis has been widely con-served across the eukaryotic kingdoms including yeast, insects,plants and mammals (Deshaies, 1997; Ingvarsden and Veierskov,2001; Tang et al., 1997). In this pathway, a protein targeted fordegradation goes through two distinct processes: firstly, a spe-cific recognition step which employs ubiquitination machinery;and secondly, a nonspecific degradation step involving theproteosome.

In the ubiquitination machinery, a small 76-amino acid poly-peptide, either ubiquitin, or a ubiquitin-like protein such as SUMO(small ubiquitin-like modifier) and RUB (related to ubiquitin; termed‘ubiquitions’) is used for tagging the protein to be degraded (del

290 M. TÖR et al.

Pozo and Estelle, 1999; Vierstra and Callis, 1999). The ubiquitinis first activated by the ubiquitin-activating enzyme (E1) and sub-sequently transferred to a member of the ubiquitin conjugatingenzymes (E2). At the final stage, the ubiquitin is bound to the tar-get protein either directly by E2 itself or with the help of anotherenzyme complex ubiquitin-ligase (E3) (Hershko and Ciechanover,1998; Wilkinson, 1995). While the sole function of the E1 and E2enzymes is to bring the charged ubiquitin near to the substrate(target protein), E3 plays a major role in determining the substratespecificity (for a general review, see Vierstra, 1996). Once the ubi-quitin molecules are linked together in chains to a protein (theprocess known as ubiquitination), the protein may undergo sev-eral alternative processes: (a) degradation into small peptides bythe 26S proteosome (Voges et al., 1999), (b) cleavage into aminoacids by enzymes such as tripeptidyl peptidase (Tomkinson, 1999),(c) autophagy (Abeliovich and Klionsky, 2001; Mizushima et al.,1998), (d) de-ubiquitination by enzymes (Zhu et al., 1996), and(e) be maintained as a stable conjugate within the cell (Davies,2001; Voges et al., 1999).

Ubiquitin ligases (E3) seem to be the ultimate arbiters of intra-cellular proteolysis. So far, several simple and complex types ofE3s have been reported in eukaryotes including plants (Callis andVierstra, 2000). For example, HECT-domain proteins form onegroup of simple ubiquitin ligases show homology at the C-terminal region to E6-AP (E6 is an oncoprotein produced by pap-illomaviruses in human and E6-AP is E6 Associated protein) whichfunctions as ubiquitin ligase to p53 (a tumour suppresser geneoriginally found in humans) (Bates and Vierstra, 1999; Scheffneret al., 1995). RING-H2 (also known as RING-finger, really inter-esting new gene) E3 ligases form another group of which COP1(constitutive photomorphogenesis) is a member (Suzuki et al.,2002). Two examples of complex E3 ubiquitin ligase enzymes arethe SKP1-Cullin-F-box protein (termed SCF-complex, del Pozoand Estelle, 2000) and the anaphase-promoting complex (APC;King et al., 1996; Peters, 1998).

The involvement of ubiquitin-mediated proteolysis in manyaspects of plant development and physiology has been docu-mented (reviewed by Ellis et al., 2002; Vierstra, 1996). Differentsignalling pathways in plants, including hormone response(Gray et al., 2001; Schwechheimer et al., 2001; Seeger et al.,2001), circadian rhythms (Somers et al., 2000), flower develop-ment (Nelson et al., 2000), senescence (Woo et al., 2001) andphotomorphogenesis (Clough et al., 1999), have been shown tobe governed by proteolysis.

The SCF–complex is of interest in the context of disease resist-ance in plants. It is composed of four primary subunits, cullin1/Cdc53, Rbx1/Roc1/Hrt1, SKP1 and an F-box protein (Gagne et al.,2002). While cullin, Rbx1 and SKP1 form the core ligase activity,with Rbx1 recruiting the E2 enzyme carrying an activated ubiqui-tin molecule to the target protein (Gagne et al., 2002), the F-boxprotein (a conserved 40–50 amino acid motif named after the

first such protein, human cyclin, identified) has been shown tofunction as a receptor for the targets (Bai et al., 1996; Pattenet al., 1998; Skowyra et al., 1997). In some cases, phosphoryla-tion of the target proteins seems to be a prerequisite for recogni-tion by the F-box protein (del Pozo and Estelle, 2000). This maybe due to the fact that phosphorylation changes the affinity of thetarget protein for docking ubiquitins. Therefore, it is reasonable toassume that in such cases, regulation is achieved at the level ofmodification of the target proteins (Kirshner, 1999).

The tremendous proteolytic capability of plants is evident fromArabidopsis, which contains more than 50 ubiquitin proteins,more than 150 RING-finger proteins, at least four proteins withHECT domains, 12 cullin-like, 19 SKP-like, and 694 F-box proteins(Gagne et al., 2002). In addition, the existence of a vast array ofkinases (Swiderski and Innes, 2001) that may be involved inphosphorylating proteins could add further to the complexity ofthe ubiquitin-mediated proteolysis.

INVOLVEMENT OF PROTEOLYSIS IN THE PLANT DEFENCE RESPONSE

Several genes have recently been characterized which suggestthat ubiquitin-mediated protein degradation may also act as aregulatory mechanism in the plant defence response. The Arabi-dopsis mutant coi1 (Coronatine insensitive; Feys et al., 1994)provided an early clue that proteolysis may be involved in theplant defence response. Mutation of this gene has been associ-ated with male sterility, reduced jasmonic acid (JA)-inducedwound responses and reduced defence against some necro-trophic fungal and bacterial pathogens (Thomma et al., 1998)and insect pests (McConn et al., 1997). The COI1 gene has anF-box and 16 LRR motifs (Fig. 1A; Xie et al., 1998). It has recentlybeen shown that COI1 interacts with Arabidopsis CUL1, RBX1and ASK1 or ASK2, two of the SKP-like proteins (Devoto et al.,2002; Xu et al., 2002). It has also been demonstrated that sup-pression of RBX1 leads to a phenotype similar to that of COI1 (Xuet al., 2002).

Similarly, the Arabidopsis SON1 (suppressor of nim1-1) genealso encodes an F-box protein and is involved in the regulationof the defence response (Kim and Delaney, 2002). However, thisprotein does not have C-terminal protein-interacting domainssuch as LRR, WD-40, or the Kelch region as demonstrated byGagne et al. (2002) for other F-box proteins. In addition, unlikeCOI1, mutation in this gene shows enhanced resistance to bac-terial and downy mildew pathogens (Kim and Delaney, 2002).This finding contrasts with the expectation that the mutation inan F-box protein should lead to the loss of resistance as has beenshown with other components of the SCF-complex (see below).However, Kim and Delaney (2002) have attributed this finding tothat the SCF–complex could have a negative or positive effect ondefence response.

Proteolysis in defence signalling 291

Another plausible link of defence to proteolysis came fromthe cloning of the barley RAR1 gene (Shirasu et al., 1999). Deter-mination of the domains of the RAR1 protein (Fig. 1B) suggestedthat it might interact with the SGT1 protein, which has beenshown in yeast to associate with the kinetochore and the SCF-complex by interacting with the SKP1 (Kitagawa et al., 1999).Azevedo et al. (2002) used antibodies raised against barleySGT1 and detected both SKP1 and CUL1 homologues within thebarley SGT1 immunoprecipitates. Interestingly, they have alsodetected several subunits of the COP9 signalosome, which isclosely linked to the 26S proteosome (Schwechheimer et al.,2001, 2002).

Arabidopsis has two homologues of the SGT1 gene, desig-nated AtSGT1a and AtSGT1b. Mutation in AtSGT1b abolishesresistance to at least seven different Peronospora parasitica iso-lates, each of which is recognized by a different R gene (Austinet al., 2002; Tör et al., 2002). In these examples, AtSGT1a doesnot compensate the loss of AtSGT1b function. However, the lossof AtSGT1b function does not affect the resistance to bacterialpathogens or resistance to downy mildew which is mediated bygenes such as RPP1, RPP8 (Austin et al., 2002) and RPP27 (Töret al., 2002).

Both AtSGT1a and AtSGT1b have three tetratrico-peptiderepeats (TPR) at the amino terminus, followed by a bipartite CS(CHORD containing proteins and Sgt, Shirasu et al., 1999)domain and an SGT1 specific domain at the carboxyl terminus

(Azevedo et al., 2002; Tör et al., 2002; Fig. 1C). The SGT1-specificdomain of SGT1 proteins has been conserved across thekingdom (Fig. 2).

The CHORD-II domain of the Arabidopsis RAR1 protein inter-acts with both AtSGT1a and AtSGT1b proteins through the CSdomain (Azevedo et al., 2002; Peart and Shirasu, 2002). In addi-tion, Azevedo et al. (2002) have successfully complemented thetemperature sensitive yeast sgt1 mutants with both the AtSGT1aand AtSGT1b genes, indicating that the functions of the SGT1genes have been conserved between plants and yeast.

Specific R gene mediated pathways have different require-ments for RAR1 and SGT1b. For example, mutation in RAR1does not affect RPP2-, RPP6- and RPP7-mediated resistance. Onthe other hand, bacterial resistance genes RPM1, RPS4 and RPS5,as well as some downy mildew resistance genes RPP1 and RPP27do not require SGT1b. In these examples, AtSGT1a does notcompensate the loss of AtSGT1b function. Interestingly, theRPP8-mediated resistance requires neither RAR1 nor AtSGT1b(Austin et al., 2002; Dodds and Schwechheimer, 2002; Tör et al.,2002).

Tobacco homologues of RAR1 (NbRAR1) and SGT1 (NbSGT1)have been isolated and shown to be required for the function ofN, a member of the TIR-NB-LRR class of R-genes that confersresistance to Tobacco mosaic virus (TMV; Liu et al., 2002a,b;Peart et al., 2002). Similar to barley and Arabidopsis studies onSGT1 interaction, NbRAR1 has been shown to physically associate

Fig. 1 Domain structures of Arabidopsis COI1, RAR1 and SGT1b proteins.

Fig. 2 The SGTS domain is conserved in SGT1 proteins across Kingdoms. SGT-specific domains of SGT1 protein sequences were aligned by Vector NTI. At, Arabidopsis thaliana; Hv, barley; Hs, human; Nc, Neurospora crassa; Os, rice; Sc, Saccharomyces cerevisia. Identical residues are displayed in black boxes. GENBANK accession numbers; AtSGT1a, CAA23023; AtSGT1b, CAB51410; HvSGT1, AAL33610; HsSGT1, NP_006695; NcSGT1, CAB88589; Os, AAF18438; Sc, S66940.

292 M. TÖR et al.

with NbSGT1 and NbSKP1 (Liu et al., 2002b). Liu et al. (2002b)reported that virus-induced gene silencing (VIGS) of NbSKP1 andCOP9 subunits suppressed N-mediated resistance to TMV intobacco. The VIGS method has also been used to show thatNbSGT1 is required for Rx- and Pto-mediated resistance inNicotiana benthamiana (Peart et al., 2002). Similarly, Peart et al.(2002) have observed that silencing NbSGT1 in N. benthamianacaused a loss of non-host resistance to bacterial pathogens.

A summary model for the involvement of proteolysis in theR-gene mediated defence signalling pathway is presented in Fig. 3.

FUTURE PROSPECTS AND CONCLUDING REMARKS

Cloning of several defence regulatory genes including COI1,RAR1 and AtSGT1b, and post-transcriptional silencing of other

Fig. 3 A model describing the involvement of proteolysis in R gene mediated defence response. Plants are frequently challenged by potential parasites that attempt to establish an intimate relationship with the host by either adhering to the plant cell (e.g. bacterial pathogens), penetrating the cell with their specialized structures (fungal pathogens), or entering entirely into the cell (e.g. viral pathogens) (1). Effector molecules (EM) of the pathogen enter the host cell (in the case of viral pathogens, domains of viral proteins function as effector molecules) (2). Some of these effector molecules may be recognized either directly by an R gene product acting as a receptor or via an interactor (I) gene product (3). This recognition then activates the downstream signalling defence response (4). In some cases, genes such as EDS1, NDR1 and RAR1 could relay this signal via SGT1 to the SCF-complex (comprised of Cullin, RBX1, SKP1 and F-box proteins). This complex may then recruit a target protein, which may act as a repressor of defence, with the help of kinase. This target protein may subsequently be ubiquitinated by the SCF- complex with the help of ubiquitin (Ub) activating (E1) and ubiquitin conjugating (E2) enzymes. The target protein may then be degraded by the 26S proteosome (5), leading to the expression of downstream genes that mediate disease resistance. However, in other cases, defence may be initiated without the ubiquitin—mediated proteolysis (6).

genes that are involved in the protein degradation pathway suchas NbSKP1, and subunits of COP9 signalosome provided evi-dence that proteolysis may play an important regulatory role inthe defence signalling pathway. Although the research is still inits infancy, these isolated genes can be used as a starting pointto uncover key components of protein degradation. For example,obvious candidates are the Arabidopsis SKP1 homologue thatinteracts with SGT1a and SGT1b, and the specific F-box proteinsthat interact with AtSGT1b and SKP1 complex.

The classification of Arabidopsis F-box proteins according totheir interaction with various Arabidopsis SKP1-like genes byGagne et al. (2002) should provide a useful guide to identify theparticular F-box protein for a given resistance response. Similarly,target proteins or substrates for proteolysis need to be identified.These proteins may be involved in de-repressing the defencemechanism. Mutants of the identified F-box and target proteinsshould be isolated so that they can be challenged with pathogensto uncover their role in R gene mediated defence responses.

Rapid degradation of the disease resistance gene RPM1 duringthe hypersensitive response (Boyes et al., 1998) was one of theearliest hints of the increased proteolytic activity in the defenceresponse. It will be interesting to find out whether this is due toubiquitin /proteosome dependent proteolysis. Since different R genesdisplay different requirements for RAR1 and SGT1b, investigatingthe different domains of RAR1 and SGT1 proteins will be helpfulin uncovering the entire role of these genes. For example, sinceonly the CHORD-II domain of RAR1 interacts with SGT1b, identi-fying CHORD-I interactors of RAR1 would contribute to an under-standing of the RAR1 requirements by specific R genes. In addition,uncovering the role of SGT1a in defence signalling is needed.

In the long term, the degradation of several functional R-genes,interactors and effector molecules by the plant degradationmachinery should be demonstrated. The search will then moveto the phosphorylation of target proteins.

ACKNOWLEDGEMENTS

We would like to thank Drs Alison Woods-Tör and Carol Jenner forcritically reading the manuscript and Lauren Warner for her helpwith the diagrams. Work on SGT1b in our laboratory has beensupported by grants from BBSRC and DEFRA.

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The role of proteolysis in R gene mediated defence in plants

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