14-3-3proteins and the response toabiotic andbiotic stress

Plant Molecular Biology1031:1031–1039,2002.

?2002Kluwer Academic Publishers.Printed in the Netherlands.

1031 14-3-3proteins and the response to abiotic and biotic stress

Michael R.Roberts1,?,Julio Salinas2and David B.Collinge3

1Department of Biological Sciences,IENS,Lancaster University,Lancaster LA14YQ,UK(?author for correspon-dence;e-mail m.r.roberts@https://www.360docs.net/doc/0e6556909.html,);2Departamento de Biotecnolog′i a,Instituto Nacional de Investigaci′o n y Tecnolog′ia Agraria y Alimentaria(INIA),Carretera de la Coru?a,Km.7,28040Madrid,Spain;3Section for Plant Pathology,Department of Plant Biology,Royal Veterinary and Agricultural University,Thorvaldsensvej40, 1871Frederiksberg C,Copenhagen,Denmark

Received6September2001;accepted in revised form7June2002

Key words:14-3-3protein,abiotic stress,biotic stress,defence responses,plant pathogen,signalling

Abstract

14-3-3proteins function as regulators of a wide range of target proteins in all eukaryotes by effecting direct protein-protein interactions.Primarily,interactions between14-3-3proteins and their targets are mediated by phosphorylation at speci?c sites on the target protein.Hence,interactions with14-3-3s are subject to environmental control through signalling pathways which impact on14-3-3binding sites.Because14-3-3proteins regulate the activities of many proteins involved in signal transduction,there are multiple levels at which14-3-3proteins may play roles in stress responses in higher plants.In this article,we review evidence which implicates14-3-3proteins in responses to environmental,metabolic and nutritional stresses,as well as in defence responses to wounding and pathogen attack.This evidence includes stress-inducible changes in14-3-3gene expression, interactions between14-3-3proteins and signalling proteins and interactions between14-3-3proteins and proteins with defensive functions.

Abbreviations:ABA,abscisic acid;CDPK,calcium-dependent protein kinase/calmodulin domain protein kinase; FC,fusicoccin;H+-ATPase,plasma membrane proton pumping ATPase;HR,hypersensitive response;NR,nitrate reductase

Introduction

Plants growing in nature constantly sense their en-vironment and adapt to changes by using a range of biochemical and molecular mechanisms.They ex-hibit both long-term responses to the physical en-vironment in the form of modi?ed growth patterns and metabolism,and short-term defence responses to counter immediate threats such as pathogen attack.In each case,the appropriate response is the result of the perception of external information and the relay-ing of this information between and within plant cells. The molecular and genetic basis for stress response signalling in plants has been the subject of intensive research over the past decade or so,with major areas for focus including responses to light,temperature, water and salt stress,atmospheric pollutants such as ozone,wounding,herbivory and pathogen infection. Recent reviews of these areas can be found in Bray (1997),de Bruxelles and Roberts(2001),Collinge et al.(2001),Dangl and Jones(2001),Hasegawa et al. (2000),Mackerness(2000),Mullineaux et al.(2000), Srivastava(1999)and Thomashow(1999).

Intriguingly,though these stresses tend to elicit a speci?c?nal response,many of the signalling inter-mediates,such as plant hormones,reactive oxygen species,calcium,etc.,are common to many path-ways.In fact this is not entirely surprising,since the plant must integrate its response to a particular stress within the context of other environmental pressures.

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Furthermore,some disparate physical environmental conditions,such as drought,osmotic stress(high salin-ity)and cold,have overlapping physiological effects on the plant.The use of overlapping signalling path-ways allows for modulation of responses via cross-talk between pathways at points of intersection(Knight and Knight,2001).

Because of their role in protein-protein interac-tions,the involvement of14-3-3proteins in plant stress responses has often been suggested,though few well de?ned examples exist.Such roles have been in-ferred from the potential of14-3-3s to regulate both signalling pathways and those proteins involved in the ?nal response.14-3-3proteins could operate in stress responses via one of several mechanisms for which precedents already exist.Most simply,they might di-rectly regulate the activity of individual proteins with either signalling or defensive functions.Alternatively, they could play roles in the formation of multi-protein complexes as a result of the ability of14-3-3s to act as adapters.They could also potentially regulate the expression of stress-inducible genes by regulat-ing the activity and or localisation of transcription factors(see Muslin and Xing,2000).Evidence from animal systems supports the concept of14-3-3pro-teins as regulators of stress responses.For example, the14-3-3σisoform is essential for cell cycle arrest to allow DNA repair after radiation-induced damage, (e.g.Chan et al.,1999)and changes in expression of the14-3-3σgene are associated with several human cancers(Ferguson et al.,2000;Iwata et al.,2000; Suzuki et al.,2000).14-3-3proteins are also known to have an anti-apoptotic function(reviewed by Fu et al., 2000).One important physiological consequence of this is that they are required to protect the heart from pressure overload(Xing et al.,2000).

Plant and animal14-3-3proteins are signi?cantly similar in amino acid sequence(commonly around 50%identity),especially in the conserved ligand-binding groove.Target sequences for14-3-3binding are equally well conserved,such that mammalian Raf-1and plant nitrate reductase for example,carry essentially identical14-3-3binding sites.It is there-fore reasonable to suggest that the biological functions of14-3-3protein-protein interactions might also be conserved between plants and animals.In the case of stress responses,support for such roles comes from observations of changes in14-3-3gene expression during stress responses and from the detection of inter-actions between14-3-3s and proteins with signalling or protective functions.

In this review,we summarise the evidence that plant14-3-3proteins are involved in biotic and abi-otic stress responses and suggest possible targets and mechanisms of action.

Targets for14-3-3proteins in stress regulation

A number of targets for14-3-3proteins have been characterized from eukaryotic systems.These include the motifs RSXpSXP(Muslin et al.,1996),RXSX-pSXP(Andrews et al.,1998),RXF/YpSXP(Yaffe et al.,1997)and YpTV(Fuglsang et al.,1999),where pS and pT represent phosphoserine and phosphothre-onine respectively.Finnie et al.(1999)used the GCG program FINDPATTERNS(Wisconsin Package,Ver-sion8.1-UNIX,Genetics Computer Group)to search for the?rst three of these motifs in SwissProt(Release 34.0:12/96)and NewSwissProt(3/3/99).This study revealed a number of potential target proteins with known or conceivable roles in stress responses and defence in addition to those proteins for which such an interactions have been demonstrated biochemically. The many potential targets for14-3-3proteins identi-?ed include both metabolic targets(e.g.,an enzyme of ethylene biosynthesis,a cytochrome P450,glutathione reductase)and more obvious regulatory targets(e.g., several protein kinases,phosphatase,phospholipase,a leucine zipper).Since14-3-3binding requires phos-phorylation of the target sequence,regulatory interac-tions are likely in many cases to be mediated by the activation of signalling pathways which result in pro-tein phosphorylation.Many stress-responsive protein kinases are known,and a challenge for the future will be to recognize which of their substrates are14-3-3 target sequences.

14-3-3proteins and abiotic stress

For the purposes of this review,we will treat the term abiotic stress in its broadest sense.Hence we will consider insults as severe as gross tissue loss or dam-age,through stresses like?uctuations in temperature or nutrient levels,down to transient metabolic per-turbations caused by,for example,changes in light quality.14-3-3proteins were?rst implicated in abiotic stress responses when their mRNAs were identi?ed in a number of screens for stress-regulated gene ex-pression.Since then,interactions with several14-3-3

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target proteins which are also associated with stress re-sponses have been identi?ed,strengthening the appar-ent link between14-3-3function and stress responses. Below,we discuss these examples in the context of speci?c abiotic and biotic challenges. Environmental stress

One of the?rst plant14-3-3genes isolated was iden-ti?ed as a transcript that accumulated in callus and seedlings of rice when exposed to high NaCl con-centration or low temperature(Kidou et al.,1993). Additional evidence connecting14-3-3to salt stress arose from a tobacco gene encoding a14-3-3pro-tein(T14-3-3)that was reported to be down-regulated during salinity adaptation(Chen et al.,1994).Interest-ingly,the levels of the corresponding transcripts were unaffected by direct treatment with NaCl,ABA or eth-ylene.The reduction of T14-3-3mRNA accumulation in salt-adapted tobacco cells was suggested to be part of an adjustment in the perception of the salt stress (Chen et al.,1994).Two genes from Arabidopsis en-coding14-3-3proteins have also been described to be regulated in response to low temperature(Jarillo et al., 1994).The expression of these genes,formerly named RCI1and RCI2(Jarillo et al.,1994)but renamed RCI1A and RCI1B,respectively(Abarca et al.,1999), is induced by cold in a development-independent way, but,in contrast to most cold-inducible genes charac-terized in plants,is not responsive to ABA,NaCl or water stress.The kinetics of RCI1A and RCI1B mRNA accumulation by low temperature is correlated with the increase in freezing tolerance that occurs during the cold acclimation process in Arabidopsis(Jarillo et al.,1994;Abarca et al.,1999),suggesting that these genes may have a role in this adaptive process. Experiments with transgenic plants containing RCI1A-promoter::reporter-gene fusions have shown that low temperature regulates the accumulation of RCI1A tran-scripts at the transcriptional level(López-Cobollo, Fernández-Calvin and Salinas,manuscript in prepa-ration).In Arabidopsis,there are at least10and as many as13genes that are known to be expressed and encode14-3-3isoforms(Wu et al.,1997;DeLille et al.,2001;Sehnke et al.,this issue).Of these genes, only RCI1A and RCI1B,which encode isoforms?and ?,respectively,seem to be induced in response to low temperature(Jarillo,Capel,Martínez-Zapater and Salinas,unpublished results)indicating that different isoforms are differentially regulated and can account for speci?c functions.Unfortunately,the functions of the14-3-3proteins described above,as well as their role in plant responses to cold and salt stresses,still remain unknown.

A redistribution of14-3-3proteins has been de-scribed in sugar beet cells exposed to cold or os-motic stress(Chelysheva et al.,1999;Babakov et al., 2000),constituting another demonstration that they have a role in abiotic stress responses.In both cases, there is an increase in14-3-3protein levels in the plasma membrane fraction,associated with an in-creased amount of H+-ATPase/14-3-3complexes and enhanced ATPase activity.It is now clear that14-3-3 proteins activate the H+-ATPase by interaction with its regulatory C-terminus in a phosphorylation-dependent manner(see Bunney et al.,this issue).Thus,it ap-pears that under low temperature and high osmolarity conditions,14-3-3proteins interact with the autoin-hibitory C-terminal domain of the plasma membrane H+-ATPase activating the proton pump that is crucial for the protective system plants have developed against external adverse in?uences(Serrano,1989).Although not strictly a stress response,the blue-light activa-tion of the plasma membrane H+-ATPase in stomatal guard cells is worthy of mention as a well charac-terised example of the regulation of plant physiology by14-3-3proteins.Work by Kinoshita and Shimazaki (1999)demonstrated blue-light-dependent phosphory-lation of the C-terminus of the H+-ATPase,resulting in14-3-3protein binding and proton pump activa-tion.The resulting membrane hyperpolarisation leads to K+in?ux,water uptake and thus stomatal opening. Interestingly,subsequent work has indicated that spe-ci?c14-3-3isoforms are responsible for this response in Vicia faba guard cells(Emi et al.,2001).

Outward-rectifying K+channels,which have been found in all plant cell types tested so far,constitute a second class of plasma membrane ion transport pro-tein potentially involved in abiotic stress responses that have been identi?ed as14-3-3binding proteins.In whole-cell patch-clamp experiments,the tomato14-3-3isoforms TFT4and TFT7were shown to modulate outward-rectifying K+channels in tomato suspension cells(Booij et al.,1999).As a consequence,the steady-state outward K+current increases two-fold in a cytoplasmic-ATP independent way.An essen-tial function of outward-rectifying K+channels is to reset the membrane potential,which is important, for instance,upon depolarisation of the membrane potential caused by changes in the external environ-ment.In fact,as we will discuss below,cold,drought and other environmental stresses open calcium chan-

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nels at the plasma membrane(Knight et al.,1991; Knight et al.,1997).The resulting inward calcium cur-rent depolarises the plasma membrane(Minorsky and Spanswick,1989)while also increasing cytoplasmic calcium concentration which furthers the depolarisa-tion(Lewis et al.,1997).The report by Booij et al. (1999)implies that in addition to H+-ATPase activa-tion,increases in cellular14-3-3concentration pro-duced in response to several abiotic stresses can also alter K+transport at the plasma membrane,restoring the membrane potential after stress situations.

As well as ion transport proteins,other factors with potential roles in abiotic stress responses have also been identi?ed as targets of14-3-3proteins.A calcium-dependent protein kinase(CDPK)isoform from Arabidopsis,CPK-1,has been reported to be bound by Arabidopsis14-3-3isoformsω,ψand , its calcium-dependent activity being stimulated almost two-fold(Camoni et al.,1998).CDPKs constitute a unique family of plant kinases which are de?ned by a C-terminal calmodulin-like regulatory domain and have been shown to be involved in stress signalling. In fact,calcium is an important second messenger in multiple signal transduction pathways in plants, and transient increases in cytosolic concentration have been shown to occur in response to different abiotic stresses,including low temperature,drought or high salt(Knight et al.,1991,1997).To elicit responses, calcium transients need to be ef?ciently linked to a protein phosphorylation signalling cascade,which is usually directly done via CDPKs(Roberts and Har-mon,1992).The results obtained by Camoni and co-workers with CPK-1raise the possibility that increases in cellular14-3-3proteins such as may result from inducible gene expression under different adverse en-vironmental conditions can contribute to the activation of CDPK signal transduction pathways in plants and induce the corresponding adaptive responses.

Protein-protein interactions between14-3-3and two further Arabidopsis proteins involved in de-fence have been identi?ed using the yeast two-hybrid system(Zhang et al.,1997a;1997b).One of these,caffeic acid/5-hydroxyferulic acid O-methyltransferase(OMT1)is an enzyme of phenyl-propanoid metabolism.Products of this biosynthetic pathway are implicated in defense against wounding, pathogens and ultra violet light(Dixon and Paiva, 1995).The second two-hybrid interacting protein identi?ed was an ascorbate peroxidase,an enzyme involved in protection against oxidative stress.More recently,Bunney et al.(2001)demonstrated that14-3-3proteins regulate the activity of mitochondrial and chloroplast F0F1ATP synthases via an interaction with the F1β-subunit.Phosphorylation-dependent binding of14-3-3to these ATP synthase complexes results in inhibition of activity.The authors suggest that14-3-3s provide a mechanism for regulation of ATP synthase activity in response to light/dark transitions,anoxia in roots and changes in nutrient supply.

Metabolism/nutrient stress

Plant cell metabolism is an area where there is more comprehensive evidence for regulation by14-3-3pro-teins,including responses to environmental condi-tions.Powerful af?nity puri?cation techniques de-veloped by MacKintosh and co-workers,coupled to mass spectrometry,allowed the classi?cation of nu-merous14-3-3interacting proteins from plant cells (Moorhead et al.,1999;Cotelle et al.,2000).Inter-estingly,many of these proteins were identi?ed as key enzymes of primary metabolism,such as nitrate re-ductase(NR),sucrose phosphate synthase,glutamine synthase and glyceraldehyde-3-phosphate dehydroge-nase,along with regulators of these enzymes,such as a CDPK.The interaction between14-3-3and NR is well characterised,with14-3-3binding to and inactivating NR in a light-dependent manner(see Huber et al., this issue,for further details).Signi?cantly,the under-standing of the14-3-3/NR interaction and those with other proteins was extended by recent studies of the regulation of these interactions under nutrient stress (Cotelle et al.,2000).Using14-3-3overlay assays and immunodetection of individual target proteins,it was demonstrated that following sugar starvation,the ability of target proteins to bind14-3-3was lost and that as a consequence,they were subject to targeted proteolysis.The role of14-3-3protein interactions with metabolic enzymes thus appears to be to main-tain those enzymes in a particular(active)state in the healthy cell.The response to sugar starvation appears to be mediated by changes in the ATP/AMP ratio, which is an indicator of the energy status of the cell. 14-3-3proteins therefore appear to have the ability to co-ordinate cells metabolic responses to nutrient stress by regulating the activity of key enzymes. Herbivory and wound stress

Tissue damage caused by mechanical wounding or in-sect chewing is another stress which results in changes in14-3-3gene expression.14-3-3transcripts from

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Figure1.Northern blot analysis demonstrating differential expres-sion of tomato14-3-3genes during abiotic and biotic stress re-sponses.A.Response to wounding.RNA was extracted at the times shown after crushing wounds to leaves and blots probed to show mRNA levels for isoforms TFT4and TFT10,which are induced and repressed respectively.B.Pathogen resistance response.RNA was extracted from leaves of plants carrying the Cf9resistance gene at the times shown after elicitation with Cladosporium fulvum Avr9 peptide.Blots were probed to show mRNA levels of isoforms TFT1, TFT4and TFT6(Roberts and Bowles,1999).Expression of TFT4 is maximal early in the response,coinciding with the end of the oxidative burst(OX).TFT6is expressed during the middle of the time course,coinciding with the period of ethylene and salicylic acid biosynthesis(C2H4&SA).TFT1is expressed maximally to-wards the end of the time course,coinciding with the appearance of a visible hypersensitive response(HR).

white spruce and hybrid poplar trees have been iden-ti?ed which are up-regulated after wounding or by treatment with chitosan or jasmonic acid,elicitors of wound-responsive gene expression(Lapointe et al., 2001a,b).In tomato,different members of the14-3-3 gene family display differing responses to wound-ing.While most genes are unaffected by mechanical wounding,some genes are down-regulated whilst oth-ers show increased mRNA levels(e.g.Figure1;G.de Bruxelles and M.R.Roberts,unpublished data).These observations suggest possible roles for speci?c14-3-3protein isoforms in wound response signalling or the regulation of defensive proteins.

As described above,the regulation of the plasma membrane H+-ATPase is one area which seems to be a target for regulation by14-3-3proteins dur-ing stress.In relation to wound stress,some years ago it was found that application of fusicoccin(FC) to tomato plants ef?ciently inhibited the induction of wound responsive gene expression(Doherty and Bowles,1990).It is now known that as a result of its ability to activate the H+-ATPase and hyper-polarize the plasma membrane,FC inhibits wound gene expression in response to wounding,oligosac-charides and the peptide systemin,which all cause plasma membrane depolarisation in the absence of FC(Messiaen and Van Cutsem,1994;Schaller and Oecking,1999;Schaller and Frasson,2001; D.J. Bowles,personal communication).Furthermore,in-hibition of the H+-ATPase by pharmacological agents induces the expression of wound-responsive genes in the absence of wounding or elicitors(Schaller and Oecking,1999;Schaller and Frasson,2001).On the basis of these data,the authors suggested that the H+-ATPase might be an important early component of wound signalling.However,the same logic cannot be applied to pathogen response signalling,since FC ac-tually enhances the expression of pathogen-responsive genes upon elicitor treatments which also normally result in plasma membrane depolarisation(Roberts and Bowles,1999),and is able to induce PR gene expression when applied alone(Roberts and Bowles, 1999;Schaller and Oecking,1999).Hence two dif-ferent responses which are both associated with initial plasma membrane depolarisations are affected by FC in opposing ways.

Plant-pathogen interactions

The defences of plants against pathogens are particu-larly complex since they do not re?ect an adaptation to physical stress alone but the result of aeons of co-evolution of many diverse types of pathogenic or-ganisms with their hosts.The nature and regulation of these stresses has been reviewed regularly(see, for example,Collinge et al.(2001)and Dangl and Jones(2001)for recent reviews).One of the earli-est reports of a14-3-3protein in a plant resulted from a subtractive cDNA library screen for transcripts accumulating in barley leaves after inoculation with the non-host powdery mildew fungus,Blumeria(syn.

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Erysiphe)graminis f.sp.tritici(Smedegaard-Petersen et al.,1992;Brandt et al.,1992;Thordal-Christensen et al.,1992).The transcript for the14-3-3protein was seen to accumulate early,though weakly,in the de-fence response concomitantly with a number of other defence-related transcripts(encoding pathogenesis-related proteins and peroxidase among others).In contrast to other interactions,no difference in the ac-cumulation of14-3-3transcripts was noted between compatible and incompatible interactions with the bar-ley powdery mildew fungus(Blumeria graminis f.sp hordei).This suggests that the transcript accumula-tion is associated with the penetration stage.Transcript and protein accumulation was most marked in the epidermis of the infected leaves,and levels in the mes-ophyll were essentially unaffected(Gregersen et al., 1997;Finnie et al.2002).Several observations sug-gest that14-3-3proteins have a role in this defence response through regulating the proton pump(H+-ATPase)to activate the hypersensitive response:the pH drops under epidermal tissue undergoing a hy-persensitive response(HR),the HR is stimulated by fusicoccin and treatment with acidic buffer and is sup-pressed by more alkaline buffer(Zhou et al.,2000). Furthermore,fusicoccin-binding activity of an epi-dermal microsomal fraction increases upon pathogen attack,and a100kDa protein which co-migrates with the H+-ATPase accumulates and binds14-3-3proteins (Finnie et al.,2002).

A different functional role for14-3-3proteins during plant pathogen resistance reponses was indi-cated following characterisation of the Arabidopsis AKR2gene(Yan et al.,2002).The AKR2protein was initially identi?ed in a yeast two-hybrid screen as a14-3-3-interacting protein.Subsequently,an-tisense expression in transgenic plants showed that AKR2negatively regulates a number of pathogen re-sistance responses.Antisense AKR2plants developed spontaneous HR-like lesions and exhibited increased H2O2generation and expression of defence-related transcripts.These phenomena were associated with in-creased resistance to a bacterial pathogen.As yet,the role of the AKR2/14-3-3interaction is not understood.

As compared with the situation in the bar-ley/powdery mildew interaction,a more complex pat-tern of14-3-3gene expression has been observed in a model pathogen resistance response in tomato. Roberts and Bowles(1999)examined the expression of ten14-3-3genes from tomato after challenge of re-sistant or susceptible plants with the avirulence elicitor Avr9from the fungal pathogen Cladosporium fulvum.Of the ten genes examined,three showed increased expression during an incompatible response elicited by Avr9,each with a different temporal pro?le of transcript accumulation(Roberts and Bowles,1999). These results are summarized in Figure1,where the temporal pattern of mRNA levels are related to the ma-jor events occurring as part of the resistance response. The patterns of14-3-3gene expression in the intact pathosystem have not yet been assessed.As yet,there is no evidence for differential expression of barley14-3-3isoforms in response to powdery mildew infection (C.H.Andersen,C.S.Finnie,P.L.Gregersen and D.B. Collinge,unpublished).

The accumulation of a14-3-3transcript is also in-duced during a race-speci?c HR of soybean(Glycine max)inoculated with Pseudomonas syringae pv. glycinea(Seehaus and Tenhaken,1998)and in cot-ton(Gossypium hirsutum)roots inoculated with the wilt pathogen Verticillium dahliae(Hill et al.,1999). In both studies,14-3-3transcripts were identi?ed along with other defence-related transcripts.In soy-bean,chalcone isomerase,ubiquitin,‘enhancer of rudimentary’(a protein implicated in protein-protein interactions;Gelsthorpe et al.,1997),glucose-6-phosphate dehydrogenase and a putative NBS-LRR resistance gene were identi?ed by differential display and their induction con?rmed by northern blotting in tissue undergoing HR.In cotton,differential screening of a cDNA library yielded ten classes of transcript, and northern analysis con?rmed their accumulation following infection.Some of these(e.g.,phenylala-nine ammonia-lyase and PR-10)represent well-known defence-related genes.Others have not previously been associated with defence pathogen attack but may be induced by pathogen as part of the pathogenicity process(e.g.,glucose-6-phosphate/phosphate translo-cator,dTDP-glucose4-6-dehydratase).As with the barley-powdery mildew study,the amount of tran-script accumulating in these interactions is not greatly elevated compared to uninoculated tissues.This is not unusual for regulatory defence-related transcripts.For instance,the Npr1transcript only accumulates about two-fold in Arabidopsis after pathogen attack,yet a two-fold constitutive elevation of the transcript results in the up-regulation of a number of antimicrobial pro-teins and enhanced resistance to several pathogens (Cao et al.,1998).

Interestingly,a region of chromosome4AL of wheat(Triticum aestivum)containing14-3-3and ox-alate oxidase genes is associated with a quantitative resistance marker(QTL)against two fungal diseases,

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tan spot(Pyrenophora tritici-repentis)and leaf rust (Puccinia recondita f.sp.tritici)(Faris et al.,1999). The signi?cance of this remains unknown.

Our understanding of the signi?cance of14-3-3 proteins in the regulation of the defence response or indeed in the pathogenicity process is still in its in-fancy.It is now clear that14-3-3proteins accumulate in interactions with bacteria and both biotrophic and necrotrophic fungal pathogens.In the case of bacteria, the accumulation has as yet only been observed in an incompatible interaction where a successful defence (as a hypersensitive response has been observed.In the case of fungi,14-3-3proteins accumulate in both susceptible and resistant plants.In the barley-powdery mildew system,it is clear that14-3-3proteins have more than one target and it is highly probable that the H+-ATPase is stimulated in the defence response by 14-3-3proteins.

In the pathogen response as in other stress re-sponses,many targets for14-3-3proteins undoubtedly remain to be identi?ed.Furthermore,it is notable that very few binding motifs have been identi?ed in target proteins from plants compared with work in animal systems.Importantly,the biological signi?cance of the regulation by14-3-3proteins of targets known and as yet unknown remains to be determined in the vast ma-jority of cases.How this will be achieved will prove to be the key challenge for future investigations because of the existence of multiple14-3-3isoforms and the even greater numbers of target proteins present in plant cells at any one time.The study of plants in which individual14-3-3isoforms have been suppressed or over-expressed may be helpful(e.g.see Wilczynski et al.,1998;Sehnke et al.,2001).However,it is more likely that for each individual case,it will be neces-sary to map the precise sites of14-3-3interaction and manipulate these sites in individual target proteins in order to fully understand the functional signi?cance of this ubiquitous class of protein-protein interaction. Acknowledgements

M.R.R.is grateful to the Royal Society and the EC 4th Framework programme for?nancial support for his research on14-3-3proteins.Funding in J.S.’s lab-oratory comes from the Comisión Interministerial de Ciencia y Tecnología,Spain(grant BIO98-0189)and the EC(grant QLK3-CT-2000-00328).14-3-3work in D.B.C.’s laboratory has been supported by the EC4th Framework CRAFTT(Central Role in Adaptation of Fourteen Three Three proteins)project BIO4-CT97-2275DG12-SSMI,several KVL PhD stipends and a Frame Programme grant‘Molecular Whole-Plant Physiology’from the Danish Veterinary and Agricul-tural Research Council,S.J.V.F.

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