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Unravelling R gene-mediated disease resistance pathways in Arabidopsis Blackwell Science, Ltd

JA N E E . PA R KE R * , B A R T J. FE Y S , E R I K A . VA N D E R B I E Z E N , L A U R E N T N O Ë L , N I C O L E A A R T S , M A R K J. A U S T I N , M I G U E L A . B O T E L L A , L O U I S E N. FR O S T , M I C H A E L J. D A N I E L S A N D J O N A T H A N D. G. J O N E S The Sainsbury Laboratory, Norwich Research Park, Colney, Norwich NR4 7UH, UK

SUMMARY Molecular genetic approaches were adopted in the model crucifer, Arabidopsis thaliana, to unravel components of RPP5and RPP1-mediated disease resistance to the oomycete pathogen, Peronospora parasitica. The products of RPP5 and three genes comprising the RPP1 complex locus belong to a major subclass of nucleotide-binding/leucine-rich repeat (NB-LRR) resistance (R) protein that has amino-terminal homology to the cytoplasmic domains of Drosophila and mammalian Toll and interleukin-1 family receptors (the so called ‘TIR’ domain). Similarities in the domain architecture of these proteins and animal regulators of programmed cell death have also been observed. Mutational screens revealed a number of genes that are required for RPP5conditioned resistance. Among these are EDS1 and PAD4. Both EDS1 and PAD4 precede the function of salicylic acid-mediated plant responses. The EDS1 and PAD4 genes were cloned and found to encode proteins with similarity to the catalytic site of eukaryotic lipases, suggesting that they may function by hydrolysing a lipid-based substrate.

I N T RO D U C T I O N Plants have evolved numerous defence systems that enable them to prevent pathogen invasion and the onset of disease. One highly specific recognition system that leads to disease resistance depends upon the expression of corresponding pairs of genes in the plant and the pathogen, known, respectively, as resistance (R ) genes and avirulence (avr ) genes (Baker et al., 1997). This type of resistance is commonly associated with localized plant cell death (the hypersensitive response or HR) and can lead to the elaboration of systemic signals that immunize

*Correspondence: E-mail: [email protected]

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distal parts of the plant to pathogen infection, a process known as systemic acquired resistance (SAR) (Ryals et al., 1996). The precise mechanisms controlling R-avr gene specified disease resistance are poorly understood, although a requirement for a phenolic derivative, salicylic acid (SA), has been demonstrated in several plant–pathogen interactions (Delaney et al., 1994; Mauch-Mani and Slusarenko, 1996). A role for the oxylipin signalling molecule jasmonic acid (JA) has also been implicated in SA-independent plant defence pathways (Bowling et al., 1997; Penninckx et al., 1996; Pieterse et al., 1998). Other studies suggest that the formation of reactive oxygen species, ion flux changes and protein kinase activation are important early events in specific pathogen recognition (Grant and Mansfield, 1999; Lamb and Dixon, 1997). The cloning of R genes from a number of plant species has provided some key insights to molecular mechanisms that underpin plant-pathogen recognition. R genes specifying resistance to several taxonomically unrelated pathogens fall into a limited number of classes based on shared structural motifs (Ellis and Jones, 1998; HammondKosack and Jones, 1997), suggesting that plant responses to different pathogens may have certain common mechanistic features. We have used a molecular genetic approach in the model crucifer, Arabidopsis thaliana, to unravel R gene-mediated pathogen recognition events and disease resistance signalling pathways. Arabidopsis is a natural host to all the major pathogen classes: viruses, bacteria, fungi and nematodes, and in many of these interactions R loci have been identified. Also, the anticipated completion of the ≈ 130 Mb Arabidopsis genome sequence in the year 2000 should permit comprehensive integration of information on different plant stress responses. Here, we describe recent progress in the cloning and structural analysis of RPP genes conferring race-specific resistance to the obligate biotroph, Peronospora parasitica, and in the isolation and characterization of several R gene pathway signalling components. Our studies reveal the existence of distinct R gene-specified pathways that appear to be conditioned by R protein structure rather than pathogen type.

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N B - L R R RE S I S T A N C E P RO T E I N A RC H I T E C T U RE The most prevalent R gene class, found in dicots and monocots, encodes large putatively cytoplasmic proteins that contain a central nucleotide-binding (NB) motif and carboxy-terminal leucine-rich repeats (LRRs). The LRR domain has been shown in several yeast and animal proteins to mediate protein–protein interactions (Kajava, 1998). This feature, together with evidence that the LRR portion of R proteins is the likely pathogen recognition domain (Botella et al., 1998; Ellis et al., 1999; McDowell et al., 1998; Parniske et al., 1997), suggests that

a complex series of molecular events accompany R proteinmediated engagement of a pathogen elicitor and induction of downstream processes. ‘NB-LRR’ type R proteins can be further divided into two structural subclasses that are distinguished by different aminoterminal portions, as shown in Fig. 1. Class I, so called TIR-NBLRR proteins, have sequence similarities in their amino termini to the cytoplasmic effector domains of the Drosophila and mammalian Toll and interleukin-1 (IL-1R) transmembrane receptor family that are involved in conserved animal innate immune responses (Hoffmann et al., 1999; Medzhitov et al., 1997; O’Neill and Green, 1998; Rock et al., 1998). In contrast, the

Fig. 1 Structural relationships between plant R proteins and animal mediators of immunity and programmed cell death. The amino-terminal ‘TIR’ domain in RPP5 and other plant R proteins is similar to the cytoplasmic effector domains of animal IL-1/Toll family of transmembrane receptors. Certain human IL-1 and Toll receptors transduce extracellular signals through homophilic TIR–TIR interactions with an adaptor protein MyD88 and, further, through homophilic Death-Domain (DD) interactions with a protein kinase (IRAK). Protein kinase activation leads to nuclear translocation of the transcription factor, NF-κB that, in turn, induces defence gene expression (Hoffmann et al., 1999; O’Neill and Greene, 1998). The modular structure of plant ‘NB-LRR’ type R proteins is reminiscent of the animal adaptor proteins Ced-4, Apaf-1 and Card-4 that regulate programmed cell death. Possible functional homologies between these proteins is reinforced by the presence of several conserved domains that lie carboxy-terminal to the kinase 1a (P-loop), kinase 2 and kinase 3a motifs of the nucleotide-binding (NB) site (Van der Biezen and Jones, 1998). In human cells, death-inducing stimuli lead to an activation of Apaf-1 through its carboxy-terminal WD40-repeat domain that, like the leucine-rich repeat (LRR) domain, mediates protein–protein associations. This creates a conformational change, permitting homophilic interactions with caspase enzymes through their respective caspase recruitment domains (CARD). The CARD domains of Ced-4, Apaf-1 and Card-4 also transduce signals through homophilic interactions with CARD-containing caspases (Aravind et al., 1999). Interestingly, Card-4 that possesses carboxy-terminal LRRs, has a role in apoptosis but also associates with a protein kinase (RICK) by CARD–CARD interactions, leading to activation of NF-κB (Bertin et al., 1999). Based on these similarities in protein architecture, it is envisaged that both conformational changes and homophilic interactions are central to the function of plant NB-LRR type R proteins in disease resistance.

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class II, designated LZ-NB-LRR, proteins possess a putative leuzine-zipper (LZ) domain that may direct protein–protein interactions through the promotion of coiled-coil structures (Hammond-Kosack and Jones, 1997). Although the functional significance of the various domains has not been demonstrated, similarities in the domain structures of NB-LRR type R proteins to animal adaptor proteins regulating programmed cell death (apoptosis) (Aravind et al., 1999; Van der Biezen and Jones, 1998) suggest mechanisms by which R proteins might transduce pathogen recognition events to a defence response (Fig. 1). Thus, it is anticipated that perception of a pathogenderived signal within the carboxy-terminal LRR domain leads to conformational activation of the amino-terminal ‘effector’ domain, probably involving ATP or GTP hydrolysis within the central NB portion of the protein. Elucidating the precise molecular events that underpin R protein function is now an area of intense study. R genes encoding class I and class II NB-LRR proteins have been cloned from Arabidopsis and their molecular characterization can be effectively integrated with genetic dissection of disease resistance pathways. In our studies we targeted for positional cloning the RPP5 (Parker et al., 1993) and RPP14 (Reignault et al., 1996) genes that confer resistance to the oomycete pathogen, Peronospora parasitica . As an obligate biotroph, P. parasitica and its natural host, Arabidopsis have evolved a high degree of genetic diversity in their interaction phenotypes and this is reflected in the number of RPP loci identified in various Arabidopsis accessions, and a correspondingly large array of genetically distinct P. parasitica isolates (Holub and Beynon, 1997). We found that RPP5, encoding a TIR-NBLRR type R protein in Arabidopsis accession Landsberg-erecta (La-er), belongs to a tightly clustered multigene family (Parker et al., 1997). Comparative sequence analysis of ≈ 90 kb comprising the RPP5 locus in La-er with the corresponding locus in the P. parasitica-compatible (rpp5 ) accession, Columbia (Col-0), revealed a staggering degree of polymorphism across the locus that was flanked on both sides by co-linear DNA (Noël et al ., 1999). Extensive sequence variation at the RPP5 locus caused by gene duplications, unequal recombination events, point mutations and retrotransposon insertions, has probably enhanced possibilities for creating novel recognition molecules. Most strikingly, domains of both functional and nonfunctional RPP homologues were found to be subject to purifying or diversifying selection pressures, suggesting that the RPP5 gene family is a dynamic locus providing a repository of sequence segments that have recently contributed to pathogen recognition (Noël et al., 1999). Although only one recognition specificity has so far been assigned to the RPP5 region, that of the RPP5 gene itself, genetic mapping places another R specificity against P. parasitica, RPP4 in accession Col-0, within or very close to the RPP5 locus (EvdB, J.E.P. and J.D.G.J., unpublished data).

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Table 1 Resistance capabilities of Arabidopsis RPP1 genes recognizing three P. parasitica isolates.

R locus

P. parasitica Isolate

R gene designation

RPP10 RPP1 RPP14

Cala2 Emoy2 Noco2

RPP1-WsA RPP1-WsA RPP1-WsA

RPP1-WsB RPP1-WsB

RPP1-WsC

Three R genes, RPP1-WsA, RPP1-WsB and RPP1-WsC, residing at the RPP1 complex locus account for disease resistance specificities previously assigned to the genetically linked R loci, RPP1, RPP10 and RPP14. Their distinct but overlapping resistance capabilities against P. parasitica isolates Noco2, Emoy2 and Cala2 suggests that each R gene recognizes a different avirulence determinant. The three predicted RPP proteins are highly related at the amino acid level, RPP1-WsA being 86% and 85% identical, respectively, to RPP1WsB and RPP1-WsC (Botella et al., 1998).

Molecular characterization of the RPP14 locus in Arabidopsis accession Wassileskija (Ws-0) has also been instructive. RPP14mediated resistance was found to be genetically linked to two other distinct RPP specificities in Ws-0, RPP1 and RPP10 (Holub et al., 1994; Reignault et al., 1996). Cloning and characterization of three out of four genes that comprise this R locus revealed that, together, these genes account for the spectrum of resistance previously assigned to RPP1, RPP10 and RPP14 and thus constitute a complex R locus, now designated the RPP1 complex locus (Table 1) (Botella et al., 1998). The predicted proteins encoded by the RPP1 genes, like RPP5, belong to the TIR-NB-LRR structural class and in addition possess either a hydrophobic or hydrophilic amino-terminal extension. Their distinct recognition capabilities but high degree of sequence similarity (Table 1) makes them ideal targets to examine the molecular basis of pathogen recognition through engineered point mutations and sequence exchanges.

R P P G E N E S I G N A L L I N G RE Q U I RE M E N T S IN ARABIDOPSIS Mutational analysis has been applied extensively in Arabidopsis to identify genes that are required for R gene-specified disease resistance (Glazebrook et al., 1997a; Parker, 1999). Forward mutational screens of chemically mutagenized, irradiated, and transposon- or TDNA-insertion populations enabling targeting of genes that are physiologically important in a particular defence response but may fail to identify redundant components of pathways. This limitation can, to some extent, be alleviated by more refined screens utilizing gene- or enhancer-trap reporter lines (Sundaresan et al., 1995). In Arabidopsis, there is still a case to be made for saturation mutagenesis using the chemical agent ethyl methanesulphonate (EMS) that can,

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Table 2 Genetic components of RPP5-specified resistance in La-er. Mutation

Mutagen

Independent alleles

Phenotype

rpp5 edsl pad4 rprl rpr2

FN/EMS FN FN FN EMS

4/2 2 1 1 1

S S* (S) S S

Mutagens used were fast neutrons (FN) or ethyl methanesulphonate (EMS). S = susceptible; S* = hypersusceptible; (S) = partially susceptible.

by causing single amino acid substitutions, create subtle and potentially more informative alterations in proteins than insertional gene knockouts obtained with transposons or TDNAs. Completion of the Arabidopsis genome sequence will greatly facilitate positional cloning of genes based on EMS-derived mutant phenotypes. We performed a number of pathology screens of EMS- and fast neutron (FN)-mutagenized La-er or Ws-0 to identify the genes required, respectively, for RPP5 and RPP1 function. In wild-type plants, RPP5- or RPP1-mediated resistance is associated with a strict limitation of pathogen growth and an HR at the point of pathogen penetration (Botella et al., 1998; Parker et al., 1997). In fully susceptible plants, P. parasitica mycelium extends rapidly from the inoculation site and colonizes the plant systemically. M2 seedlings of La-er or Ws-0 were sprayed with conidiospores of P. parasitica isolate Noco2 and susceptible seedlings selected that permitted various degrees of asexual pathogen sporulation on plant cotyledons or leaves. As shown in Table 2, the screens of La-er mutagenized populations produced multiple defective alleles of the RPP5 gene itself, indicating that resistance attributed to the RPP5 locus in La-er was indeed conferred by a single R gene. In addition, the screens revealed two eds1 mutations, a single mutant allele of PAD4, and two novel mutations, provisionally designated rpr1 and rpr2 (r equired for RPP5-mediated r esistance). Screens of EMS-mutagenized Ws-0 revealed a further five independent eds1 mutations (Falk et al., 1999). The failure to identify mutant alleles of RPP1 using the Noco2 isolate of P. parasitica was consistent with our finding that all three RPP1 genes, RPP1-WsA, RPP1-WsB and RPP1-WsC, conferred resistance to Noco2 (Table 1) (Botella et al., 1998). However, a single mutant form of RPP1-WsA was identified by Jim Beynon and colleagues at Horticultural Research International (Wellesbourne, UK) using P. parasitica isolate Cala2, in line with the unique capability of RPP1-WsA among the three RPP1 genes to interact with this pathogen isolate (Table 1) (Botella et al., 1998).

E D S 1 I S A C O M P O N E N T O F RE S I S T A N C E MEDIATED BY TIR-NB-LRR TYPE R GENES The first eds1 mutation to be isolated was eds1-1 in Ws-0 and this fully suppressed the function of all three RPP1 genes recognizing different avirulence (Avr) determinants in P. parasitica isolate Noco2 (Parker et al., 1996). Resistance could be rescued in eds1 plants after the application of a functional analogue of the plant defence signalling molecule salicylic acid (SA), indicating that the wild-type EDS1 protein was likely to function upstream of SA-inducible defences. Interestingly, eds1 also suppressed resistance mediated by an unlinked RPP gene, RPP12, and nonhost resistance to several Brassica-infecting oomycetes, but had no effect on resistance specified by RPM1 against a Pseudomonas syringae avirulence (avr ) gene, avrB. RPM1 and two other R genes, RPS2 and RPS5, that recognize, respectively, avrRpt2 and avrPphB from P. syringae, were instead shown to require a different gene, NDR1 (Century et al., 1995, 1997). A broader assessment of the signalling requirements of different Arabidopsis R genes for EDS1 and NDR1 was performed by comparing the effects of eds1 and ndr1 on the same spectrum of R genes. In some cases this necessitated crossing La-er eds1 or a Col-0 ndr1 mutation into comparable R gene backgrounds. The results of this study revealed the existence of at least two distinct R gene-mediated signalling pathways (Aarts et al., 1998). R genes that had a strong dependence on EDS1 had a weak or negligible requirement for NDR1. Conversely, NDR1dependent R genes exhibited, at most, a very weak requirement for EDS1, as shown in Fig. 2. Strikingly, the particular signalling mode was correlated with the predicted R protein structure and not with the type of pathogen attacking the plant. Thus, the TIR-NB-LRR class of R proteins appear to engage EDS1 for resistance, whereas the majority of the LZ-NB-LRR class of R proteins utilize NDR1. One exception to this scheme was observed with RPP8, a predicted LZ-NB-LRR type protein (McDowell et al ., 1998) that has no unique dependence on either EDS1 or NDR1, therefore possibly defining a third Arabidopsis defence pathway (Aarts et al., 1998). We might anticipate, based on these results and our discussion above, that the different R protein amino-terminal ‘effector’ domains activate distinct signalling processes that may then converge at one or more points downstream (Fig. 2). The PAD4 gene was originally identified by Jane Glazebrook and colleagues in a pathology based screen of mutagenized Col-0 for increased susceptibility to a virulent isolate of P. syringae pv. maculicola (Glazebrook et al., 1996) and adds to a collection of pad mutants that are deficient in phytoalexin accumulation in response to bacterial infection. Glazebrook et al. (1997b) showed that pad4 defined a regulatory component in phytoalexin production rather than a biosynthetic gene and subsequent analyses demonstrated that PAD4 (Zhou et al., 1998), like EDS1

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Fig. 2 Spectrum of requirements for EDS1 and NDR1 by different R genes in Arabidopsis. Analysis of the dependence of R genes on EDS1 or NDR1 reveals the existence of at least two disease resistance signalling pathways that are conditioned by distinct sets of R gene (Aarts et al., 1998). EDS1 functions upstream of SA-mediated defences (Falk et al., 1999) that are in part defined by the NPR1 gene (Cao et al., 1997). It is speculated that the NDR1-dependent pathway may also feed into SA-mediated responses. A mutation in another resistance signalling gene, PBS3, possibly defines a convergent pathway downstream of EDS1- and NDR1mediated processes (Warren et al., 1999).

(Falk et al., 1999), functions upstream of SA-mediated defences. In contrast to eds1, that causes a complete loss of RPP5 function, the La-er pad4 mutation ( pad4-2 ) identified in our mutant screens (Table 2) only partially compromises RPP5-specified resistance. The partial resistance phenotype associated with pad4 reveals an inability of the plant to mount an effective response. The pathogen mycelium is able to breech plant defences and this results in a trail of necrotic plant tissue associated with limited pathogen growth. An interesting phenotype associated with both eds1 and pad4 is that, in addition to suppressing R gene-conditioned responses, they cause enhanced disease susceptibility (‘eds’) in a number of compatible or partially compatible Arabidopsis– pathogen interactions (Aarts et al., 1998; Glazebrook et al., 1996). It is therefore likely that some commonality exists between mechanisms evoked during R-avr gene mediated resistance (normally associated with the HR) and the less

well-defined restriction of compatible pathogens in disease. It is possible that EDS1 and PAD4 function as basal plant resistance components that have been recruited by certain R proteins in R-avr gene-stimulated responses. Certainly, these mutations add to a battery of ‘eds’ mutant loci identified in Arabidopsis (Reuber et al., 1998; Rogers and Ausubel, 1997), consistent with the idea that growth of pathogens is actively limited by host plant defences in the absence of an HR. EDS1 and PAD4 encode lipase-like proteins The EDS1 gene was cloned from La-er plants using a I/dSpm transposon tagging approach and was shown to encode a 72-kDa putatively cytoplasmic protein (Falk et al., 1999). Database searches did not reveal sequences with extensive homology to EDS1. However, several discrete blocks of homology to eukaryotic lipases were found within the EDS1 exon 2 (Fig. 3).

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Fig. 3 The predicted EDS1 and PAD4 proteins have similarity to eukaryotic lipases. The consensus catalytic site of eukaryotic lipases spans three invariant residues, a serine (S), an aspartic acid (D) and a histidine (H), that form the catalytic triad. The catalytic motifs of EDS1 and PAD4 conform to the L-family of neutral lipases, such as R. miehei triacylglycerol lipase, for which the tertiary structure is known (Brady et al., 1990). Human platelet activating factor (PAF) acetylhydrolases, such as an intracellular form of PAF acetylhydrolase II, also conform to the signature of neutral lipase/ esterases, although they possess phospholipasee A2 activity (Stafforini et al., 1997). The positions of the predicted catalytic residues are shown for each protein and numbers on the right hand side indicate the amino acid size of the mature protein.

Significantly, the regions of amino acid similarity span three critical residues: a serine, an aspartic acid and a histidine that form a catalytic triad in a wide range of serine esterases (Schrag and Cyglar, 1997). The broader amino acid consensus across these segments between EDS1 and L-family neutral lipases, such as triacylglycerol lipases from Rhizomucor miehei or R. niveus (Derewenda and Sharp, 1993; Schrag and Cygler, 1997), suggests that EDS1 may function by hydrolysing a lipid-based substrate. Interestingly, certain secreted and intracellular forms of mammalian platelet activating factor (PAF) acetylhydrolases that have phospholipase A2 activity, conform to the neutral lipase signature (Stafforini et al., 1997). PAF acetylhydrolases hydrolyse PAF, a potent phospholipid mediator of inflammation, and are able to process other phospholipids that have been fragmented by oxidation. The latter activity of these enzymes is intriguing, since the formation of highly reactive oxygen intermediates is known to be an early event in several R-avr gene-associated plant responses (Jabs et al., 1996; Lamb and Dixon, 1997). However, we noted that a ferulic acid esterase from Aspergillus niger possesses the same pattern of conserved residues (Falk et al., 1999), raising the possibility that the EDS1 substrate is something other than a lipid molecule. Intriguingly, PAD4 encodes a lipase-like protein with the same pattern of conserved residues as EDS1, suggesting that EDS1 and PAD4 may have related activities (Jirage et al., 1999; B.J.F. and J.E.P., unpublished data) (Fig. 3). The recruitment of a lipase domain in two genetically related defence signalling components raises exciting possibilities for plant fatty acid signalling. Experiments are now being designed to elucidate more precisely the anticipated enzymatic activities of EDS1 and PAD4, their modes of expression and localization within the plant cell. We envisage that these proteins may create a lipidderived signal molecule that is specifically elaborated upon R gene-mediated pathogen recognition. Alternatively, they may

be rather less specific destroyers of membrane integrity. In the latter case their activities would have to be very tightly repressed in healthy tissue. However, EDS1 mRNA and protein were detected in unchallenged cells (Falk et al., 1999; B.J.F. and J.E.P., unpublished data) arguing against strict the regulation of expression. In addition, the EDS1- (Falk et al., 1999) and PAD4- (Zhou et al., 1998) dependent activation of PR1 mRNA (an SA-inducible defence marker) in response to bacterial infection, suggests a more subtle role than wholesale membrane destruction. One known plant lipid signalling molecule is jasmonic acid (JA), a cyclic fatty acid that has an essential role during plant wounding and in certain plant–pathogen resistance responses (Pieterse and van Loon, 1999; Reymond and Farmer, 1998). However, applications of JA did not rescue disease resistance in eds1 plants, whereas similar treatments with SA did, suggesting that if EDS1 is a lipase it must process a different lipid metabolite, leading to SA-dependent PR1 gene induction (Falk et al., 1999).

CONCLUSION The isolation and structural characterization of Arabidopsis R genes and associated signalling components provides just a first step towards unravelling the complexities of plant-pathogen recognition and defence networks. Further advances will rely on multidisciplinary approaches to scrutinize, for example, the predicted conformational attributes of NB-LRR type R proteins, the anticipated enzymatic activities of resistance signalling molecules, and the physiological relevance of protein associations during plant defence responses. Genomics-based strategies should also allow a much fuller appreciation of protein function across plant and animal species boundaries, and this in turn will furnish us with a fresh perspective on how to combat some of the world’s most destructive plant diseases.

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ACKNOWLEDGEMENTS Work at The Sainsbury Laboratory is supported by The Gatsby Charitable Foundation. We also gratefully acknowledge support from The British Biotechnology and Biological Sciences Research Council (B.J.F.), Zeneca, UK (E.v.d.B.), and a European Economic Community Training and Mobility of Researchers fellowship (N.A.).

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