Adaptive Covariation between the Coat and Movement Proteins of Prunus Necrotic Ringspot Virus

The relative functional and/or structural importance of differentamino acid sites in a protein can be assessed by evaluatingthe selective constraints to which they have been subjectedduring the course of evolution. Here we explore such constraintsat the linear and three-dimensional levels for the movementprotein (MP) and coat protein (CP) encoded by RNA 3 of prunusnecrotic ringspot ilarvirus (PNRSV). By a maximum-parsimonyapproach, the nucleotide sequences from 46 isolates of PNRSVvarying in symptomatology, host tree, and geographic originhave been analyzed and sites under different selective pressureshave been identified in both proteins. We have also performedcovariation analyses to explore whether changes in certain aminoacid sites condition subsequent variation in other sites ofthe same protein or the other protein. These covariation analysesshed light on which particular amino acids should be involvedin the physical and functional interaction between MP and CP.Finally, we discuss these findings in the light of what is alreadyknown about the implication of certain sites and domains instructure and protein-protein and RNA-protein interactions.

INTRODUCTION

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Introduction

Prunus necrotic ringspot virus (PNRSV) is a positive-sense RNAplant virus with a tripartite genome that belongs to the genusIlarvirus of the Bromoviridae family (14). PNRSV is distributedworldwide, infecting most cultivated Prunus spp. and causingsymptoms that range from no damage to necrotic spots and shotholes in young leaves and to important rugose mosaic, all causingthe plant to lose yield and vigor (51). PNRSV is easily transmittableby pollen grains and seeds and by routine propagation methods(17). Cherry PNRSV isolates were classically classified intothree serotypes (CH3, CH9, and CH30) and two pathotypes (rugosemosaic disease and mild virosis) (34), and a correlation betweensymptom severity and the primary sequence of the gene for MPor CP was observed (19). However, similar studies using PNRSVisolates from other hosts and different geographical originsshowed no clear correlation between molecular variability andbiological properties (1, 53), indicating that the results derivedfrom the cherry isolates were not necessarily valid for otherhosts. Consensus, however, exists as isolates are classifiedon the basis of their sequence homology into three groups (PE-5,PV-32, and PV-96) (1, 18, 52). Group PV-32 is characterizedby the presence of two repeated amino acids in the first thirdof CP coming from a duplication of two preceding ones (1). GroupPE-5 contains three cherry isolates (two of them producing severesymptoms) of the CH30 serotype but also contains isolates frompeach that induce mild and latent symptoms in infected trees(18). Attempts to find an association between geographical distributionand the abundance of certain serotypes have failed (1, 2, 18,52).

Ilarviruses have the same genome organization, encoding productsfunctionally similar to those of alfalfa mosaic virus (AMV)and other members of the Bromoviridae family. RNAs 1 and 2 encodethe proteins involved in replication (P1 and P2, respectively).RNA 3 is bicistronic and encodes the movement protein (MP) andcoat protein (CP) (35), although CP is translated from subgenomicRNA 4. Both MP and CP play important roles at different stagesof the infectious cycle of ilarviruses. For example, the bindingof CP into stem-loop structures flanked by AUGC sequences locatedin the 3' nontranslated region of the inoculum RNAs is requiredfor initiation of infection, a phenomenon known as genome activation(5, 6, 8, 24). PNRSV and AMV are phylogenetically close (10,11, 42). Most studies on the implication of CP in the virallife cycle have been conducted with AMV, whereas there are relativelyfew experimental data on PNRSV CP structural properties andbiological functions (3). For instance, AMV CP is required forplus-strand RNA accumulation, encapsidation, cell-to-cell movement,and systemic spread of the virus (49). This latter functionrequires the establishment of a physical interaction betweenCP and MP. It has also been suggested that, in AMV, MP may formtubular structures that traverse the cell wall through modifiedplasmodesmata, and these tubules mediate unidirectional transportof viral RNA-MP-CP complexes (41).

Tenllado and Bol (49) showed that it was possible to map thedifferent AMV CP functions into different domains of the protein.The N-terminal arm looks sufficient for genome activation withoutthe concourse of CP dimers, as demonstrated by using syntheticpeptides of this protein region in in vivo experiments (4).Mutations in the N-terminal arm also affect cell-to-cell movement.The C-terminal domain, except the last 7 to 14 amino acids,is involved in dimer formation, which is required, for example,for correct virion formation. Amino acids 17 to 20 are criticalfor plus-strand RNA accumulation. Finally, mutations at theN and C termini affect the movement of viral materials throughthe vascular system (49).

The CP N-terminal domain has been shown to be involved in cell-to-cellmovement and in systemic spread in members of the differentgenera within the family Bromoviridae (AMV [41], brome mosaicvirus [BMV] [16, 38, 39, 40], and cucumber mosaic virus [CMV][25]). Furthermore, Sánchez-Navarro and Bol (41) alsoshowed that deletions in the N- or C-terminal arm of AMV MPdid not interfere with the ability of the protein to assembleinto tubules and allow cell-to-cell and systemic movements.However, simultaneously deleting the 11 N-terminal amino acidsand 45 amino acids at the C terminus produced proteins ableto form tubules but not proficient in promoting cell-to-cellmovement. Furthermore, recently Sánchez-Navarro et al.(43) have shown that the 44 amino acids at the AMV MP C terminusphysically interact with CP to elicit movement. Apparently,the C terminus of AMV MP confers specificity on the transportprocess (41) as it does in the cases of BMV (47) and CMV (26).MPs of PNRSV, AMV, CMV, and BMV have been shown to have an RNA-bindingdomain located at the N terminus of the protein in the firsttwo cases or at the C terminus in the last two (3, 4, 7, 21).At least in the case of PNRSV, it has been shown that basicamino acids in this domain are required for RNA-binding activityand cell-to-cell movement (21). Finally, it has been reportedthat CMV MP also interacts with the 2a polymerase gene, althoughthis interaction is not relevant for the formation of the replicase1a-2a complex (23).

Although it is widely accepted that MP and CP should interactto facilitate viral cell-to-cell movement, direct evidence hasnot been provided. Here we report results from a molecular evolutionstudy seeking to identify the selective constrains acting uponCP and MP during the evolution of different isolates of PNRSV.Our aim is twofold. At the one side, using a maximum-parsimonyapproach, we have identified amino acid sites under selectionin each protein at different evolutionary time points. At theother side, we have applied statistical methods drawn from informationtheory to identify groups of amino acids that covary throughouttime as a consequence of their functional or structural relationship.The existence of covariation groups was explored both withinand between proteins.

MATERIALS AND METHODS

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Materials and Methods

Sequence alignments and phylogenetic analyses. The MP and CP sequences used in this study were downloaded fromGenBank, and the corresponding accession numbers are shown inTable 1. Only full-length sequences have been used. Proteinsequences were aligned with CLUSTAL-X version 1.8 (50). Nucleotidesequences were then aligned by concatenating triplets accordingto the amino acid sequence alignment with the program DAMBEversion 4.2.7 (53). Alignments are available upon request. Hereafter,amino acid numbering will be assigned with CH30/USA/Cherry (accessionno. AF034994) as the reference sequence for MP and E260/India/Rose(accession no. CAF4057) as the reference sequence for CP. Thesetwo isolates were chosen because no gaps were introduced intothem during alignment.

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TABLE 1. PNRSV isolates included in this study

Prior to any other evolutionary analyses by RNA alignment, theintensity of the phylogenetic signal contained was evaluatedwith the likelihood-mapping approach as implemented in the programTREEPUZZLE version 5.0 (45).

According to the likelihood ratio test implemented in MODELTESTversion 3.7 (37), the molecular evolution model that best explainsthe observed pattern of nucleotide diversity for MP alignmentwas that proposed by Tamura and Nei (48), which was modifiedto account for the heterogeneity in substitution rates amongsites (gamma parameter 0.285). Similarly, the model that bestexplained the observed sequence variation in CP alignment wasthat proposed by Kimura (27), with a transition to a transversionrate ratio of 2.291 and also incorporating heterogeneity insubstitution rates among sites (gamma parameter 0.265). Phylogeneticreconstructions were obtained by the minimum-evolution methodas implemented in the MEGA3 program (31) with the above nucleotidesubstitution models. The support of the internal nodes of thetrees was evaluated by the bootstrap method with 10,000 pseudoreplicates(15). Nodes with bootstrap support of <75% were collapsedto the nearest significant node.

Analysis of selective constraints. To uncover the signature of coevolution between CP and MP, wefirst explored the selective constraints operating upon eachindividual protein. The advantage of using parsimony methodsto determine codon regions consistently under specific selectiveconstrains relies on its robustness against methodological biases(46). In maximum-parsimony analyses, the comparison of the ratesof synonymous (d_S) and nonsynonymous (d_N) substitutions perclass of sites is used to describe the evolutionary dynamicsof protein-coding genes. The ratio of these two rates ( ${omega}$ = d_N/d_S)provides information on whether the gene has been fixing aminoacid replacements in a neutral fashion ( ${omega}$ = 1), amino acid changeshave been removed by the action of purifying selection ( ${omega}$ <1),or changes have been fixed by adaptive evolution ( ${omega}$ >1).

The particular method chosen is based on the parsimony criterion,which is appropriate here, given the conservation at the codonlevel of our alignments, and is implemented in the program SWAPSCversion 1.0 (12). SWAPSC uses a statistically optimized windowsize to detect selective constraints in specific codon regionsof the alignment at a particular branch of the phylogenetictree (13). Briefly, the method estimates the expected distributionof d_S and d_N by Li's method (32) from simulated sequence alignmentsand assuming a Poisson distribution of substitutions. A statisticallyoptimum window size is then estimated that makes the detectionof adaptive evolution independent of the window size. The empiricalvalues of d_S and d_N obtained by using the optimal window sizeare contrasted with the expected distributions, and severalhypotheses regarding the selective constraints acting on codonregions are tested. Simulated sequence alignments were obtainedwith the program EVOLVER from the PAML package version 3.14(54) with parameters estimated from the true sequence alignmentsafter running the most appropriate codon-based model in PAML(models M2 and M8 for MP and CP, respectively [55]).

The Bayesian approach implemented in the CODEML program fromPAML was also used. The sites determined to be under selectionwere the same as those found by the above-mentioned maximum-parsimonyapproach, giving robustness to the conclusion (data not shown).

Testing for coevolution between amino acid sites. The aim of this analysis was to determine whether MP and CPhave coevolved during PNRSV phenotypic radiation because oftheir putative functional link. We also tested whether coevolvingamino acids can be used to predict protein-protein contact interfaces.We realize that this type of analysis is not definitive in resolvingdocking problems, but nonetheless, results could aid in identifyingspecific amino acid sites responsible for protein interactionsand in designing new experiments. We are also interested ina more general definition of protein interaction, which canbe functional, physical, or phylogenetic. Significance of coevolutionwas tested by the mutual information criterion (MIC) approachtaken from information theory (28, 29). MIC definition involvesthe joint probability distribution, P(x_i, y_j), of the occurrenceof symbol x at position i and symbol y at position j belongingto the same protein or to two different proteins. MIC valuesrange between 0, indicating independent evolution, and a positivevalue whose magnitude depends on the strength of covariationbetween sites. Variable positions included in the analyses wereonly those parsimony informative (i.e., containing at leasttwo types of amino acids each with a minimum count of two).The statistical power of this test depends both on the samplesize and on the level of variation at the sites considered.The significance of MIC values was assessed by means of a randomizationtest in which columns in the amino acid alignment of parsimony-informativesites were shuffled in place for each protein. The MIC valuewas recalculated thereafter for each shuffle to generate theexpected probability distribution under the null hypothesisof no association. Significance P values were computed, basedon a million permutations, as the fraction of shuffles witha MIC value greater than or equal to the observed value. Tominimize the number of false positives, the significance levelwas set to 1%. Only the 15 isolates for which both full-lengthprotein sequences were available in the database were used forthese analyses (Table 1).

Threading predictions of protein tertiary structure. Tertiary structures of MP and CP were predicted by threadingwith the server http://cbsuapps.tc.cornell.edu/loopp.aspx anddefault options (33). Structure predictions are available uponrequest (see Fig. 3). So far, the native tertiary structuresof PNRSV MP and CP have not been experimentally determined.However, the structure of phylogenetically close AMV CP hasbeen obtained at 4 Å resolution (30). At this resolution,CP presents a globular structure with both the C and the N terminiemerging from it. This gives indirect support to our predictionof PNRSV CP folding as a globule with protruding ends. Evenless is known about MP structure. The PNRSV MP RNA-binding domain(amino acids 56 to 88) has been shown by circular dichroismto fold into an ${alpha}$ -helix (22). Our predicted globular structurefor MP reflects this experimental evidence. Nonetheless, giventhe scarceness of information on the in vivo three-dimensionalstructure adopted by MP, our threading prediction cannot becontrasted with experimental data and therefore a flag of cautionhas to be put on any conclusion drawn from the predicted structure.

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FIG. 3. Selected sites and covariation analysis of MP and CP. Color code: red, positively selected sites; blue, negatively selected sites; green, sites showing hypermutagenesis. Covarying amino acids are indicated and linked with yellow arrows.

Sites identified as selectively important or involved in covariationwere mapped into the predicted structures (see Fig. 3).

RESULTS

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Results

Testing of the phylogenetic signal in MP and CP genes. Likelihood-mapping analyses showed strong phylogenetic signalsin the two data sets. For the gene for MP, the tree-likeness(i.e., the percentage of all well-resolved quartets) of thedata was 87.85%, whereas for the gene for CP it was 75%; wethus concluded that our data sets contained a significant phylogeneticsignal and we proceeded with the molecular evolution study.(Because of limitations of the TREEPUZZLE software, the nucleotidesubstitution model of Tamura and Nei was used for both genesin this particular analysis.)

Figure 1 shows the minimum-evolution phylogenetic trees inferredfor the genes for MP and CP. Regardless of the gene analyzed,internal nodes are generally well supported by the bootstrapanalysis. External nodes are generally less well supported (Fig.1). More importantly, the three main groups previously definedin the literature upon sequence homology (PE-5, PV-32, and PV-96)are highly supported by bootstrap values (Fig. 1). However,the phylogenetic position of isolate ChrIt.mrs1/Italy/Cherry,previously considered a member of the PV-96 group (2), was notwell resolved. As shown in Fig. 1A, its splitting node has abootstrap support value of <75% and thus has been collapsed.Nonetheless, to keep our classification consistent with previousproposals (2), ChrIt.mrs1/Italy/Cherry has been conservativelyincluded within the PV-96 group (Fig. 1A).

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FIG. 1. Phylogenetic analysis of MP (A) and CP (B). Trees were inferred by the minimum-evolution method using the Tamura and Nei corrected nucleotide distances (48). Values at the nodes are bootstrap support values based on 10,000 pseudoreplicates. Nodes with bootstrap support of <75% were collapsed. ChrIt.mrs1/Italy/Cherry has been included in the PV-96 group for consistency with previous proposals (2).

The clusters containing isolates belonging to the PV-32 andPV-96 groups appear more closely related to each other thanthey are to isolates in group PE-5. This result is consistentwith either a more recent common ancestor of the isolates belongingto the PV groups or, alternatively, an accelerated rate of evolutionof group PE-5, this being the case for the genes for both MPand CP. The results reported in the following section supportthe second hypothesis.

Detecting selective constraints in MP and CP. The parsimony-based method found evidence of adaptive evolutionin both proteins (Table 2). The optimum window sizes estimatedby SWAPSC were five and four codons for MP and CP, respectively.A single region in MP appears to be under adaptive evolution,i.e., positive selection ( ${omega}$ >1), E252 to E257. This regionwas selected after the split occurred within the PE-5 cladeand produced the ancestor of a set of geographically and hostdiverse isolates (branch G in Fig. 2A). MP sites under purifyingselection ( ${omega}$ <1) are located in regions L61 to S65 and R212to L216. The first region has undergone purifying selectionin all branches of the phylogenetic tree, whereas the actionof purifying selection on the second region has been limitedto branches A, B, and G (Table 2 and Fig. 2A), the internaltree branches that gave rise to the different clades. Finally,the method also detected three regions of the protein that showglobally accelerated rates of amino acid substitution (Table2). The hypermutagenesis of these regions took place in thebranches leading to the PE-5 cluster (branches G and H in Fig.2A), supporting the hypothesis of an accelerated rate of evolutionduring the genesis of this clade. The above-mentioned siteswere mapped onto the predicted tertiary structure (Fig. 3).

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TABLE 2. Summary of SWAPSC maximum-parsimony analysis of adaptive evolution in PNRSV CP and MP^a

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FIG. 2. SWAPSC analysis of selective constraints in MP (panel A) and CP (panel B). In this example, the branch detected under adaptive evolution is drawn as a dotted line. The plots show the distribution of the nonsynonymous to synonymous substitution rate ratio ( ${omega}$ ) along the sequence. Peak heights are proportional to the intensities of positive selection in the corresponding regions.

Regarding CP, the picture is as follows. Two regions containamino acids under directional positive selection (S49 to G53and K139 to Q142), four regions contain amino acids under purifyingnegative selection (Table 2), and one stretch had suffered fromhypermutagenesis (Table 2). Interestingly, positive selectionacted along the deepest branches of the tree, during the diversificationof the lineage leading to PE-5 isolates. Purifying selectionacted along several branches of the tree (Table 2 and Fig. 2B),and its action during the separation of lineages PV-32 and PV-96was noticeable (Table 2 and Fig. 2B). Hypermutagenesis onlyhappened in branch D (Table 2 and Fig. 2B). The above siteswere mapped onto the predicted tertiary structure (Fig. 3).

The fact that the branch leading to the ancestor of PE-5 isolateswas found to be under adaptive evolution in both proteins providesa first indication of coevolution between MP and CP. Interestingly,this putative process of coevolution is in the root of the genesisof a new serotype.

Covariation within and between MP and CP. By the MIC approach, we have detected several covariation groups(P < 0.01) within and between proteins (Table 3). Figure3 illustrates the distribution of covariation sites on the predictedtertiary structures. In a first step, we analyzed within-proteincovariation. Regarding MP, three amino acid residues show significantcovariation (L253, E257, and I261; Table 3). Hereafter, we willcall this covariation group MP CG. For CP, two different significantcovariation groups (hereafter called CP CG1 and CG2) have beenidentified (Table 3). CP M52 belongs to both groups, and thecovariation between V48 and D141 was statistically significantat the usual 5% significance level (MIC value = 0.161, P = 0.040),suggesting that both covariation groups can be reduced to asingle one at the less stringent 5% confidence level. Nonetheless,in the following discussion we will maintain the distinctionbetween two nondisjoint groups.

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TABLE 3. Covariation groups within and between proteins

In a second step, we have explored the covariation across proteins(Table 3 and Fig. 3). Very interestingly, MP CG significantlycovaries with CP CG2. In addition, MP E256 also covaries significantlywith CP M52.

DISCUSSION

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Discussion

It has been previously reported that the variability of MP concentratesaround amino acids 232 to 285 (1, 2, 19, 52). Our analyses confirmthis observation and also show that positively selected andhypermutated sites also concentrate in this variable regionof the molecule. Our results showing evidence of adaptive evolutionwithin a region spanning amino acids 245 to 270 support thepreviously suggested antigenicity of this region (52) (Table2, E252 to E257). Some of the changes are group specific, asreplacements in amino acid positions V253, D256, D257, and P260appear always in association with both PV groups but have adifferent amino acid composition in the isolates belonging tothe PE-5 group (52). PE-5 isolates also share other group-specificmutations in this region, and interestingly, the operation ofpositive selection has been detected on the branch leading tothis serotype, revealing that the differences between the PE-5and PV groups may be due to changes in selective constraints.Furthermore, our finding that MP CG and site E256, all withinthis putative antigenic region, significantly covary with CPCG2 suggests that this region is involved in the establishmentof interactions with CP that may be important for mediatingcell-to-cell movement of PNRSV.

There are two domains of MP that are highly conserved, probablybecause of structural and/or functional constraints. These domainsrange, respectively, from L61 to S65 and from R212 to L216.In addition, amino acid sites 8 to 70 have been predicted toform a transmembrane domain and may be necessary for the proteinto interact with the cell wall (42). Supporting the existenceof such functional and/or structural constraints in this region,we have identified sites that are under the action of negativeselection (Table 2, L61 to S65). In addition, it has also beenrecently shown that MP interacts with RNA molecules throughoutamino acids 56 to 88 (21). Interestingly, this 32-amino-aciddomain includes the above-mentioned amino acids detected asnegatively selected (L61 to S65). These basic amino acids shouldbe, in accordance with our threading structural predictions(data not shown), exposed to the solvent, both properties beingtypical of RNA- and DNA-binding motifs.

The variability in CP is concentrated in two regions, includingone between N-terminal amino acids 48 and 56 (19) and anotherbetween C-terminal amino acids 139 and 145. These variable regionsalso contain most of the sites found to be under positive selection(Table 2, S49 to G53 and K139 to Q142). Amino acids 25 to 50are essential for the protein to bind PNRSV RNA in vitro (3,36). More generally, the N-terminal domain of members of thedifferent genera within the family Bromoviridae (Cucumovirus[20], Bromovirus [44], and Ilarvirus [3, 4, 7]) is involvedin capturing RNA during encapsidation and also plays an importantrole in the phenomenon of genomic activation in the case ofilarviruses (5, 6). Zhang et al. (56) predicted, on the basisof in silico experiments, that the main requirement for cowpeachlorotic mottle bromovirus RNA encapsidation should be an abundanceof positively charged amino acids at the internal surface ofthe capsid. In good agreement with this prediction, changesS50N and D141N (both positively selected in branch D leadingto group PE-5) and change G53R (positively selected in the branchleading to isolates ChrIt.lam1/Italy/Cherry and AprIt.caf1/Italy/Apricotof group PV-96) imply net gains of positive charges. Therefore,changes increasing the strength of the RNA-CP electrostaticinteraction may have been selected during PNRSV diversification.

It has also been suggested that the amino acids around M110should play an important role during the formation of CP dimers(9, 52). This importance is confirmed by our finding of a stretchof four amino acids (Table 2, L114 to L117) under the actionof negative selection.

Our maximum-parsimony analysis of CP identified sites underpositive selection at the origin of the PE-5 group (Table 2,S49 to G53). Precisely in this region of PE-5 CP, Va $s$ kováet al. (52) have predicted upon bioinformatic grounds the existenceof an antigenic site. Amino acids K139 to Q142 (Table 2) havealso been identified as under positive selection in the branchleading to isolates PchIt.mry1/Italy/Peach and ChrIt.bla1/Italy/Cherryof the PE-5 group. The biological significance of these sitesis not obvious. However, since it is quite close to the regionincluding A148 to K152, which is (i) under negative selection(and hence likely has an important function) and (ii) predictedto be in the surface of the folded protein (data not shown),we can speculate that it may be involved in the formation ofthe MP-CP complexes that allow PNRSV cell-to-cell movement.This suggestion is supported by our covariation analyses, whichreveal that the C-terminal amino acids of MP are coevolvingwith the N-terminal region of CP (CP CG1). This suggestion getsfurther support from evidence for such contact in other membersof the Bromoviridae family such as CMV (26), BMV (47), and AMV(41, 43).

Comparison of Tables 2 and 3 shows that all but one (MP I261)of the amino acids involved in covariation groups have beenshown to be the target of natural selection. MP L253, E256,and E257 are under positive selection, as is the case for CPM52 and D141, whereas CP V48 is under purifying selection. Thisalmost perfect match suggests that selection acts upon the maintenanceof the right folded structures (i.e., within-molecule interactions)and interaction between proteins. From a practical standpoint,this match between the two types of analyses enhances the robustnessof the results.

Within the inherent limitations of the threading technique usedfor predicting MP and CP tertiary structures, it is worth notingthat the amino acids covarying between the proteins are notscattered along the molecules but it is possible to fit bothmolecules in space in such a way that the predicted intermolecularcovariations start making structural sense (Fig. 3). The threeamino acids belonging to MP CG plus MP E256 are all concentratedin the same surface region of the predicted globule, whereasthe three amino acids belonging to CP CG1 and CG2 are locatedin a predicted hypothetical cavity (CP M52 occupying a centrallocation) into which MP would fit to maximize the likelihoodof physical interactions between the two predicted between-proteincovariation groups.

The present in silico study opens new research avenues for researchersinterested in experimentally exploring the in vivo interactionbetween MP and CP. In particular, it suggests which sites canbe changed by site-directed mutagenesis to preclude the formationof MP-CP complexes. Also, site-directed mutagenesis studiesmay shed light on the importance of the sites identified hereas the target of selection.

ACKNOWLEDGMENTS

We thank F. Aparicio, V. Pallás, and J. A. Sánchez-Navarrofor comments and discussion.

This work was supported by grants from the Spanish MEC-FEDER(BMC2003-00066 and BFU2005-23720-E/BMC), the Generalitat Valenciana(GV04B280 and GRUPOS03/064), and the EMBO Young InvestigatorProgram to S.F.E. and from the Irish Science Foundation underthe President of Ireland Young Researcher Award program to M.A.F.F.M.C. was the recipient of fellowships from the CSIC I3P Bioinformaticsprogram and from the ESF Functional Genomics program.

FOOTNOTES

* Corresponding author. Mailing address: Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Avenida de los naranjos s/n, 46022 València, Spain. Phone: 34 963 877 895. Fax: 34 963 877 859. E-mail: sfelena{at}ibmcp.upv.es.

REFERENCES

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