BACKGROUND
plant viruses are obligate parasites that infect plants owing to specific interactions between virus and host factors that determine the plant susceptibility to viral infection  <cit> .
mutation or loss of one such susceptibility factor may result in virus resistance.
therefore, genes encoding susceptibility factors constitute potential targets for biotechnological and genomics-assisted breeding for improvement of crops resistance to viruses  <cit> .
throughout the last decade several susceptibility factors to plant viruses have been identified and characterized using model organisms as experimental systems  <cit> .
however, among these factors, only translation initiation factors of the 4e family  and eif <dig> 4g have been found to mediate resistance in the natural diversity of crops  <cit> .
in the host cell, eif4e is a part of the eif4f protein complex, which has an essential role in the initiation step of cap-dependent mrna translation.
in eukaryotes, most cellular mrnas contain terminal structures consisting of a 5'-cap and a 3'-poly tail which are brought together through interactions with translation initiation factors to promote translation  <cit> .
significantly, positive-sense single stranded rna viruses often lack the 5'-cap, the poly tail or both of these structures, yet they need to use the host translational machinery to translate their mrnas  <cit> .
indeed, mutagenesis of model hosts  <cit>  and the characterization of some natural recessive resistance genes  <cit>  have implicated eif4e as a susceptibility factor required for plant virus multiplication.
melon  is an economically important cucurbit crop cultivated in temperate, subtropical and tropical climates.
it is a diploid species  which has an estimated genome size of  <dig> mb.
virus resistance is a major melon breeding objective, as several diseases caused by viruses have great economical impact in melon crops worldwide.
significant examples include the cucumovirus  cucumber mosaic virus , the potyviruses watermelon mosaic virus , zucchini yellow mosaic virus , the ipomovirus  cucumber vein yellowing virus , the crinivirus cucurbit yellow stunting disorder virus  and the carmovirus  melon necrotic spot virus   <cit> .
despite this, not many natural resistance genes have been identified and introgressed into commercial melon cultivars.
probably, one of the most widely used is the nsv gene, which confers recessive resistance to all known strains of mnsv except to mnsv- <dig>  <cit> .
there are at least two known sources of resistance to mnsv in melon: the cultivar gulfstream and the korean accession pi  <dig>  both controlled by nsv  <cit> .
recently, we have characterised the nsv locus demonstrating that it encodes melon eif4e  and that a single amino acid change at position  <dig> of the protein leads to resistance to mnsv  <cit> .
in this paper, we present the work done for the identification and characterization of new nsv alleles that could be responsible of resistance to mnsv.
thus, we screened a collection of cucumis spp.
accessions for mnsv susceptibility and analysed by ecotilling the diversity of eif4e in this collection.
ecotilling is a variation of tilling  which has been successfully used to examine genetic variation in arabidopsis ecotypes  <cit>  and wild populations of populus trichocarpa  <cit> .
we found a notable conservation of the exonic regions of eif4e and showed that the only non-silent nucleotide change identified in c. melo accessions perfectly correlated with a phenotypic change in susceptibility to mnsv.
interestingly, a few accessions characterised in this work were previously identified as potential sources of resistance to viruses different than mnsv  <cit> .
a comparison of data on virus susceptibility and variability in eif4e suggested that other factors different than eif4e are probably involved in these resistances.
in addition, we have characterised a new eif4e allele from c. zeyheri  which, in a functional analysis, appeared to be potentially responsible for the resistance of plants of this species to mnsv.
RESULTS
phenotyping cucumis spp.
accessions for virus susceptibility
we tested  <dig> c. melo and  <dig> wild relative accessions of the germplasm collection of estación experimental "la mayora"- csic  for their susceptibility to mnsv strains mα <dig>   <cit>  and  <dig>   <cit>  and to cucumber vein yellowing virus   <cit> .
accessions were from different geographical origins:  <dig> from africa,  <dig> from america,  <dig> from central asia,  <dig> from europe ,  <dig> from the far east and india,  <dig> from middle east and the remaining  <dig> from unknown origins .
inoculations with mnsv showed that only one accession, c- <dig> , was resistant to both mnsv-mα <dig> and mnsv- <dig>  c. melo accessions c- <dig> and c- <dig>  c. dipsaceus c- <dig>  c. meeusii c- <dig> and c. anguria c- <dig> were resistant to mnsv-mα <dig>  but susceptible to mnsv- <dig>  symptoms on mnsv-inoculated cotyledons of susceptible accessions consisted of small necrotic lesions which appeared  <dig> to  <dig> days after inoculations .
accessions c. africanus c- <dig> and c- <dig>  c. prophetarum c- <dig>  c. ficifolius c- <dig>  cucumis spp.
c- <dig> and c- <dig>  despite of being susceptible, showed a very low average number of virus-induced lesions per inoculated cotyledon .
as reported by mallor et al.
<cit> , systemic symptoms appeared only in a proportion of the inoculated plants of susceptible accessions and consisted of small chlorotic spots in leaves that become necrotic a few days after appearance , and necrotic streaks along the stems and petioles.
the frequency of symptomatic plants varied with accessions.
moreover, a clear difference in the proportion of plants showing systemic symptoms after inoculation with mnsv-mα <dig>  and mnsv- <dig>  was observed , suggesting that mnsv-mα <dig> was more efficient than mnsv- <dig> in inducing systemic symptoms on mechanically inoculated plants.
inoculations with cvyv showed that all c. melo accessions tested were susceptible, and that c. africanus c- <dig>  c. dipsaceus c- <dig> and c- <dig> and c. prophetarum c- <dig> were resistant to this virus .
no symptoms could be observed on inoculated cotyledons of all accessions, except for c- <dig>  systemic symptoms in susceptible accessions consisted of foliar mosaics and vein yellowing in young, newly emerged leaves which appeared about  <dig> to  <dig> days after inoculations .
notably, resistance of c- <dig> plants was associated with the appearance of local necrotic lesions after cvyv mechanical inoculation , suggesting an hr-like type of response.
screening of eif4e polymorphisms by ecotilling
in order to scan the complete coding region of eif4e for natural sequence variation, three primer pairs to be used in ecotilling were designed on introns and on the 5' and 3' non-coding regions of the gene .
using these primers, we analysed a cucumis spp.
collection of  <dig> accessions previously characterised for their susceptibility to mnsv and cvyv .
out of the  <dig> accessions, no pcr product was obtained from eight c. melo wild relative accessions, despite several attempts using different pcr amplification conditions.
these eight accessions were thus excluded from further analysis.
pcr products obtained from the remaining accessions were mixed with pcr products amplified from the cultivar védrantais, which was chosen as reference, and analysed by ecotilling .
polymorphisms were observed in introns and exons, but only polymorphisms in exons were recorded.
six polymorphic sites were identified.
exons  <dig> and  <dig> contained  <dig> and  <dig> polymorphisms, respectively.
no polymorphism was observed in exons  <dig>   <dig> and  <dig>  considering polymorphisms, we classified the accessions in six different haplotypes, named h. <dig> to h. <dig> .
ninety seven accessions showed no polymorphisms in comparison to the reference, and were classified as haplotype h. <dig>  in contrast,  <dig> accessions showed polymorphisms and were grouped in  <dig> different haplotypes, h <dig> to h. <dig> .
the most frequent haplotypes, apart from h. <dig>  were h. <dig>  observed for  <dig> accessions and corresponding to a polymorphism in exon  <dig>  and h. <dig>  observed for  <dig> accessions and corresponding to two polymorphisms in exon  <dig> .
h <dig> likely derives from h. <dig> as both haplotypes have in common one polymorphism  .
we demonstrated previously that nsv codes for an allele carrying a single nucleotide polymorphism  in exon  <dig> of eif4e , and that this snp is responsible for resistance to mnsv  <cit> .
to estimate the frequency of the nsv allele, we analysed further by ecotilling the cucumis spp.
collection.
exon  <dig> was pcr amplified and heteroduplex dnas were generated using accession pi  <dig>  homozygous for nsv, as reference.
in this analysis, no cleaved product in exon  <dig> was observed from individuals of the h. <dig> haplotype and, thus, the nsv allele is represented by four accessions among the  <dig> tested.
d
a polymorphisms were observed in comparison with the cultivar védrantais  <cit> .
b exon harboring the snps/c position of the snps are given in reference to the translational start site.
d accessions are referred to by estación experimental "la mayora" code's.
e a dash indicates that no polymorphisms were identified.
variation of eif4e versus virus susceptibility
the precise position and the nature of identified polymorphisms were determined by sequencing pcr products comprising exons  <dig> and  <dig> for all accessions from haplotypes h. <dig> to h. <dig> .
this also served to confirm that ecotilling was precise enough to localise polymorphisms in exons.
accessions of the same haplotype in ecotilling exhibited the same nucleotide change .
only nucleotide change t683-a of accessions of the h. <dig> haplotype was non-silent and corresponded to amino acid change leu228-his.
therefore, a high degree of conservation of the eif4e protein was observed.
significantly, all accessions of the h. <dig> haplotype were resistant to mnsv-mα <dig>  whereas all other accession grouped in haplotypes different to h. <dig> were susceptible to this virus .
thus, a perfect correlation was found between amino acid change at position  <dig> of the eif4e protein and resistance to mnsv-mα <dig>
b
a as identified by díaz et al.
<cit>  and in this work.
b melon necrotic spot virus  strains mα <dig> and  <dig>  cucumber vein yellowing virus , cucumber mosaic virus , papaya ringspot virus strain w , watermelon mosaic virus  and zucchini yellow mosaic virus .
c s and r indicate susceptible and resistant accessions, respectively.
in addition to mnsv and cvyv, most of the accessions characterised here have been tested also for their susceptibility to cmv, papaya ringspot virus strain w , wmv and zymv  <cit> .
table  <dig> also includes accessions identified by díaz et al.
<cit>  as potential sources of resistance to these viruses.
except for the above mentioned case of mnsv, no correlation was found between variation of eif4e and virus resistance .
characterization of new eif4e resistance alleles
the only accession found to be resistant to both mnsv-mα <dig> and mnsv- <dig> during the phenotypic screening was c- <dig> .
c. zeyheri eif4e  exons were pcr amplified and sequenced.
sequence comparisons showed that cz-eif4e exon  <dig> showed no variation with respect to cm-eif4e-ved, the melon allele conferring susceptibility to mnsv  <cit> .
interestingly, exon  <dig> showed  <dig> polymorphisms able to give rise to  <dig> non-conservative amino acid changes.
given the implication of eif4e of diverse species in virus susceptibility  <cit> , we hypothesized that cz-eif4e could mediate c. zeyheri susceptibility to mnsv as cm-eif4e mediates melon susceptibility to this virus  <cit> .
our previous experience indicated that the co-expression of the melon susceptibility allele with the non-resistance breaking strain of mnsv in melon resistant plants indeed complements virus accumulation  <cit> .
therefore, we carried out a functional analysis based on the prediction that the co-expression of the susceptibility allele of cm-eif4e together with mnsv in c. zeyheri plants would complement virus accumulation.
appropriate dna constructs   <cit>  were bombarded into leaves of c. zeyheri plants and virus accumulation was assessed at  <dig> days post bombardment.
in the mnsv-mα <dig> case, we could not detect the accumulation of mnsv when it was bombarded alone or in combination with the melon resistance allele, but it was detected when it was bombarded together with the melon susceptibility allele .
in the mnsv- <dig> case, we detected the presence of the virus when it was bombarded alone, indicating that this strain can multiply, at least locally, in c. zeyheri tissues .
notably, mnsv- <dig> accumulation seemed to be stimulated when it was co-bombarded with the melon susceptibility allele .
discussion
use of ecotilling as a polymorphism discovery tool in melon
we have adapted and set up for the first time ecotilling in melon.
this technology was initially used to characterise the variability of  <dig> genes within a collection of arabidopsis ecotypes  <cit> .
then, it has been successfully used in analyses of the natural variability of wild populations of populus trichocarpa  <cit> , in the identification of allelic variation in resistance genes of barley  <cit>  and it is being used for genotyping in other species  <cit> .
used in combination with sequencing, ecotilling is a very cost-effective technology: once polymorphisms are identified by ecotilling, individuals can be grouped according to haplotype and only interesting haplotypes, and/or representatives from each haplotype, can be sequenced; in addition, ecotilling allows the approximate location of the polymorphism within the locus studied and, therefore, restricts the necessity of sequencing the complete locus but only regions around the polymorphism.
in our case, these reasons together with the low number of different haplotypes found have reduced in more than 90% the number of sequencing reactions potentially required to characterise the variability of eif4e in our collection of melon accessions.
due to the limited number of accessions characterised in this work, pooling dna from individual accessions  <cit>  was not necessary.
we expect that pooling would be feasible for c. melo accessions, but probably more difficult to apply when including wild melon relatives.
in fact, one of the major problems that we have encountered is the difficulty in pcr amplifying eif4e dna from melon wild relatives, probably caused by misspriming.
once solved this problem, ecotillling can be a potent tool for genetic analyses such as the study of heterozygosity in wild species, as it has been done for populus trichocarpa  <cit> .
variation in eif4e versus virus susceptibility
factor eif4e is highly conserved in eukaryotes.
the diversity found among factors from different organisms mainly resides at the amino-terminus of the protein, a region which may even have quite different lengths and which seems not to be directly involved in cap-binding  <cit> .
in agreement with these data, we have found a very low diversity among cucumis eif4e.
taking into consideration results from the characterization of cz-eif4e, the amino-terminus of cucumis eif4e appears to be also the region where amino acid changes accumulate preferentially.
however, our ecotilling results in c. melo uncover just one amino acid change, located at the very carboxy-terminus of the protein.
moreover, this change perfectly correlated with resistance to mnsv-mα <dig>  a result coincident with our previous observations  <cit> .
the eif4e carboxy-terminus, though outside of the cap-binding pocket, seems to have a critical role for functional regulation of cap binding through interactions with nucleotides downstream the cap  <cit> .
mnsv rna is uncapped, and our data indicate that a short non-coding region at the 3'-end of the viral rna  is critical for the outcome of the melon/mnsv interaction controlled by nsv, which encodes melon eif4e  <cit> .
a direct interaction between the virulence determinant and the eif4e carboxy-terminus probably controls translation initiation of mnsv rnas  and, thus, multiplication of the virus.
interestingly, accessions used in this work have been previously tested for their susceptibility to cmv, prsv-w, wmv and zymv, and potential sources for resistance to these viruses have been identified  <cit> .
for example, the accession c- <dig>  has been described as a potential source of resistance to wmv and to cmv and zymv aphid transmission  <cit>  and the genetics of c- <dig> resistance to wmv has been characterised in detail  <cit> .
however, our work has shown that all potential sources of resistance that have been analysed here, except those resistant to mnsv, have identical eif4e proteins.
it may be that the expression of eif4e in resistant accessions is somehow altered through mutations in control regions of the gene, but this possibility seems to be unlikely given the critical role that this protein has in general translation initiation.
therefore, other factors, including translation initiation factors different than eif4e, could control these resistances.
the case of pi  <dig> constitutes another interesting example.
this accession exhibited resistance to mnsv, cmv and prsv.
it would be possible that the mutation leu228-his in eif4e controlling mnsv resistance also controls resistance to the other two viruses.
however, this is unlikely, as accessions c- <dig> and c- <dig>  with the same mutation, are fully susceptible to cmv and prsv.
therefore, different or additional factors  must be involved in the pi  <dig> resistances to cmv and prsv.
new eif4e alleles for mnsv resistance
multiallelic, recessive resistance against plant viruses seems to be frequent , therefore we hypothesized that screenings to uncover the natural diversity of eif4e might contribute to the discovery of new resistance alleles that can be incorporated into resistance breeding programs.
however, in the case of mnsv resistance studied here, all c. melo accessions resistant to mnsv-mα <dig> corresponded to a unique genetic type, and none of the c. melo accessions analysed here were resistant to mnsv- <dig>  nevertheless, we identified one melon wild relative accession , corresponding to c. zeyheri, that was resistant to both mnsv strains.
significantly, the analysis of the cz-eif4e sequence showed  <dig> polymorphisms in exon  <dig> that result into  <dig> non-conservative amino acid changes located at the amino-terminus of the protein; none of these changes had a correspondence with the snp responsible for the change of mnsv susceptibility in melon  <cit> .
therefore, we hypothesized that cz-eif4e could be a new allele for resistance to mnsv.
the complementation experiments described in this paper allow speculation in this regard.
in nsv resistant melons, co-bombardment of mnsv-mα <dig> together with the melon susceptibility allele results in virus multiplication  <cit> .
similarly, here we observed that when c. zeyheri plants are co-bombarded with mnsv-mα <dig> and the melon susceptibility allele, virus multiplication could be detected, whereas co-bombardment with the melon resistance allele does not result in virus multiplication.
assuming that cz-eif4e has an expression pattern equivalent to that of cm-eif4e, these results strongly suggest that cz-eif4e is unable to contribute to mnsv-mα <dig> multiplication and, therefore, cz-eif4e may constitute the factor controlling resistance to mnsv-mα <dig> in c. zeyheri plants.
the situation seems to be different for mnsv- <dig>  on the one hand, there is an apparent contradiction between the results of the bombardment experiments and the phenotypic screening: bombardment of c. zeyheri plants with mnsv- <dig> showed that this viral strain can accumulate in inoculated leaves of c. zeyheri plants, while results of the phenotypic screenings indicated that this accession is resistant to mnsv- <dig>  this discrepancy may be due to differences in the inoculation and detection methods used in both assays or, alternatively, mnsv- <dig> movement might be restricted to the initial infection foci in c. zeyheri plants.
on the other hand, bombardment experiments have suggested that the presence of the c. melo eif4e susceptibility allele stimulates the mnsv- <dig> multiplication in c. zeyheri tissues.
to be fully understood, results concerning mnsv- <dig> bombardments on c. zehyeri tissues require additional experiments.
CONCLUSIONS
the low variability found for melon eif4e, together with data on the importance of eif4e as a virus susceptibility factor  <cit> , recommend approaching the generation of new eif4e alleles through mutagenesis.
high throughput identification of melon eif4e mutants should be feasible, and tilling could be an appropriate technology for this purpose.
our data has also pointed to the importance of considering additional candidate genes as susceptibility factors: resistance of cucumis spp.
accessions to different viruses seemed not to rely uniquely on eif4e.
thus, identification of new susceptibility factors in model species, together with phenotypic screenings of the natural species diversity, are activities of the outmost importance to identify new sources of virus resistance.

 plant viruses are obligate parasites that infect plants owing to specific interactions between virus and host factors that determine the plant susceptibility to viral infection. mutation or loss of one such susceptibility factor may result in virus resistance. therefore, genes encoding susceptibility factors constitute potential targets for biotechnological and genomics-assisted breeding for improvement of crops resistance to viruses. throughout the last decade several susceptibility factors to plant viruses have been identified and characterized using model organisms as experimental systems. however, among these factors, only translation initiation factors of the 4e family and eif 4g have been found to mediate resistance in the natural diversity of crops. in the host cell, eif4e is a part of the eif4f protein complex, which has an essential role in the initiation step of cap-dependent mrna translation. in eukaryotes, most cellular mrnas contain terminal structures consisting of a 5'-cap and a 3'-poly tail which are brought together through interactions with translation initiation factors to promote translation. significantly, positive-sense single stranded rna viruses often lack the 5'-cap, the poly tail or both of these structures, yet they need to use the host translational machinery to translate their mrnas. indeed, mutagenesis of model hosts and the characterization of some natural recessive resistance genes have implicated eif4e as a susceptibility factor required for plant virus multiplication. melon is an economically important cucurbit crop cultivated in temperate, subtropical and tropical climates. it is a diploid species which has an estimated genome size of mb. virus resistance is a major melon breeding objective, as several diseases caused by viruses have great economical impact in melon crops worldwide. significant examples include the cucumovirus cucumber mosaic virus, the potyviruses watermelon mosaic virus, zucchini yellow mosaic virus, the ipomovirus cucumber vein yellowing virus, the crinivirus cucurbit yellow stunting disorder virus and the carmovirus melon necrotic spot virus. despite this, not many natural resistance genes have been identified and introgressed into commercial melon cultivars. probably, one of the most widely used is the nsv gene, which confers recessive resistance to all known strains of mnsv except to mnsv-. there are at least two known sources of resistance to mnsv in melon: the cultivar gulfstream and the korean accession pi both controlled by nsv. recently, we have characterised the nsv locus demonstrating that it encodes melon eif4e and that a single amino acid change at position of the protein leads to resistance to mnsv. in this paper, we present the work done for the identification and characterization of new nsv alleles that could be responsible of resistance to mnsv. thus, we screened a collection of cucumis spp. accessions for mnsv susceptibility and analysed by ecotilling the diversity of eif4e in this collection. ecotilling is a variation of tilling which has been successfully used to examine genetic variation in arabidopsis ecotypes and wild populations of populus trichocarpa. we found a notable conservation of the exonic regions of eif4e and showed that the only non-silent nucleotide change identified in c. melo accessions perfectly correlated with a phenotypic change in susceptibility to mnsv. interestingly, a few accessions characterised in this work were previously identified as potential sources of resistance to viruses different than mnsv. a comparison of data on virus susceptibility and variability in eif4e suggested that other factors different than eif4e are probably involved in these resistances. in addition, we have characterised a new eif4e allele from c. zeyheri which, in a functional analysis, appeared to be potentially responsible for the resistance of plants of this species to mnsv.  phenotyping cucumis spp. accessions for virus susceptibility we tested c. melo and wild relative accessions of the germplasm collection of estación experimental "la mayora"- csic for their susceptibility to mnsv strains mα and and to cucumber vein yellowing virus. accessions were from different geographical origins: from africa, from america, from central asia, from europe, from the far east and india, from middle east and the remaining from unknown origins. inoculations with mnsv showed that only one accession, c-, was resistant to both mnsv-mα and mnsv- c. melo accessions c- and c- c. dipsaceus c- c. meeusii c- and c. anguria c- were resistant to mnsv-mα but susceptible to mnsv- symptoms on mnsv-inoculated cotyledons of susceptible accessions consisted of small necrotic lesions which appeared to days after inoculations. accessions c. africanus c- and c- c. prophetarum c- c. ficifolius c- cucumis spp. c- and c- despite of being susceptible, showed a very low average number of virus-induced lesions per inoculated cotyledon. as reported by mallor et al., systemic symptoms appeared only in a proportion of the inoculated plants of susceptible accessions and consisted of small chlorotic spots in leaves that become necrotic a few days after appearance, and necrotic streaks along the stems and petioles. the frequency of symptomatic plants varied with accessions. moreover, a clear difference in the proportion of plants showing systemic symptoms after inoculation with mnsv-mα and mnsv- was observed, suggesting that mnsv-mα was more efficient than mnsv- in inducing systemic symptoms on mechanically inoculated plants. inoculations with cvyv showed that all c. melo accessions tested were susceptible, and that c. africanus c- c. dipsaceus c- and c- and c. prophetarum c- were resistant to this virus. no symptoms could be observed on inoculated cotyledons of all accessions, except for c- systemic symptoms in susceptible accessions consisted of foliar mosaics and vein yellowing in young, newly emerged leaves which appeared about to days after inoculations. notably, resistance of c- plants was associated with the appearance of local necrotic lesions after cvyv mechanical inoculation, suggesting an hr-like type of response. screening of eif4e polymorphisms by ecotilling in order to scan the complete coding region of eif4e for natural sequence variation, three primer pairs to be used in ecotilling were designed on introns and on the 5' and 3' non-coding regions of the gene. using these primers, we analysed a cucumis spp. collection of accessions previously characterised for their susceptibility to mnsv and cvyv. out of the accessions, no pcr product was obtained from eight c. melo wild relative accessions, despite several attempts using different pcr amplification conditions. these eight accessions were thus excluded from further analysis. pcr products obtained from the remaining accessions were mixed with pcr products amplified from the cultivar védrantais, which was chosen as reference, and analysed by ecotilling. polymorphisms were observed in introns and exons, but only polymorphisms in exons were recorded. six polymorphic sites were identified. exons and contained and polymorphisms, respectively. no polymorphism was observed in exons and considering polymorphisms, we classified the accessions in six different haplotypes, named h. to h.. ninety seven accessions showed no polymorphisms in comparison to the reference, and were classified as haplotype h. in contrast, accessions showed polymorphisms and were grouped in different haplotypes, h to h.. the most frequent haplotypes, apart from h. were h. observed for accessions and corresponding to a polymorphism in exon and h. observed for accessions and corresponding to two polymorphisms in exon. h likely derives from h. as both haplotypes have in common one polymorphism. we demonstrated previously that nsv codes for an allele carrying a single nucleotide polymorphism in exon of eif4e, and that this snp is responsible for resistance to mnsv. to estimate the frequency of the nsv allele, we analysed further by ecotilling the cucumis spp. collection. exon was pcr amplified and heteroduplex dnas were generated using accession pi homozygous for nsv, as reference. in this analysis, no cleaved product in exon was observed from individuals of the h. haplotype and, thus, the nsv allele is represented by four accessions among the tested. d a polymorphisms were observed in comparison with the cultivar védrantais. b exon harboring the snps/c position of the snps are given in reference to the translational start site. d accessions are referred to by estación experimental "la mayora" code's. e a dash indicates that no polymorphisms were identified. variation of eif4e versus virus susceptibility the precise position and the nature of identified polymorphisms were determined by sequencing pcr products comprising exons and for all accessions from haplotypes h. to h.. this also served to confirm that ecotilling was precise enough to localise polymorphisms in exons. accessions of the same haplotype in ecotilling exhibited the same nucleotide change. only nucleotide change t683-a of accessions of the h. haplotype was non-silent and corresponded to amino acid change leu228-his. therefore, a high degree of conservation of the eif4e protein was observed. significantly, all accessions of the h. haplotype were resistant to mnsv-mα whereas all other accession grouped in haplotypes different to h. were susceptible to this virus. thus, a perfect correlation was found between amino acid change at position of the eif4e protein and resistance to mnsv-mα b a as identified by díaz et al. and in this work. b melon necrotic spot virus strains mα and cucumber vein yellowing virus, cucumber mosaic virus, papaya ringspot virus strain w, watermelon mosaic virus and zucchini yellow mosaic virus. c s and r indicate susceptible and resistant accessions, respectively. in addition to mnsv and cvyv, most of the accessions characterised here have been tested also for their susceptibility to cmv, papaya ringspot virus strain w, wmv and zymv. table also includes accessions identified by díaz et al. as potential sources of resistance to these viruses. except for the above mentioned case of mnsv, no correlation was found between variation of eif4e and virus resistance. characterization of new eif4e resistance alleles the only accession found to be resistant to both mnsv-mα and mnsv- during the phenotypic screening was c-. c. zeyheri eif4e exons were pcr amplified and sequenced. sequence comparisons showed that cz-eif4e exon showed no variation with respect to cm-eif4e-ved, the melon allele conferring susceptibility to mnsv. interestingly, exon showed polymorphisms able to give rise to non-conservative amino acid changes. given the implication of eif4e of diverse species in virus susceptibility, we hypothesized that cz-eif4e could mediate c. zeyheri susceptibility to mnsv as cm-eif4e mediates melon susceptibility to this virus. our previous experience indicated that the co-expression of the melon susceptibility allele with the non-resistance breaking strain of mnsv in melon resistant plants indeed complements virus accumulation. therefore, we carried out a functional analysis based on the prediction that the co-expression of the susceptibility allele of cm-eif4e together with mnsv in c. zeyheri plants would complement virus accumulation. appropriate dna constructs were bombarded into leaves of c. zeyheri plants and virus accumulation was assessed at days post bombardment. in the mnsv-mα case, we could not detect the accumulation of mnsv when it was bombarded alone or in combination with the melon resistance allele, but it was detected when it was bombarded together with the melon susceptibility allele. in the mnsv- case, we detected the presence of the virus when it was bombarded alone, indicating that this strain can multiply, at least locally, in c. zeyheri tissues. notably, mnsv- accumulation seemed to be stimulated when it was co-bombarded with the melon susceptibility allele. discussion use of ecotilling as a polymorphism discovery tool in melon we have adapted and set up for the first time ecotilling in melon. this technology was initially used to characterise the variability of genes within a collection of arabidopsis ecotypes. then, it has been successfully used in analyses of the natural variability of wild populations of populus trichocarpa, in the identification of allelic variation in resistance genes of barley and it is being used for genotyping in other species. used in combination with sequencing, ecotilling is a very cost-effective technology: once polymorphisms are identified by ecotilling, individuals can be grouped according to haplotype and only interesting haplotypes, and/or representatives from each haplotype, can be sequenced; in addition, ecotilling allows the approximate location of the polymorphism within the locus studied and, therefore, restricts the necessity of sequencing the complete locus but only regions around the polymorphism. in our case, these reasons together with the low number of different haplotypes found have reduced in more than 90% the number of sequencing reactions potentially required to characterise the variability of eif4e in our collection of melon accessions. due to the limited number of accessions characterised in this work, pooling dna from individual accessions was not necessary. we expect that pooling would be feasible for c. melo accessions, but probably more difficult to apply when including wild melon relatives. in fact, one of the major problems that we have encountered is the difficulty in pcr amplifying eif4e dna from melon wild relatives, probably caused by misspriming. once solved this problem, ecotillling can be a potent tool for genetic analyses such as the study of heterozygosity in wild species, as it has been done for populus trichocarpa. variation in eif4e versus virus susceptibility factor eif4e is highly conserved in eukaryotes. the diversity found among factors from different organisms mainly resides at the amino-terminus of the protein, a region which may even have quite different lengths and which seems not to be directly involved in cap-binding. in agreement with these data, we have found a very low diversity among cucumis eif4e. taking into consideration results from the characterization of cz-eif4e, the amino-terminus of cucumis eif4e appears to be also the region where amino acid changes accumulate preferentially. however, our ecotilling results in c. melo uncover just one amino acid change, located at the very carboxy-terminus of the protein. moreover, this change perfectly correlated with resistance to mnsv-mα a result coincident with our previous observations. the eif4e carboxy-terminus, though outside of the cap-binding pocket, seems to have a critical role for functional regulation of cap binding through interactions with nucleotides downstream the cap. mnsv rna is uncapped, and our data indicate that a short non-coding region at the 3'-end of the viral rna is critical for the outcome of the melon/mnsv interaction controlled by nsv, which encodes melon eif4e. a direct interaction between the virulence determinant and the eif4e carboxy-terminus probably controls translation initiation of mnsv rnas and, thus, multiplication of the virus. interestingly, accessions used in this work have been previously tested for their susceptibility to cmv, prsv-w, wmv and zymv, and potential sources for resistance to these viruses have been identified. for example, the accession c- has been described as a potential source of resistance to wmv and to cmv and zymv aphid transmission and the genetics of c- resistance to wmv has been characterised in detail. however, our work has shown that all potential sources of resistance that have been analysed here, except those resistant to mnsv, have identical eif4e proteins. it may be that the expression of eif4e in resistant accessions is somehow altered through mutations in control regions of the gene, but this possibility seems to be unlikely given the critical role that this protein has in general translation initiation. therefore, other factors, including translation initiation factors different than eif4e, could control these resistances. the case of pi constitutes another interesting example. this accession exhibited resistance to mnsv, cmv and prsv. it would be possible that the mutation leu228-his in eif4e controlling mnsv resistance also controls resistance to the other two viruses. however, this is unlikely, as accessions c- and c- with the same mutation, are fully susceptible to cmv and prsv. therefore, different or additional factors must be involved in the pi resistances to cmv and prsv. new eif4e alleles for mnsv resistance multiallelic, recessive resistance against plant viruses seems to be frequent, therefore we hypothesized that screenings to uncover the natural diversity of eif4e might contribute to the discovery of new resistance alleles that can be incorporated into resistance breeding programs. however, in the case of mnsv resistance studied here, all c. melo accessions resistant to mnsv-mα corresponded to a unique genetic type, and none of the c. melo accessions analysed here were resistant to mnsv- nevertheless, we identified one melon wild relative accession, corresponding to c. zeyheri, that was resistant to both mnsv strains. significantly, the analysis of the cz-eif4e sequence showed polymorphisms in exon that result into non-conservative amino acid changes located at the amino-terminus of the protein; none of these changes had a correspondence with the snp responsible for the change of mnsv susceptibility in melon. therefore, we hypothesized that cz-eif4e could be a new allele for resistance to mnsv. the complementation experiments described in this paper allow speculation in this regard. in nsv resistant melons, co-bombardment of mnsv-mα together with the melon susceptibility allele results in virus multiplication. similarly, here we observed that when c. zeyheri plants are co-bombarded with mnsv-mα and the melon susceptibility allele, virus multiplication could be detected, whereas co-bombardment with the melon resistance allele does not result in virus multiplication. assuming that cz-eif4e has an expression pattern equivalent to that of cm-eif4e, these results strongly suggest that cz-eif4e is unable to contribute to mnsv-mα multiplication and, therefore, cz-eif4e may constitute the factor controlling resistance to mnsv-mα in c. zeyheri plants. the situation seems to be different for mnsv- on the one hand, there is an apparent contradiction between the results of the bombardment experiments and the phenotypic screening: bombardment of c. zeyheri plants with mnsv- showed that this viral strain can accumulate in inoculated leaves of c. zeyheri plants, while results of the phenotypic screenings indicated that this accession is resistant to mnsv- this discrepancy may be due to differences in the inoculation and detection methods used in both assays or, alternatively, mnsv- movement might be restricted to the initial infection foci in c. zeyheri plants. on the other hand, bombardment experiments have suggested that the presence of the c. melo eif4e susceptibility allele stimulates the mnsv- multiplication in c. zeyheri tissues. to be fully understood, results concerning mnsv- bombardments on c. zehyeri tissues require additional experiments.  the low variability found for melon eif4e, together with data on the importance of eif4e as a virus susceptibility factor, recommend approaching the generation of new eif4e alleles through mutagenesis. high throughput identification of melon eif4e mutants should be feasible, and tilling could be an appropriate technology for this purpose. our data has also pointed to the importance of considering additional candidate genes as susceptibility factors: resistance of cucumis spp. accessions to different viruses seemed not to rely uniquely on eif4e. thus, identification of new susceptibility factors in model species, together with phenotypic screenings of the natural species diversity, are activities of the outmost importance to identify new sources of virus resistance. 
translation initiation factors of the 4e and 4g protein families mediate resistance to several rna plant viruses in the natural diversity of crops. a high conservation of eif4e exonic regions was found, with six polymorphic sites identified out of ecotilling accessions. was characterised for susceptibility to mnsv and cucumber vein yellowing virus and used for the implementation of ecotilling to identify new allelic variants of eif4e. we have also characterized a new allele of eif4e from cucumis zeyheri, a wild relative of melon. sequencing of regions surrounding polymorphisms revealed that all of them corresponded to silent nucleotide changes and just one to a non-silent change correlating with mnsv resistance. this study shows the applicability of ecotilling in cucumis spp., but given the conservation of eif4e, new candidate genes should probably be considered to identify new sources of resistance to plant viruses. functional analyses suggested that this new eif4e allele might be responsible for resistance to mnsv. part of the methodology described here could alternatively be used in tilling experiments that serve to generate new eif4e alleles. except for the mnsv case, no correlation was found between variation of eif4e and virus resistance, suggesting the implication of different and/or additional genes in previously identified resistance phenotypes.
 translation initiation factors of the 4e and 4g protein families mediate resistance to several rna plant viruses in the natural diversity of crops. particularly, a single point mutation in melon eukaryotic translation initiation factor 4e controls resistance to melon necrotic spot virus in melon. identification of allelic variants within natural populations by ecotilling has become a rapid genotype discovery method.  a collection of cucumis spp. was characterised for susceptibility to mnsv and cucumber vein yellowing virus and used for the implementation of ecotilling to identify new allelic variants of eif4e. a high conservation of eif4e exonic regions was found, with six polymorphic sites identified out of ecotilling accessions. sequencing of regions surrounding polymorphisms revealed that all of them corresponded to silent nucleotide changes and just one to a non-silent change correlating with mnsv resistance. except for the mnsv case, no correlation was found between variation of eif4e and virus resistance, suggesting the implication of different and/or additional genes in previously identified resistance phenotypes. we have also characterized a new allele of eif4e from cucumis zeyheri, a wild relative of melon. functional analyses suggested that this new eif4e allele might be responsible for resistance to mnsv.  this study shows the applicability of ecotilling in cucumis spp., but given the conservation of eif4e, new candidate genes should probably be considered to identify new sources of resistance to plant viruses. part of the methodology described here could alternatively be used in tilling experiments that serve to generate new eif4e alleles. 