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Keywords:

  • eukaryotic translation initiation factor 4E;
  • viral genome-linked protein;
  • potato virus Y;
  • tobacco etch virus;
  • Capsicum annuum;
  • recessive virus resistance

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Amino acid substitutions in the eukaryotic translation initiation factor 4E (eIF4E) result in recessive resistance to potyviruses in a range of plant species, including Capsicum spp. Correspondingly, amino acid changes in the central part of the viral genome-linked protein (VPg) are responsible for the potyvirus’s ability to overcome eIF4E-mediated resistance. A key observation was that physical interaction between eIF4E and the VPg is required for viral infection, and eIF4E mutations that cause resistance prevent VPg binding and inhibit the viral cycle. In this study, polymorphism analysis of the pvr2-eIF4E coding sequence in a worldwide sample of 25 C. annuum accessions identified 10 allelic variants with exclusively non-synonymous variations clustered in two surface loops of eIF4E. Resistance and genetic complementation assays demonstrated that pvr2 variants, each with signature amino acid changes, corresponded to potyvirus resistance alleles. Systematic analysis of the interactions between eIF4E proteins encoded by the 10 pvr2 alleles and VPgs of virulent and avirulent potato virus Y (PVY) and tobacco etch virus (TEV) strains demonstrated that resistance phenotypes arose from disruption of the interaction between eIF4E and VPg, and that viral adaptation to eIF4E-mediated resistance resulted from restored interaction with the resistance protein. Complementation of an eIF4E knockout yeast strain by C. annuum eIF4E proteins further shows that amino acid changes did not impede essential eIF4E functions. Altogether, these results argue in favour of a co-evolutionary ‘arms race’ between Capsicum eIF4E and potyviral VPg.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In recent years, several studies have implicated eukaryotic translation initiation factors in resistance against specific RNA viruses in both monocots and dicots, leading to the definition of a new class of natural virus resistance genes (Maule et al., 2007; Robaglia and Caranta, 2006). They differ from the major class of disease resistance genes encoding nucleotide binding site-leucine rich repeat proteins (R genes) by their recessive inheritance. Because the small genome of plant viruses generally encodes fewer than a dozen proteins, the viral infectious cycle largely relies on the use of cellular factors, making completion of the viral cycle a complex interplay between virus-encoded and host-encoded factors. Therefore, absence or inadequacy of a single host factor leads to full or partial resistance to viruses, and these non-functional versions of host factors result in recessive inheritance.

Although many host factors are required for plant virus infection (Kushner et al., 2003), molecular cloning of recessive resistance genes in crops including pepper, lettuce, pea, melon, barley and rice has so far only revealed a group of proteins linked to the translation machinery: the eukaryotic initiation factor 4E (eIF4E), and, to a lesser extent, the eukaryotic initiation factor 4G (eIF4G). eIF4E recognizes and binds the 5′ cap structure of mRNA (m7GpppN) and also binds to eIF4G to form the eIF4F complex. Additional translation initiation factors and the 40S ribosomal subunit are then recruited to initiate mRNA translation (Browning, 2004). In plants, several genes encode two protein sub-families, eIF4E and eIF(iso)4E, that have both overlapping and isoform-specific biological roles (Combe et al., 2005; Gallie and Browning, 2001; Rodriguez et al., 1998).

The majority of the recessive resistance controlled by eIF4E functions against potyvirus infection, although eIF4E has also been implicated in natural resistance to a Bymovirus (a genus related to Potyvirus) and a Carmovirus (Nieto et al., 2006; Stein et al., 2005). The genus Potyvirus is the largest among plant viruses and includes the most common and destructive viruses for a number of crops worldwide. The potyviral RNA is polyadenylated at its 3′-end and is covalently linked to a 25 kDa virus-encoded protein (VPg, viral genome-linked protein), replacing the cap structure of mRNAs at the 5′-end (Riechmann et al., 1992). VPg is cleaved from the NIa protein, comprising N-terminal VPg and C-terminal protease domains, and plays central roles in replicative and proteolytic functions (Revers et al., 1999). A key observation is the ability of the VPg (or the NIa precursor) to bind eIF4E isoforms in yeast two-hybrid and in vitro binding assays (Leonard et al., 2000, 2004; Schaad et al., 2000; Wittmann et al., 1997).

eIF4E-mediated recessive resistance against potyviruses results from a small number of amino acid changes, most of which are non-conservative (Gao et al., 2004; Kang et al., 2005; Nicaise et al., 2003; Ruffel et al., 2002, 2005; Yeam et al., 2007). Structural models predict that these mutations are on the surface of eIF4E and clustered in two neighbouring regions, one near the cap-binding pocket (hereafter named region I), and another rotated 90° from the cap-binding pocket (named region II) (Monzingo et al., 2007; Robaglia and Caranta, 2006). Recent analysis of the expression level of eIF4E mRNAs and proteins from various pepper genotypes confirmed that phenotypic differences in potyviral infection are determined by the amino acid changes themselves rather than other components regulating the expression or accumulation of eIF4E (Yeam et al., 2007). A further common feature linking eIF4E-mediated resistance to potyviruses is the identification of VPg as the virulence determinant. Amino acid mutations in the central part of the protein are responsible for the virus’s ability to overcome the recessive resistance controlled by eIF4E and cause a compatible interaction (Borgstrøm and Johansen, 2001; Moury et al., 2004). The exact mechanism by which eIF4E mutations control resistance remains to be elucidated, but recent protein–protein interaction studies carried out using yeast two-hybrid, glutathion S. transferase (GST) pull-down and bimolecular fluorescence complementation (BiFC) assays argue in favour of disruption or impairment of the direct interaction between eIF4E and VPg proteins. Therefore, a physical interaction between eIF4E and viral VPg is necessary for viral infection, and amino acid substitutions induce an altered function with respect to VPg binding (Kang et al., 2005; Yeam et al., 2007).

Host–pathogen interactions are an important force shaping organism diversity, and, due to the extreme intimacy between plant and virus life cycles, co-evolution, with complex and dynamic selection pressures on the genes involved in the interaction, is probable. In this respect, the interaction between Capsicum spp. and potyviruses provides an excellent system for functional and evolutionary studies of eIF4E-mediated resistance, because potyvirus infection is one of the major constraints to pepper cultivation worldwide and both the resistance protein and the viral ligand (avirulence protein) have been characterized at the molecular level. Among the five potyviruses reported to infect peppers, potato virus Y (PVY) is widespread throughout most of the cultivated areas, while tobacco etch virus (TEV) occurs mainly in North and Central America and the Caribbean (Green and Kim, 1991). Facing this pathogen diversity, resistance appears to be the usual outcome of interaction between Capsicum and potyviruses. Approximately 40% of Capsicum accessions are resistant to common strains of PVY, and a number of recessive genetic factors have been described and confer various spectra and levels of resistance against potyviruses (Kyle and Palloix, 1997; Palloix and Daubèze, INRA, France; unpublished results ). Among these, the pvr2 gene has been successfully used for decades in crop breeding programs as an effective and stable source of resistance (Greenleaf, 1986). pvr2 has been demonstrated to encode an eIF4E gene, and, to date, two resistance alleles, pvr21 and pvr22, have been identified in C. annuum, and one, pvr1, in C. chinense (pvr2 and pvr1 were recently shown to be alleles of the same gene; Kang et al., 2005; Ruffel et al., 2004, 2002). Interestingly, these alleles differ not only with regard to signature amino acid substitutions localized to regions I and II of eIF4E, but also by their resistance spectra. pvr21 is only effective against common strains of PVY (i.e. pathotype PVY-0), while pvr22 is effective against PVY-0 and -0,1 and also controls common strains of TEV. Finally, the pvr1 allele from C. chinense confers broad-spectrum resistance to common strains of PVY, TEV and pepper mottle virus (Kyle and Palloix, 1997). These allelic variants lead to a common mechanism of resistance, blocking viral accumulation in inoculated leaves (Deom and He, 1997; Ruffel et al., 2002). On the virus side, amino acid substitutions in the central part of VPg have been shown to determine the ability of PVY to infect pepper genotypes homozygous for the pvr21 or pvr22 resistance alleles (Moury et al., 2004). Similarly, the avirulence determinant of TEV has been identified as the VPg (Kang et al., 2005).

In this paper, we examine the pattern of polymorphism of the pvr2-eIF4E coding sequence among a set of 25 C. annuum accessions representing a worldwide sample from diverse origins. Ten different allelic variants were found for which all nucleotide polymorphisms resulted in amino acid changes, mostly clustered in regions I and II of the eIF4E protein. We performed a systematic functional analysis of the impact of these distinct combinations of amino acid substitutions on (i) the resistance spectrum against PVY and TEV, (ii) physical interaction with the VPg of avirulent and virulent PVY and TEV strains, and (iii) the ability to complement an eIF4E knockout yeast strain. Our results show that this extensive non-synonymous variation modulates interactions with potyviruses, and also provide functional evidence for co-evolution between C. annuum eIF4E and potyviral VPg.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Sequence analysis at pvr2-eIF4E identified an extensive non-synonymous variation

Coding sequence variability at pvr2-eIF4E was surveyed in 25 C. annuum accessions representing a worldwide sample from diverse geographic regions (Figure 1). The sample included YW, YY and F, for which resistance phenotypes against potyviruses and eIF4E cDNA sequences have previously been determined and assigned to the pvr2+, pvr21 and pvr22 alleles, respectively. pvr21 and pvr22 encode eIF4E resistance proteins with signature amino acid changes at positions 67, 79 and 109 (Kang et al., 2005; Ruffel et al., 2002).

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Figure 1.  Origin of the 25 Capsicum annuum genotypes used in this study.

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The sequenced coding regions span 687 bp from the start to stop codons and encode a predicted protein of 228 amino acids. The eIF4E coding sequences are intact in all genotypes, with no indel polymorphism. Ten allelic variants were identified based on nine polymorphic nucleotide sites localized in exon 1 [six single nucleotide polymorphisms (SNPs) between nucleotides 196 and 236], exon 2 (two SNPs at positions 319 and 325) and exon 4 (one SNP at position 614). Only two different nucleotides were present at each polymorphic site. For comparison with the polymorphism in non-coding regions, the sequences of eIF4E introns 2, 3 and 4 (representing 1347 bp) were compared between the 25 genotypes. Interestingly, there was a significantly higher proportion of polymorphic sites in the coding regions in comparison with non-coding regions (nine SNPs within 687 bp versus six SNPs within 1347 bp; χ2 test of homogeneity 4.65; P = 0.03).

A major observation from this study was that all SNPs lead to amino acid changes in the deduced eIF4E proteins (Table 1). Ten accessions from diverse geographic origins shared the same nucleotide and amino acid sequences as YW, previously assigned to pvr2+. Each new allelic variant was designated pvr2, with a numerical superscript for the new allele based on chronological precedent (pvr22), according to the nomenclature proposed by Kyle and Palloix (1997). This nomenclature was maintained for C. annuum alleles and will be used throughout the paper. In comparison with pvr2+, the nine other allelic variants presented between one and four amino acid substitutions, and all nine had a glutamate substitution at either position 67 or 68 of the protein. The pvr23 allele was observed three times; pvr21, pvr22 and pvr26 were present twice, and pvr24, pvr25, pvr27, pvr28 and pvr29 were observed only once (Table 1).

Table 1.   Amino acid substitution observed between deduced eIF4E protein sequences from the 25 Capsicum annuum genotypes and their corresponding alleles
C. annuum accessionsAllelesPosition of amino acid substitutions
55666768737479107109205
  1. aHDC69 is a doubled-haploid line obtained from the F1 hybrid (Criollo de Morelos 334 X YW) that possesses only a recessive resistance allele (from Criollo de Morelos 334) for resistance to PVY-0.

  2. bamino acids identical to those of eIF4E from Yolo Wonder.

  3. cadditional sequencing of eIF4E cDNA from distinct Florida VR2 plants coming from various seed batches showed that amino acid at position 79 is either a L or a R.

Yolo Wonder (YW)pvr2+TPVAAALGDD
Flambeaupvr2+b
PI187331pvr2+
Cuba3pvr2+
Pimiento Morronpvr2+
Antiboispvr2+
Bastidonpvr2+
Doux Long Landespvr2+
Lamupvr2+
Bousso1pvr2+
H3pvr2+
Yolo Y (YY)pvr21ER
Avelarpvr21ER
Florida VR2 (F)pvr22ER/LcN
Chay Angolanopvr22ERN
Perennialpvr23EG
HD-C69apvr23EG
PI201234pvr23EG
PI322719pvr24E
SC81pvr25TED
Maroc1pvr26EDDG
LP1pvr26EDDG
Serrano Vera Cruzpvr27ERG
PI195301pvr28ER
Chile de Arbolpvr29EDG
Resistance allele from C. chinensepvr1ATR

This survey identified seven new allelic variants within C. annuum and six new polymorphic sites at positions 66, 68, 73, 74, 107 and 205 of the eIF4E protein. Those at positions 66 (pvr25) and 107 (pvr28) were also identified in the eIF4E resistance protein encoded by the pvr1 allele from C. chinense (Table 1; Kang et al., 2005). None of the amino acid substitutions, except D109N, targeted amino acids involved in m7GTP binding, amino acids important for stabilizing the structure of the protein, or amino acids conserved in plants (boxed in red, black and green, respectively, in Figure 2). Based on sequence alignments, the D109N substitution in pvr21 and pvr22 corresponds to amino acid D96 of wheat eIF4E (Protein Data Bank accession number 2IDR), which is involved in binding the cap structure by stabilization of amino acid R158 via a salt bridge. According to the three-dimensional model for plant eIF4E (Monzingo et al., 2007), all substitutions are localized at, or near, the surface of the eIF4E protein, where mutations are tolerated and do not destabilize the core structure. Moreover, all except D109N corresponded to non-conservative substitutions in the respective proteins and to a change of a hydrophobic amino acid into a hydrophilic one, but none are predicted to have a major effect on overall folding of the protein (data not shown). One important observation is that all amino acid changes, except D205G, occurred within regions I and II on different faces of the eIF4E protein where the residues have been demonstrated to be involved in potyvirus or Bymovirus resistance (Robaglia and Caranta, 2006). Amino acid substitutions P66T, V67E, A68E, A73D, A74D and L79R were localized in region I, near the cap-binding site, and substitutions G107R and D109N were localized in region II, which is rotated 90° from the cap-binding pocket.

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Figure 2.  Alignment of amino acid sequences and secondary structure for wheat (Triticum aestivum) eIF4E and the predicted eIF4E protein from the Capsicum annuum Yolo Wonder genotype. Amino acid positions are numbered according to the predicted eIF4E protein sequence from C. annuum. Polymorphic amino acids identified in this study are shown in black boxes. Blue open boxes indicate the neighbouring regions previously demonstrated to be involved in resistance against potyviruses (Robaglia and Caranta, 2006). Amino acids involved in m7GTP binding are boxed in red. Amino acids important for stabilizing the structure of the protein are shown in black open boxes. ‘Plant-specific’ amino acids (i.e. conserved in more than 95% of plant species and different from those in other eukaryotes) are boxed in green (Monzingo et al., 2007). β-sheets are represented by arrows, α-helix by oscillations and turns by open squares.

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pvr23-eIF4E to pvr29-eIF4E correspond to potyvirus resistance alleles

To test whether amino acid changes identified in the new eIF4E allelic variants could be related to potyvirus resistance, a systematic study to characterize infectivity of PVY and TEV strains was conducted (Table 2). As expected, all plants from the genotype YW mechanically inoculated with PVY-LYE84, PVY-SON41, TEV-HAT or TEV-CAA10 presented obvious typical mosaic symptoms in apical non-inoculated leaves at 7 days post-inoculation (dpi) and exhibited high double antibody sandwich (DAS)-ELISA values at 30 dpi. The 10 genotypes belonging topvr2+-eIF4E presented a phenotype similar to that of YW, i.e. susceptible to all virus strains. The other genotypes were resistant to PVY-LYE84, with no systemic symptoms and no virus detected by DAS-ELISA at 30 dpi (Table 2). Six genotypes, belonging to the pvr25, pvr26, pvr27, pvr28 and pvr29 variants, were also resistant to PVY-SON41. For TEV, all accessions were susceptible to TEV-CAA10, and only accessions F and Chay Angolano with the pvr22 allele were resistant to TEV-HAT (Table 2).

Table 2.   Resistance phenotypes against PVY and TEV strains of pepper genotypes used in this study
GenotypeseIF4E allelesPVY-LYE84PVY-SON41TEV-HATTEV-CAA10
  1. aHD-C69 is a double haploid line (obtained from the F1 hybrid between Criollo de Morelos 334 and YW) that possesses only a recessive resistance allele (from Criollo de Morelos 334) for resistance to PVY-0.

  2. R, resistant; S, Susceptible.

Yolo Wonder (YW)pvr2+SaSSS
Flambeaupvr2+SSSS
PI187331pvr2+SSSS
Cuba3pvr2+SSSS
Bousso1pvr2+SSSS
Pimiento Morronpvr2+SSSS
H3pvr2+SSSS
Doux Long Landespvr2+SSSS
Lamupvr2+SSSS
Bastidonpvr2+SSSS
Antiboispvr2+SSSS
Yolo Y (YY)pvr21RSSS
Avelarpvr21RSSS
Florida VR2 (F)pvr22RSRS
Chay Angolanopvr22RSRS
Perennialpvr23RSSS
PI201234pvr23RSSS
HD-C69pvr23RSSS
PI322719pvr24RSSS
SC81pvr25RRSS
Maroc1pvr26RRSS
LP1pvr26RRSS
Serrano Vera Cruzpvr27RRSS
PI195301pvr28RRSS
Chile de Arbolpvr29RRSS

Genetic complementation analyses were then performed to determine whether PVY and TEV resistance is controlled by pvr2 allele(s) or not. In a first step, the recessive nature of the resistance was checked by resistantX susceptible crosses, and, in a second step, allelism relationships were evaluated by resistantX resistant crosses. In this case, if resistance is controlled by distinct loci, then only susceptible offspring should be recovered in F1 progeny; in contrast, if they are allelic, the F1 progeny should be uniformly resistant. Allelic relationships between PVY-resistant genotypes (YY, which is known to carry the pvr21 resistance allele, and Perennial, HD-C69, PI322719, SC81, Maroc 1, Serrano Vera Cruz, PI195301 and Chile de Arbol) were evaluated by inoculation with PVY-LYE84, and allelic relationships between TEV-resistant genotypes (F, carrying the pvr22 resistance allele, and Chay Angolano) were evaluated by inoculation with TEV-HAT. In each assay, plants of the susceptible control YW developed bright systemic mosaic symptoms characteristic of PVY or TEV infection on newly emerged non-inoculated leaves and accumulated PVY and TEV to high levels in both inoculated and non-inoculated leaves. F1 hybrids between the PVY-LYE84-resistant genotypes Perennial, HD-C69, PI322719, SC81, Maroc 1, Serrano Vera Cruz, PI195301 and Chile de Arbol and the susceptible genotype YW (pvr2+) were all susceptible to PVY-LYE84, demonstrating fully recessive inheritance for resistance to PVY-LYE84 (Figure 3). F1 hybrids between the same genotypes and the PVY-LYE84 resistant genotype YY (pvr21) were uniformly resistant to PVY-LYE84, showing that resistance is controlled by alleles of pvr2 (Figure 3). Similarly, the F1 progeny between YW and Chay Angolano were susceptible to TEV-HAT, indicating recessive inheritance, and the F1 progeny between the TEV-HAT-resistant genotypes F (pvr22) and Chay Angolano were resistant to TEV-HAT, indicating that resistance in Chay Angolano is controlled by pvr22.

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Figure 3.  Genetic analysis to examine the recessive nature of PVY-LYE84 or TEV-HAT resistance (F1 hybrids with YW-pvr2+) and allelism relationships with pvr2 (F1 hybrids with YY-pvr21 or with F-pvr22). F1 hybrids were inoculated with PVY-LYE84 or TEV-HAT and assayed for viral coat protein accumulation by DAS-ELISA at 30 dpi in non-inoculated leaves. The assay includes a healthy control (non-inoculated YW plants) and a susceptible control (inoculated YW plants). The DAS-ELISA result was considered positive when the absorbance value at 405 nm for the sample was at least three times greater than the mean value for the healthy controls. Error bars indicate standard error.

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Genetic analysis demonstrated that the PVY and TEV resistance identified in genotypes belonging to allelic variants pvr23-, pvr24-, pvr25-, pvr26-, pvr27-, pvr28- and pvr29-eIF4E were controlled by alleles at the pvr2 locus. In addition to distinct combinations of amino acid changes, these alleles differ with regard to their resistance spectrum against PVY and TEV strains. All alleles except pvr2+ control resistance to PVY-LYE84, which belongs to pathotype 0. In contrast, PVY-SON41, which is classified as pathotype 0,1,2 due to its capacity to break down the pvr21 and pvr22 resistance alleles (Moury et al., 2004), infects plants that are homozygous for pvr23 and pvr24 but not those that are homozygous for pvr25, pvr26, pvr27, pvr28 or pvr29. With regard to TEV resistance, only the pvr22 allele controls TEV-HAT in addition to PVY-LYE84. This resistance allele is overcome by the TEV-CAA10 strain.

Amino acid substitutions in pvr2-eIF4E impair physical interactions with VPg of avirulent PVY and TEV strains

With the dual objective of testing the impact of the distinct combinations of amino acid changes identified in eIF4E proteins on interaction with the viral VPg, and obtaining insights into molecular mechanisms underlying viral adaptation to eIF4E-mediated resistance (i.e. resistance breaking), we performed a systematic study of the physical interaction between the 10 eIF4E proteins encoded by alleles at the pvr2 locus and the VPg of the two PVY and two TEV strains. Recent studies have demonstrated that viral adaptation of PVY-SON41 to pvr21 and pvr22 resistance alleles results from five amino acid changes in the central part of the VPg cistron of PVY (Table 3; Moury et al., 2004). Similarly, VPg of the virulent strain TEV-CAA10 differs from that of TEV-HAT by 13 amino acids substitutions, four of which occur in the 23-codon region in the central part of the protein that is known to be involved in resistance breaking (Table 3). As previous results have shown that physical interaction between eIF4E and VPg is necessary for viral infection and that eIF4E resistance proteins show altered function with respect to VPg binding (Kang et al., 2005; Yeam et al., 2007), it is tempting to speculate that viral adaptation to eIF4E-mediated resistance results in restoration of the interaction with the resistance protein.

Table 3.   Amino acid differences in the central part of the VPg of PVY (Moury et al., 2004) and TEV strains used in this study
 Position of amino acid substitutionsa
101105115119123
  1. aNumbers are amino acid positions in the VPg.

PVY-LYE84SKMRN
PVY-SON41GRPYS
 111112113115 
TEV-HATIEPS 
TEV-CAA10LDHD 

Expression of the 10 pvr2-eIF4E coding sequences in yeast was determined by Western blot analysis, which showed that all proteins were stably expressed at similar levels (Figure 4). In three independent protein–protein interaction experiments, each conducted in triplicate, the fusion protein translated from pvr2+ (from YW) interacted in yeast with VPg proteins from the two PVY and TEV strains. pvr2+-eIF4E interactions with TEV VPg were stronger than those with PVY VPg, as yeast transformants complemented both adenine and histidine auxotrophy (Figure 5). In contrast, the pattern of interaction of eIF4E proteins translated from the pvr21 to pvr29 alleles differed depending on the VPg used (Figure 5 and Table 4). eIF4E encoded by pvr21, pvr23 and pvr24 interacted with VPg of PVY-SON41, TEV-HAT and TEV-CAA10, but failed to interact with VPg from PVY-LYE84. eIF4E encoded by pvr22 interacted with VPg from PVY-SON41 and TEV-CAA10 but not with those from PVY-LYE84 or TEV-HAT. Finally, eIF4E proteins encoded by pvr25, pvr26, pvr27, pvr28 and pvr29 interacted with VPgs from the two TEV strains but not with VPgs from the two PVY strains. The results of the interaction assays between pvr21-, pvr22-, pvr23- and pvr24-eIF4E and VPg of PVY-LYE84 and PVY-SON41, and between pvr22-eIF4E and VPg of TEV-HAT and TEV-CAA10, are of particular interest. These proteins control resistance to PVY-LYE84 and TEV-HAT for pvr22, but are overcome by PVY-SON41 and TEV-CAA10. In all cases, the VPg of avirulent strains failed to interact with the eIF4E resistance proteins encoded by pvr21, pvr22, pvr23 and pvr24, whereas the VPg of virulent strains interacted with these resistance proteins.

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Figure 4.  Expression of eIF4E proteins in the yeast two-hybrid system. Lane 1, SDS–PAGE molecular weight standard (low range; Bio-Rad, http://www.bio-rad.com/); lane 2, yeast cells with empty vector; lanes 3–12, eIF4E fusion gene in the pGADT7 vector. (a) Yeast total protein extracts obtained using the urea/SDS method. (b) Western blot analysis using anti-HA antibody.

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Figure 5.  eIF4E protein–VPg protein interaction assay using the yeast two-hybrid system. Examples of eIF4E–VPg protein interactions detected by the yeast two-hybrid system. Yeast expressing both ‘bait’ and ‘prey’ recombinant proteins were obtained by first transforming yeast cells with the individual plasmid construction followed by separate conjugation between yeasts expressing eIF4E proteins and those expressing VPg proteins. Conjugations were then cultured on control plates (−LW) and on two selective media lacking leucine, tryptophan and histidine (−LWH) or leucine, tryptophan, histidine and adenine (−LWHA). Each plasmid combination was spotted in triplicate. Positive and negative controls from the Matchmaker GAL4 two-hybrid system 3 were used (top panel). The TEV-HAT NIa/TEV-HAT NIb interaction (Schaad et al., 2000) was also used as positive control in each experiment.

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Table 4.   Summary of interactions detected by the yeast two-hybrid system between Capsicum annuum eIF4E proteins and VPg proteins, together with the resistance phenotype against PVY and TEV strains controlled by the allelic variants at the pvr2 locus
C. annuum genotypeseIF4E haplotypesPotyvirus strains
PVY-LYE84PVY-SON41TEV-HATTEV-CAA10
PhenotypeaVPg-eIF4E interactionbPhenotypeVPg-eIF4E interactionPhenotypeVPg-eIF4E interactionPhenotypeVPg- eIF4E interaction
  1. aS, susceptible; R, resistant.

  2. b−, no interaction; +, interaction on SD-LWH medium; ++, interaction on SD-LWHA medium.

YWpvr2+S+S+S++S++
YoloYpvr21R-S+S+S+
Florida VR2pvr22R-S+R-S+
Perennialpvr23R-S+S+S+
PI322719pvr24R-S+S+S+
SC8Ipvr25R-R-S+S+
Maroclpvr26R-R-S+S+
Serrano Vera Cruzpvr27R-R-S+S+
PI195301pvr28R-R-S+S+
Chile de Arbolpvr29R-R-S+S+

In summary, these results identified four patterns of interaction between the 10 eIF4E proteins and the VPg proteins from four strains that perfectly match the resistance/susceptibility phenotypes controlled by alleles at the pvr2 locus: susceptibility correlated with interaction with the VPg of virulent PVY and TEV strains, whereas resistance correlated with impaired/disrupted interaction with the VPg of avirulent strains (Table 4). This systematic analysis of eIF4E/VPg interactions confirmed and expanded the observation that eIF4E proteins corresponding to resistance alleles showed altered function with respect to VPg binding. These data also support the hypothesis that overcoming eIF4E-mediated resistance arises from restoration of the interaction with VPg.

pvr2-eIF4E recapitulates essential eIF4E functions in yeast

To address the functional consequences of amino acid substitutions identified in pvr2-eIF4E proteins encoded by the allelic variants, we analysed whether these proteins were able to rescue the growth of the haploid yeast strain JO55 in which the sole chromosomal copy of eIF4E has been deleted and replaced by human eIF4E (Altmann et al., 1989). Because expression of the human eIF4E cDNA required for yeast growth is galactose-dependent, this system allows in vivo functional assessment of any eIF4E coding sequence under the control of a glucose-inducible promoter (Hughes et al., 1999) and has been used successfully to address the function(s) of plant eIF4E isoforms (Rodriguez et al., 1998).

Empty JO55 yeast strains or JO55 transformed with an empty p424GBP/TRP1 vector were used as negative controls. The positive control was JO55 transformed with p424GBP/TRP1:At-eIF4E (At4g18040). The 10 pvr2-eIF4E coding sequences from susceptible (pvr2+) and resistant (pvr21 to pvr29) genotypes allowed growth of the yeast strain in medium containing glucose in a similar manner to A. thaliana eIF4E cDNA (Figure 6). The ability of these exogenous eIF4Es to complement the eIF4E knockout yeast strain indicated that C. annuum eIF4E shares functions with yeast and human eIF4Es, and that amino acid substitutions identified in resistance proteins do not impede essential eIF4E functions, including those linked to mRNA translation.

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Figure 6.  Complementation of yeast strain JO55 with Capsicum annuum eIF4E cDNAs. The yeast strain JO55 was transformed with plasmids p424GBP/TRP1:eIF4E (pvr21 to pvr29). Control transformations were performed with no DNA (empty JO55) or the empty plasmid p424GBP/TRP1 (negative controls) and with p424GBP/TRP1:At-eIF4E (At4g18040) (positive control). Equal amounts of transformed yeast cells were plated on appropriate selective nutrient drop-out media containing glucose. Cells were grown for 10 days at 30°C.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This study, which combines genetic diversity and functional analyses, provides evidence for co-evolution between C. annuum eIF4E and potyviral VPg. The nucleotide variation analysis at the pvr2-eIF4E locus identified solely non-synonymous mutations and a significantly higher proportion of polymorphic sites in coding regions in comparison with non-coding regions (i.e. introns) of the gene. These two features strongly suggest that this gene is subjected to diversifying selection. Resistance assays and genetic complementation analysis demonstrated that nine of the 10 identified allelic variants correspond to potyvirus resistance alleles, each with signature amino acid substitutions. The majority of these alleles control PVY, although pvr22 also confers resistance to the common strain of TEV. This is in agreement with previous surveys for potyvirus resistance in Capsicum spp., which show a high proportion of resistance to PVY and a much lower proportion of resistance to TEV.

Each resistance protein encoded by the nine allelic variants carried a distinct combination of non-conservative amino acid changes largely localized on the surface of eIF4E in regions I and II, which have been demonstrated to be targets in virus resistance (Robaglia and Caranta, 2006). Previous studies identified one or two eIF4E resistance alleles per species with signature amino acid substitutions in regions I or II (in pepper, lettuce and pea) or both (in pepper, tomato and pea) (Gao et al., 2004; Kang et al., 2005; Nicaise et al., 2003; Ruffel et al., 2002, 2005). Our data illustrate the occurrence and maintenance of a great diversity of combinations of amino acid substitutions leading to resistance, and also emphasize the importance of amino acid changes in region I with respect to resistance in C. annuum, at least against PVY, as resistance alleles mainly control this potyvirus. Indeed, within region I, all resistance proteins shared one or two (pvr25) amino acid substitutions at positions 66–68, with additional changes at positions 73 and/or 74 (in pvr25, pvr26, pvr29) or 79 (in pvr21, pvr22, pvr27). Interestingly, the allelic variant pvr24 identified in a single accession showed a single amino acid substitution (V67E), indicating that this change alone is sufficient to compromise PVY infection. Two variants (pvr22 and pvr28) showed an additional amino acid change in region II of the protein (G107R or D109N), and only pvr22 was involved in resistance to TEV in addition to PVY. Finally, four variants shared the D205G mutation in the C-terminal region of eIF4E. Although the N- and C-termini of eIF4E are more polymorphic than the core of eIF4E (represented by approximately 170 amino acids; Joshi et al., 2005), amino acid changes in the C-terminal region have not been described in potyvirus resistance proteins from other species. However, a single amino acid change mapped to the C-terminal part of eIF4E was recently demonstrated to control recessive resistance of melon to melon necrotic spot virus (genus Carmovirus) through a mechanism distinct from the one acting against potyviruses (Nieto et al., 2006). The conservation between unrelated plant species with regard to the position of mutations leading to potyvirus resistance, together with the fact that carmoviruses can infect peppers, could suggest an involvement of D205G in another eIF4E-mediated resistance system targeted against a distinct viral genus.

Large-scale analysis of the interactions between eIF4Es and VPgs from two potyviruses demonstrates that the resistance phenotypes against PVY and TEV arise from disruption of the direct interaction between eIF4E and VPg, obviously linking amino acid mutations and altered function with respect to VPg binding. The occurrence of mutations in two regions on different faces of the eIF4E molecule suggests that VPg may have two binding sites for optimal interaction. However, the results of interaction studies performed with the eIF4E protein encoded by the pvr24 resistance allele demonstrate that disruption of a single contact in region I of the protein is sufficient to abolish the binding ability of the VPg of PVY. These data, together with the systematic occurrence of amino acid changes in region I, support the key role of mutations in this particular region with regard to VPg binding and the outcome of the interaction with PVY. A recent functional analysis conducted in Capsicum using eIF4E constructs with induced point mutations affecting various amino acids has identified the amino acid change G107R in region II (initially identified in the C. chinense pvr1 resistance allele) to be the sole change responsible for abolishing binding ability of the TEV-HAT VPg (Yeam et al., 2007). It is striking that, in our study, the pvr28 allele was shown to carry amino acid changes A68E and G107R but was fully susceptible to TEV-HAT; in yeast two-hybrid experiments, we also observed that the eIF4E protein encoded by pvr28 binds to the VPg of TEV-HAT. A possible explanation for these contradictory observations could be that the amino acid change A68E compensates for the G107R effect. Among naturally occurring eIF4E resistance alleles against potyviruses, pot1 from tomato and sbm11 from pea have amino acid substitutions in regions I and II, mo12 from lettuce and sbm12 from pea have mutations in region I only, and mo11 from lettuce has mutations in region II only (Gao et al., 2004; Nicaise et al., 2003; Ruffel et al., 2005). Altogether, these data strongly indicate that the precise contact point between eIF4E and potyviral VPg is optimized for each potyvirus, even within a single plant species, as, in pepper, the mutation V67E in region I is responsible for disruption of binding of the PVY VPg, whereas the mutation G107R in region II is responsible for disruption of binding of the TEV VPg.

One important result from the present study is that viral adaptation to eIF4E-mediated resistance through amino acid changes in the central part of the VPg results from restoration of the physical interaction with the resistance protein. As two eIF4E genes and one eIF(iso)4E homologue have been identified in the pepper genome (Kang et al., 2005; Ruffel et al., 2002, 2006), and analyses conducted in pepper and A. thaliana showed that potyviruses differ in their ability to use eIF4E isoforms (Duprat et al., 2002; Lellis et al., 2002; Ruffel et al., 2006; Sato et al., 2005), two functional models for virulence may be proposed. Amino acid changes in the VPg of PVY and TEV may allow overcoming of resistance alleles at the pvr2 locus either (i) by restoring the interaction with a mutated eIF4E resistance protein, or (ii) by de novo interaction with another eIF4E isoform or an alternative partner. Our protein–protein interaction experiments obviously argue in favour of the first hypothesis, because the VPg of avirulent PVY-LYE84 and TEV-HAT strains failed to interact with the eIF4E resistance proteins encoded by pvr21, pvr22, pvr23 and pvr24 but the VPg of virulent PVY-SON41 and TEV-CAA10 strains did interact with these resistance proteins. This is also in agreement with the demonstration that transient expression of pvr21-eIF4E in pvr22 plants promoted accumulation of PVY pathotype 0,1 (Ruffel et al., 2002). However, recent data obtained using the A. thaliana–turnip mosaic virus pathosystem showed that a single amino acid change in the central domain of the turnip mosaic virus VPg permits overcoming of the complete resistance of an A. thaliana mutant bearing a transposon-induced null allele for the eIF(iso)4E gene (German-Retana, Revers and C. Caranta, INRA, France; unpublished data). As this A. thaliana line completely lacks both eIF(iso)4E mRNA and protein (Duprat et al., 2002), and given the hypothesis that eIF4E is required for potyvirus infection, these results argue in favour of mutations conferring on VPg a new or increased affinity for another eIF4E isoform or alternative susceptibility factors. Altogether, these data indicate that potyviruses have probably developed various strategies to interact with the host’s translational apparatus and to counter eIF4E-mediated resistance through mutations in VPg.

The main function of eIF4E is cap binding during initiation of host protein synthesis, although other functions in cell growth and the cell cycle have been identified, such as the regulation of export of some mRNA from the nucleus (Culjkovic et al., 2007). Therefore, a prerequisite for the maintenance of amino acid variations leading to resistance is that the resistance gene can accumulate mutations that affect recognition of the viral VPg without imposing a significant fitness cost by impairing mRNA translation and/or other eIF4E functions. Complementation of the eIF4E knock out yeast strain by C. annuum eIF4E proteins showed that amino acid changes identified in resistance proteins do not drastically impair mRNA translation in yeast. Our results therefore extend those of Kang et al. (2005) showing that resistance proteins encoded by pvr21 and pvr22 maintain an in vitro cap-binding activity. However, the eIF4E proteins encoded by the pvr1 allele from C. chinense and the sbm1 allele from pea have been demonstrated to abolish cap binding (Gao et al., 2004; Kang et al., 2005), and the C. annuum pvr6 resistance locus was found to be a null allele of eIF(iso)4E (Ruffel et al., 2006). This suggests that a trade-off between potyvirus resistance and eIF4E functions can result in defective eIF4E proteins. A recent study of in vitro interactions indicated that the cap analogue m7GDP and the VPg of lettuce mosaic virus bind to lettuce eIF4E at two partially overlapping sites; however, the binding of one ligand reduces the affinity for the other by about 15-fold (Michon et al., 2006). Therefore, partial overlap between the VPg binding domain and the cap-binding domain, together with the observation that the precise contact point between eIF4E and potyviral VPg is optimized for each potyvirus, may explain these differences in the maintenance of cap-binding activities. Nevertheless, the occurrence of several eIF4E proteins in plants and their ability to compensate for one another (Combe et al., 2005; Duprat et al., 2002; Gallie and Browning, 2001; Rodriguez et al., 1998) probably modulates the overall impact of the decreased cap-binding ability of some eIF4E resistance proteins on plant fitness, and allow, indirectly, evolution of resistance.

In conclusion, a limited number of studies have been performed that combine polymorphism analysis and functional analysis, and these are of particular interest in obtaining insights into antagonistic relationships between plants and pathogens. In this study, the observed exclusive non-synonymous variations, associated with their great diversity, provide evidence for diversifying selection acting on the pvr2-eIF4E locus. In addition, the established functional role of this variation in potyvirus resistance suggests that this selective pressure is exerted, at least partially if not totally, by phytopathogenic potyviruses. On the virus side, amino acids of the central domain of the VPg avirulence protein have also been demonstrated to be subject to positive selection (Moury et al., 2004). Large-scale yeast two-hybrid assays indicate that the outcome of the eIF4E–VPg interaction is a key determinant of potyviral infection, and that virulence arises from restoration of the interaction with the mutated resistance protein. Altogether, our results argue in favour of a co-evolutionary ‘arms race’ between the eIF4E resistance protein and the VPg avirulence protein. Although we are considering to a particular class of resistance genes distinct from classical dominant R genes, our results are in agreement with the co-evolution model proposed for the flax L gene–Melampsora lini system (Dodds et al., 2006) and the tomato Pto gene–Pseudomonas syringae system (Rose et al., 2007). In these cases, direct interaction between resistance and avirulence proteins is the driving force for co-evolution between resistance and avirulence, leading to diversification of both genes.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant germplasm and progenies

Twenty-five accessions of C. annuum were chosen to create a worldwide sample from diverse geographic regions (Figure 1). Efforts were made to sample broadly and to avoid relationships between genotypes. The sample includes the homozygous cultivars Yolo Wonder (YW), YoloY (YY) and Florida VR2 (F), carrying the pvr2+, pvr21 and pvr22 alleles, respectively, and local populations from primary and secondary diversification centres. For local populations, seeds from single individuals were harvested to create single-seed stocks to produce the plant material used.

F1 hybrids between YW and PVY-0-resistant genotypes representing the eIF4E allelic variants (Perennial, HD-C69, PI322719, SC81, Maroc 1, Serrano Vera Cruz, PI195301 and Chile de Arbol) were produced to determine the recessive versus dominant nature of the resistance. F1 hybrids between the same genotypes and YY were produced to determine whether PVY-0 resistance was controlled by an allele of pvr2. Allelism for TEV-HAT resistance was assessed using the F1 hybrid between F and Chay Angolano.

Potyvirus strains and disease resistance evaluation

All plants were grown under greenhouse conditions and transferred into growth chambers before inoculation. The reactions of all C. annuum accessions to PVY and TEV were determined by mechanical inoculation of 20 plants per genotypes at the cotyledon stage using PVY-LYE84 (pathotype PVY-0; Moury et al., 2004), PVY-SON41 (pathotype PVY-0,1,2; Moury et al., 2004), TEV-HAT (Schaad et al., 2000) and TEV-CAA10 (a gift from B. Moury, INRA). PVY and TEV strains were maintained on C. annuum Yolo Wonder and Datura stramonium, respectively, and transferred every 4–8 weeks. Inoculum and mechanical inoculation procedures were as described previously (Caranta and Palloix, 1996). Thirty days post-inoculation (dpi), systemic infection was assayed by determining the presence/absence of symptoms on non-inoculated leaves and confirmed by DAS-ELISA using PVY or TEV antibodies. For genetic complementation analysis, F1 hybrids were evaluated for resistance to PVY-LYE84 and TEV-HAT.

Amplification, sequencing and sequence analysis of eIF4E cDNAs and partial genomic DNAs

Total RNAs were isolated from pepper leaf tissues using TRI-reagent (Sigma-Aldrich, http://www.sigmaaldrich.com/). eIF4E cDNAs from genotypes YW, YY and F (GenBank accession numbers AY122052, AF521964 and AF521965) were re-sequenced as controls. The eIF4E cDNAs from the 25 accessions were obtained by RT-PCR using primers based on the 5′- and 3′-NTR regions of the YW eIF4E cDNA (forward primer 5′-AAAAGCACACAGCACCAACA-3′; reverse primer 5′-GATTAGAAGTGCAAACACCAATAC-3′) and the following PCR conditions: 94°C for 30 sec, 53°C for 30 sec, 68°C for 1 min, for 30 cycles. In order to compare polymorphism between coding and non-coding regions, genomic DNA was isolated from leaf tissues using the CTAB extraction method (Fulton et al., 1995), and partial eIF4E genomic DNA from the 25 accessions was amplified by PCR using primers based on exons 2 and 5 (forward primer 5′-GAAGATCCTGTATGTGCCAATG-3′; reverse primer 5′-CTGTGTAACGATTCTTTGC-3′). The PCR conditions were: 94°C for 3 min, 35 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min, followed by 72°C for 5 min.

Each amplification product was sequenced twice using the INRA sequencing platform at Montpellier using an ABI 3130XL POP7 sequencer (S. Santoni, INRA Montpellier, France). Nucleotide sequences were analysed using genalys win3.3.24a software (http://software.cng.fr) and bioedit (Hall, 1999). The nucleotide and amino acid sequences were aligned using clustal w (Thompson et al., 1994). The GenBank accession numbers for the eIF4E cDNA sequences are AY122052 (pvr2+), AF521964 (pvr21), AF5211965 (pvr22), AY723737 (pvr23), AY723738 (pvr24), AY723739 (pvr25), AY723740 (pvr26), EU106863 (pvr27), EU106864 (pvr28) and EU106865 (pvr29).

A predicted two-dimensional structure of the pepper YW eIF4E protein was obtained using the known structure of wheat (Triticum aestivum) eIF4E (Protein Data Bank accession number 2IDR) as the template (Monzingo et al., 2007). It was used to map all amino acid changes observed between resistant and susceptible genotypes.

Yeast two-hybrid analysis

eIF4E, NIa, VPg and NIb (the RNA-dependent RNA polymerase) coding sequences were amplified by PCR using high-fidelity Platinum Taq polymerase (GIBCO/BRL Life Technologies; http://www.invitrogen.com) from oligo(dT)-primed reverse transcription products. Gene-specific primers were designed to introduce restriction enzyme sites. The VPg cistron was amplified from the NIa PCR product using a reverse primer incorporating a stop codon at the end of the coding sequence. PCR products were cloned into pGEMT-easy vectors (Promega, http://www.promega.com/) and sequenced. Coding sequences were then subcloned in-frame with the GAL4 activation domain or the GAL4 binding domain into the pGADT7 or pGBKT7 vectors, respectively (Clontech, http://www.clontech.com/). All pGADT7- and pGBKT7-derived vectors were sequenced using primer T7 to check orientation and in-frame insertion.

eIF4E coding sequences corresponding to pvr2+, pvr21, pvr22, pvr23, pvr24, pvr25, pvr26, pvr27, pvr28 and pvr29 alleles were amplified from YW, YY, F, Perennial, PI322719, SC81, Maroc 1, Serrano Vera Cruz, PI195301 and Chile de Arbol, respectively. NIa and VPg cistrons were amplified from PVY-LYE84, PVY-SON41, TEV-HAT and TEV-CAA10; the NIb cistrons were amplified from PVY-LYE84 and TEV-HAT.

The Matchmaker GAL4 two-hybrid system 3 (Clontech) was used according to protocols described in the Clontech Yeast Protocol Handbook. pGADT7- and pGBKT7-derived vectors were transformed into AH109 and Y187 yeast strains, respectively, which contain two independent reporter genes HIS3 and ADE2 (to confer histidine and adenine auxotrophy, respectively) driven by hybrid GAL4 promoters. After yeast mating, large double-transformed yeast colonies were resuspended in 100 μl sterile water, and 10 μl aliquots were spotted onto various selective media including synthetic medium lacking leucine and tryptophan (hereafter named −LW), medium lacking leucine, tryptophan and histidine (−LWH) and medium lacking leucine, tryptophan, histidine and adenine (−LWHA). Strong interactions are detected on medium lacking both histidine and adenine, whereas weak interactions are detected on medium lacking histidine. This feature permits a robust and reliable semi-quantitative assay of the strength of the interaction using selective media. Plates were kept at 30°C. Growth was checked daily from 2 to 7 days after spotting. For negative controls, pGADT7 and pGBKT7 plasmids were used for prey and bait, respectively. Interaction between murine p53 and SV40 large T antigen (controls from the Matchmaker GAL4 two-hybrid system 3) and iinteraction between NIa and NIb of TEV-HAT (demonstrated by Altmann et al., 1989 and Schaad et al., 2000) were used as positive controls.

For Western blots, yeast total protein extractions were performed as described in the Clontech Yeast Protocol Handbook (urea/SDS method). Equal loads of protein extracts were electrophoresed by 12% SDS–PAGE and blotted onto Hybond ECL nitrocellulose membranes (Amersham Biosciences, http://www5.amershambiosciences.com/). The membrane was first incubated with HA monoclonal antibody (12CA5, Roche; http://www.roche.com) at a dilution of 1/1000, washed and then incubated with alkaline phosphatase-conjugated rabbit anti-mouse serum (Sigma-Aldrich) at a dilution of 1/2000. After washing, the reactivity was visualized using nitroblue tetrazolium. Photographs were taken using a Nikon coolpix camera (Nikon; http://nikon.fr), and figures were processed using Adobe Photoshop 7.0 (Adobe; http://adobe.fr).

Complementation assays inSaccharomyces cerevisiae

Saccharomyces cerevisiae strain JO55 [cdc33-Δ LEU2 leu2 ura3 his3 trp1 ade2 (YCp33supex-h4E URA3)] (Hughes et al., 1999), a gift from J.M.X. Hughes (Manchester Interdisciplinary Biocentre, Manchester, UK), carries a disrupted endogenous eIF4E gene (cdc33). Its survival depends on the presence of plasmid YCp33supex-h4E URA3, which contains a copy of the human eIF4E cDNA under the control of the galactose-dependent GAL promoter. The coding sequences of each of the pvr2-eIF4E alleles were cloned into the p424GBP/TRP1 glucose-dependent vector and independently used to transform S. cerevisiae strain JO55. After transformation, yeast cells were grown in appropriate selective nutrient drop-out media containing 2% glucose and tested at 30°C for their ability to complement the lack of endogenous eIF4E (Altmann et al., 1989). Control transformations were performed with no DNA (empty JO55) and empty plasmids p424GBP/TRP1 (negative controls), and with p424GBP/TRP1:At-eIF4E (At4g18040) (positive control).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank A. Labay for very helpful comments on the manuscript, S. Santoni and A. Weber (INRA, Montpellier, France) for sequencing, A.M. Sage-Palloix for the availability of the Capsicum germplasm collection, and P. Sanchez, T. Phally and G. Nemouchi for their excellent assistance. This work was supported by grants from GENOPLANTE (GNP05003G) and the Bureau des Resources Génétiques (AO 2005–2007). C. Charron was supported by a doctoral fellowship from the French Ministry of Research.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Altmann, M., Sonenberg, N. and Trachsel, H. (1989) Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system. Mol. Cell. Biol. 9, 44674472.
  • Borgstrøm, B. and Johansen, I.E. (2001) Mutations in Pea seedborne mosaic virus genome-linked protein VPg alter pathotype-specific virulence in Pisum sativum. Mol. Plant Microbe Interact. 14, 707714.
  • Browning, K.S. (2004) Plant translation initiation factors: it is not easy to be green. Biochem. Soc. Trans. 32, 589591.
  • Caranta, C. and Palloix, A. (1996) Both common and specific genetic factors are involved in polygenic resistance of pepper to several potyviruses. Theor. Appl. Genet. 92, 1520.
  • Combe, J.P., Petracek, M.E., Van Eldik, G., Meulewaeter, F. and Twell, D. (2005) Translation initiation factors eIF4E and eIFiso4E are required for polysome formation and regulate plant growth in tobacco. Plant Mol. Biol. 57, 749760.
  • Culjkovic, B., Topisirovic, I. and Borden, K.L. (2007) Controlling gene expression through RNA regulons: the role of the eukaryotic translation initiation factor eIF4E. Cell Cycle, 6, 6569.
  • Deom, C.M. and He, X.Z. (1997) Second-site reversion of a dysfunctional mutation in a conserved region of the Tobacco mosaic tobamovirus movement protein. Virology, 232, 1318.
  • Dodds, P.N., Lawrence, G.J., Catanzariti, A.M., Teh, T., Wang, C.I., Ayliffe, M.A., Kobe, B. and Ellis, J.G. (2006) Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes. Proc. Natl Acad. Sci. USA, 103, 88888893.
  • Duprat, A., Caranta, C., Revers, F., Menand, B., Browning, K.S. and Robaglia, C. (2002) The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required for susceptibility to potyviruses. Plant J. 32, 927934.
  • Fulton, T.M., Chunwongse, J. and Tanksley, S. (1995) Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol. Biol. Rep. 13, 207209.
  • Gallie, D. and Browning, K. (2001) eIF4G functionally differs from eIF(iso)4G in promoting internal initiation, cap-independent translation and translation of structured mRNAs. J. Biol. Chem. 276, 3695136960.
  • Gao, Z., Johansen, E., Eyers, S., Thomas, C., Ellis, T. and Maule, A. (2004) The potyvirus recessive resistance gene, sbm1, identifies a novel role for translation initiation factor eIF4E in cell-to-cell trafficking. Plant J. 40, 376385.
  • Green, S.K. and Kim, J.S. (1991) Characteristics and control of viruses infecting peppers: a literature review. Asian Vegetable Development Center. Technical Bulletin, 18, 60.
  • Greenleaf, W.H. (1986) Pepper breeding. In Breeding Vegetable Crops (Basset, M.J., ed.). Westport, CT: AVI Publishing Co. Inc., pp. 67134.
  • Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 9598.
  • Hughes, J.M., Ptushkina, M., Karim, M.M., Koloteva, N., Von Der Haar, T. and McCarthy, J.E. (1999) Translational repression by human 4E-BP1 in yeast specifically requires human eIF4E as target. J. Biol. Chem. 274, 32613264.
  • Joshi, B., Lee, K., Maeder, D.L. and Jagus, R. (2005) Phylogenetic analysis of eIF4E-family members. BMC Evol. Biol. 5, 48 (doi: DOI: 10.1186/1471-2148-5-48).
  • Kang, B.C., Yeam, I., Frantz, J.D., Murphy, J.F. and Jahn, M.M. (2005) The pvr1 locus in Capsicum encodes a translation initiation factor eIF4E that interacts with Tobacco etch virus VPg. Plant J. 42, 392405.
  • Kushner, D., Lindenbach, B., Grdzelishvili, V., Noueiry, A., Paul, S. and Ahlquist, P. (2003) Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus. Proc. Natl Acad. Sci. USA, 100, 1576415769.
  • Kyle, M.M. and Palloix, A. (1997) Proposed revision of nomenclature for potyvirus resistance genes in Capsicum. Euphytica, 97, 183188.
  • Lellis, A., Kasschau, K., Whitham, S. and Carrington, J. (2002) Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for eIF(iso)4E during potyvirus infection. Curr. Biol. 12, 10461051.
  • Leonard, S., Plante, D., Wittmann, S., Daigneault, N., Fortin, M.G. and Laliberte, J.F. (2000) Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity. J. Virol. 74, 77307737.
  • Leonard, S., Viel, C., Beauchemin, C., Daigneault, N., Fortin, M.G. and Laliberte, J.F. (2004) Interaction of VPg-Pro of Turnip mosaic virus with the translation initiation factor 4E and the poly(A)-binding protein in planta. J. Gen. Virol. 85, 10551063.
  • Maule, A., Caranta, C. and Boulton, M. (2007) Sources of natural resistance to plant viruses: status and prospects. Mol. Plant Pathol. 8, 223231.
  • Michon, T., Estevez, Y., Walter, J., German-Retana, S. and Le Gall, O. (2006) The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue. FEBS J. 273, 13121322.
  • Monzingo, A.F., Dhaliwal, S., Dutt-Chaudhuri, A., Lyon, A., Sadow, J.H., Hoffman, D.W., Robertus, J.D. and Browning, K.S. (2007) The structure of translation initiation factor eIF4E from wheat reveals a novel disulfide bond. Plant Physiol. 143, 15041518.
  • Moury, B., Morel, C., Johansen, E., Guilbaud, L., Souche, S., Ayme, V., Caranta, C., Palloix, A. and Jacquemond, M. (2004) Mutations in Potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum. Mol. Plant Microbe Interact. 17, 322329.
  • Nicaise, V., German-Retana, S., Sanjuan, R., Dubrana, M.P., Mazier, M., Maisonneuve, B., Candresse, T., Caranta, C. and Le Gall, O. (2003) The eukaryotic translation initiation factor 4E controls lettuce susceptibility to the potyvirus Lettuce mosaic virus. Plant Physiol. 132, 12721282.
  • Nieto, C., Morales, M., Orjeda, G. et al. (2006) An eIF4E allele confers resistance to an uncapped and non-polyadenylated RNA virus in melon. Plant J. 48, 452462.
  • Revers, F., Le Gall, O., Candresse, T. and Maule, A. (1999) New advances in understanding the molecular biology of plant/potyvirus interactions. Mol. Plant Microbe Interact. 12, 367376.
  • Riechmann, J.L., Lain, S. and Garcia, J.A. (1992) Highlights and prospects of potyvirus molecular biology. J. Gen. Virol. 73, 116.
  • Robaglia, C. and Caranta, C. (2006) Translation initiation factors: a weak link in plant RNA virus infection. Trends Plant Sci. 11, 4045.
  • Rodriguez, C., Freire, M., Camilleri, C. and Robaglia, C. (1998) The Arabidopsis thaliana cDNAs coding for eIF4E and eIF(iso)4E are not functionally equivalent for yeast complementation and are differentially expressed during plant development. Plant J. 13, 465473.
  • Rose, L.E., Michelmore, R.W. and Langley, C.H. (2007) Natural variation in the Pto disease resistance gene within species of wild tomato (Lycopersicon): II. Population genetics of Pto. Genetics, 175, 13071319.
  • Ruffel, S., Dussault, M.H., Palloix, A., Moury, B., Bendahmane, A., Robaglia, C. and Caranta, C. (2002) A natural recessive resistance gene against Potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E). Plant J. 32, 10671075.
  • Ruffel, S., Dussault, M.H., Palloix, A., Moury, B., Revers, F., Bendahmane, A., Robaglia, C. and Caranta, C. (2004) The key role of the eukaryotic initiation factor 4E (eIF4E) in plant–potyvirus interactions. In Biology of Plant–Microbe Interactions, Vol. 4, Molecular Plant–Microbe Interaction: New Bridges Between Past And Future (Tikhonovich, I., Lugtenberg, B. and Provorov, N., eds). St Paul, MN: International Society for Molecular Plant–Microbe Interactions, pp. 8183.
  • Ruffel, S., Gallois, J.L., Lesage, M.L. and Caranta, C. (2005) The recessive potyvirus resistance gene pot-1 is the tomato orthologue of the pepper pvr2-eIF4E gene. Mol. Genet. Genomics, 274, 346353.
  • Ruffel, S., Gallois, J.L., Moury, B., Robaglia, C., Palloix, A. and Caranta, C. (2006) Simultaneous mutations in translation initiation factors eIF4E and eIF(iso)4E are required to prevent pepper veinal mottle virus infection of pepper. J. Gen. Virol. 87, 20892098.
  • Sato, M., Nakahara, K., Yoshii, M., Ishikawa, M. and Uyeda, I. (2005) Selective involvement of members of the eukaryotic initiation factor 4E family in the infection of Arabidopsis thaliana by potyviruses. FEBS Lett. 579, 11671171.
  • Schaad, M., Anderberg, R. and Carrington, J. (2000) Strain-specific interaction of the Tobacco etch virus NIa protein with the translation initiation factor eIF4E in the yeast two-hybrid system. Virology, 273, 300306.
  • Stein, N., Perovic, D., Kumlehn, J., Pellio, B., Stracke, S., Streng, S., Ordon, F. and Graner, A. (2005) The eukaryotic translation initiation factor 4E confers multiallelic recessive Bymovirus resistance in Hordeum vulgare (L.). Plant J. 42, 912922.
  • Thompson, J., Higgins, D. and Gibson, T. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 46734680.
  • Wittmann, S., Chatel, H., Fortin, M.G. and Laliberte, J.F. (1997) Interaction of the viral protein genome linked of Turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso)4E of Arabidopsis thaliana using the yeast two-hybrid system. Virology, 234, 8492.
  • Yeam, I., Cavatorta, J.R., Ripoll, D., Kang, B.C. and Jahn, MM. (2007) Functional dissection of naturally occurring amino acid substitutions in eIF4E that confers recessive potyvirus resistance in plants. Plant Cell, 19, 29132928.

The GenBank accession numbers for the eIF4E cDNA sequences are AY122052 (pvr2+), AF521964 (pvr21), AF5211965 (pvr22), AY723737 (pvr23), AY723738 (pvr24), AY723739 (pvr25), AY723740 (pvr26), EU106863 (pvr27), EU106864 (pvr28) and EU106865 (pvr29).