The Eukaryotic Translation Initiation Factor 4E Controls Lettuce Susceptibility to the Potyvirus Lettuce mosaic virus¹

Cloning and Sequence Analysis of the Lettuce eIF4E and eIF(iso)4E cDNAs

A multiple alignment of the nucleotide sequences of the eIF4E coding regions from different plant species and of their human (Homo sapiens) and murine (Mus musculus) counterparts revealed a higher degree of conservation in the central domain of each of these proteins than in the N- and C-terminal regions (data not shown). The central region of the eIF4E cDNA from the susceptible lettuce genotype Salinas was PCR amplified using oligonucleotides 4E193f and 4E408r (all oligonucleotide sequences are given in the “Materials and Methods” section), designed in reference to seven plant eIF4E sequences, including three sequences in Arabidopsis. This product was cloned, and all of the eight clones sequenced yielded the same 169-bp sequence. This sequence information was used to design the oligonucleotides Ls4E250f and Ls4E255r for 3′- and 5′-RACE amplification of the cDNA ends, respectively. Finally, the oligonucleotides Ls4E3f and Ls4E813r were used to PCR amplify the nearly full-length eIF4E cDNA, including the entire coding region, before cloning in pGEM-T Easy and sequencing. The assembled eIF4E cDNA nucleotide sequence was determined from at least five independent clones at each position. No variability was observed between the cDNA clones sequenced. The full-length sequence (GenBank accession no. AF530162) was 1,032 nucleotides in length, with a single open reading frame from positions 21 to 710, encoding a protein with a calculated molecular mass of 26.1 kD (Fig. 1). The closest matches obtained after a BLAST search in GenBank with the full-length nucleotide sequence were the eIF4E cDNAs from tomato (accession no. AF259801, E = 5.10^–27), rice (Oryza sativa; U34597, E = 5.10^–27) and Arabidopsis (Y10548, E = 8.10^–26), and the closest matches with the predicted translation product were the eIF4E amino acid sequences from Arabidopsis (E = 3.10^–81), tomato (E = 3.10^–80), and maize (Zea mays; AF076954, E = 4.10^–78), confirming that the cDNA cloned was the eIF4E cDNA. The identity between the predicted amino acid sequence and 11 eIF4E sequences from plants, vertebrates, and insects ranged between 40% and 70% (data not shown).

Similarly, a near full-length eIF(iso)4E cDNA was obtained from the susceptible genotype Salinas by a combination of 3′- and 5′-RACE PCR. The 3′ region of the cDNA was amplified by PCR using an oligo(dT)-containing primer and the degenerate oligonucleotide 4E198f, designed from a conserved central region of the eIF4E genes from tomato, tobacco, and Arabidopsis. The oligonucleotide Ls(iso)4E680r was designed from the sequence of the 3′-RACE product and used in the 5′-RACE system (Invitrogen) to amplify the complete cDNA lacking the 3′ non-coding region. The 5′RACE products were cloned into pGEM-T Easy and sequenced. The assembled sequence (GenBank accession no. AF530163) contained a single open reading frame encoding a protein of 193 amino acids with a calculated molecular mass of 22.0 kD. The closest matches obtained after a BLAST search in GenBank with the full-length nucleotide sequence were the eIF(iso)4E cDNAs from wheat (accession no. M95819, E = 6.10^–20), Arabidopsis (Y10547, E = 2.10^–13), and maize (AF076955, E = 5.10^–8), and the closest matches with the predicted translation product were the eIF(iso)4E amino acid sequences from wheat (M95819, E = 7.10^–77), rice (AAK27811, E = 8.10^–70), and maize (AF076955, E = 1.10^–69), confirming that the cDNA cloned was the eIF(iso)4E cDNA.

The eIF4E and eIF(iso)4E coding regions were 55.3% identical in nucleotide sequence and 46.8% identical in amino acid sequence. Similar levels of identity in nucleotide and amino acid sequences between the isoforms of eIF4E were found in Arabidopsis (53.9% and 41.2%, respectively), maize (62.9% and 48.9%), rice (59.9% and 46.5%), and wheat (60.2% and 47.7%).

Correlation between Sequence Variations in the eIF4E cDNA and the Presence of mo1¹ or mo1²

The region encompassing nucleotide positions 20 to 787, which includes the entire coding region of the eIF4E cDNA, was PCR amplified as described above, cloned, and sequenced for the seven additional lettuce genotypes Floribibb (mo1¹), Mantilia (mo1¹), Malika (mo1¹), Salinas 88 (mo1²), Vanguard (susceptible), Vanguard 75 (mo1²), and 87-20M (susceptible). All sequences were highly conserved, but some of them contained mutations when compared with the sequence from Salinas (Table I; Fig. 1). On the basis of their patterns of variation, the sequences were classified into three types. Type 0 sequences (Vanguard and 87-20M) have coding regions identical to that found in Salinas. Type 1 sequences (Floribibb, Malika, and Mantilia) have a silent C to T substitution at position 299, a deletion of nucleotides 344 to 349, and a non-silent G to T substitution at position 576. Type 2 sequences (Salinas 88 and Vanguard 75) have a non-silent G to C substitution at position 228 and a C to T substitution at position 730 in the 3′-non-coding region. This last sequence variation was also found in the susceptible genotype 87-20M. Types 0, 1, and 2 of lettuce eIF4E were named Ls-eIF4E°, Ls-eIF4E¹, and Ls-eIF4E², respectively.

Therefore, within this limited set of eight lettuce genotypes, there was a strict correlation between Ls-eIF4E¹ and the presence of mo1¹ and between Ls-eIF4E² and the presence of mo1², whereas the susceptible genotypes all had Ls-eIF4E°. This correlation was maintained even in the case of two independent pairs of genotypes nearly isogenic for mo1², Salinas, and Salinas 88 on one hand, and Vanguard and Vanguard 75 on the other hand. To confirm and extend this correlation, the central domain of the Ls-eIF4E cDNA from 11 additional lettuce genotypes was PCR amplified using oligonucleotides Ls4E83f and Ls4E442r and sequenced (Table I). Again, a complete correlation was observed in this central region between the type of Ls-eIF4E and the presence of mo1¹ or mo1² in the following genotypes: Fiona, Girelle, Jessy, and Mariska (Ls-eIF4E°, susceptible), Alizé, Classic, Oriana, and Presidio (Ls-eIF4E¹ and mo1¹), and Autumn Gold and Desert Storm (Ls-eIF4E² and mo1²).

The sequence of the eIF(iso)4E coding region from the susceptible genotypes Salinas and Vanguard and from their nearly isogenic mo1² genotypes Salinas 88 and Vanguard 75 was also determined. No difference was detected between the eIF(iso)4E coding sequences from these four genotypes. An identical sequence was also found in the mo1¹ genotype Mantilia. This suggests that eIF(iso)4E sequence variations are not directly correlated with the mo1-related phenotypes.

Predicted Three-Dimensional (3D) Structure of the Ls-eIF4E Protein

A 3D model of the Ls-eIF4E protein was predicted based on the known 3D structures of human (Hs-eIF4E) and murine (Mm-eIF4E) cap-bound eIF4E molecules (Marcotrigiano et al., 1999; Tomoo et al., 2002). The central domain of Ls-eIF4E°, from beta-sheet 2 to beta-sheet 6 (Fig. 1), was 50% identical in sequence to that of Mm-eIF4E (Fig. 1) and Hs-eIF4E (data not shown), suggesting that a homology-based model of the Ls-eIF4E folding would be valid within the range of 1 Å (Chothia and Lesk, 1986). Two independent algorithms (Guex and Peitsch, 1997; Bates et al., 2001) yielded structurally similar predictions (data not shown). Using Swiss-model and Mm-eIF4E as a template, the Ls-eIF4E° central domain tightly matched that of Mm-eIF4E (root mean square [RMS] deviation = 0.74 Å) and Hs-eIF4E (RMS = 1.4 Å). When Hs-eIF4E was used as the modeling template, RMS deviation values obtained with Ls-eIF4E were 1.23 Å for Hs-eIF4E and 1.35 Å for Mm-eIF4E, respectively. Using the same procedure to predict the folding of Hs-eIF4E based on its homology with Mm-eIF4E yielded a model with a 0.96-Å RMS deviation compared with its actual x-ray diffraction structure, providing a landmark for comparison of RMS deviation values.

Figure 2 shows the predicted 3D structure of Ls-eIF4E° and, superimposed to visualize the predicted structural differences, those of Ls-eIF4E¹ and Ls-eIF4E². According to this model, the amino acids that differ between the three Ls-eIF4E types were all predicted to be at or near the surface of the protein. All mapped near the cap recognition pocket, on the face of eIF4E opposite to the eIF4G-binding site (Fig. 2). Ala-70-Pro, the only amino acid different between Ls-eIF4E° and Ls-eIF4E², was predicted to be part of the loop between beta-sheets 1 and 2 (Marcotrigiano et al., 1997), which protrudes at the rim of the cap-recognition pocket itself (Fig. 2). The three amino acids Gln-108-Gly-109-Ala-110, replaced by a single His in Ls-eIF4E¹, are located in the neighboring loop (Figs. 1 and 2), between alpha-helix 1 and beta-sheet 3. Although the replacement of three amino acids by a single one in Ls-eIF4E¹ was predicted to have a strong effect on the folding of this loop (Fig. 2), the Ala-70-Pro replacement in Ls-eIF4E² was only predicted to have a minor impact because of the already existing sterical constraints on the chain in this region (Fig. 2). Similarly, the Ala-186-Ser replacement in the loop between beta-sheet 6 and alpha-helix 3 in Ls-eIF4E¹ was predicted to cause no detectable effect on the predicted folding (Fig. 2).

Genetic Cosegregation between the mo1¹ and Ls-eIF4E¹ Loci

With the objective to strengthen the correlation between sequence variation in the eIF4E cDNA and LMV resistance controlled by mo1¹, the genetic linkage between mo1¹ and the six-nucleotide deletion found at positions 344 to 349 in Ls-eIF4E¹ was studied in two F₂ progenies Mariska (susceptible) × Mantilia (mo1¹) and Girelle (susceptible) × Mantilia (mo1¹). A cleaved amplified polymorphic sequence (CAPS) marker, eIF4E-PagI, was defined on the Ls-eIF4E cDNA in relation with the presence of a PagI restriction site generated by the six-nucleotide deletion in the Ls-eIF4E¹ cDNA. Ten to 15 d after inoculation of 36 F₂ plants with LMV-0, 10 plants showed no symptoms, whereas the remaining 26 presented typical mosaic symptoms caused by LMV. This ratio, 10 resistant to 26 susceptible, is consistent with the segregation of a single recessive gene coding for LMV resistance (χ² = 0.15, P = 70%). Twenty-five of these F₂ plants, eight without symptoms and 17 with typical LMV symptoms, were further analyzed for segregation of the CAPS marker eIF4E-PagI. A cDNA fragment of 442 to 448 bp was PCR generated using oligonucleotides Ls4E250f and Ls4E697r, designed on both sides of the six-nucleotide deletion present in Ls-eIF4E¹. Electrophoretic analysis of PagI-digested PCR products revealed three distinct restriction patterns (Fig. 3; data not shown). All eight resistant plants showed a complete digestion of the PCR product, as in the Mantilia parent, and, thus, were determined to be homozygous for the presence of eIF4E-PagI. The five susceptible plants showed no detectable PagI digestion, as in the Mariska and Girelle parents, and, thus, were homozygous for the absence of eIF4E-PagI. Finally, the remaining 12 susceptible plants showed a partial digestion, as in the F₁ plant, which was characteristic of individual plants heterozygous for eIF4E-PagI. Thus, a complete map cosegregation was observed between mo1¹-associated LMV resistance and the CAPS marker eIF4E-PagI.

Agro-Infiltration of Ls-eIF4E Restores LMV Systemic Accumulation in mo1² Plants

Leaf infiltration with a suspension of Agrobacterium tumefaciens transformed with a binary plasmid containing an expression cassette between T-DNA borders (agro-infiltration) allows localized, transient expression of a foreign protein in plant tissues (Bechtold et al., 1993; Schöb et al., 1997). This strategy was used to evaluate the ability of Ls-eIF4E to restore LMV susceptibility in mo1² plants.

We have shown previously that mo1² prevents systemic accumulation of LMV-0-GFP, a green fluorescent protein-tagged LMV-0 (German-Retana et al., 2000; Candresse et al., 2002), but, unlike mo1¹, does not completely block replication in the inoculated leaf (German-Retana et al., 2000) and phloem loading (S. German-Retana, unpublished data). In our assay, lettuce plants were inoculated with LMV-0-GFP, and a few days later, their apical noninoculated leaves were agro-infiltrated with constructs designed to locally express Ls-eIF4E°, Ls-eIF4E¹, or Ls-eIF4E². A construct allowing the expression of the β-glucuronidase (GUS) was co-agro-infiltrated as an internal control. Six days after agro-infiltration, systemic accumulation of LMV-0-GFP, i.e. GFP accumulation in the noninoculated agro-infiltrated areas, was scored under UV light (Fig. 4).

The pattern and timing of viral accumulation were not detectably changed in susceptible plants of the genotype Salinas in leaf areas upon agro-infiltration with any of the three types of Ls-eIF4E. Stereomicroscopic observation under UV light showed comparable GFP fluorescence intensity in Salinas leaves agro-infiltrated with the Ls-eIF4E°, Ls-eIF4E¹, or Ls-eIF4E² constructs (Fig. 4, E–G). GUS activity was readily detected in all infiltrated areas (data not shown), confirming the success of agro-infiltration for foreign protein expression.

In Salinas 88 (mo1²), no systemic spread of GFP-tagged LMV-0 was observed up to 2 weeks after inoculation, as expected from previous results (Candresse et al., 2002). However, GFP fluorescence was observed in leaves distant from the inoculation sites that had been agro-infiltrated with any of the three forms of Ls-eIF4E (Fig. 4, A–C). The GUS construct alone did not restore the ability of LMV-0-GFP to infect these leaves (Fig. 4D). The size difference of the GFP spots that is apparent in Figure 4, A to C, was not considered significant because it was not reproduced from one leaf to another.

All three forms of Ls-eIF4E restored to some extent the ability of LMV-0-GFP to spread into agro-infiltrated areas distant from the inoculation site. To determine whether the Ls-eIF4E alleles differed quantitatively in this ability, we counted the GFP spots in each agro-infiltrated area (Table II). The analysis of the number of fluorescent spots confirmed the visual observation that all three types of Ls-eIF4E resulted in complementation for LMV-0-GFP systemic accumulation, both when the numbers of spots per agro-infiltrated area and the number of areas containing fluorescent spots were considered. For these two parameters, the χ² probability for the random hypothesis was lower than 0.01%. However, the Ls-eIF4E types differed quantitatively in their abilities to restore the systemic accumulation of LMV-0-GFP. Although GFP accumulation was overall less efficient in agro-infiltrated areas of Salinas 88 leaves than in Salinas (Fig. 4), the number of GFP spots was higher after agro-infiltration with Ls-eIF4E° than it was with Ls-eIF4E¹ or Ls-eIF4E² (Table II). More areas were GFP positive after agro-infiltration with Ls-eIF4E° than with Ls-eIF4E¹ (P = 3.05% for the random hypothesis) or Ls-eIF4E² (P = 6.06%), and a significantly higher total number of spots was counted with Ls-eIF4E° than with Ls-eIF4E¹ (P = 0.04%) or Ls-eIF4E² (P = 2.91%). The apparent differences in efficiency of Ls-eIF4E¹ and Ls-eIF4E² to restore systemic viral accumulation (Table II) were not statistically supported (P = 75% for the number of GFP-positive areas and P = 21% for the number of spots). In summary, agro-infiltration of all three forms of Ls-eIF4E restored the LMV-0-GFP systemic accumulation in the mo1² genotype Salinas 88, and Ls-eIF4E° appeared to be more efficient than Ls-eIF4E¹ and Ls-eIF4E².

Expression of Ls-eIF4E°, But Not Ls-eIF4E¹, Restores the Infectivity of a Recombinant LMV in mo1¹ or mo1² Plants

To test the ability of Ls-eIF4E to restore LMV susceptibility in mo1¹ genotypes of lettuce, the infectivity of LMV-0-4E° was evaluated. This viral construct is derived from the nonresistance breaking isolate LMV-0 and contains the Ls-eIF4E° coding region as a translational fusion between the viral P1 and Hc-Pro domains that is proteolytically processed in vivo to yield the free proteins. Similarly, LMV-0-4E¹ and LMV-0-iso4E contain the Ls-eIF4E¹ and the Ls-eIF(iso)4E coding regions, respectively.

LMV-0-4E°, LMV-0-4E¹, and LMV-0-iso4E were inoculated to three susceptible genotypes (Trocadéro, Salinas, and Vanguard), three mo1¹ genotypes (Floribibb, Malika, and Mantilia) and two mo1² genotypes (Salinas 88 and Vanguard 75), and symptoms were scored visually (Fig. 5; data not shown). As expected, LMV-0 caused symptoms only in the susceptible varieties. This was also the case of LMV-0-4E¹ and LMV-0-iso4E. However, symptoms appeared in all plants inoculated with LMV-0-4E°. The timing of symptoms appearance in mo1¹ and mo1² genotypes inoculated with LMV-0-4E° was similar to that in susceptible plants infected with LMV-0: A faint vein clearing became evident in emerging leaves 10 to 12 d after inoculation, followed by mosaic symptoms 2 to 3 d later (data not shown).

Accumulation of LMV-0-4E°, LMV-0-4E¹, and LMV-0-iso4E was assayed by ELISA (Table III; data not shown). The accumulation of nonrecombinant LMV was not detected in mo1¹ genotypes and was strongly reduced in mo1² genotypes compared with susceptible genotypes, consistent with the general idea that mo1¹ is associated with stronger LMV resistance than mo1². The same situation was observed for LMV-0-4E¹ and LMV-iso4E, except no virus accumulation was detected in mo1² genotypes. On the other hand, LMV-0-4E° accumulated in all three categories of genotypes. These results, and the persistence of the Ls-eIF4E insert in the replicating virus during the course of the experiments, were confirmed by back inoculation and by RT-PCR (data not shown). LMV-0-4E° had a similar or slightly decreased accumulation in susceptible plants compared with nonrecombinant LMV, indicating that its ability to infect mo1¹ and mo1² plants was probably not due to a nonspecific enhancement of virus accumulation by the Ls-eIF4E° insert.

Together, these results indicate that expression of Ls-eIF4E° rendered LMV able to accumulate and produce symptoms in mo1¹ and mo1² genotypes, unlike that of Ls-eIF4E¹ or Ls-eIF(iso)4E.

Plant Material and Viral Constructs

All plants were grown under greenhouse conditions. The LMV-susceptible lettuce (Lactuca sativa) genotypes Fiona, Girelle, Jessy, Mariska, Salinas, Vanguard, Trocadéro, and the early flowering accession 87-20M (Ryder, 1996), the mo1¹ genotypes Alizé, Classic, Floribibb, Malika, Mantilia, Oriana, and Presidio, and the mo1² genotypes Autumn Gold, Desert Storm, Vanguard 75, and Salinas 88 (Candresse et al., 2002) were used. The susceptible genotype Trocadéro was routinely used to propagate LMV. Two independent pairs of lettuce genotypes included in this analysis, namely Salinas/Salinas 88 and Vanguard/Vanguard 75, are nearly isogenic for mo1² (Ryder, 1979, 1991). The use of genetic markers showed that the region introgressed around mo1² in the genome of Salinas 88 and Vanguard 75 is small (Irwin et al., 1999).

All experiments were made with LMV-0, an LMV isolate unable to produce symptoms in mo1¹ and mo1² plants (Revers et al., 1997). The recombinant construct LMV-0-GFP carries an insertion of the Aequorea victoria gfp gene between the regions coding for P1 and Hc-Pro of the viral genome, with an engineered NIa-Pro cleavage site, so that the GFP protein is processed from Hc-Pro in vivo (S. German-Retana, unpublished data). LMV-0-4E°, LMV-0-4E¹, and LMV-0-iso4E were constructed similarly by inserting the Ls-eIF4E°, Ls-eIF4E¹, and Ls-eIF(iso)4E coding regions, respectively, in frame between the P1 and Hc-Pro domains of LMV, with an NIa-Pro cleavage site resulting in the addition of eight amino acids (PGDEVYHQ) at the C terminus of the eIF4E sequence and five amino acids (SDVPG) at its N terminus. LMV-0-clv is an empty viral vector with only these additional amino acids but no eIF4E or other gene inserted. Lettuce plants primarily inoculated by biolistics (Helios Gene-Gun, Bio-Rad, Hercules, CA) were homogenized in 25 mm Na₂HPO₄ containing 2% (w/v) diethyldithiocarbamate and used to rub inoculate plants as described previously (Revers et al., 1997). Detection of the viral progeny by RT-PCR was performed as described previously (Krause-Sakate et al., 2002). Double-antibody sandwich ELISA was performed as described after homogenization of plant tissues in 4 volumes of extraction buffer (Revers et al., 1997).

F₁ hybrids between Mantilia (mo1¹/mo1¹) and the susceptible genotypes Mariska and Girelle were self-pollinated to obtain F₂ progenies. The parental lines, the F₁ hybrids, and the F₂ individuals were evaluated under greenhouse conditions for LMV resistance. Plants were rub inoculated 3 weeks after sowing at the five- to six-leaves stage. Symptoms were recorded 10 to 15 d after inoculation. These conditions ensured symptom appearance in 100% of Mariska and Girelle, whereas no symptoms were visible in Mantilia.

RT-PCR Amplification, Cloning, and Sequencing of cDNAs

Total RNA was extracted from 100 to 200 mg of leaf tissues using TRI Reagent (Sigma-Aldrich, St. Louis). Total cDNA was synthesized from 5 μg of total RNA using 15 units of AMV RT (Amersham Biosciences, Uppsala) and 1 μm oligo(dT) in a total volume of 50 μL, incubated for 1 h at 42°C.

PCR amplification was routinely performed using 1 μL of the total cDNAs in 50-μL reactions containing 0.5 units of Extra-Pol I Taq DNA polymerase (Eurobio, Les Ulis, France) or Platinum Taq DNA Polymerase High Fidelity (Invitrogen), using 1 μm oligonucleotide primers. For these reactions, 40 cycles (30 s of denaturation at 95°C, 30 s of annealing at different temperatures according to the primers used, and 1 min of elongation at 72°C) were performed in a thermal cycler (iCycler, Bio-Rad) after an initial denaturation of the RNA-cDNA duplex at 95°C for 2 min. The SMART RACE cDNA amplification kit (CLONTECH) was used according to the recommendations provided by the supplier to amplify the cDNA 5′ and 3′ ends of eIF4E, and, similarly, the 3′- and 5′-RACE Systems (Invitrogen) were used to amplify the 5′ and 3′ ends of eIF(iso)4E cDNA. The oligonucleotides used are listed in Table IV.

The pGEM-T Easy vector system (Promega, Madison, WI) was used to clone cDNAs after PCR amplification. Automated DNA sequencing of cloned fragments or PCR products was performed at Genaxis (St-Cloud, France) or Genome Express (Grenoble, France), using ABI PRISM 373XL or 377 sequencers (Applied Biosystems, Foster City, CA).

Generation of the CAPS Marker eIF4E-PagI

Total RNA was extracted from leaves of genotypes Mantilia, Mariska, and from the F₁ hybrids and the F₂ progenies as described above. From each RNA preparation, an eIF4E cDNA fragment of about 442 to 448 bp was amplified by RT-PCR using the oligonucleotides Ls4E250f and Ls4E697r (Table IV). The amplified cDNAs were subjected to digestion by the PagI endonuclease (Fermentas, Vilnius, Lithuania). The digestion products were resolved by electrophoresis in a 1.5% (w/v) agarose gel. Digestion at an additional PagI site present in all three types of Ls-eIF4E sequences provided an internal control for PagI activity by generating a 35-bp fragment not visualized in this electrophoresis system and a 407- to 413-bp fragment further digested in Ls-eIF4E¹ to yield two fragments of 312 and 95 bp.

Functional Complementation Assay Using Agro-Infiltration

For transient expression in planta by agro-infiltration (Bechtold et al., 1993; Schöb et al., 1997), cDNAs were inserted in a 35S-nos expression cassette in the binary vector pGreenII (Hellens et al., 2000). Agrobacterium tumefaciens C58C1 was transformed by electroporation and grown overnight at 28°C in Luria-Bertani medium containing 5 μg mL^–1 tetracycline, 50 μg mL^–1 kanamycine, 10 mm MES, and 20 μm acetosyringone. The bacteria were collected by centrifugation and resuspended in 10 mm MES and 10 mm MgCl₂ containing 150 μm acetosyringone to an OD₆₀₀ of 0.6. They were then incubated at room temperature for 4 h and infiltrated using a syringe in apical leaves of plants that had been inoculated 5 to 6 d earlier with LMV-0-GFP on their lower leaves. Agro-infiltration took place when GFP fluorescence began to be visible under UV light near the veins in upper noninoculated leaves in the susceptible genotypes. Four to 6 d after infiltration, GFP expression was monitored as described by German-Retana et al. (2000) and using a fluorescence stereomicroscope (MZ FLIII, Leica Microsystems, Heerburg, Switzerland) equipped with a filter with an excitation window at 470 ± 20 nm and an arrest window at 525 ± 25 nm.

Sequence Analysis

The following eIF4E cDNA and protein sequences were retrieved from GenBank: human (Homo sapiens; NM_004846, XM_017925), mouse (Mus musculus) (M61731), Spodoptera frugiperda (AF281654), rice (Oryza sativa; U34597), maize (Zea mays; AF076954), wheat (Triticum aestivum; Z12616), tomato (Lycopersicon esculentum; AF259801), and Arabidopsis (Y10548, NM_102695 and NM_102699). The eIF(iso)4E sequences were: rice (U34598), maize (AF076955), and Arabidopsis (Y10547). Homology-based database searches were made in GenBank using the program BLAST (Altschul et al., 1990). The nucleotide and amino acid sequences were aligned using ClustalW (Thompson et al., 1994), which generates and uses a distance dendrogram (Saitou and Nei, 1987) to construct multiple sequence alignments, or with Align (Myers and Miller, 1988) for pair-wise alignments.

The 3D structures of cap-bound eIF4E of human (Hs-eIF4E) and mouse (Mm-eIF4E) were retrieved from the Protein Data Bank (http://www.rcsb.org/pdb) with the codes 1IPB and 1EJH, respectively. Comparative protein modeling was elaborated online using Swiss-model and Swiss-PdbViewer (Guex and Peitsch, 1997) and 3D-Jigsaw (Bates et al., 2001). The fit between 3D structure models was evaluated in Swiss-PdbViewer by calculating the RMS deviation (Chothia and Lesk, 1986) after iterative fitting.