The eukaryotic translation initiation factor 4E controls lettuce susceptibility to the Potyvirus Lettuce mosaic virus

Abstract

The eIF4E and eIF(iso)4E cDNAs from several genotypes of lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce mosaic virus (LMV; genus Potyvirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E(1) and Ls-eIF4E(2) were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1(1) and mo1(2). Ls-eIF4E(1) and mo1(1) cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1(1) genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1(2) plants and a recombinant LMV expressing Ls-eIF4E degrees from its genome, but not Ls-eIF4E(1) or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1(1) or mo1(2) genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in lettuce and that mo1(1) and mo1(2) are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyviruses in plants, probably through a general mechanism yet to be clarified.

Figure 1.

Amino acid sequence alignment of murine and lettuce eIF4E. The amino acid sequence of the eIF4E proteins from mouse (Mm-eIF4E) and the lettuce genotype Salinas (Ls-eIF4E) were aligned using ClustalW (Thompson et al., 1994). Asterisks, Identical amino acids; semicolons, similar amino acids; hyphens, deletions in one sequence compared with the other. The structural elements of Mm-eIF4E, as determined by x-ray crystallography (Marcotrigiano et al., 1997), are indicated: S1 to S8, eight beta-sheets (delineated with hyphens); and H1 to H4, four alpha-helices (delineated with omegas). The amino acids differing in Ls-eIF4E¹ (¹) and Ls-eIF4E² (²) are shown below the Ls-eIF4E° sequence. The central domain of Ls-eIF4E, sequenced for 11 additional genotypes as described in the text, is shown in bold font. Amino acids are numbered on the right.

Figure 2.

Stereo view of the predicted 3D structure of Ls-eIF4E. The structure of Ls-eIF4E° was predicted based on its homology with Mm-eIF4E, using Swiss-model (Guex and Peitsch, 1997) and shown in a ribbon representation. The positions of the cap analog 7-methyl-GDP (cap, yellow) and an eIF4G-derived peptide (eIF4GII, yellow) are derived from those observed in a cocrystal of these molecules with the murine eIF4E (Marcotrigiano et al., 1999). At variable positions, the side chains of the amino acid present in Ls-eIF4E° are displayed in green, and those in Ls-eIF4E¹ or Ls-eIF4E² are in red (labeled X¹Y and X²Y, respectively, where X is the residue in Ls-eIF4E° and Y in Ls-eIF4E¹ or Ls-eIF4E²). At the variable positions, the ribbon is colored using the same code.

Figure 3.

Cosegregation of the LMV resistance phenotype and Ls-eIF4E¹ in the F₂ progeny from a cross between Mantilia (mo1¹) and Mariska (susceptible). PCR products of 448 bp (442 bp for Ls-eIF4E¹) were generated by reverse transcriptase (RT)-PCR from RNAs isolated from leaves of the F₂, F₁, and the parental lines Mantilia (P1) and Mariska (P2) using oligonucleotides Ls4E250f and Ls4E697r. These cDNA fragments were digested by PagI and resolved in a 1.5% (w/v) agarose gel. Arrows indicate the molecular weights of the various fragments. The resistant (R) or susceptible (S) phenotype of each individual plant was scored in parallel 10 to 15 d after inoculation with LMV-0.

Figure 4.

Functional complementation of the systemic accumulation of LMV-0-GFP in mo1² plants by transient Ls-eIF4E expression. GFP fluorescence stereomicroscopy was visualized in leaf areas of an mo1² (Salinas 88, A–D) and a susceptible (Salinas, E–H) genotype after co-agro-infiltration with constructs allowing expression of the GUS and Ls-eIF4E. A and E, Areas agro-infiltrated with a construct allowing expression of Ls-eIF4E°. B and F, Areas agro-infiltrated with a construct allowing expression of Ls-eIF4E¹. C and G, Areas agro-infiltrated with a construct allowing expression of Ls-eIF4E². D and H, Areas agro-infiltrated with the GUS construct only. Bar = 1 cm.

Figure 5.

LMV-0-4E° and LMV-0-4E¹ symptoms in lettuce mo1¹ or mo1² genotypes. Lettuce seedlings were rub inoculated with LMV-0, LMV-0-4E°, or LMV-0-4E¹, and detached leaves were photographed 3 weeks later. Lettuce genotypes are: A to C, Salinas (susceptible); D to F, Mantilia (mo1¹); and G to I, Salinas 88 (mo1²). LMV constructs are: A, D, and G: LMV-0; B, E, and H: LMV-0-4E°; and C, F, and I: LMV-0-4E¹.