Simultaneous mutations in translation initiation factors eIF4E and eIF

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Simultaneous mutations in translation initiation factors eIF4E and eIF Powered By Docstoc
					Journal of General Virology (2006), 87, 2089–2098                                                                 DOI 10.1099/vir.0.81817-0

                                         Simultaneous mutations in translation initiation
                                         factors eIF4E and eIF(iso)4E are required to
                                         prevent pepper veinal mottle virus infection of
                                         Sandrine Ruffel,1 Jean-Luc Gallois,1 Benoıt Moury,2 Christophe Robaglia,3
                                                      1                    1
                                         Alain Palloix and Carole Caranta
 Correspondence                             INRA, Genetics and Breeding of Fruits and Vegetables1 and Plant Pathology2, Domaine St
 Carole Caranta                             Maurice, BP 94, F-84143 Montfavet, France            3
                                                            ´ ´                                                    ´
                                            Laboratoire de Genetique et Biophysique des Plantes, CEA-CNRS-Universite Aix-Marseille II,
                                            Faculte des Sciences de Luminy, F-13009 Marseille, France

                                         Capsicum resistance to Pepper veinal mottle virus (PVMV) results from complementation between
                                         the pvr2 and pvr6 resistance genes: recessive alleles at these two loci are necessary for resistance,
                                         whereas any dominant allele confers susceptibility. In line with previous results showing that
                                         pvr2 resistance alleles encode mutated versions of the eukaryotic translation initiation factor 4E
                                         (eIF4E), the involvement of other members of the eIF4E multigenic family in PVMV resistance
                                         was investigated. It was demonstrated that pvr6 corresponds to an eIF(iso)4E gene, predicted
                                         to encode the second cap-binding isoform identified in plants. Comparative genetic mapping
                                         in pepper and tomato indicated that eIF(iso)4E maps in the same genomic region as pvr6.
                                         Sequence analysis revealed an 82 nt deletion in eIF(iso)4E cDNAs from genotypes with the
                                         pvr6 resistance allele, leading to a truncated protein. This deletion was shown to co-segregate with
                                         pvr6 in doubled haploid and F2 progeny. Transient expression in a PVMV-resistant genotype of
                                         eIF(iso)4E derived from a genotype with the pvr6+ susceptibility allele resulted in loss of resistance
                                         to subsequent PVMV inoculation, confirming that pvr6 encodes the translation factor eIF(iso)4E.
                                         Similarly, transient expression of eIF4E from a genotype with the pvr2+-eIF4E susceptibility
                                         allele also resulted in loss of resistance, demonstrating that wild-type eIF4E and eIF(iso)4E are
                                         susceptibility factors for PVMV and that resistance results from the combined effect of mutations in
 Received 6 January 2006                 the two cap-binding isoforms. Thus, whilst most potyviruses specifically require one eIF4E isoform
 Accepted 17 March 2006                  to perform their replication cycle, PVMV uses either eIF4E or eIF(iso)4E for infection of pepper.

INTRODUCTION                                                                 eIF4G, a scaffold for other components of the complex, to
                                                                             form the eIF4F complex. Plants contain two distinct cap-
Viruses depend on the host cellular machinery to achieve
                                                                             binding proteins, eIF4E and eIF(iso)4E, which assemble
plant infection and completion of the viral cycle is the result
                                                                             into different eIF4F complexes [eIF4F and eIF(iso)4F]
of a complex interplay between virus-encoded and host-
                                                                             (Browning, 1996). In Arabidopsis thaliana, three genes
encoded factors. Identification of the host-encoded factors
                                                                             named eIF4E1 (At4g18040), eIF4E2 (At1g29590) and eIF4E3
involved and understanding how they are implicated in viral
                                                                             (At1g29550) encode the proteins of the eIF4E subfamily
genome amplification and cell-to-cell and long-distance
                                                                             and one gene (At5g35620) encodes eIF(iso)4E (http://
movement represent major challenges to our knowledge of
                                                                   ; Robaglia &
plant–virus interactions (reviewed by Whitham & Wang,
                                                                             Caranta, 2006). The structure of the eIF4E gene family in
2004). Among the host factors required for viral infection,
                                                                             other plant species is not known precisely, but in all cases
eukaryotic translation initiation factor 4E (eIF4E) has been
                                                                             several loci have been identified. Overall, differences in
demonstrated to play an essential role. eIF4E provides
                                                                             transcription patterns, in binding affinities depending on
the cap-binding function during formation of translation
                                                                             the 59 mRNA structure and/or the presence of a cap and in
initiation complexes of most mRNA and is associated with                     ability to rescue the growth of a yeast mutant in which the
                                                                             eIF4E gene was disrupted strongly suggest that isoforms
The GenBank/EMBL/DDBJ accession numbers for the sequences                    encoded by eIF4E1 and eIF(iso)4E have complementary
determined in this work are DQ022080–DQ022084.                               biological roles (Gallie & Browning, 2001; Rodriguez et al.,

0008-1817 G 2006 SGM              Printed in Great Britain                                                                               2089
S. Ruffel and others

1998). It remains to be determined whether eIF4E2 and                 demonstrated to control resistance to Pepper veinal mottle
eIF4E3 encode proteins with translation initiation function.          virus (PVMV) only when combined with the pvr21 or pvr22
                                                                      resistance alleles, whereas pvr2 and pvr6 do not separately
Arabidopsis mutant lines bearing either transposon- or ethyl          confer any resistance to PVMV and pvr6 alone has no
methanesulfonate-induced null alleles for the eIF(iso)4E              detectable effect on the potyvirus infection process (Caranta
gene have been shown to be resistant to infection by several          et al., 1996).
viruses from the genus Potyvirus including Turnip mosaic
virus (TuMV), Lettuce mosaic virus (LMV) and Tobacco etch             In the present study, we investigated the involvement of
virus (TEV) (Duprat et al., 2002; Lellis et al., 2002). More          the second cap-binding isoform, eIF(iso)4E, in pepper
recently, it was demonstrated that the knockout mutation of           resistance against PVMV. Our data showed that pvr6
eIF4E1 prevented Clover yellow vein virus (ClYVV) replica-            corresponds to eIF(iso)4E and that simultaneous mutations
tion, indicating that potyviruses selectively use eIF4E iso-          in two genes belonging to the eIF4E family are required to
forms (Sato et al., 2005). Potyviral RNA differs from host            prevent the PVMV infectious cycle. Therefore, while PVY
mRNAs in that it lacks a 59 m7GpppX cap structure and is              and TEV require one specific eIF4E isoform to achieve
instead covalently linked at its 59 end to a viral-encoded            pepper infection, PVMV can use both eIF4E and eIF(iso)4E
protein, VPg (viral protein genome-linked) (Urcuqui-                  isoforms.
Inchima et al., 2001). Experiments using the yeast two-
hybrid system and an ELISA have shown that the VPg (or
its precursor, NIa) interacts with eIF4E or eIF(iso)4E                METHODS
(Leonard et al., 2000; Schaad et al., 2000; Wittmann et al.,          Plant material. The C. annuum homozygous lines used were Yolo
1997). Loss of interaction of VPg with eIF4E is correlated            Wonder (YW), Yolo Y (YY), FloridaVR2 (F), Perennial (P) and
with a loss of infectivity of the virus, suggesting that the          DH801. YW contains the pvr2+ dominant allele for susceptibility to
interaction is critical for virus production (Leonard et al.,         potyviruses and pvr6+, which does not confer resistance to PVMV
2000; Schaad et al., 2000).                                           in combination with pvr2 alleles; YY has the recessive allele pvr21 for
                                                                      complete resistance to PVY pathotype 0 and the pvr6+ allele; F has
Interestingly, the molecular characterization of naturally            the recessive allele pvr22 for complete resistance to PVY pathotypes
                                                                      0 and 0,1 and to TEV and the pvr6+ allele; P has the recessive allele
occurring recessively inherited resistance loci also converged
                                                                      pvr23 for partial resistance to PVY and the pvr6 allele. YW, YY, F
towards identification of the translation initiation factor 4E         and P are susceptible to PVMV. DH801 is a doubled haploid (DH)
(reviewed by Robaglia & Caranta, 2006). Four recessive                line homozygous for pvr6 (from P) and pvr22 (from F). It is com-
resistance genes against potyviruses have been shown to               pletely resistant to PVMV (Caranta et al., 1996; this study). The pvr6
encode defective forms of eIF4E: pvr2 and pvr1 from pepper            segregating population was composed of 22 DH lines obtained by
(recently shown to be alleles of the same gene; Kang et al.,          another culture from an F1 hybrid between P and YW. The pro-
2005; Ruffel et al., 2002, 2004) and its orthologue, pot-1,           cedure to determine the allele at the pvr6 locus has been described
                                                                      by Caranta et al. (1996). Briefly, the genotype at the pvr6 locus was
from tomato for Potato virus Y (PVY) and TEV resistance
                                                                      inferred through PVMV resistance evaluation of 22 F2 progeny
(Ruffel et al., 2005), mo1 for lettuce resistance to LMV              plants (200 plants per F2) obtained by crossing the 22 DH lines with
(Nicaise et al., 2003) and sbm-1 for pea resistance to Pea seed-      F, homozygous for pvr22. The F2 progeny (182 plants) segregating
borne mosaic virus (PSbMV) (Gao et al., 2004). Resistance             for both pvr22 and pvr6 were generated from the F1 hybrid between
resulted from a small number of amino acid changes in the             DH218 (known to be homozygous for pvr6; Caranta et al., 1996)
eIF4E proteins encoded by the recessive resistance alleles.           and F (homozygous for pvr22). This F2 progeny was assessed for
Moreover, in the four species, most of the resistance-related         PVMV resistance. The same F2 plants were also genotyped for dele-
                                                                      tion at eIF(iso)4E and for alleles at the pvr2-eIF4E locus. The tomato
changes corresponded to non-conservative amino acid sub-              introgression line (IL) population was used to map eIF(iso)4E. The
stitutions and were clustered in two neighbouring regions             IL population was composed of 75 Lycopersicon esculentum cv. M82
and at the surface of the predicted eIF4E 3D structure (Gao           lines, each containing a single genomic fragment introgressed from
et al., 2004; Nicaise et al., 2003; Ruffel et al., 2005). Recently,   the wild species Lycopersicon pennellii LA716 (Liu & Zamir, 1999).
the rym4/5 gene for barley resistance to strains of the Barley
yellow mosaic virus complex from the genus Bymovirus                  Virus strain and detection of virus accumulation. Inoculation
                                                                      experiments were carried out under growth chamber conditions
was also demonstrated to correspond to eIF4E (Stein et al.,
                                                                      (22 uC, 12 h photoperiod). Experiments were performed using the
2005).                                                                PVMV-IC isolate from the Ivory Coast, provided by J. C. Thouvenel
                                                                      (IRD, Montpellier, France). PVMV-IC was maintained on suscepti-
Numerous recessive resistance genes characterized by an               ble pepper cultivar YW and transferred every 4 weeks. Inoculum
important diversity of resistance spectra and resistance levels       was prepared from 1 g (fresh weight) infected foliar tissues ground
against potyviruses have been described in pepper, making             with 4 ml 0?03 M potassium phosphate buffer (pH 7) containing
this species a useful model for the study of host factors             0?2 % diethyldithiocarbamate, 80 mg active charcoal and 80 mg
required for viral infection. In addition to pvr2 resistance          Carborundum (400 mesh). For inoculation, leaves of 4-week-old
against PVY and TEV mediated by mutations in eIF4E, two               plants (two- to three-leaf stage) were rubbed manually with inocu-
                                                                      lum extract and rinsed with water for 5 min after rubbing. Purity
other recessive resistance loci have been identified. pvr3,            was monitored regularly with differential host index tests. PVMV-IC
from Capsicum annuum cv. Avelar, confers resistance to                accumulation in inoculated and apical non-inoculated pepper leaves
long-distance movement of Pepper mottle virus (Guerini &              was checked by double-antibody sandwich ELISA (DAS-ELISA)
Murphy, 1999). pvr6, from C. annuum cv. Perennial, was                and RT-PCR. Polyclonal antibodies raised against the PVMV coat

2090                                                                                                        Journal of General Virology 87
                                                                                        Two eIF4E isoform mutations for PVMV resistance

protein (CP) were supplied by R. Nono-Wondim (AVRDC, Arusha,             post-inoculation (p.i.), N. benthamiana-inoculated leaves were used
Tanzania). Samples were considered to be virus-positive when the         as an inoculum source for a transient expression assay in pepper. In
ELISA absorbance value was at least three times greater than the         three independent experiments, pepper genotypes YW (homozygous
mean value of the healthy controls. RT-PCR for PVMV was                  for pvr2+ and pvr6+ susceptibility alleles) and DH801 (homozygous
performed with primers specific for the coding sequences of NIb           for both pvr22 and pvr6 resistance alleles) were inoculated with PVX
(forward, 59-GGIAARGCNCCNTAYAT-39) and CP (reverse, 59-                  plasmids and 10 days later, the same leaves were inoculated with
CGCGCTAATGACATATCGGT-39) (Moury et al., 2005).                           PVMV-IC. PVX and PVMV accumulation in inoculated pepper
                                                                         leaves was checked at 10 days p.i. by DAS-ELISA and RT-PCR. A
Restriction fragment length polymorphism (RFLP) analysis.                sample was considered to be positive for virus when the ELISA
Plant genomic DNA isolation and RFLP were done as described              absorbance value at 405 nm was at least three times greater than the
previously with hybridization at 65 uC (Caranta et al., 1996). The       mean value of the healthy controls. RT-PCR for PVX was performed
tomato eIF(iso)4E cDNA probe for RFLP mapping on tomato                  with primers 59-CCGATCTCAAGCCACTCTCCG-39 (forward) and
and pepper progenies was obtained by RT-PCR using primers                59-CCTGAAGCTGTGGCAGGAGTTG-39 (reverse) specific for either
59-TGGACTGCTACTAGCAGCAG-39 (forward) and 59-GCAGAC-                      side of the cloning site to check the stability of the constructs.
AAGCGCAAAAAAGTG-39 (reverse), which amplified an 882 bp
eIF(iso)4E sequence (TIGR accession no. TC126316).

Amplification, cloning and sequencing of eIF(iso)4E cDNA                  RESULTS
from pepper. Total RNA from YW, YY, F, P and DH801 plants
was isolated from 100 to 200 mg leaf tissue using TRI Reagent            Detection of PVMV-IC in C. annuum genotypes
(Sigma-Aldrich). The 39 end of YW eIF(iso)4E cDNA was obtained           with distinct allelic combinations at the pvr2
using the Gibco-BRL Life Technologies 39RACE system (version
                                                                         and pvr6 loci
2.0). The primer 59-AAGTGGACTGTTACGAGCAGCAG-39 specific
for the eIF(iso)4E gene was designed from alignment of the A.            All YW, YY, F and P plants inoculated with PVMV-IC
thaliana (GenBank accession no. Y10547), Lactuca sativa (GenBank         presented mosaic symptoms in apical, non-inoculated
accession no. AF530163) and Lycopersicon esculentum (TIGR acces-
                                                                         leaves (referred to as apical leaves) and exhibited high
sion no. TC126316) eIF(iso)4E cDNAs and used together with the
adapter primer (AUAP) in the kit. Full-length YW eIF(iso)4E cDNA         DAS-ELISA values at 15 days p.i., whereas no systemic
was amplified by RT-PCR with a primer designed from the sequence          infection was observed and no virus CP was detected by
of the 39RACE product (59-ATTGCTGGAACTTGGGGAGGG-39)                      DAS-ELISA in inoculated or apical leaves of DH801 plants
and a primer designed from the 59 untranslated region of TIGR            carrying both pvr22 and pvr6 resistance alleles (Table 1). To
accession no. TC126316 (59-AAAACAATGGCCACCGAAGCA-39).                    determine precisely how infection progresses in the various
All amplifications were performed with High Fidelity Platinum             host genotypes and to check whether pvr2 or pvr6 resistance
Taq polymerase (Gibco-BRL Life Technologies). The pGEM-T
Easy vector system (Promega) was used to clone cDNAs after PCR
                                                                         alleles alone had any detectable effect on PVMV accumula-
amplification. At least three independent positive clones were            tion in inoculated leaves, further virus detection on inocu-
sequenced from both ends by Genome Express (Grenoble, France).           lated leaves was performed by RT-PCR and DAS-ELISA at 2,
The Genetics Computer Group (Madison, WI, USA) software pack-            3, 4 and 7 days p.i. Viral RNA and CP accumulation were
age was used for nucleic and protein sequence analysis.                  detected in inoculated leaves of YW, YY, F and P plants
PCR markers. Total RNA was extracted from the leaves of geno-
                                                                         challenged with PVMV-IC at 2, 3, 4 and 7 days p.i., but
types P, F and YW, from the 22 DH lines segregating for pvr6             were not detected in inoculated leaves of DH801 at any
and from the 182 F2 progeny (DH2186F) segregating for both pvr6          time point during two independent experiments (Table 1).
and pvr22 as described above. The eIF(iso)4E cDNA of 527 or              Thus, these data, together with previous genetic analysis
609 bp, depending on the presence/absence of the 82 bp deletion,         (Caranta et al., 1996), indicate that resistance to PVMV,
was amplified by RT-PCR with the primers 59-ATGGCCACCGAAG-                expressed as undetectable accumulation of the virus in
                                                                         inoculated leaves, results from the combined effect of the
TCTTAGCT-39 (reverse). The eIF4E cDNA corresponding to the pvr2
locus was amplified with the primers 59-AAAAGCACACAGCA-                   pvr22 and pvr6 alleles.
(reverse). A cleaved amplified polymorphic sequence (CAPS) differ-        eIF(iso)4E maps in the same genomic region as
ential cleavage site revealed by the restriction endonuclease MvnI was
based on a single nucleotide polymorphism of TRG at position 236
                                                                         the pvr6 locus
of the cDNA that distinguishes pvr22 from the pvr2+ and pvr23 alleles.   Genetic mapping of eIF(iso)4E was conducted as a first step
After amplification of the 687 bp product with the eIF4E cDNA-            in tomato because of the availability of IL progeny and
specific primers, products were digested with MvnI, generating two
fragments of 453 and 234 bp only for the pvr22 allele from F. PCR
                                                                         expressed sequence tags that facilitate mapping of multiloci
products were resolved by electrophoresis in a 2 % (w/v) agarose gel.    components. The tomato eIF(iso)4E probe generated from
                                                                         TIGR accession no. TC126316 identified RFLPs between
Functional complementation assays. Functional complementa-               Lycopersicon pennellii LA716 and Lycopersicon esculentum
tion was performed using a Potato virus X (PVX)-mediated transient       M82 with HindIII- and XbaI-digested DNA. For both
expression assay as described by Ruffel et al. (2002). eIF4E ORFs
from YW and F were cloned into pPVX201 (provided by D. C.
                                                                         restriction enzymes, an RFLP was assigned to a genomic
Baulcombe, Sainsbury Laboratory, UK; Baulcombe et al., 1995) to          region of 23?6 cM spanning ILs 9.3 to 9.3.2 with overlapping
produce plasmids pPVXeYW and pPVXeF, respectively. Similarly,            introgression fragments (Fig. 1).
eIF(iso)4E ORFs from YW and P were cloned into pPVX201 to pro-
duce plasmids pPVX(iso)eYW and pPVX(iso)eP. Plasmids were                The syntenic relationship between tomato and pepper
then manually inoculated to Nicotiana benthamiana; at 10 days            genomes makes it possible to infer the location of a pepper                                                                                                             2091
S. Ruffel and others

Table 1. PVMV-IC accumulation assessed by DAS-ELISA and RT-PCR in inoculated or apical non-inoculated leaves of C.
annuum genotypes with distinct alleles at the pvr2 and pvr6 loci

                                                                   C. annuum genotype and alleles at the pvr2 and pvr6 loci

                                                         YW                  YY                  F                  P             DH801
                                                        +      +             1     1            2     2            3        3
                                                    pvr2 /pvr2           pvr2 /pvr2         pvr2 /pvr2         pvr2 /pvr2       pvr22/pvr22
                                                    pvr6+/pvr6+         pvr6+/pvr6+        pvr6+/pvr6+          pvr6/pvr6        pvr6/pvr6

 ELISA on inoculated leaves at 15 days p.i.*         1?44±0?18           1?46±0?16          1?45±0?23          1?56±0?22         0?13±0?02
 ELISA on apical leaves at 15 days p.i.*             1?74±0?20           1?58±0?40          1?62±0?33          1?56±0?26         0?14±0?01
 RT-PCR on inoculated leaves at 2 days p.i.D             +                   +                  +                  +                 2
 RT-PCR on inoculated leaves at 3 days p.i.D             +                   +                  +                  +                 2
 RT-PCR on inoculated leaves at 4 days p.i.D             +                   +                  +                  +                 2
 RT-PCR on inoculated leaves at 7 days p.i.D             +                   +                  +                  +                 2

*Means±SD of DAS-ELISA values of A405 calculated from 10 leaves in two independent experiments. ELISA was considered to be positive when
the A405 of the sample was at least three times greater than the mean value of the healthy controls (i.e. A405¢0?36 in these experiments). The
mean A405 of healthy plants was 0?12±0?01.
DAs negative controls, RT-PCR was performed on PVMV-IC-inoculated leaves of the non-host plants Cucumis melo and Vinca rosea at 2, 3, 4 and
7 days p.i. Ten inoculated leaves were assessed for each genotype in two independent experiments. +, RT-PCR amplification of a 737 bp
fragment corresponding to the NIb/CP region of PVMV-IC (Moury et al., 2005); 2, no amplification.

locus on the tomato genome (Livingstone et al., 1999). In                 that contained a single open reading frame of 609 nt
pepper, the pvr6 locus was localized in a genomic region of               encoding a protein of 202 aa. The closest matches obtained
chromosome P3 defined by RFLP markers CT220, TG591                         after a BLAST search with the full-length nucleotide sequence
and CD008, 9?4 cM from TG591 (Caranta et al., 1996;                       were the eIF(iso)4E cDNA from lettuce (GenBank accession
Lefebvre et al., 2002) (Fig. 1). These three RFLP markers                 no. AF530163, E=1?10259), pea (GenBank no. AY423377,
are linked on the tomato genetic linkage map and localize to              E=2?10224), maize (GenBank no. AF076955, E=4?10210),
a chromosome 9 region that overlaps the chromosomal                       Arabidopsis (GenBank no. Y10547, E=6?1029) and wheat
region defined by ILs 9.3 and 9.3.2 (Liu & Zamir, 1999;                    (GenBank no. M95818, E=9?1028). The closest matches
Tanksley et al., 1992) (Fig. 1). Taken together, these data               with the predicted translation product were the eIF(iso)4E
suggested that pvr6 and the RFLP marker generated using                   amino acid sequences from lettuce (GenBank protein no.
the tomato eIF(iso)4E probe were localized to the same                    AAP86603, E=8?10277) and wheat (GenBank protein no.
genomic region.                                                           AAA34296, E=1?10273), confirming that the cloned cDNA
                                                                          corresponded to eIF(iso)4E. The pepper eIF4E (GenBank
To delimit further the map location of eIF(iso)4E in com-                 accession no. AY122052) and eIF(iso)4E coding regions
parison with pvr6, the tomato eIF(iso)4E probe was hybri-                 were 57?5 % identical in nucleotide sequence and 48?3 %
dized on pepper genotypes YW, F and P with distinct alleles               identical in amino acid sequence.
at the pvr6 locus. With EcoRV-digested DNA, hybridization
revealed an RFLP between the pvr6 and pvr6+ genotypes.                    To determine whether sequence variation could be associ-
This marker was mapped on the pepper progeny of 22 DH                     ated with pvr6 or pvr6+ alleles, nucleotide and amino acid
lines that were used by Caranta et al. (1996) for the demon-              sequences of eIF(iso)4E cDNAs from YW, YY, F, P and
stration of complementation between pvr22 and pvr6 for                    DH801 plants were compared. Nucleotide sequence align-
PVMV resistance. These lines segregated for pvr6 alleles into             ment showed that the pvr6+ genotypes YW, YY and F were
eight pvr6+ lines and 14 pvr6 lines. Genetic co-segregation               100 % identical in their eIF(iso)4E nucleotide sequence. The
between pvr6 and the RFLP generated using the eIF(iso)4E                  pvr6 genotypes P and DH801 were also 100 % identical in
cDNA probe was observed among the 22 DH lines, pro-                       their eIF(iso)4E nucleotide sequence but presented, in com-
viding additional data in favour of linkage between the two               parison with pvr6+ genotypes, a deletion from nt 89 to 170,
loci.                                                                     a GRA substitution at position 268, a CRA substitution
                                                                          at position 483 and a CRT substitution at position 537
                                                                          (Fig. 2). The complete eIF(iso)4E genomic sequence was
eIF(iso)4E cDNAs from pvr6 genotypes have a
                                                                          amplified from YW and P DNA and the first exon was
deletion of 82 nt
                                                                          sequenced to confirm that the deletion occurred on geno-
An eIF(iso)4E cDNA was obtained from the potyvirus-                       mic DNA and was not the result of a mRNA splicing event
susceptible pepper genotype YW using 39RACE PCR and                       (data not shown). The 82 nt deletion modified the open
sequence data available for the tomato eIF(iso)4E cDNA                    reading frame by insertion of a stop codon after aa 51.
(TIGR accession no. TC126316). A 660 nt cDNA was deduced                  Alignment of predicted eIF(iso)4E protein sequences from

2092                                                                                                           Journal of General Virology 87
                                                                                 Two eIF4E isoform mutations for PVMV resistance

                                                                    the 82 nt deletion in the eIF(iso)4E cDNA was tested in 182
                                                                    F2 progeny plants segregating for both the pvr6 and pvr22
                                                                    resistance alleles. In the first step, F2 plants were phenotyped
                                                                    for PVMV-IC resistance. The segregation ratio observed for
                                                                    PVMV resistance (proportion of resistant and susceptible
                                                                    plants assessed by DAS-ELISA) was consistent with two
                                                                    recessive genes involved in resistance [observed ratio
                                                                    14R : 168S; x21df(1R:15S)=0?6461 and P=42?2 %]. The
                                                                    same 182 F2 plants were then genotyped with the PCR
                                                                    marker based on the presence/absence of the 82 nt deletion
                                                                    in the eIF(iso)4E cDNA and with the CAPS marker based on
                                                                    a single nucleotide polymorphism within the coding region
                                                                    of the pvr2-eIF4E locus that distinguishes pvr22 from other
                                                                    pvr2 alleles segregating in the progeny. Among the 182 F2
                                                                    plants, all of the 14 PVMV-resistant plants produced both a
                                                                    PCR fragment of 527 bp corresponding to the 82 nt deletion
                                                                    in eIF(iso)4E and two fragments of 453 and 234 bp follow-
                                                                    ing MvnI digestion of the 687 bp PCR product obtained
                                                                    using eIF4E cDNA-specific primers, a pattern specific for
                                                                    the pvr22 allele (Fig. 3a). In contrast, all PVMV-susceptible
                                                                    plants displayed PCR profiles producing at least one non-
                                                                    deleted eIF(iso)4E cDNA fragment and/or at least one PCR
                                                                    fragment corresponding to pvr2 alleles distinct from pvr22
                                                                    (Fig. 3a).

                                                                    Segregation of the PCR marker based on the presence/
                                                                    absence of the 82 nt deletion in the eIF(iso)4E cDNA was
                                                                    also assessed in the 22 DH lines segregating for pvr6. All of
                                                                    the DH lines known to be homozygous for pvr6+ produced
                                                                    a PCR fragment of 609 bp, whereas all those homozygous
                                                                    for pvr6 produced a PCR fragment of 527 bp (Fig. 3b).
                                                                    Taken together, the observed co-segregation between pvr6
                                                                    and the 82 nt deletion in the eIF(iso)4E cDNA in both F2 and
Fig. 1. Map location of an RFLP marker generated by the tomato
                                                                    DH progeny strengthens the possibility that this locus
eIF(iso)4E cDNA probe on tomato chromosome 9 and compara-
                                                                    corresponds to eIF(iso)4E.
tive mapping with pepper. (a) Lycopersicon esculentum chromo-
some 9 is represented on the left superimposed with the eight
introgression fragments (IL9.1, IL9.1.2, IL9.1.3, IL9.2, IL9.2.5,
                                                                    Transient expression in a PVMV-resistant
IL9.2.6, IL9.3 and IL9.3.2) from Lycopersicon pennellii LA716 in    genotype of either pvr6+-eIF(iso)4E or
the Lycopersicon esculentum M82 recipient lines (Liu & Zamir,       pvr2+-eIF4E restores PVMV susceptibility
1999). The approximate map location of the eIF(iso)4E marker        Previously published results (Caranta et al., 1996) together
and RFLP markers CD008, TG591 and CT220 are indicated. A            with RT-PCR detection of PVMV RNA in inoculated leaves
segment of C. annuum chromosome 3 is represented on the right       of C. annuum genotypes with distinct allelic combinations
with the linkage between RFLP markers CD008, TG591 and              at the pvr2 and pvr6 loci showed that complete resistance to
CT220 and the pvr6 locus. The alignment of markers between
                                                                    PVMV resulted from the combined effect of the pvr6 and
tomato and pepper is indicated by dotted lines. One centimetre
                                                                    pvr22 resistance alleles. Genetic and sequence analysis also
represents approximately 25 cM. (b) The XbaI RFLP fragment
                                                                    suggested that the pvr6 locus corresponded to eIF(iso)4E
generated with the eIF(iso)4E cDNA probe from LA716 and
retrieved in ILs 9.3 and 9.3.2 is indicated by an arrow.
                                                                    and that the recessive resistance allele encoded a truncated
                                                                    protein lacking key domains for cap-binding activity. In
                                                                    order to confirm the involvement of the two cap-binding
pvr6+ and pvr6 genotypes showed that only the N-terminal            isomers in the pepper/PVMV interaction, eIF(iso)4E and
29 aa of the 202 aa of wild-type protein were conserved             eIF4E cDNAs from pvr6+ and pvr2+ genotypes were
                                                                    expressed independently in a PVMV-resistant genotype to
(Fig. 2).
                                                                    check whether their expression could lead to loss of resis-
                                                                    tance. This was achieved through PVX-mediated transient
The 82 nt deletion within eIF(iso)4E
                                                                    expression of eIF(iso)4E or eIF4E in the resistant genotype
co-segregates with pvr6
                                                                    DH801, followed by PVMV inoculation. Previous results
To confirm the genetic linkage between the pepper                    have clearly demonstrated that PVX is an efficient expres-
eIF(iso)4E cDNA and pvr6, the linkage between pvr6 and              sion vector in Capsicum (Ruffel et al., 2002). PVMV                                                                                                   2093
S. Ruffel and others

                                                                                Fig. 2. Alignment of nucleotide eIF(iso)4E
                                                                                open reading frame sequences (a) and pre-
                                                                                dicted eIF(iso)4E protein sequences (b) from
                                                                                pepper genotypes homozygous for pvr6+ or
                                                                                pvr6 alleles. Genotypes homozygous for the
                                                                                pvr6+ susceptibility allele are YW, YY and F
                                                                                and genotypes homozygous for the pvr6
                                                                                resistance allele are P and DH801. Black
                                                                                boxes indicate nucleotide differences observed
                                                                                between pvr6+ and pvr6 genotypes.

susceptibility was monitored using RT-PCR and DAS-           encoded by the pvr2+ susceptibility allele, DH801 plants
ELISA. We first verified that PVX systemic infection of        were inoculated with pPVXeYW and PVMV-IC. Of 30
pepper genotypes DH801 and YW followed by inoculation        double-inoculated leaves, 13 displayed significant accumu-
with PVMV-IC did not modify the infectivity of PVMV-IC       lation of PVMV CP and NIb-CP RNA (Table 2; Fig. 4, lane
compared with plants inoculated with PVMV-IC alone           12), whereas expression of eIF4E encoded by the pvr22
(Table 2; Fig. 4, lanes 3 and 10).                           resistance allele did not support PVMV accumulation in
                                                             DH801 plants (Table 2; Fig. 4, lane 11).
To see whether PVMV infection of resistant DH801 plants
could be supported by expression of eIF(iso)4E from the      Thus, functional complementation experiments demon-
susceptible pvr6+ genotype, DH801 plants were inocu-         strated that transient expression of the pvr6+-eIF(iso)4E or
lated with pPVX(iso)eYW and PVMV-IC. Of 40 double-           pvr2+-eIF4E allele in the resistant genotype DH801 resulted
inoculated leaves, eight displayed significant accumulation   in loss of resistance to subsequent PVMV infection. These
of PVMV CP and NIb-CP RNA (Table 2; Fig. 4, lane 14).        experiments confirmed that pvr6 corresponds to eIF(iso)4E
Conversely, DH801 leaves inoculated with pPVX(iso)eP         and that both eIF4E and eIF(iso)4E wild type are suscept-
expressing eIF(iso)4E from resistant pvr6 plants and PVMV-   ibility factors for PVMV infection. In three independent
IC did not support PVMV accumulation (Table 2; Fig. 4,       experiments, the expression of the resistance allele pvr22-
lane 13).                                                    eIF4E or pvr6-eIF(iso)4E did not restore PVMV suscept-
                                                             ibility, clearly indicating that loss of resistance results from
To determine whether PVMV infection of resistant DH801       amino acid changes identified in proteins encoded by resis-
plants could also be supported by expression of eIF4E        tance and susceptibility alleles.

2094                                                                                           Journal of General Virology 87
                                                                                    Two eIF4E isoform mutations for PVMV resistance

      Fig. 3. Genetic co-segregation between pvr6 and the 82 nt deletion in the eIF(iso)4E cDNA in two progeny pepper plants.
      (a) Genetic co-segregation in the F2 progeny segregating for both pvr6 and pvr22 resistance alleles. The 14 PVMV-resistant
      plants produced a PCR fragment of 527 bp corresponding to the 82 nt deletion in eIF(iso)4E and two fragments of 453 and
      234 bp following MvnI digestion of the 687 bp PCR product obtained using eIF4E cDNA-specific primers. This pattern is
      characteristic of pvr22 homozygous plants. PVMV-susceptible plants produced at least one non-deleted eIF(iso)4E fragment
      and/or a PCR pattern for pvr2-eIF4E corresponding to a homozygous susceptible or heterozygous state. (b) Genetic
      co-segregation in the DH progeny segregating for pvr6. A RT-PCR fragment of 609 bp was amplified in YW, F and the eight
      DH lines known to be homozygous for the susceptibility allele pvr6+ (6+), whereas a fragment of 527 bp was amplified in P
      and the 14 DH lines known to be homozygous for the resistance allele pvr6.

DISCUSSION                                                            results showing that resistance alleles at the pvr2 locus
                                                                      encode mutated versions of eIF4E, we investigated the
In pepper, resistance to PVMV is digenic and results from
                                                                      involvement of the second cap-binding isoform, eIF(iso)4E,
complementation between the pvr6 and pvr22 recessive
                                                                      in PVMV resistance. Genetic mapping, sequence analysis
resistance alleles (Caranta et al., 1996). pvr6 alone has no
                                                                      and functional complementation assays demonstrated that
detectable effect on potyvirus infection, whereas pvr22
                                                                      pvr6 encodes eIF(iso)4E and that complete resistance to
controls both PVY and TEV resistance. In line with previous
                                                                      PVMV resulted from the combined effect of mutations in
                                                                      translation initiation factors eIF4E and eIF(iso)4E. This
                                                                      provides the first molecular interpretation of complementa-
Table 2. Number of PVMV-infected leaves/number of                     tion for resistance between two naturally occurring recessive
PVMV-inoculated leaves assessed by DAS-ELISA in a PVX-
based transient expression assay
                                                                      genes. The systematic analysis of alleles of the eIF4E gene
                                                                      family therefore appears to be a powerful means of
A leaf was considered to be infected when the DAS-ELISA absor-        characterizing molecularly the nature of recessive resistance
bance value was at least three times greater than the mean value      genes towards poty- and related viruses.
of the healthy controls (A405¢0?39 in these experiments).
                                                                      In contrast to previously characterized naturally occurring
 Inoculum                           C. annuum genotype                recessive resistance genes against potyviruses, all of which
                                                                      depend on a discrete number of amino acid changes in eIF4E
                                     YW             DH801
                                                                      homologues, the pvr6 resistance allele was predicted to
                                (pvr2+/pvr6+)     (pvr22/pvr6)
                                                                      encode a truncated protein with only the N-terminal 29 aa.
 PVMV-IC                            20/20             0/20            Because none of the features considered to be hallmarks of
 pPVX201*+PVMV-IC                   20/20             0/20            functional eIF4E proteins is present, including the eight
 pPVX(iso)eYW+PVMV-IC               20/20             8/40            conserved tryptophan residues, three of which are required
 pPVX(iso)eP+PVMV-IC                20/20             0/40            for cap-binding activity (Marcotrigiano et al., 1997; Matsuo
 pPVXeYW+PVMV-IC                    20/20            13/30            et al., 1997), we suggest that pvr6 encodes a non-functional
 pPVXeF+PVMV-IC                     20/20             0/30            cap-binding factor. Similarly to the Arabidopsis mutant line
                                                                      completely lacking both eIF(iso)4E mRNA and protein
*pPVX201, empty PVX vector; pPVX(iso)eYW, PVX with pvr6+-             (Duprat et al., 2002), plants homozygous for pvr6 do not
eIF(iso)4E cDNA from the YW genotype; pPVX(iso)eP, PVX with           display any obvious growth and development phenotype,
pvr6-eIF(iso)4E cDNA from the P genotype; pPVXeYW, PVX                suggesting that this eIF(iso)4E activity is not essential. This
with pvr2+-eIF4E cDNA from the YW genotype; pPVXeF, PVX               is strengthened further by the fact that the pvr6 allele has
with pvr22-eIF4E cDNA from the F genotype.                            been deployed in the field without any reduction in crop                                                                                                         2095
S. Ruffel and others

                                                                  probably combine a non-functional eIF(iso)4E and an
                                                                  eIF4E differing from the wild-type protein by three amino
                                                                  acid substitutions, V67E, L79R and D109N (Ruffel et al.,
                                                                  2002; this study). The D109N mutation affects a highly
                                                                  conserved residue involved in stabilization of cap binding
                                                                  (Marcotrigiano et al., 1997). The fact that PVMV-resistant
                                                                  plants are phenotypically normal in every respect suggests
                                                                  that point mutations at pvr22-eIF4E have no functional
                                                                  consequences for translation initiation in planta. This hypo-
                                                                  thesis is supported by the recent demonstration that pvr22-
                                                                  eIF4E mutations did not abolish in vitro cap-binding activity
                                                                  (Kang et al., 2005). However, amino acid changes in eIF4E
                                                                  proteins encoded by other resistance alleles against poty-
                                                                  viruses in pea and pepper have been demonstrated to abolish
                                                                  cap-binding activity (Gao et al., 2004; Kang et al., 2005).
                                                                  Taken together, these observations are of particular interest
                                                                  for the management of eIF4E resistance factors in breeding
                                                                  programmes. The diversity of mutations in several eIF4E
                                                                  factors could be exploited for broad-spectrum resistance
                                                                  against poty- and related viruses if the cellular function of
                                                                  one or the other cap-binding isoforms is preserved.

                                                                  Recent analysis of Arabidopsis mutant lines demonstrated
Fig. 4. Complementation experiments using PVX-based transient     that potyviruses differ in their ability to use eIF4E isoforms
expression of the eIF4E (pvr2+) or eIF(iso)4E (pvr6+) cDNA from   from a given host plant. The knockout mutation of the
PVMV-susceptible pepper genotypes in PVMV-resistant pepper        eIF(iso)4E gene prevents infection by TuMV, TEV and LMV
plants. Lanes 1–7 correspond to the susceptible genotype YW       (Duprat et al., 2002; Lellis et al., 2002) without preventing
and lanes 8–14 to the PVMV-resistant genotype DH801. Lanes 1      infection by ClYVV (Sato et al., 2005). Conversely, the
and 8 correspond to healthy tissue. Lanes 2 and 9 were inocu-
                                                                  knockout mutation of eIF4E1 allows TuMV but not ClYVV
lated with PVMV-IC and lanes 3 and 10 were double inoculated
                                                                  replication (Sato et al., 2005), indicating that potyviruses
with pPVX201 (201) and PVMV. Lanes 4–7 and 11–14 were
                                                                  selectively use either eIF4E1 or eIF(iso)4E to infect
double inoculated with the PVX construct pPVXeF (eF),
pPVXeYW (eYW), pPVX(iso)eP [(iso)eP] or pPVX(iso)eYW
                                                                  Arabidopsis. This study therefore identifies a new situation
[(iso)eYW] and PVMV. (a) Detection of PVMV in inoculated          regarding the specificity of use of eIF4E isoforms by poty-
leaves by RT-PCR using primers as defined by Moury et al.          viruses. PVMV, unlike other potyviruses, can use either
(2005). The expected size of the amplification product was         eIF4E or eIF(iso)4E for pepper infection, indicating that the
737 bp. The results of the RT-PCR are from individual leaves      two isoforms are functionally interchangeable in this parti-
representative of each treatment. (b) Detection of PVX by RT-     cular plant/potyvirus pair. The molecular mechanism that
PCR assay. The expected size of the amplification product for      determines this specificity remains to be elucidated. From
the PXV vector without an insert was 0?23 kb, with the eIF4E      the well-documented ability of the potyviral VPg to bind to
ORF insert was 0?92 kb and with the eIF(iso)4E ORF insert         eIF4E proteins (Schaad et al., 2000; Wittmann et al., 1997)
was 0?84 or 0?76 kb depending on the allele (pvr6+ or pvr6).      and published data supporting a key role for the eIF4E–VPg
The results of the RT-PCR are from the same leaves and same       interaction with respect to the outcome of viral infection
RNA sample as those shown in (a).                                                      ´
                                                                  (Kang et al., 2005; Leonard et al., 2000), a simple hypothesis
                                                                  would be that this specificity relies on differential binding
                                                                  affinities between the potyviral VPg and eIF4E isoforms.
yield. Similarly, eIF4E1 also appears to be dispensable for       Yeast two-hybrid assays and in vitro assays of VPg–
normal A. thaliana growth (Yoshii et al., 2004). These            eIF4E interactions support this hypothesis. In Arabidopsis,
observations reinforce the hypothesis that, despite having        eIF(iso)4E is required for TuMV infection and eIF(iso)4E
idiosyncratic properties, these factors can compensate for        interaction with the TuMV VPg is stronger than with eIF4E
one another in cellular functions.                                   ´
                                                                  (Leonard et al., 2000). Similarly, in tomato, resistance
                                                                  against TEV is controlled by eIF4E (Ruffel et al., 2005)
Interestingly, whereas stable antisense depletion of either       and yeast two-hybrid assays have identified interactions
eIF4E or eIF(iso)4E in tobacco had no obvious effect,             between the TEV VPg and eIF4E and a failure to bind to
depletion of both eIF4E and eIF(iso)4E resulted in plants         eIF(iso)4E (J. L. Gallois & C. Caranta, unpublished data).
with a semi-dwarf phenotype and an overall reduction in           However, in another system where eIF4E is known to be the
polyribosome loading, demonstrating that the two isoforms         resistance gene, no interaction between the viral avirulent
contribute additively to translation and plant growth             determinant VPg of PSbMV and eIF4E from a susceptible
(Combe et al., 2005). PVMV-resistant pepper genotypes             pea genotype was demonstrated (Gao et al., 2004). It is likely

2096                                                                                               Journal of General Virology 87
                                                                                          Two eIF4E isoform mutations for PVMV resistance

that interaction of eIF4E and VPg in planta are influenced                  ´
                                                                          Leonard, S., Plante, D., Wittman, S., Daigneault, N., Fortin, M. G. &
by interactions with other plant or viral proteins and                    Laliberte, J.-F. (2000). Complex formation between potyvirus VPg
nucleic acids. Taken together with our current results, these             and translation eukaryotic initiation factor 4E correlates with virus
                                                                          infectivity. J Virol 74, 7730–7737.
observations illustrate the diversity of molecular mechan-
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We thank D. Headon (University of Manchester, UK) for improving           Marcotrigiano, J., Gingras, A.-C., Sonenberg, N. & Burley, S. K.
the English of the manuscript and also A. Maule (John Innes Centre,       (1997). Cocrystal structure of the messenger RNA 59 cap-binding
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T. Phally, A. M. Daubeze and A. Moretti for their excellent assistance.   Matsuo, H., Li, H., McGuire, A. M., Fletcher, C. M., Gingras, A.-C.,
This work was supported by grants from GENOPLANTE (CI-2001-05)            Sonenberg, N. & Wagner, G. (1997). Structure of translation
and the French Ministry of Research (ACI Jeunes Chercheurs 2003-          initiation factor eIF4E bound to m7GDP and interaction with
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2098                                                                                                                Journal of General Virology 87

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