Download A natural recessive resistance gene against potato virus

The Plant Journal (2002) 32, 1067–1075
A natural recessive resistance gene against potato virus Y
in pepper corresponds to the eukaryotic initiation factor
4E (eIF4E)
Sandrine Ruffel1, Marie-Hélène Dussault1, Alain Palloix1, Benoı̂t Moury2, Abdelhafid Bendahmane3,
Christophe Robaglia4 and Carole Caranta1,
1
Genetics and Breeding of Fruits and Vegetable, Dom. St Maurice, BP94, F-84143 Montfavet,
2
Plant Pathology, Dom. St Maurice, BP94, F-84143 Montfavet,
3
Plant Genomics Research Unit, 2 rue G. Cremieux, CP 5708, F-91057 Evry, and
4
Lab. Métabolisme Carboné, UMR 163 CNRS CEA, Univ. Méditerranée UMR 163, F-13108 St Paul-lez-Durance,
Cedex, France
Received 12 August 2002; revised 2002; accepted 4 October 2002.
For correspondence (fax þ 334 327 227 02; e-mail [email protected]).
Summary
We show here that the pvr2 locus in pepper, conferring recessive resistance against strains of potato virus Y
(PVY), corresponds to a eukaryotic initiation factor 4E (eIF4E) gene. RFLP analysis on the PVY-susceptible
and resistant pepper cultivars, using an eIF4E cDNA from tobacco as probe, revealed perfect map cosegregation between a polymorphism in the eIF4E gene and the pvr2 alleles, pvr21 (resistant to PVY-0)
and pvr2 2 (resistant to PVY-0 and 1). The cloned pepper eIF4E cDNA encoded a 228 amino acid polypeptide
with 70–86% nucleotide sequence identity with other plant eIF4Es. The sequences of eIF4E protein from two
PVY-susceptible cultivars were identical and differed from the eIF4E sequences of the two PVY-resistant
cultivars Yolo Y (YY) (pvr21 ) and FloridaVR2 (F) (pvr2 2 ) at two amino acids, a mutation common to both
resistant genotypes and a second mutation specific to each. Complementation experiments were used to
show that the eIF4E gene corresponds to pvr2. Thus, potato virus X-mediated transient expression of eIF4E
from susceptible cultivar Yolo Wonder (YW) in the resistant genotype YY resulted in loss of resistance to
subsequent PVY-0 inoculation and transient expression of eIF4E from YY (resistant to PVY-0; susceptible to
PVY-1) rendered genotype F susceptible to PVY-1. Several lines of evidence indicate that interaction
between the potyvirus genome-linked protein (VPg) and eIF4E are important for virus infectivity, suggesting that the recessive resistance could be due to incompatibility between the VPg and eIF4E in the resistant
genotype.
Keywords:eIF4E, VPg, virus vectors, potyvirus, Capsicum annuum.
Introduction
Virus resistance genes, long a mainstay of the plant breeder, provide a highly efficient barrier to virus infection in
plants. The best characterized resistance genes include
dominant genes (R-genes) that trigger a hypersensitive
response (HR) or extreme resistance (ER). These two resistant phenotypes are conferred through matched specificity
between the R-gene and a pathogen avirulence gene, and
operate on the basis of ‘gene-for-gene’ mechanism (Keen,
1990). Four virus R-genes have been cloned (N, Rx1, Rx2
and Sw-5) and all belong to the nucleotide binding site,
leucine-rich repeat (NBS-LRR) super-family of R-genes
ß 2002 Blackwell Publishing Ltd
(Whitham et al., 1994; Bendahmane et al., 1999; Bendahmane et al., 2000; Brommonschenkel et al., 2000).
Other types of genetic resistance include virus resistance
genes that are not associated with HR or ER (Fraser, 1990).
The two dominant genes RTM1 and RTM2, involved in
restriction of long-distance movement of tobacco etch virus
(TEV) in Arabidopsis thaliana fall into this class (Chisholm
et al., 2000; Whitham et al., 2000). Resistance in this case is
not caused by activation of known defense pathways and
it remains unclear how these genes operate. Recessive
resistance genes also belong to this class but, up to now,
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1068 Sandrine Ruffel et al.
most information about the molecular mechanisms
involved in recessive resistance was obtained using mutagenesis approach. The recessive mlo mutation in barley
which controls broad spectrum resistance against several
isolates of the fungus Erysiphe graminis f.sp. hordei, and
the Arabidopsis recessive mutant, edr1, which provokes
resistance to some bacterial and fungal pathogens
(Büschges et al., 1997; Frye and Innes, 1998), illustrate
mutations affecting the control of the defense response
and/or cell death. Concerning viruses, it is widely believed
that incompatibility occurs when the plant lacks one or
several genes required for virus infection. Such recessive
mutations suppressing efficient multiplication of tobamoviruses and potyviruses were recently identified in Arabidopsis (Yamanaka et al., 2000; Yamanaka et al., 2002; Lellis
et al., 2002).
Plant–potyvirus interactions constitute an interesting
model to investigate recessive resistance because an unusually high frequency of occurrence of genes conferring
recessive resistance to potyviruses has been observed (40%
versus 20% for resistance against other viruses) (Provvidenti and Hampton, 1992). pvr2-mediated resistance of
pepper (Capsicum spp.) to potato virus Y (PVY) falls into
this type of resistance. The pvr2 resistance locus consists of
two alleles, pvr21 and pvr22 (Kyle and Palloix, 1997): pvr21 is
effective only against PVY-0, while pvr22 is effective against
PVY-0 and -1 but is overcome by PVY-1,2. Further studies
revealed another characteristic feature of potyvirus resistance genes: resistance is often not restricted to a single
potyvirus. Thus, pvr22 is also associated with resistance to a
second potyvirus, the TEV. Whether a single locus or two
tightly linked genes controls PVY and TEV resistance
remains to be determined.
In this paper, we describe isolation of the pvr2 gene using
a candidate gene approach, an alternative to map-based
cloning and insertional mutagenesis when assumptions
can be made regarding the biological function of the gene
of interest (Pflieger et al., 2001). We demonstrate map cosegregation between the pvr2 resistance locus and a eukaryotic translation initiation factor 4E (eIF4E) gene. eIF4E was
chosen as a plausible candidate gene because (i) eIF4E
proteins bind with the viral protein genome linked (VPg)
to turnip mosaic virus (TuMV) and TEV (Wittmann et al.,
1997; Léonard et al., 2000; Schaad et al., 2000); (ii) the resistance-breaking determinants for several recessive potyvirus resistance genes localize in the VPg-coding domain
(Nicolas et al., 1997; Keller et al., 1998; Masuta et al., 1999;
Hjulsager et al., 2002, Ramajäki and Valkonen, 2002) ; and
(iii) the VPg–eIF4E interaction is necessary for virus infectivity and upregulates genome amplification (Léonard et al.,
2000; Schaad et al., 2000). Moreover, it was recently shown
that disruption of the eIF(iso)4E gene in Arabidopsis
resulted in resistance to potyviruses (Lellis et al., 2002).
Confirmation that pvr2 is indeed an allele of eIF4E was
obtained using a potato virus X (PVX)-based transient
expression assay. Two nucleotide substitutions inducing
amino acid differences between alleles in resistant and
susceptible plants were identified.
Results
An eIF4E gene co-segregates with the pvr2 locus
eIF4E belongs to a small multigenic family (Rodriguez et al.,
1998; Manjunath et al., 1999). Therefore, conventional RFLP
was chosen as the mapping strategy because it facilitates
mapping of multiloci components. To look for map cosegregation between eIF4E and the pvr2 locus, tobacco
eIF4E cDNA was used as a probe on genomic DNA of
pepper cultivars with distinct alleles at the pvr2 locus. With
EcoRI-digested DNA, hybridization revealed an RFLP of
approximately 7 kb between the PVY-susceptible cultivar,
Yolo Wonder (YW) and the PVY-resistant cultivars, Yolo Y
(YY) and Florida VR2 (F) (Figure 1). Four other major EcoRI
fragments, monomorphic between YW, YY and F, were also
detected confirming the occurrence of several eIF4E genes
in the genome.
Segregation observed for PVY-0 resistance in the BC1
progeny of 440 plants was consistent with a single recessive gene model (224 resistant : 216 susceptible; w2 for
1 : 1 ¼ 0.145; P ¼ 0.7). DNA from each BC1 was digested with
EcoRI and subjected to Southern blot hybridization with the
tobacco eIF4E cDNA probe. The 224 BC1 plants resistant to
Figure 1. Map co-segregation between the pvr2 locus and a RFLP marker
detected with a tobacco eIF4E cDNA probe.
Yolo Wonder (YW) contains the pvr2þ allele for susceptibility to PVY; Yolo Y
(YY) contains the pvr21 recessive allele for resistance to PVY-0 and FloridaVR2 (F) contains the pvr22 recessive allele for resistance to PVY-0 and -1.
F1 corresponds to the F1 hybrid between YW and YY. Lanes R correspond to
DNA from PVY-0 resistant plants of the BC1 progeny (YW YY) YY and
lane S to DNA from PVY-0 susceptible plants from the BC1. The EcoRI DNA
fragment linked to the resistant alleles is indicated by A and the EcoRI DNA
fragment linked to the susceptible allele is indicated by B. MW indicates DNA
size markers.
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1067–1075
A natural recessive resistance gene corresponding to the eukaryotic initiation factor 4E
PVY (homozygous for pvr21) presented the same RFLP
pattern as YY whereas the 216 BC1 plants susceptible to
PVY (heterozygous pvr2þ/pvr21) presented the same RFLP
pattern as the F1 (YW YY), indicating perfect map cosegregation between the eIF4E related-sequence and the
pvr2 locus.
Molecular cloning of the pepper eIF4E gene
30 and 50 RACE experiments were used to obtain the fulllength sequence of an eIF4E cDNA from YW. The resulting
cDNA was 870-nucleotides long, with a 50 untranslated
region of 28 nucleotides preceding the first in-frame
AUG. The open reading frame (ORF) is 687 nucleotides in
length and encodes a protein of 228 amino acids, assuming
that translation initiates at the first AUG codon, as in
tobacco and tomato eIF4E mRNA. Moreover, the sequence
AA3CAAUGGþ4C flanking the putative initiation codon
exactly matches the consensus sequence reported for
initiation codons in plants (Lutcke et al., 1987). The pepper
eIF4E ORF displays 73.4% nucleotide sequence identity
with the tobacco eIF4E sequence and 86.1% identity with
that of tomato (GenBank accession nos. AF259801). The
pepper eIF4E sequence is more similar to the Arabidopsis
eIF4E ORF sequence (EMBL accession no. Y10548) than it is
to the Arabidopsis eIF (iso) 4E ORF sequence (EMBL accession no. Y10547) with 70.4 and 55.3% identity, respectively,
suggesting that the cDNA codes for a protein of the eIF4E
subfamily.
To determine whether the cloned cDNA corresponds to
the eIF4E gene that co-segregates with pvr2, a pepper BAC
library was screened using primers P1 and P2. PCR amplified a single fragment of 493 bp from YW cDNA and a single
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fragment of 1800 bp on YW genomic DNA, indicating the
presence of at least one intron in this part of the gene. If the
BAC clones identified with these primers contain the eIF4E
gene which co-segregates with pvr2, they should contain a
7-kb EcoRI fragment corresponding to the RFLP (Figure 1)
and this 7-kb fragment should contain at least part of the
eIF4E gene. Four BAC clones were identified, a number
consistent with the library’s estimated representation
based on the average insert size and the total number of
clones. Restriction analysis indicated that the four BAC
clones correspond to the same locus in the genome. EcoRI
digestion of the clones produced a 7-kb fragment. This
fragment was cloned and amplified using primers P1 and
P2. The sequence of the resulting 1800-bp PCR product
demonstrated that the eIF4E gene which co-segregated
with the pvr2 locus had been cloned.
Two amino acid substitutions within the eIF4E protein
distinguish the susceptible and resistant pepper
genotypes
Nucleotide sequences of the eIF4E cDNA of YW, DDL, YY
and F were obtained from three independent RT-PCR
experiments. Amino acid sequence alignment revealed that
the two unrelated susceptible genotypes YW and DDL were
100% identical in their eIF4E sequences (Figure 2). Compared to the susceptible genotypes, the two resistant
genotypes, YY and F, each contained two amino acid substitutions. A Glu instead of a Val was present at position 67
of both resistant genotypes (Figure 2). In YY (pvr2-1), a
second specific mutation occurred at position 79 (Arg for
Leu) whereas F (pvr22) contained an Asn for Asp mutation
at position 109 (Figure 2). Alignment of the pepper eIF4E
Figure 2. Alignment of predicted eIF4E protein sequences from pepper genotypes Yolo Wonder (YW), Doux Long Des Landes (DDL), Yolo Y (YY) and FloridaVR2
(F).
Black boxes indicate amino acid differences observed between PVY-resistant and PVY-susceptible genotypes. Strictly conserved amino acids, based upon the
alignment of eIF4E proteins of Arabidopsis thaliana (EMBL accession no. Y10548), Saccharomyces cerevisiae (GenBank accession no. P07260), Mus musculus
(GenBank accession no. NP_031943) and Homo sapiens (Genbank accession no. XP_017925), are highlighted. Amino acids implicated in binding of the mRNA
cap structure are marked by arrowheads and amino acids implicated in recognition of eIF4G and 4E binding proteins by open boxes (Marcotrigiano et al., 1997).
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1067–1075
1070 Sandrine Ruffel et al.
protein sequences with eIF4E sequences of Arabidopsis
(EMBL accession no. Y10548), Saccharomyces cerevisiae
(GenBank accession no. P07260), Mus musculus (GenBank
accession no. NP_031943), and Homo sapiens (GenBank
accession no. XP_017925) showed that these mutations do
not target highly conserved amino acids, or amino acids
implicated in recognition of the mRNA 50 cap structure
(m7GpppN, where N is any nucleotide), or in interactions
with the eIF4G and 4E binding proteins (Marcotrigiano
et al., 1997).
A PVX-mediated transient expression assay confirms
that pvr2 corresponds to the eIF4E gene
The ORF corresponding to eIF4E from susceptible YW
and resistant YY were cloned into PVX vector pPVX201
(Figure 3). To obtain a high-titer inoculum for application
to pepper leaves, pPVXeYW, pPVXeYY and empty vector
pPVX201 were propagated in Nicotiana benthamiana.
Because genetic instability of the PVX vector insert was
reported by Sablowsky et al. (1995), inoculum from
N. benthamiana inoculated leaves and systemically
infected leaves was tested on pepper plants. PCR analysis
indicated that PVX eIF4E inserts were maintained in all parts
of N. benthamiana. However, pepper plants inoculated with
sap from systemically infected tissue developed severe
symptoms and PCR analysis revealed PVX vectors without
inserts or with shortened inserts. In contrast, inoculum
from inoculated N. benthamiana leaves resulted in only
weak symptoms on pepper and RT-PCR revealed that the
eIF4E inserts were largely maintained in full-length form.
Therefore, only inoculated leaves of N. benthamiana were
used as inoculum source in experiments with pepper.
Figure 3. PVX constructs used for transient expression assay of the pepper
eIF4E gene.
The PVX genes are RdRp required for PVX replication, M1, M2 and M3
required for movement and coat protein (CP). 35S indicates the CaMV 35S
promoter and NOS indicates the transcriptional terminator. CS indicates the
position of adjacent ClaI, and SalI restriction sites (Baulcombe et al., 1995).
Positions of the primers used for RT-PCR experiments are indicated by
arrows. eIF4E YW and eIF4E YY correspond to the coding sequence of eIF4E
cloned from YW (PVY susceptible) and YY (PVY resistant) pepper, respectively.
Based on the hypothesis that the dominant eIF4E allele is
required for susceptibility to PVY, we predicted that transient expression of the dominant susceptible eIF4E allele in
pepper plants resistant to PVY would lead to PVY genome
amplification in the PVX-infected cells expressing the fulllength eIF4E insert. In preliminary experiments, the effect of
double-inoculation of pPVX201 and PVY on the phenotype
of the pvr2 alleles was tested. Both RT-PCR and doubled
antibody sandwich enzyme-linked immunosorbent assay
(DAS-ELISA) analysis of PVY accumulation indicated that
PVX systemic infection of pepper genotypes YW, YY and F
followed by infection with PVY, did not alter infectivity of
PVY-0 and PVY-1, compared to plants infected with PVY-0
or PVY-1 alone (Figure 4; Table 1).
To test if PVY-0 infection of resistant YY (pvr21) could be
supported by expression of eIF4E from susceptible YW, YY
plants were inoculated with pPVXeYW and PVY-0. Of 20
double-inoculated leaves, four displayed significant accumulation of PVY coat protein and RNA (Figure 4a,b, lane
10). Similarly, PVY-0 accumulation was observed in four of
17 resistant genotype F (pvr22) plants double-inoculated
with pPVXeYW and PVY-0 (Table 1). In parallel experiments, it was shown that expression of eIF4E from PVY-0
resistant YY did not support PVY-0 infection in YY plants
(Figure 4a,b, lane 9). Susceptible genotype YW inoculated
with PVY-0 or with pPVX201, pPVXeYW or pPVXeYY followed by inoculation with PVY-0, accumulated PVY capsid
protein and RNA (Figure 4, lanes 3–6). These results indicate
that PVX vector expression of eIF4E from the dominant
allele pvr2þ from YW in resistant genotypes YY (pvr21) and
F (pvr2 2 ) can lead to a high level of virus accumulation.
YY is resistant to PVY-0 but susceptible to PVY-1. Thus,
we hypothesized that transient expression of the YY eIF4E
allele in F, which is resistant to PVY-1, should lead to PVY-1
accumulation in double-inoculated F leaves. DAS-ELISA
and RT-PCR experiments showed an accumulation of
PVY-1 in six inoculated leaves among the 30 assessed; F
inoculated with only PVY-1 never presented PVY accumulation and YY inoculated with PVY-1 or inoculated with
pPVXeYY and PVY-1 presented a high level of accumulation
of the PVY capsid antigen (Figure 4; Table 1). Thus, complementation experiments demonstrate that expression of
the eIF4E susceptible alleles in resistant pepper genotypes
is both necessary and sufficient to restore susceptibility to
PVY.
PVY accumulation was never observed in upper uninoculated leaves of double-inoculated resistant pepper
plants, suggesting that PVX expression of the eIF4E susceptible alleles did not support systemic infection with PVY.
In inoculated leaves, the absorbance values observed in
leaves from resistant genotypes expressing eIF4E susceptibility allele were significantly lower than in the susceptible
controls (Figure 4a, lane 10 and 16). This can be explained
by the requirement that the preliminary infection by PVX
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1067–1075
A natural recessive resistance gene corresponding to the eukaryotic initiation factor 4E
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Figure 4. Complementation experiments using transient expression (via PVX) of the eIF4E allele from susceptible pepper in PVY-resistant pepper genotypes.
Lanes 1 and 2 correspond to healthy tissue and mock-inoculated tissue, respectively. Lanes 1–6 correspond to the susceptible genotype YW, lanes 7–13
correspond to the PVY-0 resistant genotype YY and lanes 14–16 to the PVY-1 resistant genotype F. Lanes 3 and 7 were inoculated with PVY-0, lanes 4 and 8 were
double-inoculated with pPVX201 (201) and PVY-0, and lanes 5 and 9 were double-inoculated with pPVXeYY (YY) and PVY-0. Lanes 6 and 10 were doubleinoculated with PVXeYW (YW) and PVY-0, 11 and 14 were inoculated with PVY-1, lanes 12 and 15 were double-inoculated with pPVX201 and PVY-1, and lanes 13
and 16 were double-inoculated with pPVXeYY and PVY-1. MW corresponds to the 1 kb Lambda Marker (Promega) and sizes are given in kilobases (kb).
(a) Detection of PVY coat protein by DAS-ELISA. For lanes 1–10, absorbance values correspond to an average from 20 leaves from distinct plants except for lane
10 where it corresponds to an average from four leaves from four distinct plants (these four leaves correspond to those presenting accumulation of PVY, among
the 20 double-inoculated with pPVXeYW and PVY-0). For lanes 11–16, absorbance values correspond to an average from 30 leaves from distinct plants except for
lane 16 where absorbance value correspond to an average from six leaves from distinct plants (these six leaves correspond to those presenting accumulation of
PVY, among the 30 double-inoculated with pPVXeYY and PVY-1). Vertical bars show the standard deviation with a type I error of 5%. The dotted line designates
the absorbance value above which samples were considered as infected (three times the average value of the healthy control).
(b) Detection of PVY by RT-PCR assay. Primers based on the PVY-capsid sequence were used. The expected size of the amplification product is 0.82 kb. Results of
RT-PCR are from individual leaves representative of the treatment.
(c) Detection of PVX by RT-PCR assay. A pair of primers binding on each side of the cloning site were used for amplification. The expected size of the amplification
product for the PVX vector without insert is 0.23 kb and with insert is 0.92 kb. Results of RT-PCR are from the same leaves (and same RNA sample) as those shown
in panel (b).
Table 1 Number of PVY-infected leaves/number of
PVY-inoculated leavesin the PVX-based transient expressionassay
Pepper genotypes
Inoculated with
YW
YY
F
PVY-0
pPVX201 þ PVY-0
pPVXeYW þ PVY-0
pPVXeYY þ PVY-0
PVY-1
pPVX201 þ PVY-1
pPVXeYW þ PVY-1
pPVXeYY þ PVY-1
20/20
20/20
20/20
20/20
20/20
20/20
20/20
20/20
0/20
0/20
4/20
0/20
30/30
30/30
n.d.
30/30
0/20
0/20
4/17
n.d.
0/30
0/30
n.d.
6/30
n.d.: not determined.
and subsequent eIF4E expression must occur in the same
cells which are infected by PVY. Furthermore, even if a
given cell becomes infected with both the PVX vector and
PVY, it is evident that loss of the eIF4E insert from the vector
in such a cell will abolish PVY accumulation. Presumably,
the low percentage of resistant plants which become PVYinfected in the above experiments is due to this sort of
phenomenon.
It is well known that PVX carrying host-derived sequence
inserts can silence expression of the corresponding gene in
infected plants in a process known as virus-induced gene
silencing (VIGS, Burton et al., 2000). If infectivity of PVY-0
on YY plants depended on silencing of eIF4E by pPVXeYW,
it would be expected that pPVXeYY would also induce
silencing of eIF4E in YY plants and thus allow PVY-0 infection. However, virus accumulation was never detected by
RT-PCR or by DAS-ELISA assays in YY leaves inoculated
with pPVXeYY and PVY-0 (Figure 4a,b). RT-PCR and sequence analysis of YY plants inoculated with pPVXeYW
and PVY-0 confirmed the presence of eIF4E mRNA corresponding to the YW allele expressed from pPVXeYW (data
not shown). Thus, it was concluded that PVX functioned as
an expression vector rather than a silencing vector.
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1067–1075
1072 Sandrine Ruffel et al.
Discussion
We reported the molecular characterization of the first
natural recessive resistance gene against a plant virus
and showed that the pvr2 locus of pepper corresponds to
a gene encoding a protein of the eIF4E subfamily. This
work, thus, underlines the key role of eukaryotic initiation
factor 4E not only in the viral life cycle as suggested by the
known interaction between eIF4E and VPg (Wittmann et al.,
1997; Léonard et al., 2000; Schaad et al., 2000), but also in
the resistance strategy developed by the host to combat
potyvirus infection.
eIF4E is a component of the eIF4F complex and provides
the 50 cap-binding function during formation of translation
initiation complexes on most eukaryotic mRNAs. In plant
cells, this complex is composed of only two proteins, eIF4E
and eIF4G (Browning, 1996). Another specific feature of
plants is the existence of an additional cap-binding complex, eIF (iso) 4F, in which a second cap-binding protein (eIF
(iso) 4E) binds with eIF (iso) 4G (Bailey-Serres, 1999). In
Arabidopsis, different genes code for these isoforms; three
genes code for proteins of the eIF4E subfamily and one for
eIF (iso) 4E (Rodriguez et al., 1998). Southern hybridization
experiments in pepper also show that eIF4E encoding
genes belong to multigenic family.
Potyviral RNA differs from host mRNAs in that the 50 cap
structure is replaced by the 50 covalently linked VPg. As
noted above, the VPg has been shown to bind to eIF4E
proteins in several plant–potyvirus systems (Wittmann
et al., 1997; Léonard et al., 2000; Schaad et al., 2000). The
VPg may intervene at several steps in the virus infection
cycle, including viral RNA replication, and viral cell-to-cell
and long-distance movement (Revers et al., 1999), but the
biological significance of the interaction between eIF4E
proteins and the VPg remains to be determined. The most
likely hypothesis is that interaction is important for translation and/or replication of the viral genome. Thus, pvr2resistant plants never developed visible symptoms when
challenged with the virus, and virus accumulation in inoculated leaves could not be detected by ELISA, back-inoculation, or RT-PCR (data not shown). Furthermore, pvr2
resistance is active at the protoplast level (Deom et al.,
1997) and following graft inoculation. We cannot rule out
the possibilities that eIF4E–VPg interaction could also interfere with translation of host mRNAs by sequestration of
translation initiation factor, as observed during infection of
animal cells by adenovirus and poliovirus (Feigenblum and
Schneider, 1993).
Only two amino acid substitutions in the eIF4E protein
determine the resistance phenotype against PVY: a substitution common to both resistant genotypes and two
genotype-specific substitutions. The three substitutions
do not involve highly conserved amino acids among the
eukaryotes, nor amino acids implicated in the recognition
of the mRNA 50 cap structure or in recognition of eIF4G and
4E binding proteins (Figure 2), suggesting that the key
functions of eIF4E are not disrupted. However, a direct
effect on the structure of protein is possible because both
YY mutations involve replacement of an amino acid containing a non-polar side chain with an amino acid having an
acidic or basic side chain. Thanks to the high conservation
of the eIF4E sequence among eukaryotes and to the availability of 3D structures of eIF4E from mouse and yeast
(Marcotrigiano et al., 1997; Matsuo et al., 1997), the positions of the three substitutions can be predicted to be
physically close and at the surface of the protein. The
two alleles display different levels of resistance towards
potyvirus strains and potyviruses (both pvr21 and pvr22
control the PVY pathotype 0, but pvr22 also controls resistance to PVY-1 and to TEV). Moreover, the YW allele confers
susceptibility to PVY-0 in both YY and F, and the YY allele
confers susceptibility to PVY-1 in F, matching the specificity
of the pvr2 alleles. Thus, it can be hypothesized that the
genotype-specific substitutions are correlated with the
resistance phenotype conferred by the locus. Further structure–function analysis should provide insight into the
importance of the amino acid differences detected between
the different pvr2 alleles.
An interesting possibility, in accordance with the generally accepted hypothesis about the nature of recessive
resistance genes against viruses, is that the eIF4E amino
acid substitutions in YY and F abolish or decrease the
affinity between the pepper eIF4E protein and the VPg of
PVY, leading to resistance. The recent finding that PVYcontaining amino acid substitutions in the VPg can overcome the pvr21 and pvr22 alleles from pepper and the pot-1
resistance gene from tomato corroborates this hypothesis
(Moury et al., submitted). pot-1 confers a recessive resistance against PVY and TEV in tomato and was shown to
map in a colinear genomic region of the pepper genome,
suggesting that resistance to the same pathogen could be
attributed to evolutionarily related loci (Parrella et al., 2002).
These results, together with the recent demonstration that
eIF(iso)4E is required for susceptibility to TuMV, TEV and
lettuce mosaic virus (LMV) in Arabidopsis (Lellis et al., 2002;
Duprat et al.,2002), indicate that eIF4E-mediated resistance
occurs in several other plant–potyvirus interactions. This
conclusion is strengthened by the unusually high frequency
of recessive resistance genes against potyviruses in plants
and by the key role of the central domain of the VPg in
overcoming several unrelated host resistance genes from
distinct plant families. Finally, it is of interest to note that the
eIF4E and eIF (iso) 4E isoforms are functionally distinct in
their interaction with the mRNA 50 cap structure (Gallie and
Browning, 2001) and in their ability to promote viral genome amplification. Thus, a protein from the eIF4E subfamily is involved in PVY resistance of pepper (this work)
whereas a protein from the eIF (iso) 4E subfamily promotes
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1067–1075
A natural recessive resistance gene corresponding to the eukaryotic initiation factor 4E
TuMV, TEV and LMV amplification in Arabidopsis (Duprat
et al.,2002). In conclusion, the demonstration that pvr2
corresponds to an isoform of eIF4E is a major step in
understanding plant–potyvirus interactions; it should contribute to the elucidation of the molecular mechanisms
underlying plant susceptibility to potyviruses, the frequent
occurrence of recessive resistance and the main features of
this class of natural resistance genes.
Experimental procedures
Plants, virus isolates and disease resistance scoring
C. annuum cultivars used were Yolo Wonder (YW), Doux Long Des
Landes (DDL), Yolo Y (YY) and FloridaVR2 (F). Both YW (from
California) and DDL (from southern France) contain the pvr2þ
dominant allele for susceptibility to PVY and are unrelated cultivars; YY contains the pvr21 recessive allele for resistance to PVY
pathotype 0 and F contains the pvr22 allele for resistance to PVY
pathotypes 0 and 1 (Kyle and Palloix, 1997). A back-cross progeny
(BC1) of 440 plants originating from the F1 hybrid (YW YY) backcrossed with YY was developed to search for map co-segregation
between the candidate gene eIF4E and the pvr2 locus. The parental
lines, the F1 hybrid and the 440 BC1 plants were evaluated under
growth chamber conditions for resistance to PVY isolate LYE72
(pathotype 0). Inoculum and mechanical inoculation procedures
were as described previously (Caranta and Palloix, 1996). Thirty
days post-inoculation (dpi), plants without obvious symptoms
were evaluated for presence/absence of PVY coat protein by
DAS-ELISA.
Genomic DNA extraction and mapping
Plant genomic DNA isolation and restriction fragment length
polymorphism (RFLP) were performed as described previously
(Lefebvre et al., 1995). Hybridization was done at 558C. DraI, EcoRI,
EcoRV, HindIII and XbaI (GIBCO/BRL, Life Technologies) were used
to digest genomic DNA. The tobacco eIF4E cDNA probe used for
RFLP mapping was kindly provided by D. Twell (University of
Leicester, UK).
Isolation and analysis of the eIF4E cDNA from pepper
Total RNA was isolated from YW, DDL, YY and F leaf tissue using
Tri-Reagent (Sigma). The full-length eIF4E cDNA of YW was
obtained by rapid amplification of cDNA ends (RACE) using the
GIBCO/BRL Life Technologies 30 and 50 RACE System (Version 2.0).
The 30 end of YW eIF4E cDNA was obtained using two degenerate
oligonucleotides designed from an alignment of the tobacco
(sequence from D. Twell) and tomato eIF4E cDNA (GenBank
accession no. AF259801), in combination with the adapter primer
(AUAP) of the kit. The full-length YW eIF4E cDNA was amplified
with oligonucleotides designed from the sequence of the 30 RACE
product. All amplifications were performed with High Fidelity
Platinum Taq polymerase (GIBCO/BRL, Life Technologies). The
RACE products were cloned into the pGEM-T easy vector (Promega) and 10 independent 50 and 30 terminal clones were
sequenced by Genome Express (Grenoble, France). Two primers
based on sequence in the 50 and 30 NTR regions of the YW eIF4E
cDNA were used in RT-PCR experiments to obtain the full length
1073
eIF4E cDNA of DDL, YY and F. All primer sequences are available
from the authors on request. Genetics Computer Group (Madison,
WI, USA) software package version 10.3 was used for protein and
nucleic acid sequence analysis. The pepper eIF4E cDNA sequences
have been deposited in the GenBank database (accession nos.
AY122052, AF521963, AF521964, AF521965).
Identification and analysis of the BAC clones
A doubled haploid line, HD208, containing the pvr2þdominant
allele of YW was the source of genomic DNA for the library
construction. High molecular weight DNA was separately digested
with EcoRI, BamHI and HindIII (GIBCO/BRL, Life Technologies) and
cloned into the BAC vector pCUGIBAC1. The library consisted of
239,232 clones with an average insert size of 125 kb, representing
approximately ten haploid genome equivalents (Ruffel, S., Caranta, C. and Bendahmane, A., unpublished). Two oligonucleotides,
designed from the full-length eIF4E cDNA sequence of YW (P1:
50 -AGACTTTCATTGTTTCAAGCATAA-30 and P2: 50 -GATTAGAAAGTGCAAACACCAATAC-30 ) were used to screen the pepper BAC
library as described (Kanyuka et al., 1999). Maxipreps of four
candidate BAC clones were performed as described (http://www.
plpa.agri.umn.edu/neviny/labsite/protocolsf/bacmini.html). For
isolation of the 7-kb fragment, 30 ml of BAC DNA was digested
using EcoRI (GIBCO/BRL, LifeTechnologies). Following electrophoresis, fragments of approximately 6–8 kb were eluted and
cloned into pGEM3Zfþ (Promega) digested with EcoRI and dephosphorylated. Positive clones were selected by PCR using primers
P1 and P2.
PVX-mediated transient expression assay
Primers corresponding to the 50 and 30 ends of the YW eIF4E ORF
and containing ClaI and XhoI sites were used to amplify the YW
and YY eIF4E ORFs. The digested PCR products were transferred to
ClaI- and SalI-digested pPVX201 (kindly provided by DC
Baulcombe, Sainsbury Laboratory, UK, Baulcombe et al., 1995)
to produce plasmids pPVXeYW and pPVXeYY. The plasmids
were manually inoculated to carborundum-dusted leaves of
N. benthamiana (2.5 mg of DNA per leaf). Ten dpi, the inoculated
N. benthamiana leaves were used as inoculum source for transient
expression assays in pepper. First, YW, YY and F leaves were
manually inoculated with the N. benthamiana leaf extract. Ten
days later, the same leaves were manually inoculated with PVYLYE72 (pathotype 0) or PVY-CAA16 (pathotype 1). In each experiment, pepper genotypes with specific behavior against PVY pathotypes were inoculated to check that no contamination occurred.
Stability of the PVX constructs was checked by RT-PCR with
primers sited on both sides of the cloning site (50 -CCGATCTCAAGCCACTCTCCG-30 and 50 -CCTGAAGCTGTGGCAGGAGTTG30 ). PVY accumulation in inoculated pepper leaves was determined
10 dpi by DAS-ELISA using PVY capsid antigen and RT-PCR with
two primers corresponding to the PVY capsid sequence (50 -CATCGATTATGGCAAATGATACAATTGATGC-30 and 50 -TGTCGACATTCACATGTTCTTGACTCC-30 ).
Acknowledgements
We thank K. Richards and H. Lecoq for very helpful comments on
the manuscript, T. Desnos for help in RACE methodology and M.L.
Lesage, G. Nemouchi, T. Phally and A.M. Daubèze for their excellent assistance. We are very grateful to David Twell for providing
the unpublished tobacco eIF4E sequence. This work was
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1067–1075
1074 Sandrine Ruffel et al.
supported by grants from GENOPLANTE and the French Ministry
of Agriculture and Fishing (CI-1999-019 and CI-2001-005). SR was
supported by a doctoral fellowship from the "Provence-Alpes Côte
d’Azur Region" and Clause S.A. Experiments were carried out in
compliance with current French guidelines concerning genetically
modified organisms.
References
Bailey-Serres, J. (1999) Selective translation of cytoplasmic
mRNAs in plants. Trends Plant Sci. 4, 142–148.
Baulcombe, D.C., Chapman, S. and Santa-Cruz, S. (1995) Jellyfish
green fluorescent protein as a reporter for virus infections. Plant
J. 7, 1045–1053.
Bendahmane, A., Kanyuka, K. and Baulcombe, D.C. (1999) The Rx
gene from potato controls separate virus resistance and cell
death responses. Plant Cell, 11, 781–791.
Bendahmane, A., Querci, M., Kanyuka, K. and Baulcombe, D.C.
(2000) Agrobacterium transient expression system as a tool for
the isolation of disease resistance genes: application to the Rx2
locus in potato. Plant J. 21, 73–81.
Brommonschenkel, S.H., Frary, A., Frary, A. and Tanksley, S.D.
(2000) The broad-spectrum tospovirus resistance gene Sw-5 of
tomato is a homolog of the root-knot nematode resistance gene
Mi. Mol. Plant Microbe Interact. 13, 1130–1138.
Browning, K.S. (1996) The plant translational apparatus. Plant Mol.
Biol. 32, 107–143.
Burton, R.A., Gibeaut, D.M., Bacuc, A., Findlay, K., Roberts, K.,
Hamilton, A., Baulcombe, D.C. and Fincher, G.B. (2000) Virusinduced silencing of a plant cellulose synthase gene. Plant Cell,
12, 691–706.
Büschges, R., Hollricher, K., Panstruga, R. et al. (1997) The barley
Mlo gene: a novel control element of plant pathogen resistance.
Cell, 88, 695–705.
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, 15–20.
Chisholm, S.T., Mahajan, S.K., Whitham, S.A., Yamamoto, M.L.
and Carrington, J.C. (2000) Cloning of the Arabidopsis RTM1
gene, which controls restriction of long-distance movement of
tobacco etch virus. Proc. Natl. Acad. Sci. USA, 97, 489–494.
Deom, C.M., Murphy, J.F. and Paguio, O.R. (1997) Resistance to
tobacco etch virus in Capsicum annuum: inhibition of virus RNA
accumulation. Mol. Plant Microbe Interact. 7, 917–921.
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, 927–934.
Feigenblum, D. and Schneider, R.J. (1993) Modification of eukaryotic initiation factor 4F during infection by Influenza virus.
J. Virol. 67, 3027–3035.
Fraser, R.S.S. (1990) The genetics of plant–virus interactions:
mechanisms controlling host range, resistance and virulence.
In Recognition and Response in Plant–Virus Interactions (Fraser,
R.S.S., ed.). Berlin: Springer Verlag, 71–93.
Frye, C.A. and Innes, R.W. (1998) An Arabidopsis mutant
with enhanced resistance to powdery mildew. Plant Cell, 10,
947–956.
Gallie, D.R. and Browning, K.S. (2001) eIF4G functionally differs
from eIFiso4G in promoting internal initiation, cap-independent
translation, and translation of structure mRNAs. J. Biol. Chem.
276, 36951–36960.
Hjulsager, C.K., Lund, O.S. and Johansen, I.E. (2002) A new pathotype of pea seed-borne mosaic virus explained by properties of
the P3-6K1 and viral genome-linked (VPg) coding regions. Mol.
Plant Microbe Interact. 15, 169–171.
Kanyuka, K., Bendhamane, A., Rouppe van der Voort, J.N.A.M.,
van der Vossen, E.A.G. and Baulcombe, D.C. (1999) Mapping of
intra-locus duplications and introgressed DNA: aids to mapbased cloning of genes from complex genomes illustrated by
physical analysis of the Rx locus in tetraploid potato. Theor.
Appl. Genet. 98, 679–689.
Keen, N.T. (1990) Gene-for-gene complementarity in plant–pathogen interactions. Annu. Rev. Genet. 24, 447–463.
Keller, K.E., Johansen, E., Martin, R.R. and Hampton, R.O. (1998)
Potyvirus genome-linked protein (VPg) determines pea seedborne mosaic virus pathotype-specific virulence in Pisum sativum. Mol. Plant Microbe Interact. 2, 124–130.
Kyle, M.M. and Palloix, A. (1997) Proposed revision of nomenclature for potyvirus resistance genes in Capsicum. Euphytica, 97,
183–188.
Lefebvre, V., Palloix, A., Caranta and Pochard, E. (1995) Construction of an intraspecific integrated linkage map of pepper using
molecular markers and doubled-haploid progenies. Genome,
38, 112–121.
Lellis, A.D., Kasschau, K.D., Whitham, S.A. and Carrington, J.C.
(2002) Loss of susceptibility mutants of Arabidopsis thaliana
reveal an essential role for eIF (iso) 4E during potyvirus infection.
Curr. Biol. 12, 1046–1051.
Léonard, S., Plante, D., Wittmann, S., Daigneault, N., Fortin, M.G.
and Laliberté, J.F. (2000) Complex formation between potyvirus
VPg and translation eukaryotic initiation factor 4E correlates
with virus infectivity. J. Virol. 74, 7730–7737.
Lutcke, H.A., Chow, K.C., Mickel, F.S., Moss, K.A., Kern, H.F. and
Scheele, G.A. (1987) Selection of AUG initiation codons differs in
plants and animals. EMBO J. 6, 43–48.
Manjunath, S., Williams, A.J. and Bailey-Serres, J. (1999) Oxygen
deprivation stimulates Ca2þ-mediated phosphorylation of
mRNA cap-binding protein eIF4E in maize roots. Plant J. 19,
21–30.
Marcotrigiano, J., Gringras, A.C., Sonenberg, N. and Burley, S.K.
(1997) Co-crystal structure of the messenger RNA 50 capbinding protein (eIF4E) bound to 7-methyl-GDP. Cell, 89, 951–
961.
Matsuo, H., Hanjun, L., McGuire, A.M., Fletcher, C.M., Gingras, AC., Sonenberg, N. and Wagner, G. (1997) Structure of translation
factor eIF4E bound to m7-GDP and interaction with 4E-binding
protein. Nat. Struct. Biol. 4, 717–724.
Masuta, C., Nishimura, M., Morishita, H. and Hataya, T. (1999) A
single amino acid change in viral genome associated protein of
potato virus Y correlates with resistance breaking in ’Virgin A
Mutant’ tobacco. Phytopathol. 89, 118–123.
Nicolas, O., Dunnington, S., Gotow, L.F., Pirone, T.P. and Hellmann, G.M. (1997) Variations in the VPg protein allow a potyvirus to overcome va gene resistance in tobacco. Virology, 237,
452–459.
Parrella, G., Ruffel, S., Moretti, A., Morel, C., Palloix, A. and
Caranta, C. (2002) Recessive resistance genes against potyviruses are localized in colinear genomic regions of the tomato
(Lycopersicon spp.) and pepper (Capsicum spp.) genomes.
Theor. Appl. Genet. 105, 855–861.
Pflieger, S., Lefebvre, V. and Causse, M. (2001) The candidate gene
approach in plant genetics: a review. Molecular Breeding, 7,
275–291.
Provvidenti, R. and Hampton, R.O. (1992) Sources of resistance to
viruses in the Potyviridae. Arch. Virol. 5, 189–211.
Ramajäki, M.L. and Valkonen, J.P.T. (2002) Viral genome-linked
protein (VPg) controls accumulation and phloem-loading of a
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1067–1075
A natural recessive resistance gene corresponding to the eukaryotic initiation factor 4E
potyvirus in inoculated potato leaves. Mol. Plant Microbe Interact. 15, 138–149.
Revers, F., Le Gall, O., Candresse, T. and Maule, A.J. (1999)
New advances in understanding the molecular biology of
plant–potyvirus interactions. Mol. Plant Microbe Interact. 12,
367–376.
Rodriguez, C.M., Freire, M.A., 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, 465–473.
Sablowsky, R.W.M., Baulcombe, D.C. and Bevan, M. (1995) Expression of a flower-specific Myb protein in leaf cells using a viral
vector causes ectopic activation of a target promoter. Proc. Natl.
Acad. Sci. USA, 92, 6901–6905.
Schaad, M.C., Anderberg, R.J. and Carrington, J.C. (2000) Strainspecific interaction of the tobacco etch virus NIa protein with the
translation initiation factor eIF4E in the yeast two-hybrid system.
Virology, 273, 300–306.
Whitham, S., Dinesh-Kumar, S.P., Choi, D., Hehl, R., Corr, C. and
Baker, B. (1994) The product of the tobacco mosaic virus
1075
resistance gene N: similarity to toll and interleukin-1 receptor.
Cell, 78, 1011–1015.
Whitham, S.A., Anderberg, R.J., Chisholm, S.T. and Carrington,
J.C. (2000) Arabidopsis RTM2 gene is necessary for specific
restriction of tobacco etch virus and encodes an unusual small
heat shock-like protein. Plant Cell, 12, 569–582.
Wittmann, S., Chatel, H., Fortin, M.G. and Laliberté, 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, 84–92.
Yamanaka, T., Ohta, T., Takahashi, M., Meshi, T., Schmidt, R.,
Dean, C., Naito, S. and Ishikawa, M. (2000) TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus,
encodes a putative transmembrane protein. Proc. Natl. Acad.
Sci. USA, 97, 10107–10112.
Yamanaka, T., Imai, T., Satoh, R., Kawashima, A., Takahashi, M.,
Tomita, K., Kubota, K., Meshi, T., Naito, S. and Ishikawa, M.
(2002) Complete inhibition of tobamovirus multiplication by
simultaneous mutations in two homologous host genes. J.
Virol. 76, 2491–2497.
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1067–1075