Download Allele mining in the pepper gene pool provided new

Journal of General Virology (2009), 90, 2808–2814
DOI 10.1099/vir.0.013151-0
Allele mining in the pepper gene pool provided new
complementation effects between pvr2-eIF4E and
pvr6-eIF(iso)4E alleles for resistance to pepper
veinal mottle virus
Manuel Rubio,3 Maryse Nicolaı̈, Carole Caranta and Alain Palloix
Correspondence
Alain Palloix
INRA, Centre d’Avignon, UR1052, Unité de Génétique et Amélioration des Fruits et Légumes,
BP 94, 84143 Montfavet cedex, France
[email protected]
Received 6 May 2009
Accepted 24 July 2009
Molecular cloning of recessive resistance genes to potyviruses in a large range of host species
identified the eukaryotic translation initiation factor 4E (eIF4E) as an essential determinant in the
outcome of potyvirus infection. Resistance results from a few amino acid changes in the eIF4E
protein encoded by the recessive resistance allele that disrupt the direct interaction with the
potyviral protein VPg. In plants, several loci encode two protein subfamilies, eIF4E and eIF(iso)4E.
While most eIF4E-mediated resistance to potyviruses depends on mutations in a single eIF4E
protein, simultaneous mutations in eIF4E (corresponding to the pvr2 locus) and eIF(iso)4E
(corresponding to the pvr6 locus) are required to prevent pepper veinal mottle virus (PVMV)
infection in pepper. We used this model to look for additional alleles at the pvr2-eIF4E locus that
result in resistance when combined with the pvr6-eIF(iso)4E resistant allele. Among the 12 pvr2eIF4E resistance alleles sequenced in the pepper gene pool, three were shown to have a
complementary effect with pvr6-eIF(iso)4E for resistance. Two amino acid changes were
exclusively shared by these three alleles and were systematically associated with a second amino
acid change, suggesting that these substitutions are associated with resistance expression. The
availability of new resistant allele combinations increases the possibility for the durable
deployment of resistance against this pepper virus which is prevalent in Africa.
INTRODUCTION
Improving the knowledge of the functional basis of resistance
specificity and defence pathways provides increasing candidate sequences to explore available gene pools in cultivated
plants and related species, but also enables further engineering of new resistance alleles and expansion of the gene pool
for breeding (Michelmore, 2003). Moreover, functional
markers that are derived from DNA polymorphisms directly
responsible for the resistance phenotype provide optimal
tools to track the resistance genes in breeding progenies
(Andersen & Lübberstedt, 2003). However, relating phenotype variations to sequence information remains challenging
when considering the major class of resistance genes
encoding the nucleotide-binding sites–leucine rich repeat
(NBS–LRR) proteins because of their great number in plant
genomes, their organization in clusters and their sequence
homology, which make the development of allele-specific
amplification difficult (Hammond-Kosack & Parker, 2003).
3Present address: CEBAS-CSIC, Department of Plant Breeding, PO
Box 164, 30100 Espinardo (Murcia), Spain.
Supplementary material showing an amino acid sequence alignment of
eIF4E is available with the online version of this paper.
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Among resistance genes, the eukaryotic translation initiation factors eIF4E and eIF(iso)4E control conserved
resistance mechanisms in several genera of RNA viruses
and offer facilities for allele mining in plant gene pools and
tracking targeted alleles in breeding programmes (Robaglia
& Caranta, 2006). In eukaryotic cells, eIF4E binds to the
m7GpppX cap as the first step in recruiting mRNA into the
translation initiation complex. In plants, eIF4E belongs to a
small multigenic family encoding two protein isoforms,
eIF4E and eIF(iso)4E. Although these two isoforms are
considered equivalent for in vitro translation of some
mRNA, they differ in their in vivo expression pattern and
show some specificity for different capped mRNAs (Gallie
& Browning, 2001).
In a range of plant species, eIF4E-mediated resistance to
viruses belonging to the genus Potyvirus results from a
small number of nonconservative amino acid changes in
the protein encoded by the recessive resistance allele (Ruffel
et al., 2002; Robaglia & Caranta, 2006; Maule et al., 2007
for review). These amino acid changes are clustered in two
neighbouring regions at the surface of the predicted eIF4E
3D structure (Monzingo et al., 2007). Resistance genes
control multiple specificities targeted against distinct
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Allele mining for virus resistance
potyviruses or pathotypes of the same virus and these
specificities are explained by the occurrence of small allelic
series where distinct alleles are represented as a combination of distinct amino acid substitutions. The exact
mechanism by which eIF4E mutations control resistance
remains to be elucidated, but protein–protein interaction
studies have shown that the physical interaction between
eIF4E and the potyviral protein VPg is required for viral
infection, and that eIF4E mutations that cause resistance
prevent VPg binding and inhibit the viral infectious cycle
(Yeam et al., 2007; Charron et al., 2008). Reciprocally, the
resistance breakdown by the virus results from amino acid
substitutions in VPg that restore the physical interaction
with the mutated eIF4E protein (Moury et al., 2004; Ayme
et al., 2007; Charron et al., 2008), so that co-evolution
between the two partners has led to the diversification of
both genes.
The largest allelic series is found at the pvr2-eIF4E locus in
pepper (Capsicum spp.), with 11 alleles of the eIF4E gene in
which mutations have related resistance specificities to
diverse strains of potato virus Y (PVY) and tobacco etch
virus (TEV) strains (Charron et al., 2008; Kang et al.,
2005a). Characterization of the molecular mechanisms
underlying digenic recessive resistance to another pepper
potyvirus pepper veinal mottle virus (PVMV) (Brunt et al.,
1978) suggests a more complex pattern of interaction.
Complete resistance to PVMV results from the combination of recessive alleles at two distinct loci: the pvr2-eIF4E
locus on the pepper chromosome P4 and the pvr6 locus on
the chromosome P3 that corresponds to the isoform
eIF(iso)4E (Ruffel et al., 2006). The resistant allele pvr6eIF(iso)4E differs from the susceptible one (pvr6+eIF(iso)4E) by an 82 nt deletion and encodes a truncated
protein with only the N-terminal 29 aa, suggesting a nonfunctional cap-binding factor. Up to now, only the pvr22eIF4E allele, differing from the pvr2+-eIF4E allele by three
amino acid substitutions, has been shown to complement
pvr6-eIF(iso)4E for resistance to PVMV. The other pvr2
alleles have not been assessed for their complementary
effect with pvr6 for PVMV resistance. In this context, new
allele combinations would be useful, since the high
prevalence of PVMV all over sub-Saharan Africa and the
diversity of PVMV strains can threaten the durability of
this unique digenic resistance source (Moury et al., 2005).
With the objective to look for new sources of resistance to
PVMV, we generated all the possible combinations
between the partially deleted pvr6-eIF(iso)4E allele and
the 10 recessive pvr2-eIF4E alleles previously identified in
pepper germplasm, and tested their complementation for
PVMV resistance. Two new allele combinations were
identified that provide alternatives to the single resistance
source available up to now. Comparisons between pvr2eIF4E protein sequences enabling or not the complementary effect with pvr6-eIF(iso)4E brought insights into amino
acid changes within the pvr2-eIF4E protein that determine
PVMV resistance.
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METHODS
Plant material. Eleven strains of Capsicum spp. were chosen for their
original alleles at the pvr2-eIF4E locus (Table 1, Charron et al., 2008):
Capsicum annuum genotypes ‘Yolo Wonder’, ‘Yolo Y’, ‘HD801’,
‘Perennial’, ‘PI322719’, ‘SC81’, ‘Maroc 1’, ‘Serrano VC’, ‘PI195301’
and ‘Chile de árbol’, which are homozygous for the alleles pvr2+,
pvr21, pvr22, pvr23, pvr24, pvr25, pvr26, pvr27, pvr28 and pvr29,
respectively, and Capsicum chinense ‘PI159336’ homozygous for pvr1,
which was shown to be a C. chinense original allele at the pvr2 locus
(Ruffel et al., 2004; Kang et al., 2005b). All these genotypes are
homozygous at the pvr6-eIF(iso)4E locus and possess the susceptible
allele pvr6+, except ‘Perennial’ and ‘HD801’ which possess the
recessive resistance allele pvr6.
In order to test the ability of the different pvr2-eIF4E alleles to confer
resistance to PVMV when combined with pvr6-eIF(iso)4E, F2
progenies were produced between ‘Perennial’ and the genotypes
representing the different pvr2-eIF4E haplotypes (Table 1). To check
the combinations between pvr6-eIF(iso)4E and the C. chinense pvr1eIF4E allele, breeding progenies previously developed to introduce
PVMV resistance from HD801 into C. chinense cultivars were used:
two plants, heterozygous pvr1/pvr22 and homozygous pvr6/pvr6, were
selected using molecular markers described below from the F2
(‘HD801’6‘PI159336’) and BC1 [(‘HD801’6‘PI159336’)6‘HD801’]
progenies, respectively (Table 1). These plants were self-pollinated to
generate, respectively, F3 and BC1-I1 progenies segregating for the
pvr1/pvr22 alleles.
PVMV resistance evaluation. Seeds were sown in disinfected peat
and grown to the expanded cotyledon stage under greenhouse
conditions and transferred into growth chambers before inoculation.
Six to 20 plants for parental lines and 121–200 plants for segregating
progenies were mechanically inoculated with the virus as described
previously (Caranta & Palloix, 1996). The PVMV strain used
originated from the Ivory Coast (PVMV-IC) (Moury et al., 2005).
At 21–30 days post-inoculation (p.i.), systemic infection was assayed
by the presence/absence of symptoms on non-inoculated leaves and
confirmed by DAS-ELISA using specific PVMV antibodies, as
described previously (Moury et al., 2005). Samples were considered
positive when absorbance values were at least three times greater than
the negative control.
Amplification, sequencing and sequence analysis of eIF4E and
eIF(iso)4E cDNAs and gDNAs. Total RNA was isolated from
pepper leaf tissues using the TRI-reagent protocol (Sigma-Aldrich).
The eIF(iso)4E cDNA of 527 or 609 bp, depending on the presence/
absence of the 82 bp deletion, was amplified by RT-PCR using the
primers 59-ATGGCCACCGAAGCACCACCACCGG-39 (forward)
and 59-TCACACGGTGTATCGGCTCTTAGCT-39 (reverse), as
described by Ruffel et al. (2006).
DNA was extracted according to the micro-extraction method
described by Fulton et al. (1995). Tetra-primer ARMS-PCR was used
to differentiate the alleles pvr21, pvr22, pvr23 and pvr2+, as described
by Rubio et al. (2008).
To distinguish pvr22 from pvr1, a cleaved amplified polymorphic
sequence (CAPS) marker was used. The differential cleavage site
revealed by MvnI was based on a single nucleotide polymorphism of
T236G of cDNA that distinguishes pvr22 from the pvr1 allele
[described by Ruffel et al. (2006)] after amplification of the 687 bp
product with the eIF4E cDNA-specific primers (forward 59AAAAGCACACAGCACCAACA-39 and reverse 59-TATTCCGACATTGCATCAAGAA-39).
To distinguish the allele pvr27 from the others, we analysed the
sequence of the eIF4E cDNAs from the F2 ‘Perennial’
(pvr23)6‘Serrano VC’ (pvr27). RT-PCR was performed using eIF4E
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M. Rubio and others
Table 1. Genotype of the parental lines and segregating progenies at the pvr2 and pvr6 loci, and assessment of
the resistant or susceptible phenotype after DAS-ELISA tests of individual plants inoculated with the PVMV-CI
strain
Virus
Inbreed lines
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
annuum Yolo Wonder
annuum HD801
annuum Perennial
annuum Yolo Y
annuum PI322719
annuum SC81
annuum Maroc 1
annuum Serrano VC
annuum PI195301
annuum Chile de árbol
chinense PI159336
Allele
pvr2
+
F2 (Perennial 6 Yolo Y)
F2 (PI322719 6 Perennial)
F2 (SC81 6 Perennial)
F2 (Maroc 1 6 Perennial)
F2 (Serrano VC 6 Perennial)
F2 (PI195301 6 Perennial)
F2 (Chile de árbol 6 Perennial)
F3 (HD801 6 PI159336)
BC1-I1 (HD801 6 PI159336) 6 HD801
S*
0
16
0
0
0
0
0
0
0
0
0
14
0
11
10
11
6
13
13
12
8
20
9
0
0
0
14
0
0
68
42
159
172
159
121
160
162
179
126
147
pvr6
+
pvr2 /pvr2
pvr22/pvr22
pvr23/pvr23
pvr21/pvr21
pvr24/pvr24
pvr25/pvr25
pvr26/pvr26
pvr27/pvr27
pvr28/pvr28
pvr29/pvr29
pvr1/pvr1
Segregating populations
R*
pvr6+/pvr6+
pvr6/pvr6
pvr6/pvr6
pvr6+/pvr6+
pvr6+/pvr6+
pvr6+/pvr6+
pvr6+/pvr6+
pvr6+/pvr6+
pvr6+/pvr6+
pvr6+/pvr6+
pvr6+/pvr6+
Allele segregation
3
pvr2 /pvr21
pvr24/pvr23
pvr25/pvr23
pvr26/pvr23
pvr27/pvr23
pvr28/pvr23
pvr29/pvr23
pvr22/pvr1
pvr22/pvr1
pvr6/pvr6+
pvr6+/pvr6
pvr6+/pvr6
pvr6+/pvr6
pvr6+/pvr6
pvr6+/pvr6
pvr6+/pvr6
pvr6/pvr6
pvr6/pvr6
*Number of plants with positive (S) or negative (R) DAS-ELISA response.
cDNA-specific primers (forward primer 59-AAAAGCACACAGCACCAACA-39 and reverse primer 59-TATTCCGACATTGCATCAAGAA-39). Each amplification product was sequenced by GenomeExpress (Grenoble, France). Nucleotide sequences were analysed with
Chromas Lite 2.01 software (http://www.Technelysium.com.au). The
nucleotide and amino acid sequences were aligned with the eIF4E
cDNA sequences [GenBank accession nos AY723737 (pvr23) and
EU106863 (pvr27)] using CLUSTAL W (1.82) (Thompson et al., 1994).
RESULTS
PVMV resistance assessment in the parental lines
and segregating progenies
Phenotypic evaluation of resistance to PVMV is recorded
in Table 1. All the plants from the susceptible parental lines
‘Yolo Wonder’, ‘Yolo Y’, ‘Perennial’, ‘PI322719’, ‘SC81’,
‘Maroc 1’, ‘Serrano VC’, ‘PI195301’, ‘Chile de árbol’ and
‘PI159336’ presented the characteristic symptoms of
systemic vein clearing and mottling in apical, noninoculated leaves, and exhibited high DAS-ELISA values
at 21 days p.i. Only the double haploid line ‘HD801’ did
not display any symptoms and remained ELISA-negative
up to 30 days p.i., attesting its resistant phenotype as
expected from its homozygous genotype (pvr22/pvr22; pvr6/
pvr6) and from earlier studies (Caranta et al., 1996; Ruffel
et al., 2006).
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Among the seven F2 progenies, five were totally susceptible
to PVMV (i.e. no resistant plants at 21 days p.i.) and only
the F2 (‘Perennial’6‘Yolo Y’) and the F2 (‘Serrano
VC’6‘Perennial’) segregated for resistance, with 9 and 14
plants, respectively, remaining healthy (i.e. no symptoms
and ELISA-negative at 30 days p.i.). The observed ratios of
9R : 159S and 14R : 160S fitted well the ratio of 1R : 15S
expected from the frequency of the double recessive
resistant genotypes (x250.228 P50.63 and x250.958
P50.33, respectively). Because of the susceptibility of the
parental lines, this suggested that PVMV resistance resulted
from complementary effects between recessive alleles from
both parental origins.
In the interspecific F3 and BC1-I1 progenies, different ratios
between resistant and susceptible plants were expected, since
these progenies do not segregate for the alleles at the pvr6
locus (the mother plants were homozygous for the recessive
pvr6 allele), but segregate at the pvr2 locus, with the pvr22
allele from HD801 which is already known to complement
with pvr6 for PVMV resistance, and pvr1 from C. chinense
which is under analysis for its complementary effect with
pvr6. Following the hypothesis that pvr1 does not
complement with pvr6 for PVMV resistance, only the pvr22
homozygous plants should display a PVMV-resistant phenotype resulting in a ratio of 1R : 3S. Following the hypothesis
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Journal of General Virology 90
Allele mining for virus resistance
that pvr1 and pvr6 have a complementary effect for PVMV
resistance, all the plants (100 %) should display a PVMVresistant phenotype. The segregation ratio observed (Table 1)
fitted the theoretical ratio of 1R : 3S for the BCI-I1 progeny
(42R : 147S, x250.78, P50.378) but not for the F3 progeny
(68R : 126S, x2510.4, P50.0012), which showed an excess of
resistant individuals. This suggests that pvr1 does not confer
PVMV resistance when combined with pvr6 and that only the
pvr22 homozygous plants were resistant. The excess of
resistant plants in the F3 progeny may result from skewed
segregations that are commonly observed in interspecific
crosses between C. annuum and C. chinense (DjianCaporalino et al., 2006). However, the genotypes of the
resistant plants remain to be assessed to confirm the
hypotheses.
Genotypes of the resistant plants
To determine the alleles at the pvr2 and pvr6 loci that lead
to PVMV resistance, all the resistant plants and samples of
susceptible plants from the three progenies segregating for
resistance were genotyped at these two loci.
Genotyping the pvr6 locus confirmed that all the resistant
plants were homozygous for the recessive allele (pvr6/pvr6),
with the characteristic 527 bp fragment that corresponds to
the deleted allele of eIF(iso)4E (Fig. 1). All the individuals
that were genotyped as heterozygous (pvr6/pvr6+) or
homozygous (pvr6+/pvr6+) at this locus were susceptible.
A sample of these individual patterns is shown in Fig. 1.
One plant (lane 7 in Fig. 1) was marked as susceptible
despite its homozygous (pvr6/pvr6) genotype.
Considering the pvr2 locus, in the F2 progeny
(‘Perennial’6‘Yolo Y’), all of the nine resistant individuals
were genotyped as homozygous for the pvr21 allele. The
individual that was marked as susceptible despite its
homozygous (pvr6/pvr6) genotype (lane 7 in Fig. 1) was
shown to be homozygous (pvr23/pvr23) (lane 11 in Fig. 2). In
the F2 progeny (‘Serrano VC’6‘Perennial’), the cDNA
sequences from all of the 14 resistant plants were identical to
the ‘Serrano VC’ allele pvr27 (Supplementary Fig. S1,
available in JGV Online) and the amino acid sequences
deduced from their cDNA sequence displayed the arginine
substitution at position 79 instead of leucine for the pvr23
allele from ‘Perennial’. In summary, all the PVMV-resistant
plants were homozygous for the pvr6 and the pvr21 or pvr27
alleles, whereas all the genotyped susceptible plants displayed heterozygosity at one of these loci or homozygosity
for the pvr6+ or pvr23 alleles.
Because of the unexpected segregation in the F3 interspecific
progeny (‘HD801’6‘PI159336’), 93 individuals were randomly sampled in order to explore the frequency of their
genotypes at the pvr2 locus (all the plants from this progeny
are homozygous pvr6/pvr6). Thirty-seven of these individuals
were resistant and 56 were susceptible. The 37 resistant
individuals proved to be homozygous (pvr22/pvr22). Amongst
the 56 susceptible individuals, 48 were heterozygous (pvr22/
pvr1) and 8 were homozygous (pvr1/pvr1). This confirmed
that pvr1 did not complement pvr6 for resistance to PVMV.
The genotypic ratios of 37 (pvr22/pvr22), 48 (pvr22/pvr1) and 8
(pvr1/pvr1) also indicate a skewed segregation compared to
the 1 : 2 : 1 ratio expected from the F3 progeny, with an excess
of the resistant homozygous (pvr22/pvr22) genotype and a
deficit in susceptible homozygous (pvr1/pvr1) genotype
(x2518.18, P50.000113).
DISCUSSION
Allele mining based on the knowledge of sequence diversity
provided two new sources of resistance to PVMV in pepper
Fig. 1. Genotypes of the parental lines and parts of the segregating individuals at the pvr6/pvr6+ locus. The pvr6+-eIF(iso)4E
susceptible allele displayed a 609 bp amplicon, whereas the deleted resistant allele (pvr6) displayed a 527 bp amplicon. The
susceptible individual in lane 7 displayed homozygosity at pvr6, but heterozygosity at pvr2. S, Susceptible phenotype; R,
resistant phenotype; –, untested plant. Lane M: 1 kb ladder (Promega).
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M. Rubio and others
Fig. 2. Genotypes of the parental and F2
individuals at the pvr2 locus revealed by the
allele-specific tetra-primer ARMS-PCR. The
251 bp fragment corresponds to the common
amplicon, 176 bp corresponds to the pvr23
allele and 132 bp to the pvr21allele. Lane M:
50 bp marker (Promega). All the F2 individuals
shown are homozygous (pvr21/pvr21) except
those in lanes 11 and 12, one of which is
homozygous (pvr23/pvr23) and one of which is
heterozygous (pvr21/pvr23). S, Susceptible
phenotype; R, resistant phenotype.
and permitted the optimization of the exploitation of
genetic resources for resistance traits resulting from
complementation effects. This complementary effect was
previously known between pvr6 from the C. annuum
cultivar ‘Perennial’ and pvr22 from ‘Florida VR2’ (Caranta
et al., 1996; Ruffel et al., 2006). We extended this effect to
two new gene combinations involving the same pvr6 allele
and two new alleles: pvr21 from ‘Yolo Wonder’ and pvr27
from ‘Serrano VC’. Indeed, as 9 and 14 resistant plants,
respectively, proved to be homozygous for both pvr6 and
pvr21 or pvr27 alleles, the probability that these doublerecessive genotypes do not control the targeted resistance
are P5(1/16)9–10210 and P5(1/16)14–10216 respectively.
The 10 pvr2 alleles tested for complementation with pvr6
were screened on the basis of their original sequence
among a core collection of 25 pepper genotypes (Charron
et al., 2008). This core collection itself resulted from a
sampling of 25 individuals among the hundreds of strains
of the INRA pepper germplasm, based on geographical
origin and absence of parental relationships (Sage-Palloix
et al., 2007). Looking for individual effects of these alleles,
pvr1 confers resistance to the same strains of PVY and TEV
as pvr22 (Kang et al., 2005b), and may have been a good
candidate, but it did not complement pvr6 for PVMV
resistance. The pvr27 allele displayed the same resistance
spectrum as pvr25, pvr26, pvr28 and pvr29 (Charron et al.,
2008) but only the former provides resistance to PVMV
when combined with pvr6. Moreover, none of these alleles
displays individual effects regarding PVMV resistance,
since the parental lines are fully susceptible to this virus.
The expected complementation effects between these alleles
were not predictable from their individual effects, and a
tremendous number of segregating progenies should be
generated and tested to detect resistant combinations. The
advantage of focussing research on alleles with distinct
sequences is particularly significant in reducing the effort
required to generate the allele combination for phenotype
expression.
The recessive resistance allele pvr6-eIF(iso)4E results from
an 82 nt deletion in the eIF(iso)4E cDNAs, leading to a
truncated and non-functional eIF(iso)4E protein (Ruffel et
al., 2006). This deletion had no perceptible phenotypic
effect, since the cultivar ‘Perennial’ displayed a normal
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phenotype, supporting the hypothesis that eIF4E and
eIF(iso)4E proteins can compensate for one another in
cellular functions (Duprat et al., 2002; Yoshii et al., 2004).
However, combination with mutated forms of pvr2-eIF4E
might alter the plant phenotype, since simultaneous
depletion of eIF4E and eIF(iso)4E in tobacco through
antisense strategy resulted in plants with a semi-dwarf
phenotype and an overall reduction in polysome loading,
demonstrating that the two isoforms contribute additively
to translation and plant growth (Combe et al., 2005). Such
alterations were not expected in plants combining pvr6eIF(iso)4E with the C. annuum alleles pvr21–pvr29-eIF4E,
since amino acid substitutions characterizing the proteins
encoded by these eIF4E alleles were shown not to impede
essential eIF4E functions, including those linked to mRNA
translation (Charron et al., 2008). However, phenotype
alterations were expected from the combination of pvr6eIF(iso)4E and pvr1-eIF4E, since the amino acid substitution G107R, the signature of the pvr1-eIF4E protein,
abolished in vitro cap-binding activity (Kang et al., 2005b;
Yeam et al., 2007). Despite the deficit in double-recessive
homozygous genotypes in the F3 interspecific progeny (i.e.
pvr1/pvr1 and pvr6/pvr6), the occurrence of both pvr1
heterozygous and homozygous plants indicated that the
combination of both depleted isoforms is viable at the
gamete level. Moreover, the plants’ phenotypes were not
significantly affected, indicating that the allele combination
is also viable at the zygote level. This lack of phenotype
alteration may indicate that other eIF4E and eIF(iso)4E
genes may compensate the depleted isoforms, since at least
two eIF4E genes and two eIF(iso)4E genes were characterized in pepper (Charron, 2007). The possible number of
combinations of mutated forms of eIF4E and eIF(iso)4E is
larger than expected and a large range of allele combinations can be exploited to enlarge the spectrum and
durability of resistance to potyviruses.
Compared with the eIF4E protein encoded by the
susceptible allele pvr2+, only the three eIF4E proteins that
contribute to PVMV resistance, i.e. pvr21, pvr22 and pvr27,
share the two amino acid substitutions V67E and L79R
(Table 2). V67E was also present in other eIF4E proteins as
a single substitution (pvr24) or associated with other
substitutions (pvr23, pvr25), indicating that this substi-
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Allele mining for virus resistance
Table 2. Amino acid substitutions observed between deduced eIF4E protein sequences from different C. annuum and C. chinense
genotypes and their corresponding alleles
The resistance phenotypes of the lines were extracted from Charron et al. (2008). The extension of resistance to PVMV when the pvr2 allele is
combined with the pvr6 allele is indicated in parentheses. The L79R substitution discussed in the text is indicated by bold type.
Inbred line
C. annuum
Yolo Wonder
Yolo Y
HD801
Perennial
PI322719
SC81
Maroc 1
Serrano VC
PI195301
Chile de árbol
C. chinense
PI159336
Allele
Resistance phenotype
51
66
67
68
71
73
74
79
107
109
205
pvr2+
pvr21
pvr22
pvr23
pvr24
pvr25
pvr26
pvr27
pvr28
pvr29
Susceptible
PVY (+PVMV)
PVY, TEV (+PVMV)
PVY
PVY
PVY
PVY
PVY (+PVMV)
PVY
PVY
T
2
2
2
2
2
2
2
2
2
P
2
2
2
2
T
2
2
2
2
V
E
E
E
E
E
2
E
2
2
A
2
2
2
2
2
E
2
E
E
K
2
2
2
2
2
2
2
2
2
A
2
2
2
2
D
D
2
2
D
A
2
2
2
2
2
D
2
2
2
L
R
R
2
2
2
2
R
2
2
G
2
2
2
2
2
2
2
R
2
D
2
N
2
2
2
2
2
2
2
D
2
2
G
2
2
G
G
2
G
pvr1
PVY, TEV
A
T
2
2
R
2
2
2
R
2
2
tution alone cannot be directly related to PVMV resistance.
L79R was present only in the pvr21, pvr22 and pvr27
proteins but was always associated with the V67E
substitution, meaning that its individual effect cannot be
assessed. Both V67E and L79R are localized in region I of
the core eIF4E protein, at the surface of the predicted 3D
structure where several mutations were shown to disrupt
the interaction with VPg from PVY and confer resistance to
different strains of this virus (Robaglia & Caranta, 2006;
Charron et al., 2008). The D205G substitution from pvr27,
which is located in the C-terminal region of the protein
and is shared with pvr23, pvr26 and pvr29, cannot be
related to PVMV resistance. The D109N substitution is
located in region II of the protein but is specific to the
pvr22 protein. Its occurrence appears to be independent of
the complementation with pvr6 for PVMV resistance.
Thus, only the V67E and L79R substitutions in the pvr2
proteins can be closely associated with PVMV resistance.
The breakdown of eIF4E-mediated resistance by potyviruses was shown to occur via amino acid substitutions in
VPg (Moury et al., 2004; Lecoq et al., 2004; Charron et al.,
2008). Two modes of resistance breakdown were hypothesized: substitutions in the VPg could (i) enable the
interaction with a new isoform of the plant translation
initiation factor or (ii) recover the interaction with the
mutated eIF4E protein. Up to now, only the second
mechanism was experimentally demonstrated in the cases
of PVY and TEV variants breaking down pvr2-mediated
resistance. It is very unlikely that the resistance breakdown
by PVMV will occur via the recovery of the interaction
with the truncated pvr6-eIF(iso)4E protein. If we hypothesize that PVMV, like PVY or TEV, breaks down the
resistance through the recovery of the interaction between
http://vir.sgmjournals.org
Position of aa substitution
its VPg and the mutated pvr2-eIF4E protein, it may be
argued that the breakdown of one of these alleles may
result in the breakdown of the others. Against this
argument, earlier studies have shown that one single
mutation in the VPg of PVY caused the breakdown of pvr21
by the virus, in contrast with pvr22, which required 2–3
mutations in the VPg, explaining the higher durability of
the resistance due to this allele (Ayme et al., 2007). It could
be expected that the combination between pvr22 and pvr6
may provide the most durable alternative to the other
combinations. However, considering the PVMV ability to
alternatively use the susceptible alleles at the pvr2 or pvr6
loci, PVMV may also adapt to resistant peppers through
the use of another isoform. Polymorphisms in pvr6 alleles
across pepper germplasm showed that non-synonymous
mutations resulting in polymorphisms of the eIF(iso)4E
protein were very low and were under a strong negative
selection, in contrast with pvr2-eIF4E (Charron, 2007).
Looking for alternative alleles to the partially deleted pvr6
allele from ‘Perennial’ is not promising compared with
pvr2. Presently, PVMV strains adapted to the digenic
recessive resistance were not isolated in the field, but the
resistance is still not largely found in the infected regions.
Based on the interaction pattern between PVY–pvr2 and
VPg–eIF4E, the mechanisms of resistance breakdown can
be explored further and additional resistance alleles need to
be searched for in a larger gene pool.
ACKNOWLEDGEMENTS
The authors acknowledge G. Nemouchi, T. Phaly and G. Bommel for
technical assistance. M. R. was supported by a grant from the Spanish
Ministry of Education and Science (Ref: EX2006-1689).
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M. Rubio and others
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