Download Molecular dissection of the potato virus Y VPg virulence factor

Journal of General Virology (2007), 88, 1594–1601
DOI 10.1099/vir.0.82702-0
Molecular dissection of the potato virus Y VPg
virulence factor reveals complex adaptations to the
pvr2 resistance allelic series in pepper
Valérie Ayme,1,2 Julien Petit-Pierre,1 Sylvie Souche,1 Alain Palloix2
and BenoÎt Moury1
Correspondence
BenoÎt Moury
[email protected]
Received 10 November 2006
Accepted 16 January 2007
1
INRA, UR407 Pathologie Végétale, F-84143 Montfavet, France
2
INRA, UR1052 Génétique et Amélioration des Fruits et Légumes, F-84143 Montfavet, France
The virulence properties of potato virus Y (PVY) towards an allelic series at the pvr2 locus in
pepper genotypes are related to variations in the genome-linked viral protein (VPg). Eleven amino
acid substitutions in the central part of the VPg were identified in strains differing by their
virulence properties and were introduced, either singly or in combination, in an infectious PVY
clone to get an in-depth genetic analysis of the virulence determinant. The virulence spectrum of
these mutants was evaluated by inoculation of four pepper genotypes carrying different alleles
at the pvr2 locus. The mutations introduced had complex effects on virulence, including
antagonistic epistasis and trade-offs for virulence towards different pvr2 alleles. In addition,
several mutants showed new virulence properties that were unknown in the natural environment.
Such complex effects of mutations on plant virus virulence are unprecedented. They provide a
better understanding of the variable levels of durability of the resistance conferred by the different
pvr2 alleles, and have important consequences for a durable management of the resistances.
INTRODUCTION
The efficiency of genetic resistance in plants is impaired by
the possible emergence of resistance-breaking pathogen
genotypes that can increase in frequency after large-scale
growing of cultivars carrying the resistance factors (e.g.
Pelham et al., 1970). However, when several resistance
genes to a pathogen are available in a host species, their
combination (gene pyramiding) can reduce resistance break
down (Lindhout, 2002). In addition, spatial or temporal
management of these resistances could increase their
efficiency and durability (Kiyosawa, 1982). Comparing the
probability of breakdown of a single gene to sets of genes
remains an important challenge for an efficient and durable
use of genetic resources.
In pepper (Capsicum annuum L.), several alleles at the pvr2
locus, differing by their resistance spectrum, control
recessive resistances to potato virus Y (PVY) (Kyle &
Palloix, 1997; Robaglia & Caranta, 2006). All these pvr2
alleles were shown to code for variants of the eukaryotic
translation initiation factor 4E (eIF4E) and differ by a small
number of amino acid substitutions (Ruffel et al., 2002,
2004; Fig. 1). The sequence of the genome-linked viral
protein (VPg) cistron is the unique virulence determinant
towards pvr2. Moury et al. (2004) and Ayme et al. (2006)
showed that non-synonymous mutations in a 23-codonlong region in the central part of the VPg cistron of PVY
determined virulence towards the three resistance alleles
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pvr21, pvr22, and pvr23. Note that throughout this paper,
virulence is an antonym for avirulence and is defined as
the genetic ability of a pathogen to cause a compatible
interaction (disease) in a host carrying a genetically
determined resistance (Shaner et al., 1992), and pathotype
is defined as a subspecies pathogen entity that is controlled
by a host gene specific for that entity (Hampton &
Provvidenti, 1992). The fact that a few codon positions of
the VPg cistron of PVY are subjected to positive selection
during their evolution strongly suggests that the encoded
protein, and not the corresponding RNA segment (apart
from its encoding capacity), determines virulence in
pepper (Moury et al., 2004). This hypothesis also fits with
functional molecular models that involve eIF4E–VPg
physical interaction to determine resistance or virulence
properties in plant–virus interactions (Miyoshi et al., 2006;
Moury et al., 2004; Robaglia & Caranta, 2006; Whitham &
Wang, 2004). It is important to note that, in these plant–
virus interactions, resistance is recessive and is the result of
incompatibility between virus and plant factors. This is
distinct from elicitation of resistances where pathogens
trigger host defence responses.
The mechanisms of adaptation of viruses that are
confronted with series of alleles of resistance in plants are
poorly understood. In this context, two other pathosystems
have been explored. The tomato mosaic virus movement
protein is the avirulence determinant to the Tm-2 and
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Mutagenesis of a PVY virulence gene
(Flo) and HD285. YW, YY and Flo possess, respectively, the pvr2+
dominant allele for susceptibility to PVY, the pvr21 and the pvr22
recessive alleles for resistance to PVY, which are the three alleles
classically used to define PVY pathotypes (0), (0,1) and (0,1,2) (Gébré
Sélassié et al., 1985; Kyle & Palloix, 1997). In the pathotype
nomenclature, figures correspond to the different pvr2 alleles to
which the PVY isolate is virulent (0 stands for virulence to pvr2+). YY
is isogenic to YW except at the pvr2 locus (Cook, 1961). Flo is closely
related to both YW and YY since it differs from them by 4 % of
around 2000 molecular markers (Lefebvre et al., 2001). HD285 is a
doubled haploid line possessing the pvr23 resistance allele and selected
for susceptibility alleles in the genetic background with the use of
molecular markers (Caranta et al., 1997; Ayme et al., 2006). The pvr23
allele was initially mapped as a major resistance quantitative trait
locus (QTL) near the pvr2 locus (Caranta et al., 1997) and was
further demonstrated to be an allele at this locus (Ruffel et al.,
2004). These four pvr2 alleles were shown to encode eIF4Es differing by a small number of amino acid substitutions (Ruffel et al., 2004
and Fig. 1).
Fig. 1. Amino acid sequence alignment of the eIF4Es corresponding to the pvr2+, pvr21, pvr22 and pvr23 alleles in pepper
(GenBank accession nos AY122052, AF521964, AF521965 and
AY723737, respectively).
Tm-22 resistance alleles in tomato. However, different
domains of the protein are involved in adaptation to each
resistance (Meshi et al., 1989; Weber et al., 1993, 2004) and
gain of virulence towards either Tm-2 or Tm-22 does not
affect virulence towards the other allele (Fraser, 1985). The
coat protein of tobamoviruses is the avirulence determinant to an allelic series of resistances at the L locus in
pepper (Berzal-Herranz et al., 1995; Gilardi et al., 2004). In
that pathosystem, adaptation of the virus to cultivars
carrying the different resistance alleles occurred mainly
through replacement of tobamovirus populations belonging to different species, rather than through point
modifications of the avirulence gene within a virus species
(except for the L3 allele) (Takeuchi et al., 2005). In this
context, the pepper–PVY interaction is a unique model to
study the adaptation of a virus to a resistance allelic series,
notably by evaluating whether PVY can accumulate
virulences towards all pvr2 alleles or is specialized to
particular pvr2 alleles. To unravel these mechanisms of
adaptation, we studied the effect of amino acid substitutions in the central part of the VPg of PVY on its virulence
properties towards four pvr2 alleles.
METHODS
Plant genotypes and PVY isolates. The C. annuum inbred lines
used in this work were Yolo Wonder (YW), Yolo Y (YY), Florida VR2
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PVY isolates LYE84, SON41p (Moury et al., 2004) and To72 (Gébré
Sélassié et al., 1985) were collected in Lycopersicon esculentum in the
Canary Islands in 1984, in Solanum nigrum in France in 1982 and in
L. esculentum in France in 1972, respectively. Isolates CAA21, CAA82,
CAA141 and EP03 were obtained from field-infected C. annuum
plants in Turkey (1994), Israel (1982) and France (1999, 2003),
respectively.
Sequencing of the PVY VPg cistron and mutagenesis of a fulllength cDNA clone. The cDNA corresponding to the VPg cistron of
each PVY isolate was cloned and sequenced as previously described
(Moury et al., 2004). Based on the sequence diversity of the central
part of the VPg sequence, mutants with single mutations or
mutation combinations were then produced. The infectious cDNA
clone of isolate SON41p (EMBL accession no. AJ439544) (Moury
et al., 2004) was mutated using a QuikChange site-directed mutagenesis kit (Stratagene). Cloning experiments were carried out using
homologous recombination in yeast as described by Ayme et al.
(2006).
PVY inoculation tests and detection. Inoculation experiments
were carried out under greenhouse conditions or in climatic rooms.
Primary inoculations with the different PVY cDNAs were made by
DNA-coated tungsten particle bombardments of Nicotiana clevelandii
or Nicotiana benthamiana plants, since direct bombardment of
pepper plants is not efficient (Moury et al., 2004). Pepper plants
were mechanically inoculated on their two cotyledons approximately
3 weeks after sowing (first leaf stage), using crude extracts from
infected Nicotiana spp., according to Moury et al. (2004). Detection
of the virus in the inoculated plants was performed by the doubleantibody sandwich ELISA (DAS-ELISA) as described by Legnani
et al. (1995) at various time points after inoculation. The relative
concentration of PVY in Nicotiana spp. and pepper plants was
determined by semi-quantitative DAS-ELISA as described by Ayme
et al. (2006). Each experiment involved at least 10 plants per virus
clone or isolate and per pepper genotype and was performed at least
twice.
Sequence analysis of viral progeny. Total RNAs from leaves of
systemically infected plants were purified with a TRI Reagent kit
(Molecular Research Center Inc.) and used for RT-PCR for
amplification of the entire VPg cistron (Moury et al., 2004). Viral
progeny of VPg mutants were checked by sequencing RT-PCR DNA
fragments produced from at least two plants per virus–genotype
combination using the same primers as described by Moury et al.
(2004).
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V. Ayme and others
RESULTS
differences in their relative accumulation could be revealed
in semi-quantitative DAS-ELISA (Ayme et al., 2006; data
not shown). These 17 mutants were tested for their
virulence towards the four pepper genotypes (Table 2).
Since these pepper genotypes essentially differ by their
allele at the pvr2 locus (see Methods), virulence differences
observed would be caused by specificities of interaction
between mutations in the VPg of the virus and the pvr2
alleles in the plants. All the infected plants developed a
systemic mosaic (YW, YY and Flo) or systemic necrotic
symptoms (HD285) 10–15 days post-inoculation (days
p.i.) and yielded high absorbances at 405 nm (A405) in
DAS-ELISA tests, while no PVY could be detected by DASELISA in plants that showed no symptoms. For most
pepper genotype–PVY mutant combinations, 0 or 100 % of
the plants became infected, and the VPg sequence of the
progeny in infected plants was identical to that of the PVY
clone used to prepare the inoculum. In four combinations
(T115V/YW, D119H/Flo, H121N/YY and T115M-H121N/YW),
only some of the plants were infected and infection was
delayed (20 days p.i. or later). Sequencing of the PVY
progeny in a random set of these plants (11 out of 29)
always revealed additional amino acid substitutions in the
central part of the VPg (Table 2). These additional amino
acid substitutions were observed at positions shown to be
critical for virulence (Ayme et al., 2006) and are
presumably involved in PVY adaptation. For the T115V
and T115M-H121N mutants in particular, different additional amino acid substitutions were observed depending
on the plant analysed (Table 2). For these last two mutants,
the virus isolates extracted from the YW plants were backinoculated to new YW plants. As a control, the original
virus populations (from N. benthamiana) were also
inoculated to YW plants. All back-inoculated plants, i.e.
9/9 for T115V and 15/15 for T115M-H121N, showed high
A405 values in DAS-ELISA and developed systemic
symptoms at 10 days p.i., while significantly lower
proportions of YW accumulated virus in DAS-ELISA after
inoculation with isolates from N. benthamiana (2/9 and
4/15, respectively; P50.001 and P,0.001, Fisher’s exact
tests). We concluded that infection of the YW plants by the
initial PVY mutants was the result of a gain of virulence of
the original virus that occurred during the resistance tests.
Pathotypes and sequence of the VPg of PVY
isolates
As a first step to characterize the PVY virulence determinants
in relation to the pvr2 alleles in pepper, the virulence
properties of seven PVY isolates to four pvr2 alleles were
assessed by inoculation of YW, YY, Flo and HD285 pepper
plants, homozygous for pvr2+ (the reference susceptibility
allele), pvr21, pvr22 and pvr23, respectively. Isolates LYE84,
CAA21, To72, CAA82 and EP03 were shown to belong to
pathotype (0), isolate CAA141 to pathotype (0,1) and
SON41p to pathotype (0,1,2) (Ayme et al., 2006; Moury
et al., 2004; data not shown; see the Methods section for the
pathotype nomenclature). In order to estimate the amino
acid diversity of the VPg of these isolates, the VPg cistron of
isolates CAA21, To72, CAA82, EP03 and CAA141 was
sequenced and compared to that of SON41p and LYE84
(Moury et al., 2004; Table 1). CAA21 had the same nucleotide
sequence as LYE84 and differed from SON41p by five amino
acids in the central part of the VPg, while To72, EP03 and
CAA82 shared identical nucleotide sequences and differed
from SON41p by two amino acids (each corresponding to a
single nucleotide substitution) (Table 1). CAA141 was
relatively distant from SON41p, To72 and LYE84 (six, five
and four amino acid substitutions in the central part of the
VPg, respectively). Based on these sequences, several VPg
mutants of SON41p were created by introducing in SON41p
single-nucleotide substitutions corresponding to the
observed amino acid differences (Tables 1 and 2). Only
mutations Asp119Ser and Ser123Lys, present in CAA141, were
not introduced into SON41p. SON41p mutants with
combinations of some of these substitutions were also created
and five single-nucleotide mutants of SON41p previously
selected for virulence towards pvr23 (Ayme et al., 2006) were
also included in the study. These are the mutants S101G,
T115R, T115K, D119N and S120C (Table 2).
Virulence properties of the VPg mutants
All SON41p mutants were infectious following bombardment of N. clevelandii and N. benthamiana and no
Table 1. Amino acid sequence of the central part of the VPg of PVY isolates or clones
Numbers are amino acid positions in the VPg.
Isolate
Pathotype
SON41p
LYE84
CAA21
To72
CAA82
EP03
CAA141
(0,1,2)
(0)
(0)
(0)
(0)
(0)
(0,1)
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Amino acid sequence of the VPg
101
S
-
E
-
V
-
R
-
105
R
K
K
K
K
-
M
-
V
-
E
-
D
-
D
-
E
-
I
-
E
-
115
T
M
M
V
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Q
-
A
-
L
-
119 120 121
D S
H
H N
H N
N N
N N
N N
S
C N
T
-
123
S
N
N
K
Journal of General Virology 88
Mutagenesis of a PVY virulence gene
Table 2. Virulence properties of VPg mutants of PVY SON41p towards the pvr2 resistance alleles in pepper
S101G, T115R, T115K, D119N and S120C are SON41p mutants previously selected for virulence towards pvr23 (Ayme et al., 2006).
PVY isolate/mutant
SON41p
S101G
R105K
T115R
T115K
T115M
T115V
D119N
D119H
S120C
H121N
S123N
R105K-S123N
T115M-D119H
T115M-H121N
D119H-H121N
D119N-H121N
T115M-D119H-H121N
C. annuum genotype*
Pathotype
YW
YY
Flo
HD285
pvr2+/pvr2+
pvr21/pvr21
pvr22/pvr22
pvr23/pvr23
20/20
20/20
20/20
40/40
20/20
20/20
13/22d
20/20
30/30
20/20
30/30
20/20
20/20
20/20
10/33
20/20
20/20
20/20
20/20
20/20
20/20
37/37
20/20
20/20
30/30
20/20
30/30
20/20
1/39I
20/20
20/20
20/20
0/28
0/20
0/20
0/20
20/20
20/20
20/20
0/40
0/20
0/20
0/30
0/20
5/30§
20/20
0/28
20/20
20/20
0/20
0/20
0/20
0/20
0/20
29/79D
20/20
20/20
40/40
20/20
20/20
0/28
20/20
30/30
20/20
20/20
0/20
0/20
20/20
0/25
20/20
0/20
20/20
(0,1,2)
(0,1,2,3)
(0,1,2,3)
(0,1,3)
(0,1,3)
(0,1,3)
(1)
(0,1,3)
(0,1,3)
(0,1,2,3)
(0,3)
(0,1,2)
(0,1,2)
(0,1,3)
(2)
(0,3)
(0)
(0,3)
*Number of plants infected at the systemic level/number of inoculated plants.
DData from Ayme et al. (2006). Infected plants correspond to resistance-breaking events.
dThe additional mutations 119N (in three plants), 120C (in one plant) and 115M+119N (in one plant) were observed in the VPg of the virus
progeny, compared to the original mutant. Only a number of randomly chosen infected plants were tested for the presence of additional mutations
in the virus progeny.
§The additional mutation 101C (in three plants) was observed in the VPg of the virus progeny, compared to the original mutant. Only a number of
randomly chosen infected plants were tested for the presence of additional mutations in the virus progeny.
||The additional mutation 115R was observed in the VPg of the virus progeny, compared to the original mutant.
The additional mutations 119N (in one plant) and 119D+120C (in one plant) were observed in the VPg of the virus progeny, compared to the
original mutant. Only a number of randomly chosen infected plants were tested for the presence of additional mutations in the virus progeny.
In the case of D119H/Flo and H121N/YY combinations,
systemic infections were observed in a small number of
inoculated plants (5/30 and 1/39, respectively) and in one
of three independent experiments only. Finally, we
considered that mutants T115V, D119H, H121N and
T115M-H121N belonged to pathotypes (1), (0,1,3), (0,3)
and (2), respectively (Table 2). From these data, we could
classify the PVY clones into seven pathotypes: (2), (0),
(0,1,2), (0,1,3), (0,1,2,3), (1) and (0,3), in addition to
pathotype (0,1) defined by CAA141 (see above).
Analysis of the effect of single amino acid substitutions
allows to draw a map of virulence determinants towards
the different pvr2 alleles in the context of SON41p (Fig. 2).
Among single-amino acid mutants, only H121N lost
virulence towards pvr21, suggesting that this amino acid is
essential for conferring virulence towards this allele
to SON41p. For the pvr22 and pvr23 alleles, different
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Fig. 2. Map of virulence changes towards alleles at the pvr2 locus
conferred by single amino acid substitutions in the VPg of PVY
SON41p [pathotype (0,1,2)]. Numbers correspond to amino acid
positions in the VPg. Gains and losses of virulence are indicated
above or below the line, respectively.
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V. Ayme and others
substitutions at the same position modified the virulence
properties in an opposite manner. Amino acid substitutions
at three positions [115 (4 of 4), 119 (2 of 2) and 121]
abolished virulence towards pvr22, while amino acid
substitutions at six positions [101, 105, 115 (3 of 4),
119 (2 of 2), 120 and 121] resulted in a gain of virulence
towards pvr23. Only the single mutant S123N had a
pathotype similar to that of SON41p.
Atypical PVY pathotypes
All PVY isolates already described in natural infection
conditions worldwide belong to pathotypes (0), (0,1) and
(0,1,2) (Luis-Arteaga & Gil-Ortega, 1986; Boiteux et al.,
1996; Gébré Sélassié et al., 1985; Palloix et al., 1994), since
pvr23 was not taken into account in previous works.
Through mutagenesis, we generated new pathotypes. One
mutant (T115M-H121N) could infect Nicotiana spp. plants,
but was unable to infect YW or the other C. annuum
genotypes. We do not know whether this mutant has lost
the capacity to be infectious in peppers or whether other
pepper genotypes would still be susceptible to it. In
addition, description of pathotype (1) (T115V mutant) was
not expected and provides the first evidence for PVY
variants virulent towards pvr21 but avirulent towards
pvr2+. The poor infectivity of mutants T115V and T115MH121N in YW is not due to deleterious effects of the
mutations introduced into the VPg on fitness, since these
mutants accumulated to the same titre as SON41p, T115M
or H121N in N. benthamiana or N. clevelandii as measured
in semi-quantitative DAS-ELISA (data not shown). Also,
mutant T115V accumulated to the same titre as T115M in
YY (data not shown). Since YW and YY are isogenic except
for the pvr2 locus (Cook, 1961), this is also the first
evidence that the pvr2+ allele can exert a resistance towards
particular PVY variants.
Trade-offs between virulence properties
The effect of amino acid substitutions examined on the
whole spectrum of virulence rather than on individual pvr2
alleles reveals trade-offs between virulence properties, i.e.
situations where an amino acid substitution induces a gain
of virulence towards a particular pvr2 allele and simultaneously a loss of virulence towards one or several other
pvr2 allele(s). Particularly, among the nine single amino
acid mutations that induced the virulence towards pvr23,
six induced avirulence towards pvr22 and one also induced
avirulence towards pvr21 (Table 2). Trade-offs are, however, conditional to particular mutations, since three
single-amino acid mutants (S101G, R105K and S120C) have
the ability to infect all pepper genotypes. It should be
noticed, however, that two of these mutants were
previously studied (i.e. S101G and S120C) and displayed a
poor relative ability to accumulate and to compete in
pvr23-carrying peppers (Ayme et al., 2006). This loss of
fitness may represent the cost of possessing a wide
spectrum of virulence, as hypothesized in theoretical and/
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or experimental studies (Garcı́a-Arenal & McDonald, 2003;
Harrison, 2002).
Antagonistic epistasis between mutations
Complex effects of substitutions can be observed when
comparing the virulence spectra of single vs multiple
mutants. When two or three amino acid substitutions were
introduced simultaneously into SON41p, the virulence
spectrum of the multiple mutant usually included only the
virulences shared by all corresponding single mutants
(R105K-S123N, T115M-D119H, D119H-H121N and T115MD119H-H121N). In two occurrences, however, the virulence
spectrum of the multiple mutant included fewer virulences
than those shared by all corresponding single mutants.
Single mutants T115M, D119N and H121N infect pvr2+ and
pvr23-carrying genotypes. However, the double mutant
D119N-H121N is avirulent towards pvr23 and the double
mutant T115M-H121N is avirulent towards both pvr2+ and
pvr23 (Table 2), indicating occurrence of antagonistic
epistasis, as defined by Wilke et al. (2003), between
substitutions within the VPg.
DISCUSSION
Explaining the higher durability of pvr22
In our study, we focussed on major, qualitative fitness
variations (i.e. virulence spectra in pepper genotypes)
determined by mutations in the VPg of PVY. Quantitative
fitness variations also act on the evolution of PVY
populations and may affect resistance durability. Semiquantitative DAS-ELISA did not reveal any quantitative
fitness differences between SON41p and its VPg mutants in
N. benthamiana, N. clevelandii or YW (for those mutants
virulent to YW) in single infections (data not shown). In
the other pepper genotypes, only limited differences in
fitness were observed in DAS-ELISA (Ayme et al., 2006;
data not shown). Although competition between mutants
within plants could potentially reveal more subtle fitness
differences invisible in single infections, it should be noted
that fitness measurements in single infections fitted
experimental data of resistance breaking better than
competition fitness measurements from a previous study
(Ayme et al., 2006). Moreover, the VPg of potyviruses is
not a determinant of plant-to-plant transmission efficiency
by the aphid vectors. As a consequence, if these mutants
appeared, they would have the potential to spread in
pepper fields or in the field environment (weeds).
Our analysis of the effect of mutations in the virulence
determinant of PVY towards the pvr2 allelic series in
pepper contributes to explain the observed durability of
resistances conferred by these alleles and to help manage
these resistances. In spite of wide cultivation of pvr21- or
pvr22-carrying pepper cultivars for decades (Greenleaf,
1986), PVY virulence towards pvr21 was rather frequent but
not predominant, and virulence towards pvr22 was only
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Journal of General Virology 88
Mutagenesis of a PVY virulence gene
exceptionally observed in the field (Boiteux et al., 1996).
This suggests a higher durability of the resistance conferred
by the latter allele. The pvr23 allele has not been exploited
in pepper hybrid cultivars but is present in landraces grown
locally. PVY populations virulent to pvr23 can easily be
selected for (Ayme et al., 2006), which suggests a low
durability. Three reasons could explain the higher durability of pvr22.
First, fewer amino acid combinations in the central part of
the VPg conferred virulence towards pvr22 in comparison
to the other pvr2 alleles. To compare the frequencies of
virulence towards pvr21, pvr22 and pvr23, one should first
remove the mutants whose virulence is biased towards
some of these alleles because of the experimental design, i.e.
(i) because the virulence to a particular allele pvr2 was
selected during the experiment, (ii) because the original
PVY clone used for mutagenesis is virulent towards
particular pvr2 alleles but not towards others and (iii)
because the isolate whose sequence was used to design the
substitutions is virulent towards particular pvr2 alleles but
not towards others. As a consequence, to compare the
frequencies of virulence towards pvr21 and pvr22, all clones
can be used except T115V, because the original clone
(SON41p) and the isolate used to design the substitution
(CAA141) are both virulent to pvr21 but not to pvr22.
Among these 17 PVY clones, six were virulent towards
pvr22 while 12 were virulent towards pvr21, which is
significantly higher (P50.047, Fisher’s exact test). To
compare the frequencies of virulence towards pvr22 and
pvr23, one cannot use the five mutants which were selected
for their virulence towards pvr23 (i.e. S101G, T115R, T115K,
D119N and S120C), or the two mutants which belong to the
same pathotype as the original clone SON41p (S123N and
R105K-S123N), since they would be expected to induce a
bias in favour of the virulence to pvr22 and against the
virulence to pvr23 and, for the same reason, SON41p itself.
Among the ten remaining mutants, only one was virulent
towards pvr22, while significantly more (seven) were
virulent towards pvr23 (P50.008, Fisher’s exact test). No
significant differences were observed between the frequencies of virulences towards pvr21 and pvr23. Thus, in the
range of variation examined, PVY should accumulate
several mutations at precise positions and of precise types
to become virulent towards pvr22, whereas numerous
possibilities of amino acid combinations confer virulence
towards pvr21 or pvr23. Consequently, the probability to
gain virulence towards pvr21 or pvr23 is expected to be
higher than that towards pvr22.
Second, due to epistatic relationships between mutations,
all sequential mutation pathways are not equivalent to
accumulate the different substitutions required for virulence towards pvr22. For example, in order to gain virulence
towards pvr22, the triple mutant T115M-D119H-H121N has
to accumulate the three reverse mutations M115T, H119D
and N121H. If we consider the six possible pathways for the
sequential acquisition of these three mutations, two of
them, beginning with H119D, lead to a ‘dead end’ for the
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virus, since mutant T115M-H121N is not infectious in all
four pepper genotypes. This would contribute to decrease
the probability of fixation of these substitutions. These
antagonistic epistases between mutations are similar to
those mentioned in other viruses by Sanjuan et al. (2004),
Burch & Chao (2004) and Bonhoeffer et al. (2004), who
evaluated quantitative fitness variations instead of virulence spectra. Sanjuan et al. (2004) showed antagonistic
epistasis of mutations chosen at random or of mutations
beneficial to the fitness of vesicular stomatitis virus. Burch
& Chao (2004) and Bonhoeffer et al. (2004) found evidence
for antagonistic epistasis of deleterious mutations in
bacteriophage W6 and human immunodeficiency virus
type 1, respectively. In these three examples, there was a
clear predominance of antagonistic over synergistic epistatic mutations. In our case, the mutants were confronted
with host resistance genes and the effects of antagonistic
epistasis on virus fitness are much more drastic since they
consist of a lack of virulence towards particular host
genotypes.
Third, among the natural isolates analysed so far, those
displaying the closest VPg sequence compared to SON41p
and its mutants virulent towards pvr22 are To72, CAA82
and EP03, differing by two amino acids. Since they were
collected in France or Israel, from 1972 to 2003, isolates
with such VPg sequences seem to be common. In order to
gain virulence towards pvr22, these isolates require at least
two nucleotide substitutions while they only require a
single nucleotide substitution to become virulent to pvr21
or pvr23. Similarly, a high durability of virus resistances has
been previously related to the necessity for the virus to
accumulate at least two substitutions to gain virulence
(Harrison, 2002). This hypothesis could be further
explored by gaining insight into the variability of the
VPg cistron of field pepper PVY isolates.
Management of pepper cultivars for durable PVY
resistance
Accumulating (pyramiding) different resistance genes in a
cultivar and temporal or spatial deployment of different
resistances are frequently proposed as promising strategies
to preserve the durability of plant resistance towards their
pathogens. The efficiency of such strategies was demonstrated in several instances (Pink, 2002) and may be due to
the difficulties for the pathogen to acquire multiple
virulences. Occurrence of trade-offs between virulences
towards pvr22 and pvr23 alleles could suggest to alternate
temporally and/or spatially cultivars carrying these alleles.
However, the occurrence of a few mutants accumulating
virulences towards all pvr2 alleles examined here suggests
that these strategies are risky. Moreover, the selective
pressures exerted by the pvr21 and pvr23 alleles could act as
‘springboards’ for the acquisition of the virulence towards
pvr22, since they could accelerate the fixation of the
first mutations required for virulence towards pvr22.
Considering the isolates that share a VPg cistron identical
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1599
V. Ayme and others
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Fig. 3. Mutation pathways between a PVY VPg mutant avirulent to
pvr22 and SON41p which is virulent to pvr22. Mutants are named
after the amino acid substitutions in the VPg in comparison with
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ACKNOWLEDGEMENTS
We thank G. Nemouchi and P. Mistral for technical assistance in the
laboratory work and the greenhouse staff for support in plant
experimentation. We thank also C. Caranta and S. Ruffel for sharing
unpublished data and M. Jacquemond, D. Fargette and E. Hébrard for
critical review of the manuscript.
pepper inbred lines for cultivar protection purposes: comparison of
AFLP, RAPD and phenotypic data. Theor Appl Genet 102, 741–750.
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inheritance of the Lycopersicum hirsutum resistance against potato
virus Y. Euphytica 86, 219–226.
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