Download Cucumber mosaic virus satellite RNAs that induce similar symptoms

Journal of General Virology (2011), 92, 1930–1938
DOI 10.1099/vir.0.032359-0
Cucumber mosaic virus satellite RNAs that induce
similar symptoms in melon plants show large
differences in fitness
Mónica Betancourt,3 Aurora Fraile and Fernando Garcı́a-Arenal
Correspondence
Fernando Garcı́a-Arenal
[email protected]
Received 21 March 2011
Accepted 6 May 2011
Centro de Biotecnologı́a y Genómica de Plantas (UPM-INIA) and E.T.S.I. Agrónomos, Universidad
Politécnica de Madrid, Campus Montegancedo, 28223 Pozuelo de Alarcón, Madrid, Spain
Two groups of Cucumber mosaic virus (CMV) satellite RNAs (satRNAs), necrogenic and nonnecrogenic, can be differentiated according to the symptoms they cause in tomato plants, a host
in which they also differ in fitness. In most other CMV hosts these CMV-satRNA cause similar
symptoms. Here, we analyse whether they differ in traits determining their relative fitness in melon
plants, in which the two groups of CMV-satRNAs cause similar symptoms. For this, ten
necrogenic and ten non-necrogenic field satRNA genotypes were assayed with Fny-CMV as a
helper virus. Neither type of CMV-satRNA modified Fny-CMV symptoms, and both types
increased Fny-CMV virulence similarly, as measured by decreases in plant biomass and lifespan.
Necrogenic and non-necrogenic satRNAs differed in their ability to multiply in melon tissues;
necrogenic satRNAs accumulated to higher levels both in single infection and in competition with
non-necrogenic satRNAs. Indeed, multiplication of some non-necrogenic satRNAs was
undetectable. Transmission between hosts by aphids was less efficient for necrogenic satRNAs
as a consequence of a more severe reduction of CMV accumulation in leaves. The effect of CMV
accumulation on aphid transmission was not compensated for by differences in satRNA
encapsidation efficiency or transmissibility to CMV progeny. Thus, necrogenic and nonnecrogenic satRNAs differ in their relative fitness in melon, and trade-offs are apparent between
the within-host and between-host components of satRNA fitness. Hence, CMV-satRNAs could
have different evolutionary dynamics in CMV host-plant species in which they do not differ in
pathogenicity.
INTRODUCTION
Cucumber mosaic virus (CMV), the type species of genus
Cucumovirus, is a plant virus with isometric particles and a
tripartite, single-stranded, messenger-sense RNA genome.
CMV is the helper virus for a single-stranded, linear, noncoding satellite RNA (satRNA), which depends on CMV
for its replication, encapsidation and transmission. The
presence of a satRNA modulates CMV replication and
pathogenicity. The outcome of the CMV–satRNA interaction depends on the genotypes of CMV and satRNA
involved, and on the infected host plant species. Hence,
CMV-satRNA is a molecular parasite that spreads epidemically in CMV populations (Alonso-Prados et al., 1998).
However, once a CMV strain becomes satRNA-infected,
the satRNA can also be considered as an additional genetic
element in the segmented virus genome. The molecular
biology of the interaction of CMV-satRNA with its helper
virus, and its genetic variability and evolution, has received
3Present address: Facultad de Ciencias Agrı́colas, Universidad Santa
Rosa de Cabal, UNISARC, Campus el Jazmı́n, Km 4 vı́a Chinchiná-Santa
Rosa, Colombia.
1930
much attention in the past (reviewed by Palukaitis et al.,
1992; Garcı́a-Arenal & Palukaitis, 1999; Palukaitis &
Garcı́a-Arenal, 2003). In contrast, the role of CMVsatRNA in modulating the genetic structure of CMV
populations and the evolution of CMV virulence has
remained underexplored.
CMV is the plant virus with the broadest host range in
nature, infecting more than 1000 species in about 80
monocotyledonous and dicotyledonous plant families, and
it is efficiently transmitted by more than 80 aphid species
in a non-persistent manner (Palukaitis et al., 1992;
Palukaitis & Garcı́a-Arenal, 2003). Hence, CMV causes
epidemics with important yield losses in many crops,
particularly in vegetable crops in temperate regions (Tien &
Wu, 1991; Jordá et al., 1992; Alonso-Prados et al., 1997;
Gallitelli, 2000). Occasionally, CMV genotypes causing
unusually severe disease symptoms emerge and, in all
reported instances, hypervirulence has been demonstrated
to be due to the presence of satRNAs associated with
less virulent CMV strains (Kaper & Waterworth, 1977;
Takanami, 1981; Gonsalves et al., 1982; Coi et al., 2001).
The emergence of hypervirulent CMV genotypes results in
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Fitness differences among CMV-satRNA in melon plants
devastating epidemics, best exemplified by those of tomato
necrosis that occurred in France, the Mediterranean basin
and Japan (Marrou, et al., 1973; Gallitelli et al., 1988;
Kosaka et al., 1989; Jordá et al., 1992). While most CMVsatRNAs attenuate or do not modify CMV symptoms in
most plant species, in tomato two main types can be
distinguished: those that attenuate or do not modify CMV
symptoms (non-necrogenic satRNAs), and those that aggravate them to a systemic necrosis (necrogenic satRNAs).
These two types of satRNA do not cause different
symptoms in other host plant species (Roossinck et al.,
1992; Garcı́a-Arenal & Palukaitis, 1999). Much attention
has been directed at understanding the mechanisms by
which some satRNA genotypes induce a systemic necrosis
in tomato plants, but not in other host species (Taliansky
et al., 1998; Xu & Roossinck, 2000; Xu et al., 2004; Irian
et al., 2007).
The frequency of satRNAs in field populations of CMV
seems to be low usually (Kearney et al., 1990; Grieco
et al., 1997; Alonso-Prados et al., 1998), hence, understanding the causes that lead to the invasion of CMV
populations by satRNAs necrogenic for tomato, which
have resulted in epidemics of tomato necrosis, may help
to explain the emergence of viral diseases. With this goal
in mind, Escriu et al. (2000a, b, 2003) analysed in tomato
plants the parameters of between-host transmission,
within-host accumulation and virulence of CMV strains,
either without satRNAs or supporting CMV-satRNAs
necrogenic or non-necrogenic for tomato. Estimates of
these fitness components were introduced into deterministic models of virulence evolution, which predicted that
the emergence of CMV genotypes supporting satRNAs
necrogenic for tomato would only occur in mixed infections with non-necrogenic satRNAs, and under conditions of high density aphid-vector populations (Escriu
et al., 2003). While these predictions agreed with field
observations during the tomato necrosis epidemic that
occurred in Eastern Spain in the 1980s–1990s, the
analyses of Escriu et al. (2003) had the limitation of not
considering the role in the evolution of CMV virulence of
hosts other than tomato, which could be reservoirs or,
alternatively, reduce satRNA population levels (i.e. be
sink hosts for CMV-satRNAs).
The aim of the present work is to fill this gap by estimating
the fitness components of CMV-satRNAs that are necrogenic and non-necrogenic for tomato, in a host-plant
species in which these two types of satRNA do not cause
different symptoms. For this we chose melon (Cucumis
melo L.), which is a major crop affected by CMV epidemics
and often shares the horticultural areas of tomato (AlonsoPrados et al., 2003). Our results show that even though
both types of satRNA do not differ in their virulence for
melon plants, they differ in their ability to accumulate and
in their effect on CMV multiplication and transmission.
These unexpected results imply different evolutionary
dynamics for CMV and CMV-satRNAs over their host
range, and not only in the differential host, tomato.
http://vir.sgmjournals.org
RESULTS
Accumulation of CMV-satRNA in single and mixed
infections and its effect on CMV accumulation
To estimate the within-host multiplication rate of
satRNAs that are necrogenic and non-necrogenic for
tomato, and their competitive ability in mixed-infected
hosts, an experiment was set up in which melon plants
were inoculated with Fny-CMV along with one of ten
necrogenic or ten non-necrogenic satRNA genotypes (see
Methods). In addition, 20 mixed-infection treatments
were included in which plants were inoculated with FnyCMV along with each necrogenic or non-necrogenic
satRNA in combination with one satRNA of the opposite
type; in mixed infections, each necrogenic satRNA was
confronted with two randomly sampled non-necrogenic
satRNAs, and vice versa. Treatments of plants inoculated
with only Fny-CMV or mock inoculated with buffer, were
also included. Ten replicated plants were included for
each of the 42 treatments. Tissue from systemically
infected leaves was harvested 25 days post-inoculation
(p.i.) and satRNA accumulation was quantified in each
plant.
All assayed necrogenic satRNAs multiplied to detectable
levels in systemically infected leaves of melon plants when
supported by Fny-CMV. However, four of ten nonnecrogenic satRNAs assayed were not detected by dot-blot
hybridization in the systemically infected leaves of FnyCMV-infected plants (Table 1). Two of these satRNAs (90/
17.1 and 92/10.1) were not detected by RT-PCR, while
satRNAs 89/15.2 and 89/23.1 were detected by RT-PCR in
1 of 10 assayed plants in each of three independent
experiments. We denote these satRNA genotypes as ‘nonnecrogenic*’. The data in Table 1 show that there were no
significant differences in accumulation among the various
necrogenic satRNAs (H9,100513.75, P50.131) nor among
those non-necrogenic satRNAs that were detected in
systemically infected leaves (H5,5952.83, P50.727). On
average (mean), necrogenic satRNAs accumulated to
significantly higher levels than non-necrogenic satRNAs
(W10,10548.00, P50.0003). This was also true when nonnecrogenic* satRNAs were excluded from the analysis
(W6,10528.00, P50.003). In mixed infections, necrogenic
satRNAs also accumulated to significantly higher levels
than non-necrogenic ones (W6,10527.0, P50.004). The
accumulation of necrogenic satRNAs was lower in mixed
infections with non-necrogenic ones than in single
infections (W10,10534.0, P50.011), while the presence of
necrogenic satRNAs in mixed infections did not affect the
accumulation of those non-necrogenic satRNAs that were
detected in systemically infected leaves (W6,659.0,
P50.173). Thus, necrogenic satRNAs accumulate in melon
plants to higher levels than non-necrogenic satRNAs, both
in single and in mixed infections, but non-necrogenic
satRNAs compete with necrogenic ones in mixed infection
more effectively than necrogenic satRNAs compete with
non-necrogenic ones.
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M. Betancourt, A. Fraile and F. Garcı́a-Arenal
Table 1. Accumulation of CMV-satRNA in melon leaves
Data are micrograms of satRNA per gram fresh leaf in nucleic acid
extracts from systemically infected leaves, wherein each satRNA
genotype was inoculated alone with Fny-CMV (single infection, mean
of 10 plants±SEM) or in combination with each of two satRNA
genotypes of the other type (mixed infection, mean of 20 plants±SEM,
ten plants for each combination).
satRNA genotype
mg satRNA (g fresh leaf)”1
Single infection Mixed infection
Necrogenic
89/15.1
89/24.1
89/42.4
90/8.2
90/19.1
90/22.1
91/3.1
91/3.2
91/5.1
94/32.1
All necrogenicD
Non-necrogenic
89/20.1
90/14.1
90/16.1
90/19.2
91/2.2
92/4.1
89/15.2*
89/23.1*
90/17.1*
92/10.1*
All non-necrogenicD
Excluding non-necrogenic*D
0.357±0.051
0.549±0.075
0.429±0.087
0.368±0.058
0.308±0.061
0.494±0.150
0.540±0.089
0.502±0.075
0.621±0.088
0.470±0.057
0.464±0.026 a
0.355±0.066
0.374±0.066
0.318±0.051
0.317±0.050
0.257±0.044
0.336±0.054
0.293±0.048
0.480±0.079
0.410±0.074
0.354±0.075
0.349±0.014 b
0.273±0.048
0.234±0.053
0.320±0.070
0.229±0.041
0.309±0.067
0.257±0.044
0.0
0.0
0.0
0.0
0.161±0.018 c
0.271±0.022 d
0.049±0.052
0.207±0.055
0.190±0.034
0.254±0.068
0.292±0.054
0.268±0.049
0.0
0.0
0.0
0.0
0.135±0.012 c
0.227±0.015 d
*Non-necrogenic satRNAs not detected by dot-blot hybridization.
DMean±SEM of all necrogenic, all non-necrogenic or all nonnecrogenic* genotypes. The same letter indicates that there are no
significant differences at the 95 % confidence interval in satRNA
accumulation by a Wilcoxon test.
To estimate the effect of satRNA on CMV accumulation,
virus particles were purified from systemically infected
leaves of five plants for each single-infection treatment in
the experiment before. Data are presented in Table 2, and
show that CMV accumulation was significantly different in
the absence of satRNA and in the presence of either
necrogenic or non-necrogenic satRNAs (H3,21511.67,
P50.008). The presence of satRNAs resulted in a significant decrease in CMV particle accumulation, which
was more severe for necrogenic satRNAs (W10,6527.0,
P50.004). Thus, the accumulation of CMV particles is
decreased in the presence of satRNA, the decrease being
more severe for necrogenic satRNAs. Interestingly, in plants
in which Fny-CMV had been inoculated with those nonnecrogenic satRNAs that were not detected in systemically
1932
infected leaves (non-necrogenic* satRNAs), a decrease
in the accumulation of CMV particles was observed
that was similar to that associated with the presence
of multiplying non-necrogenic satRNAs (W6,4522.0,
P50.749).
Efficiency of CMV-satRNA encapsidation and
transmission to CMV progeny
RNA was extracted from Fny-CMV particles that were
purified for each single-infection treatment, and the
relative proportion of satRNA in particles was quantified
by densitometry after electrophoresis in 1.2 % agarose
gels and ethidium bromide staining. Only those nonnecrogenic satRNAs that were detected in nucleic acid
extracts of systemically infected leaves were included in this
analysis, as non-necrogenic* satRNA genotypes were not
Table 2. Effect of CMV-satRNA in the accumulation of
Fny-CMV particles in melon leaves
Data are micrograms of particles per gram of fresh leaves in virion
preparations from systemically infected leaves (mean±SEM of five plants).
satRNA genotype
None
Necrogenic
89/15.1
89/24.1
89/42.4
90/8.2
90/19.1
90/22.1
91/3.1
91/3.2
91/5.1
94/32.1
All necrogenicD
Non-necrogenic
89/20.1
90/14.1
90/16.1
90/19.2
91/2.2
92/4.1
All non-necrogenicD
Non-necrogenic*
89/15.2*
89/23.1*
90/17.1*
92/10.1*
All non-necrogenic*D
mg satRNA (g fresh leaf)”1
956.558±39.31 a
317.94±83.87
33.44±9.26
92.84±54.08
148.35±30.86
427.96±33.42
491.47±138.57
263.88±38.24
104.97±26.07
426.22±152.45
181.37±28.82
248.52±31.55 c
703.88±120.88
402.80±69.79
503.58±67.44
699.23±107.05
602.36±36.60
634.76±71.46
593.17±41.47 b
658.80±57.01
337.69±107.30
787.87±118.69
338.54±129.57
485.34±66.08 b
*Non-necrogenic satRNAs not detected by dot-blot hybridization.
DMean±SEM of all necrogenic, all non-necrogenic or all nonnecrogenic* genotypes. The same letter indicates that there are no
significant differences at the 95 % confidence interval in CMV
accumulation by a Wilcoxon test.
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Journal of General Virology 92
Fitness differences among CMV-satRNA in melon plants
detected in virus particles (not shown). The fraction of
encapsidated RNA represented by satRNA did not differ
significantly for necrogenic (20.96±0.68) and for nonnecrogenic (19.22±1.11) satRNAs variants (Z50,30526.90,
P50.490). However, the presence of necrogenic and nonnecrogenic satRNAs affected the amount of CMV RNAs
encapsidated differently. The fraction of encapsidated CMV
RNAs 1+2 (31.18±0.26 in the absence of satRNA) was
significantly smaller in the presence of necrogenic satRNAs
(23.46±0.69) than in the presence of non-necrogenic
satRNAs (26.06±0.67) (W10,6525.0, P50.008). The fraction of encapsidated RNA made by CMV RNA3 and RNA4
(35.90±0.18 and 32.70±0.20, respectively, in the absence of
satRNA) was reduced similarly by necrogenic (27.74±0.36
and 20.69±0.32 for RNA 3 and RNA 4, respectively) and
non-necrogenic (25.61±0.60 and 19.45±0.61 for RNA 3
and RNA 4, respectively) satRNAs (P.0.237).
To analyse whether the different efficiencies of encapsidation of necrogenic and non-necrogenic satRNAs would
affect the probability of their transmission to CMV
progeny, the above preparations of virus particles were
used to inoculate fully expanded leaves of Chenopodium
quinoa. The presence of satRNA in the resulting necrotic
local lesions (nll) was assessed by molecular hybridization
of nll prints in nylon membranes. At least 20 nll were
analysed for each satRNA genotype. The data show (Table
3) that the fraction of nll in which satRNA was detected
was significantly smaller for the necrogenic satRNAs
(x25139.47, P,0.0001). Thus, even though necrogenic
satRNAs are as efficiently encapsidated as non-necrogenic
ones they infect CMV progeny less efficiently.
Effect of CMV-satRNA on aphid transmission of
Fny-CMV
To test the effect of satRNA on CMV aphid transmission,
three necrogenic and three non-necrogenic satRNAs were
randomly chosen from among those that were detected in
systemically infected melon leaves, i.e. non-necrogenic*
satRNAs were not considered. Melon plants were inoculated and systemically infected leaves from two melon
plants infected by each satRNA or from four plants infected
only with Fny-CMV were used as source leaves for
acquisition by Aphis gossypii. In all cases, transmissions
were done with three aphids per test plant. Fifteen days
later, test plants were assayed for CMV and satRNA
infection.
Table 4 shows that the presence of both necrogenic and
non-necrogenic satRNAs resulted in a significant reduction
of Fny-CMV transmission (x2516.31, P50.002), by
approximately 37 and 29 % for necrogenic and nonnecrogenic satRNAs, respectively. The efficiency of CMV
transmission from plants infected by Fny-CMV, or by FnyCMV plus necrogenic and non-necrogenic satRNAs
paralleled differences in CMV particle accumulation that,
as in the previous experiment, was highest for Fny-CMV
and lowest for Fny-CMV+necrogenic satRNAs (Table 4).
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Table 3. Frequency of transmission of CMV-satRNA to
Fny-CMV progeny
satRNA genotype
Necrogenic
89/15.1
89/24.1
89/42.4
90/8.2
90/19.1
90/2.1
91/3.1
91/3.2
91/5.1
94/32.1
All necrogenic
Non-necrogenic
89/20.1
90/14.1
90/16.1
90/19.2
91/2.2
92/4.1
89/15.2*
89/23.1*
90/17.1*
92/10.1*
All non-necrogenic
Excluding non-necrogenic*
Frequency of satRNA-infected nllD
18.2
8.3
21.7
4.5
72.7
37.5
41.7
45.8
70.8
4.2
32.6±7.6
37.5
25.0
87.0
50.0
5.0
58.3
0.0
0.0
0.0
0.0
26.3±9.7
43.8±11.5
*Non-necrogenic satRNAs not detected by dot-blot hybridization.
D¢20 necrotic local lesions (nll) analysed per genotype.
However, differences in CMV transmission efficiency from
plants infected with either necrogenic or non-necrogenic
satRNAs were not significant (x2531.30, P50.537).
The frequency of transmission of necrogenic and nonnecrogenic satRNAs was also similar, of 86.8, and 75.6 %,
respectively, (x251.85, P50.764).
Effect of CMV and CMV-satRNA infection on host
plant growth and survival
The effect of infection by Fny-CMV, or by FnyCMV+satRNA on plant growth was estimated by
comparing the above-ground biomass of all infected and
mock-inoculated plants from the experiment in which
CMV and CMV-satRNA accumulation was quantified at
25 days p.i.
The biomass of infected plants was always smaller than the
biomass of mock-inoculated plants (H5,430526.96,
P50.0001), but biomass was smaller for plants infected
by CMV+satRNA than for plants infected only by FnyCMV (P,0.05) (Table 5). The effect of infection by
necrogenic and non-necrogenic satRNAs on plant biomass
was similar (W10,6525.0, P50.625). Interestingly, inoculation with non-necrogenic* satRNAs had a significant
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M. Betancourt, A. Fraile and F. Garcı́a-Arenal
Table 4. Effect of CMV–satRNA on the transmissibility of Fny-CMV by A. gossypii
satRNA genotype
None
Necrogenic
89/15.1
89/24.1
90/19.1
All necrogenic§
Non-necrogenic
90/16.1
91/2.2
92/4.1
All non-necrogenic§
CMV accumulation*
CMV transmissionD
satRNA transmissiond
801.94±150.68 a
130/205 (63.4) a
–
108.96
50.16
167.76
108.96±36.34 c
34/ 90 (37.7)
46/126 (36.5)
41/ 88 (46.5)
121/304 (39.8) b
32/34 (94.1)
38/46 (82.6)
35/41 (85.4)
105/121 (86.8) a
428.15
306.36
373.55
368.51±31.04 b
35/ 81 (43.2)
32/ 78 (41.0)
19/ 33 (57.5)
86/192 (44.79)b
25/35
26/32
14/19
65/86
(71.4)
(81.3)
(73.7)
(75.6) a
*Data are micrograms of particles per gram of fresh leaves in virion preparations from systemically infected leaves averaged (mean) for all source
leaves.
DData are (no. of Fny-CMV-infected plants)/(total inoculated plants). Percentages are shown in parentheses.
dData are (no. of satRNA-infected plants)/(total Fny-CMV-infected plants). Percentages are shown in parentheses.
§Mean±SEM for source leaves infected with either any necrogenic or any non-necrogenic satRNAs. The same letter indicates that there are no
significant differences at the 95 % confidence interval in satRNA accumulation in a Wilcoxon test (second column) or a x2 test (third and fourth
columns).
effect on plant biomass, as the biomass of plants inoculated
with Fny-CMV+non-necrogenic* satRNAs were not
significantly different from the biomass of plants infected
by necrogenic satRNAs or by those non-necrogenic satRNA
that efficiently multiplied in systemically infected leaves
(P.0.240). Thus, biomass reduction by satRNA was the
same for single infections of necrogenic or non-necrogenic
satRNAs. It was also the same for mixed infections of
necrogenic and non-necrogenic satRNAs (data not shown).
The reduction of plant growth would have a direct
relationship with CMV and CMV-satRNA fitness if it
affected plant or leaf survival, i.e. the duration of the
infectious period. To test this hypothesis, an experiment
was carried out in which melon plants were inoculated
with Fny-CMV or with Fny-CMV and each of five
randomly chosen necrogenic or non-necrogenic satRNAs.
Non-necrogenic satRNAs were chosen from among those
that were detected in systemically infected melon leaves.
Each of the 11 infection treatments, plus the mockinoculated control, included five plant repetitions. Plants
were grown for 75 days p.i. and during this period leaves
were evaluated daily for senescence, and leaf survival (LS)
was rated as the number of days elapsed since the leaf was
1 cm long at the primary vein until total senescence (i.e.
leaf abscission). The biomass of all senescent leaves per
plant (senesced biomass, SB) was also determined, and the
regression between LS for each leaf and SB of all the older
leaves was analysed for each plant. During the 75 days p.i.
of the experiment a mean of ten leaves per plant underwent
total senescence. A positive correlation between LS and the
square root of SB was significant from the fourth leaf on,
and the significance was highest for the tenth leaf. For the
tenth leaf, the regression of LS against the square root of SB
1934
was not significantly different for plants infected by
Fny-CMV or Fny-CMV and either necrogenic or nonnecrogenic satRNAs (data not shown), but was significantly different in slope for mock-inoculated (LS5
210.21+25.75!SB, r50.68, p50.29) and for all infected
(LS58.23+14.45!SB, r50.48, p50.0001) plants (F1,6154.31,
P50.043). Thus, the lifespan of plants depended on their
biomass, and the higher the reduction of plant growth due to
infection, the shorter the lifespan of the plants and, hence, the
infectious period.
DISCUSSION
CMV-satRNAs can be classified into two groups, necrogenic and non-necrogenic, according to the symptoms
that they induce in tomato plants (Roossinck et al., 1992).
These two types of satRNA also differ broadly in their
fitness in this host (Escriu et al., 2000a, Escriu et al., 2003).
The aim of this study was to analyse whether satRNAs that
are necrogenic or non-necrogenic for tomato plants also
present a different phenotype in host plants in which they
do not differ in terms of modulation of CMV symptoms.
To exemplify these hosts, which are the vast majority of
CMV hosts (Roossinck et al., 1992), melon was chosen.
satRNAs necrogenic or non-necrogenic for tomato do not
differ terms of CMV symptom modulation in melon
(Collmer & Howell, 1992; Roossinck et al., 1992), and
melon, as other cucurbits, has been described as a poor
host of satRNA, which multiplies to much lower levels than
in solanaceous plants such as tomato (Kaper & Tousignant,
1977; Jacquemond & Leroux, 1982; Moriones et al., 1991;
Garcı́a-Arenal & Palukaitis, 1999). Also, melon is an
important host of CMV in horticultural areas in which
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Journal of General Virology 92
Fitness differences among CMV-satRNA in melon plants
Table 5. Effect of infection by CMV and CMV+CMV-satRNA
on the biomass of melon plants
Data are mean±SEM for 20 mock-inoculated plants or for ten plants
infected by Fny-CMV or Fny-CMV and each different satRNA genotype.
Treatment
Dry weight (mg)
Mock-inoculated
Fny-CMV
Fny-CMV plus:
Necrogenic satRNA
89/15.1
89/24.1
89/42.4
90/8.2
90/19.1
90/22.1
91/3.1
91/3.2
91/5.1
94/32.1
All necrogenicD
Non-necrogenic satRNAs
89/20.1
90/14.1
90/16.1
90/19.2
91/2.2
92/4.1
All non-necrogenicD
Non-necrogenic* satRNAs
89/15.2*
89/23.1*
90/17.1*
92/10.1*
All non-necrogenic*D
1540.3±57.56 a
1057.8±100.60 b
453.54±71.95
504.36±39.90
528.45±95.46
530.58±41.38
457.00±77.73
861.30±107.24
840.18±143.65
1192.44±163.49
817.02±98.73
657.99±145.72
686.84±39.81 c
736.02±102.90
465.31±56.71
968.70±145.95
645.48±85.48
398.84±67.21
518.18±84.34
621.69±44.97 c
1086.80±138.96
493.80±107.37
918.59±138.29
783.06±73.21
810.97±66.70 c
*Non-necrogenic satRNAs not detected by dot-blot hybridization.
DMean±SEM of all necrogenic, all non-necrogenic or all nonnecrogenic* genotypes. The same letter indicates that there are no
significant differences at the 95 % confidence interval in satRNA
accumulation in a Wilcoxon test.
tomato is grown (Alonso-Prados et al., 2003), and hence
CMV+satRNA infections in melon could have a role on
the evolution of CMV and satRNA populations leading to
epidemics of lethal necrosis in tomato crops. Thus, for
necrogenic and non-necrogenic satRNAs, we analysed the
following factors in melon plants that may determine the
within- and between-host components of the fitness of a
parasite: within-host multiplication, competitive ability in
mixed-infected hosts, effect on the within-host multiplication of the helper virus CMV, efficiency of encapsidation
and of transmission to CMV progeny, efficiency of
between-host transmission by aphid vectors of the helper
virus and the satRNA, and virulence. Infectivity was not
analysed in this work as we had previously shown that the
set of necrogenic and non-necrogenic satRNA genotypes
http://vir.sgmjournals.org
analysed here did not differ for this trait (Escriu et al.,
2000a). All experiments used Fny-CMV as the helper virus,
as we had also previously shown that the interaction of the
assayed satRNAs with CMV did not depend on the CMV
genotype for ten assayed Subgroup I field isolates (Escriu
et al., 2000a).
Our results confirmed previous reports, in that necrogenic
and non-necrogenic satRNAs did not differ in the
modulation of CMV symptoms in melon plants. In our
greenhouse conditions, all plants infected by Fny-CMV, by
themselves or with any of 20 assayed satRNA genotypes,
showed strong mosaic and leaf lamina deformation (not
shown). However, despite this similarity of symptoms,
necrogenic and non-necrogenic satRNAs differed in melon
in terms of several important traits. All assayed satRNA
genotypes multiplied in melon to quite low levels, in
agreement with previous reports (Jacquemond & Leroux,
1982; Palukaitis, 1988; Roossinck & Palukaitis, 1995), their
accumulation in infected leaves being approximately 100fold lower than in tomato for the same set of CMV-satRNA
genotypes (Escriu et al., 2000a). However, multiplication of
necrogenic satRNAs was more efficient than multiplication
of non-necrogenic satRNAs, and four of ten nonnecrogenic satRNAs assayed were not detected in systemically infected leaves of most infected melon plants, or after
amplification in tobacco plants inoculated with those
leaves (not shown), indicating a host-associated differential
selection of satRNA genotypes, as reported for other host
species (Kurath & Palukaitis, 1990; Moriones et al., 1991;
Roossinck & Palukaitis, 1991). Necrogenic satRNAs also
multiplied to higher levels in mixed infections with nonnecrogenic satRNAs, although levels were lower than in
single infections, indicating effective competition by nonnecrogenic satRNAs. Thus, necrogenic satRNAs were at an
advantage over non-necrogenic ones for traits relative to
the within-host fitness.
This advantage was countered by factors determining
the efficiency of between-host transmission: necrogenic
satRNAs caused a larger depression of the multiplication of
CMV than non-necrogenic ones, which resulted in lower
rates of CMV aphid transmission, which is positively
correlated with the accumulation of CMV particles in the
source leaf (Escriu et al., 2000b; this work). Both necrogenic and non-necrogenic satRNAs increased the virulence
of CMV, estimated as the effect of infection on plant
growth and survival. A shorter life span would result in a
shorter infectious period for CMV and, hence, for satRNA
transmission from satRNA-infected plants. Interestingly,
inoculation with those non-necrogenic satRNAs that were
not detected in systemically infected leaves (non-necrogenic* satRNAs) resulted in a decrease of Fny-CMV
accumulation and an increase in virulence. These results
could be explained if non-necrogenic* satRNAs multiplied
very inefficiently in inoculated melon leaves at the earliest
stages of infection, and if the effects on CMV multiplication and virulence would require very low levels of satRNA
and/or depend strongly on early stages of infection. The
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1935
M. Betancourt, A. Fraile and F. Garcı́a-Arenal
decrease in plant growth upon infection is a common
estimate of virulence in plant pathology (Sacristán &
Garcı́a-Arenal, 2008). An important result from this work
is that there is a positive correlation between plant biomass
and survival, indicating that a decrease of plant growth is
indeed a good correlate for increased mortality, which is
the most usual form to express virulence in models of
virulence evolution (Day, 2002).
The results of the encapsidation and transmission experiments are apparently not consistent and lead to some
interesting questions relating to the present lack of
knowledge about the distribution of satRNA among
CMV particles and its possible effect on particle stability.
These little-understood aspects of satRNA biology have not
been addressed recently (Lot & Kaper, 1976; Palukaitis et
al., 1992; Roossinck et al., 1992; Palukaitis & Roossinck,
1996). First, the rate of satRNA transmission to CMV
progeny was higher for necrogenic satRNAs than for those
non-necrogenic-satRNAs that multiplied efficiently, in
spite of a similar rate of encapsidation. This unexpected
result could be explained by the small but significant
difference in interference between both types of satRNAs
and the encapsidation of CMV genomic RNAs 1 and 2.
This difference indicates a differential distribution of
necrogenic and non-necrogenic satRNAs over the three
particles of CMV, encapsidating RNA1, RNA2, and RNAs 3
and 4 together, which are necessary to start an infection. A
differential distribution of satRNAs in CMV particles could
result in a different probability of a satRNA genotype being
present in an infectious particle set. Interference between
necrogenic and non-necrogenic satRNAs with the encapsidation of CMV genomic RNAs was different in tomato
(Escriu et al., 2000a) and melon plants, suggesting that the
host-plant environment has an effect on satRNA encapsidation. Second, differences in the rate of transmission to
the CMV progeny did not result in differences in the rate of
aphid transmission of necrogenic and non-necrogenic
satRNAs. Again, this result is difficult to explain as it has
been reported that aphid transmission of CMV involves
severe population bottlenecks (Ali et al., 2006), and the
effective number of founders starting a CMV infection by
aphid transmission is very low, approximately 1–2
(Betancourt et al., 2008). This value is similar to that for
initiating a necrotic local lesion by mechanical inoculation
(Garcı́a-Arenal & Fraile, 2011). We could speculate on a
differential stability of CMV particles containing satRNA in
inoculation buffer and in the aphid’s stylet to explain this
result.
In summary, we show here that CMV-satRNAs that are
necrogenic and non-necrogenic for tomato exhibit important fitness differences in melon plants. While necrogenic
satRNAs show a higher fitness within the infected host than
non-necrogenic ones, their higher negative effect on CMV
multiplication, i.e. the effect of the higher virulence of the
satRNA parasite on its CMV host, will affect between-host
transmission of necrogenic satRNAs more severely than that
of non-necrogenic satRNAs. Similar virulence in the CMV
1936
melon host will not differentially affect between-host
transmission of both types of satRNA. Thus, there is a
trade-off between the within- and between-host components
of CMV-satRNA fitness. Except for virulence, the estimates
of fitness components rank CMV, CMV+necrogenic
satRNAs and CMV+non-necrogenic satRNAs similarly in
melon and in tomato plants (Escriu et al.; 2003, this work).
As in tomato, necrogenic satRNAs will be favoured in melon
populations. Melon could act as a reservoir of necrogenic and
some non-necrogenic satRNAs, but as a sink for those nonnecrogenic satRNAs that were not recovered from systemically infected leaves. More significantly, our results show
that the evolutionary dynamics of satRNAs necrogenic and
non-necrogenic for tomato may differ in hosts in which these
two types of satRNA do not cause different symptoms.
Hence, analyses of the emergence of necrogenic satRNAs in
CMV populations leading to devastating epidemics (Escriu
et al., 2000a, 2003) will be incomplete without considering
other, apparently non-differential hosts of CMV, as is the
case for melon.
METHODS
CMV and CMV-satRNA isolates. Fny-CMV and 20 satRNA
genotypes sampled in the field in eastern Spain, ten of them
necrogenic (89/15.1, 89/24.1, 89/42.4, 90/8.2, 90/19.1, 90/22.1, 91/3.1,
91/3.2, 91/5.1 and 94/32.1) and ten non-necrogenic (89/15.2, 89/20.1,
89/23.1, 90/14.1, 90/16.1, 90/17.1, 90/19.2, 91/2.2, 92/4.1 and 92/10.1)
for tomato, were derived from transcripts of biologically active cDNA
clones (Rizzo & Palukaitis, 1990; Escriu et al., 2000a) by transcription
with T7 RNA polymerase (New England Biolabs). RNA transcripts of
Fny-CMV genomic RNAs, or of Fny-CMV and each of the 20 satRNA
genotypes, were used to inoculate Nicotiana clevelandii A. Gray plants
for virus multiplication. CMV virions were purified from infected
leaves as described by Lot et al. (1972), and virion RNA was extracted
by disruption with phenol and SDS.
Biological assays. Melon plants (Cucumis melo L. var. saccharinus
Naud. ‘Piel de Sapo’) were inoculated with Fny-CMV RNA with or
without satRNA. Fny-CMV was inoculated at 100 mg ml21 of RNA in
0.1 M Na2HPO4; for RNA preparations of Fny-CMV+CMV-satRNA
the fraction of CMV-satRNA was estimated after agarose gel
electrophoresis and ethidium bromide-staining. Plants were inoculated with 20 mg satRNA ml21 in 0.1 M Na2HPO4, regardless of FnyCMV RNA concentration, which in all cases exceeded 100 mg ml21.
Ten microlitres of inoculum was rubbed onto the totally expanded
cotyledons of melon plants and the plants were maintained in a
greenhouse at 20–25 uC with 16/8 h light/dark cycle.
Quantification of CMV RNA and CMV-satRNA accumulation in
infected plants. Accumulation of CMV or satRNA was quantified as
micrograms of viral or CMV-satRNA per gram of fresh tissue by dotblot hybridization as previously described (Escriu et al., 2000a). Total
nucleic acid extracts from systemically infected leaves harvested
25 days p.i. were spotted onto nylon membranes and hybridized with
32
P-labelled oligonucleotide probes. Probes were complementary to nt
1933–2215 of Fny-CMV RNA3 (GenBank accession no. D10538),
which hybridizes with the 39-UTR of the three genomic, and
subgenomic, RNAs of Fny-CMV (Palukaitis et al., 1992), or
complementary to nt 250–260 of satRNA variants necrogenic (59GCGTCATGACTCATA-39) or non-necrogenic (59-CGTCATCCACGATAC-39) for tomato (sequences specific for each group of satellites
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Journal of General Virology 92
Fitness differences among CMV-satRNA in melon plants
are underlined; Sleat et al., 1994). In each blot, internal standards of
satRNA were included as a twofold dilution series of purified RNA
(2–0.015 mg) in nucleic acid extracts from non-inoculated melon
plants. Different amounts of nucleic acid extracts from each sample to
be analysed were blotted to ensure that the hybridization signal was in
the linear portion of the RNA concentration–hybridization signal
curve. RNA hybridization was detected using a Typhoon 9400 scanner
(GE Healthcare) after exposure of the Eu+2 store phosphor screens to
the labelled samples, and quantified using ImageQuant version 5.2
(Molecular Dynamics). Hybridizations were done at 65 uC (for CMV
RNA probe) or 40 uC (for satRNA probes) overnight in 66 SSC (16
SSC is 0.15 M NaCl, 0.015 M sodium citrate) 56 Denhardt’s mix,
0.1 % SDS and 250 mg ml21 yeast tRNA. For RNA-loading controls,
dot-blot membranes were rehybridized at 65 uC with a cDNA probe
(800 nt) complementary to barley 18S rRNA (Gerlach & Bedbrook,
1979).
Quantification of Fny-CMV particles and encapsidated RNA in
infected plants. CMV was also quantified as micrograms of virus
particles per gram of fresh tissue. Virus particles were purified from
systemically infected leaves 25 days p.i. as described by Lot et al. (1972),
except that only one cycle of differential centrifugation was made. Pellets
were resuspended in 400 ml of 0.5 M sodium citrate (pH 7.0) per gram
of tissue. The amount of CMV coat protein in these preparations was
quantified by double-antibody sandwich ELISA using a polyclonal
antibody raised against Fny-CMV (Thompson & Garcı́a-Arenal, 1998).
A 1.2-fold dilution series of known amounts of Fny-CMV particles
(5 mg–19 ng) were included as internal standards in each ELISA plate.
control plants was determined after maintenance of plants at 65 uC
until constant weight. Leaf survival, expressed in days, was the time
from the leaf being 1 cm long over the primary vein until total
senescence, identified by leaf abscission.
Statistical analyses. All statistical analyses were as described in
Sokal & Rohlf (1995) and were performed using SPSS version 13.0.
None of the variables analysed in this work was normally distributed
or showed homogeneity of variances (Hartley FMax test), hence, nonparametric tests were used for analyses. Data of CMV and satRNA
accumulation were analysed using Bonferroni-corrected Kruskal–
Wallis (H) tests. Differences between treatments were determined by
using a Wilcoxon (W) test with a normal-distribution correction (Z)
when the number of samples was ¢20. The subscripted figures
associated with the statistical results are the degrees of freedom and
the value of n, respectively. Transmission frequencies were compared
by using a x2 test on contingency tables. Regressions of plant
senescence versus biomass, were compared by analysis of covariance.
Relation parameters were calculated by linear regression analysis.
ACKNOWLEDGEMENTS
This work was in part funded by the Ministerio de Ciencia e
Innovación, Spain, grant AGL2008-02458, to F. G.-A. M. B. was under
a commission for studies from Universidad Rosa de Cabal, Colombia.
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