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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 Downloaded from www.microbiologyresearch.org by 032359 G 2011 SGM IP: 88.99.165.207 On: Sat, 13 May 2017 16:10:09 Printed in Great Britain 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 13 May 2017 16:10:09 1931 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 13 May 2017 16:10:09 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). http://vir.sgmjournals.org 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 13 May 2017 16:10:09 1933 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 13 May 2017 16:10:09 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 13 May 2017 16:10:09 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 13 May 2017 16:10:09 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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