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Genetic variability of plant virus populations

Fernando García-Arenal, Aurora Fraile and José M. Malpica¹
Depto. de Biotecnología, E.T.S.I. Agrónomos, Universidad Politécnica de Madrid, 28040 Madrid, Spain, and
¹ Area de Protección Vegetal, CIT-INIA, 28040 Madrid, Spain.

Introduction
Plant viruses are the cause of important diseases of crop plants, which result in diminished production and may even compromise food supply in large areas. The only direct way of controlling virus-induced diseases of plants is by the use of genetic resistance, a strategy in which success can be compromised by the evolution in the virus populations of virulence on the used resistant factors. A vast majority of plant viruses have single-stranded RNA genomes, and since the late 1970s work with RNA viruses that infect bacteria and mammals has shown the large potential for variation of RNA genomes (Domingo and Holland, 1988). Hence the importance of analyzing the genetic variability and structure of virus populations under natural conditions. While there is a fairly large amount of data on the evolution of plant viruses under experimental conditions (Roossinck, 1997), work on natural populations is much scarcer (García-Arenal et al., 1999a). Here we present some of our past and current results on the genetic structure and variation of plant virus populations that may be of general interest to those interested in virus evolution.

Plant virus populations are not very diverse
Work at our laboratory with the tobamovirus pepper mild mottle virus (PMMV), infecting pepper crops, and tobacco mild green mosaic virus (TMGMV), infecting the wild plant Nicotiana glauca Grah. showed that their populations in South Eastern Spain were not very diverse but, rather, indicated high genetic conservation for these viruses (Rodríguez-Cerezo et al., 1989, 1991). Population diversity values were similar for both viruses (Table 1), in spite of the fact that PMMV infects an annual crop plant in which resistance genes are used for its control, and resistance-breaking strains occur in the population, while TMGMV infects a perennial wild plant. Moreover, the analysis of TMGMV populations infecting N. glauca in regions as distant as Spain, California or Australia showed that population diversity did not increase with the geographic scale of the sample. In all cases diversities were low, similar to those reported for bacteria, and much lower than for some animal viruses such as influenza A or foot and mouth disease viruses (Fraile et al., 1996). When the limited data available for other plant viruses are analyzed, it is apparent that genetic conservation is the rule for plant viruses, irrespective of the nature of their genome (RNA or DNA), the replication strategy or the life history (Table 1). Moreover, since complementation of deleterious variants may be very effective (Moreno et al., 1997) these figures are upper estimates.

Low genetic diversity might result from low mutation rates or/and small population size. No estimate for the mutation rate of a plant virus has been published. Our preliminary results on the estimation of the mutation rate of tobacco mosaic virus (TMV), a virus closely related to PMMV and TMGMV, show a mutation rate in the range of those reported for animal or bacterial RNA viruses (Drake et al., 1998). Thus, low mutation rates seem not to be the cause for low genetic diversity in plant virus populations.

Functional constraints to the variation of virus-encoded proteins
There are no estimates on the effective population size for a plant virus. There are data, though, on a second factor that may restrict genetic diversity, i.e. purifying selection associated to the maintenance of a functional RNA structure or to a functional encoded protein. Table 2 shows the value of the ratio of nucleotide diversity at non-synonymous and synonymous sites, an estimate of the degree of purifying selection, for several proteins encoded by plant RNA and DNA viruses. Ratios vary largely for different genes, irrespective of their function, but are in the range of those for moderately conserved eukaryotic proteins (García-Arenal et al., 1999). Thus, in spite of higher mutation rates, viral proteins are not more variable than those of their eukaryotic hosts. It could well be speculated that since viral proteins interact with those of their hosts for different functions, this results in limits to their variation.

Constraints to variation may be such that plant RNA viruses, as has been reported for some animal viruses (Domingo and Holland, 1997), might be near a mutational meltdown threshold. This is suggested by early experiments on the frequency of lethal mutants of TMV after treatment with nitrous acid (Gieber and Mundry, 1958). Mutational meltdown may also occur in nature and have an important role in virus evolution. This is what indicates our work with TMV and TMGMV infecting N. glauca plants in New South Wales (Fraile et al., 1997a). Both TMV and TMGMV were isolated from herbarium specimens of this host plant collected prior to 1950, but only TMGMV was isolated from more recent specimens. Gene sequences of these isolates showed that although the TMGMV population did not increase in genetic diversity with time, the TMV population did. It was also shown that TMGMV accumulation in N. glauca was the same in single infections and in mixed infections with TMV, while TMV attained only one tenth the concentration in plants doubly-infected with TMGMV than in single infected plants. Thus, the drop of TMV concentration in doubly infected plants may go below a threshold in which elimination of deleterious mutations became increasingly difficult, so that its population will progressively be dominated by less fit genomes, and will succumb.

Genetic exchange may also be limited in virus populations
Phylogenetic analyses show that genetic exchange by recombination or by reassortment of genomic segments may have an important role in virus evolution (Rossinck, 1997, García-Arenal et al., 1999a). Reassortment has also been invoked as a main process to avoid the accumulation of deleterious mutations, which could compensate in RNA viruses for the biological cost of segmented genomes (Chao, 1988). Data from experiments with different plant viruses with segmented genomes show that reassortment is not random, indicating selection for or against some genetic types (reviewed in García-Arenal et al., 1999). Our work with natural populations of cucumber mosaic virus (CMV) shows that genetic exchange is not favored for this virus (Fraile et al., 1997b; García-Arenal et al., 1999b). The analysis of about 300 field CMV isolates, representing 17 subpopulations in different locations and years shows that the population is largely built of two main genetic types, belonging to subgroup I of CMV strains. Isolates representing mixed infections between these two main types are 16% of total, what is a lower estimate of mixed infections in the field. Reassortants, and recombinants at RNA 3, between both genetic types are about 4% of the population, in both cases. In addition, the different reassortant and recombinant types appear just in only one subpopulation, and never become fixed in the CMV population. Thus, data indicate that reassortment is not a process of genetic exchange preferred to recombination for this virus with a segmented genome. Also, heterologous genetic combinations are not favored in the CMV population. The mechanisms underlying this fact are presently under analysis at our laboratory.

Conclusions
The limited available data on the genetic structure and variation of plant virus populations gives a picture of genetic stability, rather than diversity, for both RNA and DNA viruses. A main factor in low genetic diversity seems to be negative selection associated to the maintenance of the sequence of the encoded proteins. Selective constraints may also limit genetic exchange by reassortment or recombination. More data on these subjects and, particularly on the estimation of other basic parameters in virus evolution, such as mutation and recombination rates, population numbers or bottlenecks in virus epidemiology are needed.

References
Chao, L. 1988. Evolution of sex in RNA viruses. J.Theor.Biol. 133:99-112.

Domingo E., and J.J. Holland. 1997. RNA virus mutations and fitness for survival. Ann.Rev.Microbiol. 51: 151-178.

Domingo E., and J.J. Holland. 1988. High error rates, population equilibrium, and evolution of RNA replication systems. In: E.Domingo, J.J.Holland & P.Ahlquist (Eds.) "RNA genetics, Vol III", pp. 3-35. CRC Press, Boca Raton, FL.

Drake J.W., B. Charlesworth, B. Charlesworth, and J.F. Crow (1998). Rates of spontaneous mutation. Genetics 148:1667-1686.

Fraile A., J.M. Malpica, M.A. Aranda, E. Rodríguez-Cerezo, and F. García-Arenal. 1996. Genetic diversity in tobacco mild green mosaic tobamovirus infecting the wild plant Nicotiana gauca. Virology 223:148-155.

Fraile A., F. Escriu, M.A. Aranda, J.M. Malpica, J.M. Gibbs, and F. García-Arenal. 1997a. A century of tobamovirus evolution in an Australian population of Nicotiana glauca. J.Virol. 71:8316-8320.

Fraile A., J.L. Alonso-Prados, M.A. Aranda, J.J. Bernal, J.M. Malpica, and F. García-Arenal. 1997b. Genetic exchange by recombination or reassortment is infrequent in natural populations of a tripartite RNA plant virus. J.Virol. 71:934-940.

García-Arenal F., A. Fraile, J.M. Malpica (1999a). Genetic Variability and Evolution. In: C.L.Mandahar (ed.), "Molecular Biology of Plant Viruses", pp. 143-159, Kluwer Academic Publishers, Boston.

García-Arenal F., F. Escriu, M.A. Aranda, J.L. Alonso-Prados, J. M. Malpica, and A. Fraile.1999b. Molecular epidemiology of cucumber mosaic virus and its satellite RNA. Virus Res. (in press).

Gierer A., and K.W. Mundry. 1858. Production of mutants of tobacco mosaic virus by chemical alteration of its ribonucleic acid in vitro. Nature 182:1457-1458.

Moreno I., J.M. Malpica, E. Rodríguez-Cerezo, and F. García-Arenal. 1997. A mutation in tomato aspermy cucumovirus that abolishes cell-to-cell movement is maintained to high levels in the viral RNA population by complementation. J.Virol. 71:9157-9162.

Rodríguez-Cerezo E., A. Moya, and F. García-Arenal. 1989. Variability and evolution of the plant RNA virus pepper mild mottle virus. J.Virol 63: 2198-2203.

Rodríguez-Cerezo E., S.F. Elena, A. Moya, and F. García-Arenal. 1991. High genetic stability in natural populations of the plant RNA virus tobacco mild green mosaic virus. J.Mol.Evol. 32:328-332.

Roossinck, M.J. (1997). Mechanisms of plant virus evolution. Annu.Rev.Phytopathol. 35:191-209.

Table 1.- Intrapopulation nucleotide diversities in some plant viruses ^a)

^a) From García-Arenal et al (1999a)
^b) PMMV = Pepper mild mottle virus, TMGMV = Tobacco mild green mosiac virus, WSMV = wheat streak mosaic virus, CMV-satRNA = satellite RNA of cucumber mosiac virus, BCTV = Beet curly top virus, CLCuV = Cotton leaf curl virus.

Table 2.- Nucleotide diversities in some virus genes ^a) 

^a) From García-Arenal et al (1999a).
^b) TMGMV = tobacco mild green mosaic virus, CMV = Cucumber mosaic virus, PVA = Potato virus A, PVY = Potato virus Y, CLCuV= Cotton leaf curl virus, CaMV = casliflower mosaic virus.

Abstract - Presented at the Virus Evolution Workshop
Ardmore, OK
October 21 - 24th, 1999

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Dr. Marilyn Roossinck
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