*For offprint requests: email: Chris_Kearney@baylor.edu, fax: (817)755-2969

Key words: Virus, plants, evolution, mutation, population

The potential is evident for recombination between plant mRNA and RNA viruses (Green and Allison, 1994). If recombination between field viruses and certain transgenes resulted in vigorous, competitive hybrid viruses, this could lead to the development of novel viruses which could have severe effects on natural and agricultural ecosystems. However, more than just the initial recombination event is needed to develop such a novel virus. The virus must possess enough evolutionary plasticity to allow for the step-wise development of a functional virus from the starting material of the initial recombinant, resulting in a novel virus which can become established in the parental viral population, usually without the help of favorable selective pressures. Thus, we chose to measure the ability of populations of tobacco mosaic virus (TMV) to accrue point mutations in order to estimate this evolutionary potential.

Many TMV variants have been reported in the literature, and the development of new variants is usually host dependent in some way. Some TMV strains are in association with specific hosts (Bald, 1960), while other TMV variants can be selected for by host shift (Zitter and Murakishi, 1969; Pelham et al., 1970) or even by a change in the host's growth pattern, specifically bolting of whole plants (Khan and Jones, 1989a) or shoot generation in tissue culture (Khan and Jones, 1989b). Many TMV variants may be present in a greenhouse culture (Jensen, 1936), even after local lesion "cloning" (Garcia-Arenal et al., 1984). However, these studies do not necessarily demonstrate high levels of mutability. These "new" strains may have come to dominate the virus culture due to the introduction of new selective pressures rather than developing de novo via mutation. This was in fact demonstrated by one of these studies (Khan and Jones, 1989a).

To measure populational mutability, we started out with a clonal TMV culture, represented by a single tobacco plant infected with a TMV genomic in vitro transcript. We presumed that this culture would be much closer to homogeneity than a field isolate. A long-term host shift experiment was started by transferring the initial tobacco culture to 7 different host species with 1 to 4 replicate plants representing each species, for a total of 22 separate populations passaged in parallel for 413-515 days. These hosts were greatly varied in terms of phylogeny and pathogenic response, including necrotic ringspots, localized, nonsymptomatic infection, and systemic mosaic. A random sampling of members of these viral populations was performed by RT-PCR cloning of a 3' portion of TMV from virion RNA. The individual bacterial clones were sequenced, with each clone representing an individual viral RNA sequence. Two segments were sequenced: 190 bases of the 3' end of the movement protein ORF (representing also a subgenomic promoter) or 200 bases of the 5' end of the coat protein ORF.

Mutations accumulated during the passaging period, but in different patterns according to the gene sequenced. The coat protein gene segment accrued approximately 6 mutations per TMV genome, while the movement protein gene segment accumulated 1 mutation per TMV genome by the end of the 413+ day passaging period. This can be expressed as mutation frequencies of 7.5 x 10^-5 and 1.5 x 10^-5 mutations per base per passage, respectively. The type of mutation seen also differed. The two mutations detected in the movement protein gene were both nonsilent, while 6 of the 7 mutations in the coat protein gene were silent.

No patterns were seen between mutations and host type. No one host accumulated an especially large number of mutations, so comparisons cannot be made between specific hosts. When the host species are divided into two groups, systemic or localizing host response, no difference in mutation frequency was seen. Specifically, 3.1 x 10^-5 mutations per base per passage were seen in systemic hosts, compared to 5.4 x 10^-5 for localizing hosts.

The mutation frequencies of both neutral genes and native genes have now been assayed for TMV. For a selectively neutral foreign sequence replicated in a TMV vector, the mutation frequency was found to be lower than the limit of the mutation assay, which was 10^-4 mutations per base per passage (Kearney et al., 1993). Thus, for the extreme case of presumably unrestrained genetic drift under nonselective conditions, TMV populations do not seem to intrinsically accumulate mutations beyond this frequency during host passaging. The figures of 7.5 x 10^-5 and 1.5 x 10^-5 mutations per base per passage in the present work fall within this limit. Unfortunately, one cannot compare the mutation frequencies of neutral sequences of the previous study and with those of the native TMV genes of this study, since the frequency of the former was too low to measure.

No host in this study generated an unusually high frequency of viral mutations compared to the other hosts. In addition, no differences were noticed between localizing and systemic hosts. This is in contrast to the many studies noting the "appearance" of new TMV strains in virus cultures after host shifts. These published instances of rapid evolution may have been due to the selection of pre-existing minority variants rather than to de novo generation of mutants. This, of course, has implications for the mechanisms of viral evolution in the field.

We must emphasize that these are preliminary data. Sequencing of further samples is now in progress in order to create a larger sample size. In addition, though the long-term passages in tobacco may serve as an adequate control in this study, we are also sequencing clones derived from the original tobacco population from which all the other 22 population were derived. The fact that a large evolutionary differential occurred between the movement and coat genes in terms of mutation frequency and the silent:nonsilent mutation ratio suggests that more than just PCR errors are being detected in the current data set.

ACKNOWLEDGEMENT

This study was funded by the Biotechnology Risk Assessment Research Grants Program (USDA-CSRS Grant No. 94-39210-0371).

REFERENCES

Bald JG. 1960. Forms of tobacco mosaic virus. Nature 188: 645-647.

Garcia-Arenal F, Palukaitis P, Zaitlin M. 1984. Strains and mutants of tobacco mosaic virus are both found in virus derived from single-lesion-passaged inoculum. Virology 132:131-137.

Greene AE, Allison RF. 1994. Recombination between viral RNA and transgenic plant transcripts. Science 263:1423-1425.

Jensen JH. 1936. Studies on the origin of yellow-mosaic viruses. Phytopathology 26:266-277.

Kearney CM, Donson J, Jones GE, Dawson WO. 1993. Low level of genetic drift in foreign sequences replicating in an RNA virus in plants. Virology 192:11-17.

Khan IA, Jones GE. 1989a. Selection for a specific tobacco mosaic virus variant during bolting of Nicotiana sylvestris. Can J Bot 67:88-94.

Khan IA, Jones GE. 1989b. Accumulation of necrotic lesion inducing variants in TMV-infected plantlets derived from leaf disks of Nicotiana sylvestris. Can J Bot 67:984-989.

Pelham J, Fletcher JT, Hawkins JH. 1970. The establishment of a new strain of tobacco mosaic virus resulting from the use of resistant varieties of tomato. Ann Appl Biol 65:293-297.

Zitter TA, Murakishi HH. 1969. Nature of increased virulence in tobacco mosaic virus after passage in resistant tomato plants. Phytopathology 59:1736-1739.