Genetic bottlenecks facilitate the fixation and extinction of variants in populations, and viral populations are no exception to this theory. To examine the existence of genetic bottlenecks in cell-to-cell movement of plant RNA viruses, we prepared constructs for Soil -b orne wheat mosaic virus RNA2 vectors carrying two different fluorescent proteins, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). Coinoculation of host plant leaves with the two RNA2 vectors and the wild-type RNA1 showed separation of the two vector RNA2s, mostly within seven to nine cell-to-cell movements from individual initially coinfected cells. Our statistical analysis showed that the number of viral RNA genomes establishing infection in adjacent cells after the first cell-to-cell movement from an initially infected cell was 5.97 ± 0.22 on average and 5.02 ± 0.29 after the second cell-to-cell movement. These results indicate that plant RNA viruses may generally face narrow genetic bottlenecks in every cell-to-cell movement. Furthermore, our model suggests that, rather than suffering from fitness losses caused by the bottlenecks, the plant RNA viruses are utilizing the repeated genetic bottlenecks as an essential element of rapid selection of their adaptive variants in trans -acting genes or elements to respond to host shifting and changes in the growth conditions of the hosts.

Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1–20 genomes). This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus. The model showed that stochastic processes that govern the replication or degradation of individual genomes result in the infection by a small number of genomes, while a large number of infectious genomes are introduced in the cell. It also predicted two interesting characteristics regarding cell infection patterns: stochastic variation among cells in the number of viral genomes that establish infection and stochastic inequality in the accumulation of their progenies in each cell. Both characteristics were validated experimentally by inoculating tobacco cells with a library of nucleotide sequence–tagged ToMV and analyzing the viral genomes that accumulated in each cell using a high-throughput sequencer. An additional simulation model revealed that these two characteristics enhance selection during tissue infection. The cell infection model also predicted a mechanism that enhances selection at the cellular level: a small difference in the replication abilities of coinfected variants results in a large difference in individual accumulation via the multiple-round formation of the replication complex (i.e., the replication machinery). Importantly, this predicted effect was observed in vivo. The cell infection model was robust to changes in the parameter values, suggesting that other viruses could adopt similar adaptation mechanisms. Taken together, these data reveal a comprehensive picture of viral infection processes including replication, cell-to-cell transmission, and evolution, which are based on the stochastic behavior of the viral genome molecules in each cell. Viruses rapidly adapt to environmental changes, complicating our efforts to control the spread of viral infections. Adaptation occurs in two steps: the generation of adaptive variants by random mutations and the subsequent selection of those adaptive variants. High mutation rates in viruses have been well documented; however, little is known about how adaptive variants are rapidly selected, especially given that most viral gene products are shared among viral populations within an infected cell, thereby diluting the direct benefits of each variant. Here we used an RNA virus—tomato mosaic virus—to examine the selection mechanisms hidden in its infection processes. First, we simulated the cell infection process computationally. We found that stochastic behaviors of viral genome molecules result in characteristic patterns of offspring accumulation within cells: (i) predominant replication of offspring from only a handful of founder viral genome molecules in each cell, (ii) variation in the number of offspring originating from each founder, and (iii) variation in the number of founders among cells. We then found that these characteristics enable rapid selection of adaptive variants of the virus during parallel and repeated cell infections, i.e., tissue infections. We speculate that similar selection mechanisms could also be employed by other viruses.

Significance: Replication of many positive-strand RNA viruses is cis -preferential: i.e., viral replicase proteins replicate genomic RNA molecules that have served as translation templates for their own synthesis, but not the other molecules in the same cell. Here, we show that tobacco mosaic virus replicase cotranslationally binds the 5′ untranslated region of genomic RNA and that this binding inhibits further translation and leads to genomic RNA replication. Intriguingly, full-length replicase protein could not bind genomic RNA posttranslationally due to autoinhibition by the C-terminal domain. These results reveal an elegant viral strategy to enable cis -preferential replication and phase switching from translation to replication at once.

During antagonistic coevolution between viruses and their hosts, viruses have a major advantage by evolving more rapidly. Nevertheless, viruses and their hosts coexist and have coevolved, although the processes remain largely unknown. We previously identified Tm-1 that confers resistance to Tomato mosaic virus (ToMV), and revealed that it encodes a protein that binds ToMV replication proteins and inhibits RNA replication. Tm-1 was introgressed from a wild tomato species Solanum habrochaites into the cultivated tomato species Solanum lycopersicum. In this study, we analyzed Tm-1 alleles in S. habrochaites. Although most part of this gene was under purifying selection, a cluster of nonsynonymous substitutions in a small region important for inhibitory activity was identified, suggesting that the region is under positive selection. We then examined the resistance of S. habrochaites plants to ToMV. Approximately 60% of 149 individuals from 24 accessions were resistant to ToMV, while the others accumulated detectable levels of coat protein after inoculation. Unexpectedly, many S. habrochaites plants were observed in which even multiplication of the Tm-1-resistance-breaking ToMV mutant LT1 was inhibited. An amino acid change in the positively selected region of the Tm-1 protein was responsible for the inhibition of LT1 multiplication. This amino acid change allowed Tm-1 to bind LT1 replication proteins without losing the ability to bind replication proteins of wild-type ToMV. The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra. In the LT1-resistant S. habrochaites plants inoculated with LT1, mutant viruses emerged whose multiplication was not inhibited by the Tm-1 allele that confers resistance to LT1. However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1. Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1. Viruses rapidly evolve and adapt to their host organisms, and the evolutionary processes can be reproduced in the laboratory (experimental evolution). In contrast, cellular organisms (that can be viral hosts) evolve much more slowly than viruses, but the fact that they have antiviral systems suggests that viruses and their hosts have coevolved. To explore the coevolutionary histories of viruses and their hosts, we focused on Tm-1, a Solanum habrochaites gene that confers resistance to Tomato mosaic virus (ToMV). Based on analyses of the Tm-1 gene sequences in S. habrochaites, we demonstrated that a part of the gene has been under positive selection. Biochemical studies suggested that Tm-1 has evolved to strengthen its inhibitory activity rather than to diversify recognition spectra. In addition, experimental evolution analyses suggested that overcoming the Tm-1-mediated resistance by ToMV is associated with fitness costs. Based on these results, we discuss how ToMV and the plant resistance gene have coevolved.