Single-cell mutation rate of turnip crinkle virus (-)-strand replication intermediates.

Viruses with single-stranded, positive-sense (+) RNA genomes incur high numbers of errors during replication, thereby creating diversified genome populations from which new, better adapted viral variants can emerge. However, a definitive error rate is known for a relatively few (+) RNA plant viruses, due to challenges to account for perturbations caused by natural selection and/or experimental set-ups. To address these challenges, we developed a new approach that exclusively profiled errors in the (-)-strand replication intermediates of turnip crinkle virus (TCV), in singly infected cells. A series of controls and safeguards were devised to ensure errors inherent to the experimental process were accounted for. This approach permitted the estimation of a TCV error rate of 8.47 X 10-5 substitution per nucleotide site per cell infection. Importantly, the characteristic error distribution pattern among the 50 copies of 2,363-base-pair cDNA fragments predicted that nearly all TCV (-) strands were products of one replication cycle per cell. Furthermore, some of the errors probably elevated error frequencies by lowering the fidelity of TCV RNA-dependent RNA polymerase, and/or permitting occasional re-replication of progeny genomes. In summary, by profiling errors in TCV (-)-strand intermediates incurred during replication in single cells, this study provided strong support for a stamping machine mode of replication employed by a (+) RNA virus.

Intracellular bottlenecking permits no more than three tomato yellow leaf curl virus genomes to initiate replication in a single cell.

Viruses are constantly subject to natural selection to enrich beneficial mutations and weed out deleterious ones. However, it remains unresolved as to how the phenotypic gains or losses brought about by these mutations cause the viral genomes carrying the very mutations to become more or less numerous. Previous investigations by us and others suggest that viruses with plus strand (+) RNA genomes may compel such selection by bottlenecking the replicating genome copies in each cell to low single digits. Nevertheless, it is unclear if similarly stringent reproductive bottlenecks also occur in cells invaded by DNA viruses. Here we investigated whether tomato yellow leaf curl virus (TYLCV), a small virus with a single-stranded DNA genome, underwent population bottlenecking in cells of its host plants. We engineered a TYLCV genome to produce two replicons that express green fluorescent protein and mCherry, respectively, in a replication-dependent manner. We found that among the cells entered by both replicons, less than 65% replicated both, whereas at least 35% replicated either of them alone. Further probability computation concluded that replication in an average cell was unlikely to have been initiated with more than three replicon genome copies. Furthermore, sequential inoculations unveiled strong mutual exclusions of these two replicons at the intracellular level. In conclusion, the intracellular population of the small DNA virus TYLCV is actively bottlenecked, and such bottlenecking may be a virus-encoded, evolutionarily conserved trait that assures timely selection of new mutations emerging through error-prone replication.

Natural Selection, Intracellular Bottlenecks of Virus Populations, and Viral Superinfection Exclusion.

Natural selection acts on cellular organisms by ensuring the genes responsible for an advantageous phenotype consistently reap the phenotypic advantage. This is possible because reproductive cells of these organisms are almost always haploid, separating the beneficial gene from its rival allele at every generation. How natural selection acts on plus-strand RNA viruses is unclear because these viruses frequently load host cells with numerous genome copies and replicate thousands of progeny genomes in each cell. Recent studies suggest that these viruses encode the Bottleneck, Isolate, Amplify, Select (BIAS) mechanism that blocks all but a few viral genome copies from replication, thus creating the environment in which the bottleneck-escaping viral genome copies are isolated from each other, allowing natural selection to reward beneficial mutations and purge lethal errors. This BIAS mechanism also blocks the genomes of highly homologous superinfecting viruses, thus explaining cellular-level superinfection exclusion.

The Gustavus Gene Can Regulate the Fecundity of the Green Peach Aphid, Myzus persicae (Sulzer).

Myzus persicae (Sulzer), commonly known as the green peach aphid, is a notorious pest that causes substantial losses to a range of crops and can transmit several plant viruses, including potato virus Y (PVY). Chemical insecticides provide only partial control of this pest and their use is not environmentally sustainable. In recent years, many genes related to growth, development, and reproduction have been used as targets for pest control. These include Gustavus (Gus), a highly conserved gene that has been reported to play an essential part in the genesis of germline cells and, hence, in fecundity in the model insect Drosophila melanogaster. We hypothesized that the Gustavus (Gus) gene was a potential target that could be used to regulate the M. persicae population. In this study, we report the first investigation of an ortholog of Gus in M. persicae, designated MpGus, and describe its role in the fecundity of this insect. First, we identified the MpGus mRNA sequence in the M. persicae transcriptome database, verified its identity with reverse transcription-polymerase chain reaction (RT-PCR), and then evaluated the transcription levels of MpGus in M. persicae nymphs of different instars and tissues with real-time quantitative PCR (RT-qPCR). To investigate its role in regulating the fecundity of M. persicae, we used RNA interference (RNAi) to silence the expression of MpGus in adult insects; this resulted in a significant reduction in the number of embryos (50.6%, P < 0.01) and newborn nymphs (55.7%, P < 0.01) in the treated aphids compared with controls. Interestingly, MpGus was also significantly downregulated in aphids fed on tobacco plants that had been pre-infected with PVY N , concomitant with a significant reduction (34.1%, P < 0.01) in M. persicae fecundity. Collectively, these data highlight the important role of MpGus in regulating fecundity in M. persicae and indicate that MpGus is a promising RNAi target gene for control of this pest species.