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. 2010 Feb;84(4):1828-37.
doi: 10.1128/JVI.01890-09. Epub 2009 Dec 2.

Estimation of the size of genetic bottlenecks in cell-to-cell movement of soil-borne wheat mosaic virus and the possible role of the bottlenecks in speeding up selection of variations in trans-acting genes or elements

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Estimation of the size of genetic bottlenecks in cell-to-cell movement of soil-borne wheat mosaic virus and the possible role of the bottlenecks in speeding up selection of variations in trans-acting genes or elements

Shuhei Miyashita et al. J Virol. 2010 Feb.

Abstract

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-borne 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.

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Figures

FIG. 1.
FIG. 1.
Genome structure of SBWMV and cDNA constructs for fluorescent protein vector RNA2s used in this study. ORFs are indicated by rectangles. MT, methyltransferase domain of replicase; HEL, helicase domain of replicase; POL, RNA polymerase domain of replicase; MP, cell-to-cell movement protein; TLS, tRNA-like structure; CP, capsid protein; RT, readthrough region translated by readthrough of UGA stop codon for CP; p19, 19-kDa protein with RNA silencing suppressor activity.
FIG. 2.
FIG. 2.
Observation of the spatial separation between SBWMV vectors carrying different fluorescent proteins in local lesion host C. quinoa (rows A to D) and natural host wheat (row E) leaves. (A) An example of preparation of a merged image. The YFP image (first panel) and CFP image (second panel) were taken separately, and the CFP image was converted to magenta (third panel). Then, the YFP image and converted CFP image were merged (fourth panel). Rows B to D show merged images. (B) Infected sites at 22 hpi. The first and second panels show sites exclusively infected by the YFP vector and CFP vector, respectively. The third and fourth panels show coinfected sites, and the white arrows in the fourth panel indicate the cells showing exclusive infection by one of the two vectors. (C) Time course observation of a coinfected site at 22, 28, 48, and 72 hpi. (D) Coinfected sites at 72 hpi. (E) Coinfected sites in wheat at 48 hpi. All the bars represent 100 μm.
FIG. 3.
FIG. 3.
RT-PCR and PCR amplifications of RNA2 vector and control templates. (A) Primers used for RT-PCR and PCR amplification. (B) RT-PCR (left halves) and PCR (right halves) products of RNA or DNA templates using primer sets TP292 and TP41 (upper panel) and TP292 and TP290 (lower panel). Templates (lanes): 1, total RNA isolated at 28 hpi; 2, total RNA isolated at 48 hpi; 3, total RNA isolated at 72 hpi; 4, total RNA isolated from healthy plant; 5, inoculated RNA transcript; 6, template DNA used for transcription. M, λDNA/HindIII marker with 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kbp from the top. Arrowheads indicate the expected RT-PCR or PCR product sizes, 1,854 bp (upper panel, white arrowhead) and 1,070 bp (lower panel, black arrowhead).
FIG. 4.
FIG. 4.
Fundamental ideas and definitions in this work. (A) Schematic representation of classification of cells by distance from initially infected cell. (B) Settlement of founder genomes was regarded as stochastic event described by a binominal distribution between two alleles, YFP and CFP. (C) Average numbers of genomes to establish infection in Cell-0, Cell-1, and Cell-2 were defined as λ0, λ1, and λ2, respectively.
FIG. 5.
FIG. 5.
Simulation of the decrease of coinfected cells corresponding to the development of locally infected regions. The dashed line represents simulated results, while the solid line represents observed results.
FIG. 6.
FIG. 6.
Idea and simulation results of effective selection on trans-acting elements due to narrow genetic bottlenecks. (A) Schematic representation of a mechanism for effective selection on trans-acting elements. Narrow genetic bottlenecks in cell-to-cell movements stochastically isolate adaptive genomes from defective genomes, resulting in rapid selection for adaptive genomes. (B) Modeling of competition among intracellular populations by describing their probabilities of succession (S) with respect to the proportion of adaptive genomes in the intracellular population formula image. (C) Simulation of the exclusion of defective genomes with different bottleneck sizes (Ne). Note that initial ratios of adaptive and defective genomes are 80% and 20%, respectively. (D) Simulation of the selection of adaptive genomes with different bottleneck sizes (Ne). Initial ratios of adaptive and defective genomes are 20% and 80%, respectively.

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