Estimation of population bottlenecks during systemic movement of tobacco mosaic virus in tobacco plants

Abstract

More often than not, analyses of virus evolution have considered that virus populations are so large that evolution can be explained by purely deterministic models. However, virus populations could have much smaller effective numbers than the huge reported census numbers, and random genetic drift could be important in virus evolution. A reason for this would be population bottlenecks during the virus life cycle. Here we report a quantitative estimate of population bottlenecks during the systemic colonization of tobacco leaves by Tobacco mosaic virus (TMV). Our analysis is based on the experimental estimation of the frequency of different genotypes of TMV in the inoculated leaf, and in systemically infected leaves, of tobacco plants coinoculated with two TMV genotypes. A simple model, based on the probability that a leaf in coinoculated plants is infected by just one genotype and on the frequency of each genotype in the source, was used to estimate the effective number of founders for the populations in each leaf. Results from the analysis of three leaves per plant in plants inoculated with different combinations of three TMV genotypes yielded highly consistent estimates. Founder numbers for each leaf were small, in the order of units. This would result in effective population numbers much smaller than the census numbers and indicates that random effects due to genetic drift should be considered for understanding virus evolution within an infected plant.

FIG. 1.

Schematic representation of a tobacco plant at inoculation, at 3 dpi, and at harvest time (6 dpi). Leaves L0, L1, and L2 are indicated.

FIG. 2.

Quantification of TMV genotypes by dot blot hybridization. RNA extracts from 72 leaves were loaded together with TMV-wt (⧫—⧫) and 20/72 (•—•) standards (2 to 0.015 μg in a series of 12 dilutions) and hybridized with ³²P-labeled oligonucleotide probes specific for wt (WT PROBE) and 20/72 (20/72 PROBE). The absorbances were determined by densitometry of blots and plotted against the standards.