Competition among variants is predictable and contributes to the antigenic variation dynamics of African trypanosomes
Fig 2
RNA-seq confirms that switched cells retain common ESAGs.
(A) An immunofluorescence microscopy time-course assay, beginning on day-5; after three days of growth in tetracycline, to induce Cas9 with sgRNA-3, and a further two days after removal of tetracycline. Three technical replicates. n > 90 cells for each sample at each time-point. RNA was extracted from these samples for RNA-seq analysis. (B) The schematic shows the active telomeric VSG-ES (BES1) and the site targeted by sgRNA-3. Recombination with a silent bloodstream ES triggers replacement of VSG-2 with a new ES-VSG, while retaining the BES1 ESAGs. (C) RNA-seq analysis of VSG-ESs. Only the VSGs (3, 6 and 8) are activated, but not their respective ESAGs (grey boxes below each read-mapping profile, in BES7, 3 and 12), as illustrated in B. Values expressed in RPMs (reads per million). Flags indicate VSG-ES promoters; grey blocks indicate the extent of the 70-bp repeats. (D) The graph shows VSG expression levels as determined by RNA-seq on day-5 as jittered dots; averages of three replicates. The number of VSGs is: metacyclic ES-VSGs (M-ES) = 5, bloodstream ES-VSGs (B-ES) = 11, minichromosomal VSGs (MC) = 20. The horizontal bars indicate the mean for each group. Values from S1 Data. (E) The graph shows the length of the activated VSG set as jittered dots. ES-VSGs, n = 16; MC-VSGs n = 20. The summary of the data is shown as a boxplot, with the box indicating the IQR, the whiskers showing the range of values that are within 1.5*IQR and a horizontal line indicating the median. The notches represent for each median the 95% confidence interval (approximated by 1.58*IQR/sqrt(n)). The p value was derived using a two-tailed t-test.