Fig 1.
Experimental strategy for production of Ostreococcus tauri clonal lines susceptible or resistant to OtV5.
A single colony of O. tauri was used to produce two 1-litre cultures of O. tauri cells, one used to prepare DNA for genome re-sequencing and to produce 46 independent clonal lines and another that was lysed by clonal OtV5. The viral lysate was subsequently used to inoculate 38 small flasks. Fresh medium (small blue flasks) was added to each lysate or control flask, and after ~1 week OtV5-resistant cells grew. A single colony from all lines was randomly chosen after plating and maintained in liquid culture for transcriptome sequencing and further analyses.
Fig 2.
Proportion of resistant producer (RP) and resistant non-producer (RNP) lines over the course of the study.
Percentage of RP and RNP in resistant lines on the ordinate and date when virus production test was performed on the abscissa.
Fig 3.
Differential gene expression in OtV5-resistant compared to susceptible control O. tauri.
Up (orange) and Down (purple) regulated genes in virus resistant lines. (A) Counts of differentially transcribed genes per chromosome. (B) Plot of the log2 fold change values of differentially transcribed genes per chromosome. (C) Predicted glycosyltransferase gene counts per chromosome based on matches to InterPro domains and families associated with glycosyltransferases. Genes with no difference in regulation are shown in grey. (D) Counts of differentially transcribed genes grouped according to functional categories shown on the ordinate. Labels describe the genes in each category (full gene descriptions in S2 and S4 Tables). Genes located on the large inverted duplicated region on chromosome 19 were only counted once.
Fig 4.
Bipartite structure of chromosome 19.
Genes over-transcribed in virus-resistant lines were all located in a region of ~180 kb (on the left) containing long inverted repeats while under-transcribed genes were on a ~100 kb region (on the right). Tracks from top to bottom: alignment of RNA-Seq reads from susceptible (S2a–S5a) and resistant (R5, R6, R16 and R13a) samples (read depths have the same scale) onto chromosome 19 assembled from PacBio reads; scale in kb; (rpts) rectangles of the same colour on a grey background represent large duplicated blocks with black chevrons indicating inverted relative orientation and small arrowheads and chevrons of the same colour and style represent short repeats and their relative orientation; at the bottom, genomic maps zooming-in on blocks encoding genes under-transcribed (right) and over-transcribed (left) in virus-resistant lines (blue—methyltransferase, red—glycosyltransferase, green—epimerase/dehydratase, purple—sugar phosphate transporter, orange—other sugar metabolism, light pink—cell surface repeat lipoprotein, yellow—transposon-related, white—ORFan or other functions). N.B. the coloured rectangles on the grey background are not related to those used in the gene maps beneath. Significantly differentially transcribed genes are outlined in black (see S2 Table for full annotations of these genes) and those not differentially regulated are outlined in light grey. The gene map region underlined by a zigzag is part of a series of tandem repeats shown by light blue chevrons (see S3 Fig). a, b, x: Genes with the same subscript letter are putative paralogues. *These genes are duplicated as they span the ends of the adjacent inverted repeated blocks but the first gene (ostta19g00040) includes a short tract of unique intervening sequence.
Fig 5.
Pulsed-field gel electrophoresis (PFGE) analysis of susceptible and resistant lines ethidium bromide stained (top) and corresponding hybridization (bottom) using a probe for a gene on chromosome 19.
Chromosome number and size are indicated on the left. Symbols on the upper gel images show, at positions observed by the ethidium-bromide fluorescence: − : absence of a band that was present in controls, +: presence of a band that was not present in controls, v: presence of a band at the expected size of the OtV5 genome. To aid the comparison, symbols from the upper images of ethidium-stained gels are shown at the same positions on the bottom Southern blot autoradiograph images. Note that since the probe hybridizes to a specific part of chromosome 19, occasionally the band of differing mobility does not correspond to the band identified by radioactive labelling, witnessing fragmentation of chromosome 19 after deletions, insertions, or translocations that may have occurred.
Fig 6.
Virus production in two O. tauri resistant strains.
Very few cells (less than 0.5%) show visible viral particles in their cytoplasm. Morphology of lysing RP cells is similar to that of susceptible cells. (A) Most of the cells of O. tauri RP2 strain without visible viral particles. (B) Dividing O. tauri RP2 strain cell. (C) Dividing O. tauri RP3 strain cell with visible intracellular viral particles (vi). (D) O. tauri RP3 strain cell with visible intracellular viral particles (vi). E. Lysis of an O. tauri RP3 strain cell. (F) Lysis of an O. tauri RP2 strain with visible viral particles (vi). The scale bar is 500 nm long.
Table 1.
General characteristics of small outlier chromosomes (SOC) in Mamiellales.