Fig. 1: Extracellular vesicles from natural E. huxleyi populations.

A bloom of E. huxleyi was induced in a mesocosm setup at the Marine Biological Station Espegrend, Norway (60°16′11 N; 5°13′07E) in May-June 2018. Abundance of calcified E. huxleyi cells (a) was measured by flow cytometry, and EhV concentration (b) was measured by qPCR targeting the major capsid protein gene (mcp). Average ± SE of the four mesocosm bags are presented for (a) and of bags 2 and 4 in (b). Abundance of cells and virions was also measured in the surrounding fjord water as a “blank” control (empty circles). An asterisks indicate the times at which samples were taken for vesicle extraction; arrows indicate sampling times for the experiments presented in Fig. 2a, b; cross indicates time of sampling of EhV for the experiment presented in Fig. 2c. c Workflow for vesicle sRNA profiling. Sampling (1): 25 l water samples were collected on the days marked with an asterisk in (a) from each bag using a peristaltic pump and a 200 µm nylon pre-filter. Concentration (2): the water samples were combined and filtered through two subsequent filters (GF/C and 0.45 µm PVDF). Samples were then concentrated to ~500 ml on a 100 kDa TFF cartidge and stored at 4 °C in the dark. Separation and analysis (3): Once at the home lab, the samples were further concentrated on 100 kDa Amicon-ultra filters, and separated on an Optiprep gradient. After speparation and cleaning, vesicles were subjected to RNase treatment to eliminate extra-vesicular RNA. sRNA within the vesicles was then extracted and sequenced. Workflow was created with BioRender.com. d Vesicles were collected from the natural assemblages at the time points indicated by an asterisk in (a) and the sRNA cargo was sequenced. sRNA sequences were aligned to E. huxleyi target genes as indicated. sRNAs that target the same genes were also found in vesicles from lab cultures of (i) uninfected E. huxleyi CCMP2090, (ii) infected cultures, (iii) Both uninfected and infected cultures or (iv) not found in vesicles from lab cultures of E. huxleyi CCMP2090. Read counts were scaled to one million reads mapped to the E. huxleyi transcriptome, log2 transformed and compared across time points.