Rac2 is not required for spore acidification or killing. rac2+/− adult fish were crossed to each other, and the resulting larvae were injected with YFP-expressing and AlexaFluor633-cell wall-conjugated TBK1.1 (Af293) spores. At 2 dpi, larvae were stained with Lysotracker Red and live imaged and then genotyped. Average number of spores enumerated per larva via microscopy at 2 dpi = 40. Data represent 3 pooled replicates of 17–18 larvae per replicate. (A) Experimental schematic. (B) Example images showing killed spores in non-acidified compartments (YFP−, AF+, lyso−; purple arrow), killed spores in acidified compartments (YFP−, AF+, lyso+; pink arrow), alive spores in non-acidified compartments (YFP+, AF+, lyso−; green arrow), and alive spores in acidified compartments (YFP+, AF+, lyso+; brown arrow). Each image shows a single z slice from a confocal image stack. (C) The percentage of alive (YFP+) spores was calculated for each larva. (D) The percentage of spores present in acidified compartments (lyso+) was calculated for each larva. (E) The average percentage of spores in each type of compartment was calculated across all larvae. (F) The percentage of killed (YFP−) spores that are found in acidified compartments (lyso+) was calculated for each larva. For (C, D, and F) each symbol represents one larva, color coded by replicate. For C–F, lines represent emmeans ± SEM. P values were calculated by ANOVA.
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