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Dram1 deficiency leads to increased susceptibility to Mm infection.a Schematic representation of the zebrafish dram1/Dram1(ENSDARG00000045561/ENSDARP0 0000066996.3) genetic and protein domain architecture and CRISPR/Cas9 target site. Dram1 (240 amino acids) contains six transmembrane domains (indicated with boxes and labeled T1–T6 with amino acid numbers indicated above). The gene is depicted with coding exons as boxes and introns as solid black lines (introns not drawn to scale). The position of the CRISPR/Cas9 target site and the predicted truncated protein is indicated. b Confirmation of dram1 mutation by Western blotting analysis. Protein samples were extracted from 4 dpf dram1∆19n/∆19n and dram1+/+ larvae (>10 larvae/sample). The blots were probed with antibodies against Dram1 and Actin as a loading control. c Representative confocal micrographs of sections from the tail region showing TUNEL staining performed on dram1∆19n/∆19n and dram1+/+ larvae at 3 dpf. Scale bar, 10 μm. d Quantification of TUNEL-positive cells in the indicated region of the tail of dram1∆19n/∆19n and dram1+/+ larvae at 3 dpf (≥7 larvae/group). Data are represented by scatter and boxplots as detailed in the “Methods” section. e Representative stereo images of infected dram1∆19n/∆19n and dram1+/+ larvae at 3 dpi. The arrowhead indicates the accumulation of bacteria in intersegmental veins. f and g Quantification of bacterial burdens at 3 dpi for dram1 mutants, wild type siblings, and unrelated wild types f or for dram1 and dram1∆19n mRNA injected individuals g. The data are accumulated from two independent experiments (>42 larvae/group for f and >62 larvae/group for g) and represented by scatter and boxplots as detailed in the methods section.
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