Fig. 2

Identification of novel dense granule proteins by NcGRA17-BioID. a Schematic illustration of endogenous gene tagging at the C-terminus by CRISPR/Cas9-mediated site-specific insertion. Tagging plasmids were generated with smHA tag (red box) flanked by common ends (purple and yellow boxes) and including a common stop codon (gray box) followed by the HXGPRT 3′ untranslated region (UTR) (green box) and the selectable marker DHFR-TS. Amplification of this central region with primers that contained short homology regions HR1 (black box) and HR2 (blue box) together with the common flanks (purple and yellow boxes) generated products for gene-specific tagging. Co-transfection of these amplicons with a CRISPR/Cas9 plasmid bearing the gene-specific gRNA was used to add a smHA tag (red box) at the C-terminus of the endogenous locus. b IFA with rabbit anti-HA antibodies shows strong staining of the dense granules for each novel NcGRA that co-localizes with NcGRA6, demonstrating that these are novel dense granule proteins. Gene numbers and novel designations are as follows: NcLIV_227280, NcGRA3; NcLIV_012020, NcGRA11b; NcLIV_042680, NcGRA25; NcLIV_043760, NcGRA27; NcLIV_055980, NcGRA38; NcLIV_058760, NcGRA45; and NcLIV_057330, NcGRA61. (Intracellular, scale bar = 5 μm; extracellular, scale bar = 2 μm)