Fig. 1: Identification of capping enzymes in Toxoplasma.

A Immunofluorescence analysis of m⁷G mark in tachyzoites and bradyzoites (n = 3). Tz tachyzoite and Bz bradyzoite. Scale bar 5 µm. B Immuno-dot blot analysis of m⁷G levels in the indicated amount of RNA and Toxoplasma genomic DNA (gDNA) (n = 3). EtBr staining serves as a loading control. C Predicted three capping genes of Toxoplasma (TgCet, TgCeg, and TgCmt) with ToxoDB number and CRISPR score. D Schematic depicting TgCet: the triphosphatase tunnel (364–728 aa) with conserved amino acids, TgCeg: the catalytic domain (111–255 aa) with conserved amino acids and C-terminal domain 330–480 aa), and TgCmt: the conserved SAM binding motif. E–H T. gondii capping enzymes functionally complement yeast (S. cerevisiae) counterparts (n = 3). S. cerevisiae Δcet1, or Δceg1, or Δabd1 strain was transformed with pYES3 (TRP1) plasmid containing TgCet, TgCeg, or TgCmt gene. Triple deletion yeast strain was transformed with TgCet, TgCeg, and TgCmt genes containing plasmids. A control transformation was performed with the pYES3 plasmid lacking an insert. Single Trp⁺ transformants were patched to agar plates lacking tryptophan (−Trp) and then patched on agar medium containing FOA (−Trp+FOA). FOA-resistant colonies were picked and streaked on −Trp+FOA agar medium.