Fig. 2

The tyrosine-rich protein encoded by ETH_00000115 is a sporocyst wall specific protein in Eimeria tenella. a In order to determine the localisation of the tyrosine-rich protein encoded by ETH_00000115 within sporulated oocysts, a reporter plasmid pETH_00000115-mCherry was engineered using the mCherry core construct-1 as a parental plasmid. As there is no predicted intron for ETH_00000115, the putative promoter (982 bp upstream of the predicted start codon) and coding sequence of ETH_00000115 could be PCR amplified from E. tenella genomic DNA as one contiguous product (136,055 to 134,402 from supercontig HG675721) using the forward primer, MluI-ETH_00000115_F (5'-GGGGATTTTTTGGGATGG-3'), and the reverse primer, SalI-ETH_00000115_R (5'-GCAGGGCAAGCAAGGC-3'). The PCR product was cloned into the mCherry core construct-1 via MluI and SalI, replacing the etmic1 promoter and allowing read-through from the ETH_00000115 coding sequence to the mCherry coding sequence. Amplification of DNA for cloning was carried out by polymerase chain reaction using Pfu DNA Polymerase (Thermo Scientific) according to the manufacturer’s instructions. Transfection of E. tenella sporozoites was carried out as described previously [11]. b, c, d Brightfield (upper panels) and red fluorescence (wavelength = 590 nm, lower panels) microscopy of pETH_00000115-mCherry sporulated oocysts of E. tenella confirms that the tyrosine-rich protein encoded by ETH_00000115 is expressed specifically in the sporocyst wall. Recombination resulting from pairing of transfected and non-transfected gametes means that one, two or four sporocysts within any single oocyst may display the mCherry signal; b shows a single sporocyst of four, c shows two sporocysts of four and d shows all four sporocysts exhibiting fluorescence. Microscopy was done on a Zeiss Axiovert 200 microscope equipped with the Apotome imaging system. Images were generated and analysed using the Axiovision Software (Carl Zeiss SA)