Miniprepped

RPS3 coding sequence was cloned into pLENTI-C-MYC-DDK-IRES-puro plasmid (Origene, Rockville, MD, USA), transformed into Stbl3 bacterial cells, and selected on chloramphenicol plates (50 µg/mL) following standard protocols. Colonies were picked, grown, and miniprepped (Qiagen, Hilden, Germany) for sequence verification by Eton Bioscience. Lentiviral particles were produced in 293T cells with VSVG and Δ8.9 plasmids following standard protocols. Cells were transduced and selected on puromycin (1 µg/mL) for stable line expression. pcDNA3.1 MCS-BirA (R118G)-HA plasmid for RPS3 BioID was purchased from Addgene (Watertown, MA, USA) (#36047). MYC-DDK (mDDK; DDK is also known as FLAG) tagged RPS3 was cloned into pCDNA3.1 plasmid for mutagenesis. Mutagenesis was carried out using the Q5 Site-Directed Mutagenesis Kit following the manufacturer’s instructions (NEB, Ipswich, MA, USA).
Primers for constructing RPS3 Δ129-160:
5-GGAGACCCTGTTAACTAC-3
5-CTCCATGATGAACCGCAG-3

Site-specific point mutation of the pGRA-ROP17-3xHA plasmid was performed by creating primers 5′-GCGATATTTGTTCAACGGGTGTTGAGCAAT-3′ and 5′-CAGCGCGAATGGTTGCCCTGTGGTGGG-3′, 5′-GCTGTGAAACTGCAAAATTTTCTTGTTGAT-3′ and 5′-GCCATGAACAAGTCCGAACGCGTGGAA-3′, and 5′-GcCTTCACTCAAATTCTTCGTACGAATG-3′ and 5′-AGAAAGTAGAAGCAATCCCGATTTATC-3′ to mutate residues 312, 436, and 454, respectively, to an alanine codon within the ROP17 open reading frame. The “Round-the-horn” site-directed mutagenesis approach was used to introduce point mutations at the aforementioned residues using the ROP17-3xHA plasmid. The PCR products were then individually ligated using a KLD Enzyme reaction kit (New England Biolabs) for 3 h and subsequently transformed into ElectroMAX DH10B E. coli (Invitrogen). Single colonies for each point mutant were miniprepped (Qiagen) and sequence verified using either 5′-GCCATGAACAAGTCCGAACGCGTGGAA-3′ or 5′-GCGATATTTGTTCAACGGGTGTTGAGCAAT-3′.
To generate parasite lines complemented with the catalytically inactive ROP17, RHΔrop17 parasites were transfected with the pGRA1-ROP17_K312A_-3xHA, pGRA1-ROP 17_D436A_-3xHA, or pGRA1-ROP17_D454A_-3xHA plasmid and then subsequently selected for 6 days with MPA/XAN and singly cloned as previously described.

Two PCR fragments representative of each Eimeria species detected were sequenced to confirm amplicon identity and validate PCR detection, resulting in 14 sequences from 31 positive reactions (45 %). Amplicons were purified using a Qiagen PCR purification kit, cloned using pGEM-T Easy (Promega, Madison, USA) in XL1-Blue MRF Escherichia coli (Stratagene, La Jolla, USA), miniprepped (Qiagen) and sequenced (GATC Biotech, Konstanz, Germany) as described by the respective manufacturers. Sequence assembly, annotation and interrogation were undertaken using CLC Main Workbench v6.0.2 (CLC Bio, Katrinebjerg, Denmark) and sequences were identified using BLASTn against the GenBank non-redundant database with default parameters. The sequences have been submitted to GenBank under the accession numbers LT549029-LT549042.