Tagging of proteins

For the eYFP tagging of the L. mexicana proteins, the corresponding ORFs and UTRs were cloned into the pLEnTv2-YB plasmid, as previously described (Dean et al., 2015). To tag FAZ1, FAZ2, ClpGM6, FAZ10 and LRRP1 at the N-terminus with eYFP, ∼500 bp of the 5′ end of the gene directly after the start codon and ∼500 bp of 5′ UTR directly upstream of the start codon was amplified from genomic DNA. An XbaI site was added to the forward primer and a NotI site added to the reverse primer for the amplification of the ORF. A NotI site was added to the forward primer and a BamHI site added to the reverse primer for the amplification of the 5′ UTR. The resulting PCR fragments were digested with the appropriate restriction enzymes and cloned into the pLEnTv2-YB plasmid that had been digested with XbaI and BamHI (New England Biolabs). To tag FLA1BP, FAZ8 and FAZ5 at the C-terminus with eYFP, ∼500 bp of the 3′ end of the gene directly before the stop codon and ∼500 bp of 3′ UTR directly downstream of the stop codon was amplified from genomic DNA. A NotI site was added to the forward primer and a SpeI site added to the reverse primer for the amplification of the ORF. A HindIII site was added to the forward primer and a NotI site added to the reverse primer for the amplification of the 3′ UTR. The resulting PCR fragments were digested with the appropriate restriction enzymes and cloned into the pLEnTv2-YB plasmid that had been digested with SpeI and HindIII (New England Biolabs). The resulting plasmids were linearised with NotI (NEB, Hitchin, UK) and then ethanol precipitated before transfection.

For Myc-tagging of the L. mexicana BILBO1 homolog, a fusion PCR approach was taken, as previously described (Dean et al., 2015). To tag BILBO1 at the N-terminus with a Myc tag, ∼500 bp of the 5′ end of the gene directly after the start codon and ∼500 bp of 5′ UTR directly upstream of the start codon was amplified from genomic DNA. The forward primer for the amplification of the BILBO1 ORF had a 27-bp region of homology to the 3′ end of the actin 5′ UTR at its 5′ end followed by a Myc-epitope tag that was in-frame with the ORF. The reverse primer for the amplification of the 5′ UTR had a 25-bp region of identity to the start of the blasticidin-resistance gene. Three pieces of DNA, the 500 bp UTR and ORF fragments amplified from genomic DNA, and the region from the pPOTv2 plasmid containing the blasticidin-resistance gene followed by the aldolase 3′ UTR and then the actin 5′ UTR, released using an EcoRI HindIII (New England Biolabs) digest were combined in a fusion PCR. The PCR required 30 amplification cycles, five of which were without any primers, followed by 25 cycles using nested primers that annealed 40 bp from the 5′ end of the UTR fragment and 40 bp from the 3′ end of the gene fragment. After amplification, the construct was purified and then used for the transfection.