Generation of transgenic parasites

The transgenic lines for NDC80 (PBANKA_1115700) were created using single homologous recombination as shown in Fig. S1. The oligonucleotides used to generate transgenic lines are provided in Table S1. For GFP tagging, a 1153 bp region of Ndc80 without the stop codon was inserted upstream of the gfp sequence in the p277 plasmid vector using KpnI and ApaI restriction sites, as described previously (Tewari et al., 2010). The p277 vector contains the human dhfr cassette, conveying resistance to pyrimethamine. Before transfection, the sequence was linearised using EcoRV. The P. berghei ANKA line 2.34 was used for transfection by electroporation (Janse et al., 2006). Immediately, electroporated parasites were mixed with 100 μl of reticulocyte-rich blood from a phenylhydrazine (6 mg/ml, Sigma-Aldrich) treated, naïve mouse and incubated at 37°C for 30 min before intraperitoneal injection. Pyrimethamine (70 mg/l, Sigma-Aldrich) was supplied in the drinking water from 1 d.p.i. to 4 d.p.i. Infected mice were monitored for 15 d, and drug selection was repeated after passage to a second mouse. Integration PCR and western blotting were performed to confirm successful generation of the transgenic line. For integration PCR, primer 1 (IntT259) and primer 2 (ol492) were used to confirm integration of the GFP targeting construct. Primer 1 and primer 3 (T2592) were used as a control. We also generated an mCherry-tagged NDC80 transgenic parasite line as shown in the schematic provided in Fig. S1.