Files S1 - Plasmodium falciparum Erythrocytic Stage Parasites Require the Putative Autophagy Protein PfAtg7 for Normal Growth

Figure S1, Alignments show similarity between yeast and P. falciparum Atg proteins. Atg8 (A), Atg4 (B), Atg7 (C) and Atg3 (D) alignments between P. falciparum and S. cerevisiae performed through ClustalW. PfAtg protein sequences identified by a PlasmoDB blastp using ScAtg protein sequences as described in M&M in the main manuscript. Figure S2, Southern blot confirms integration into correct locus. DNA was purified from transgenic parasites: PfATG7-RFA (clone A1) and PfATG7-HA (clone C1). Southern blot analyzed 1.5 ug of DNA and single cross-over integrations were screened by AvrII/BclI digestion and probed using PfAtg7 ORF 3′ end (1 kb). The expected fragment for 3D7 and PM1 parasites is 4039 bp and the expected size fragments for successful integration of PfATG7-HA are 7653 bp and 3635 bp and for PfATG7-RFA parasites are 7528 bp and 3635 bp. Lack of wildtype locus in PfAtg7-HA and PfAtg-RFA clones indicates successful integration. High intensity band in the PfAtg7-RFA clone possibly represents concatamerization of the plasmid prior to integration into the genome. Figure S3, Semi-quantitative PCR confirms lower expression levels of PfAtg7 in PB-57 parasites. Standard PCR conditions were used to establish expression levels of PfAtg7 (A) and the ribosomal protein upstream of PfAtg7 (B) across a dilution series of cDNA (nanogram amounts) for 3D7 and PB-57 parasites, with quantification also shown (C,D). The ribosomal protein is used as a control as the transposable element was inserted between it and PfAtg7. Predicted sizes of amplified products: PfATG7: 566 bp for cDNA, 745 bp for gDNA; ribosomal protein: 464 bp for cDNA and 700 bp for gDNA. Table S1, Results of bioinformatic analysis of ATG genes in P. falciparum. See M&M in main manuscript for search parameters. (PDF)