Genetic Disruption of Genes Encoding Exported Proteins
(A) Strategy to delete genes as exemplified for PF14_0758. The vectors used were pHHT-TK (TK) or pCC1 (CDUP). The restriction enzyme used for PF14_0758 was EcoRI (E). The flanks for recombination are shaded gray or black. These flanks were probes for Southern blots and the size of DNA fragments based on 3D7 sequence are indicated in kilobases (kb).
(B) Example of a Southern blot to verify disruption for PF14_0758 (all disrupted genes are shown in Fig. S1). (C) Western blots to confirm absence of protein expression in parasites lines in which the genes PFA0110w, PFB0106c, PFE0060w, MAL7P1.172, PF10_0159, PF11_0037, PF13_0275, PF14_0018 and PF14_0758 had been disrupted. CS2 is shown in each panel and equal loading demonstrated with Hsp70 antibodies (bottom panel).

Identification of Proteins Required for Display and Function of PfEMP1 on the Surface of P. falciparum-Infected Erythrocytes
(A) Screening of mutant parasite strains to identify those with altered reactivity to anti-var2csa antibodies by FACS. Mutant parasite lines with specific gene disruptions were tested for reactivity with serum antibodies from malaria-exposed multigravid women (blue bars) and non-exposed controls (red bars). Reactivity was expressed as relative to the parental line CS2, which was set at 100%. Gene names in blue signify candidates for a trypsin cleavage assay (Figure 3B) and gene names in italics indicate a subsequently identified influence on PfEMP1 transport (Figure 7). Error bars indicate % range.
(B) Trypsin treatment of P. falciparum-infected erythrocytes to determine presence of PfEMP1 on the host erythrocyte. The full-length PfEMP1 and cytoplasmic tail were detected using antibodies to the cytoplasmic acidic terminal segment (ATS). Full-length PfEMP1 was a > 300 kDa band. Surface pool of PfEMP1 was detected by a trypsin-resistant band between 70 and 90 kDa. The lanes in each panel show Triton-X insoluble/SDS soluble extractions of parasite-infected erythrocytes: first lane, untreated (−); second lane, trypsin-treated (+); third lane, trypsin plus soybean trypsin inhibitor (i). The parasite lines shown are those that when screened by antibodies against var2csa PfEMP1 were less than 30% reactive compared to CS2 or knob-deficient (panel A). The red cell control is shown in the last panel. The anti-ATS antibody cross-reacts with spectrin (Maier et al., 2007; Rug et al., 2006). Lack of a band between 70 and 90 kDa in the trypsin-treated lanes shows absence of PfEMP1 on the erythrocyte surface. Full-length PfEMP1 is observed because there is a large pool of internal protein resistant to trypsin (Waterkeyn et al., 2000).
(C) Adherence of mutant P. falciparum-infected erythrocytes to CSA under flow conditions. Each of the P. falciparum mutant strains was tested for binding to CSA under flow conditions and parasitised cells counted as bound infected red blood cells/mm². 95% confidence intervals for CS2 wild-type binding is presented (dashed lines).

Genes Involved in P. falciparum-Infected Erythrocyte Rigidity and Properties of Proteins that Play a Role in Trafficking or Function of PfEMP1
(A) Rigidity as measured using the LORCA for all generated mutants compared to CS2. The four highest shear stress points (see Figure 5B) for each cell line was used to calculate the deformability ratio and compared to the ratio of CS2.
(B) Examples of LORCA measurements comparing membrane rigidity of P. falciparum-infected erythrocytes. The erythrocyte rigidity (expressed as elongation index [EI]) conferred on the host cell by each mutant P. falciparum line (green) compared to parent CS2 (blue) and uninfected red cells (red) at increasing shear stress measured in pascal (Pa). Parasites were synchronised and concentrated to 40% parasitaemia to increase sensitivity of the measurement. Error bars indicate standard deviation.
(C) Structure of the proteins that play a role in trafficking and function of PfEMP1. The gene number is shown for each protein from PlasmoDB (www.plasmodb). Yellow refers to a proposed signal sequence while red signifies the presence of a PEXEL required for export. Black shading corresponds to a proposed transmembrane region. Green refers to a DnaJ domain and blue a TIGRFAM01639 domain.
(D) Diagrammatic representation of a P. falciparum-infected erythrocyte signifying the localization of the protein (green symbols) or their functional position with respect to effects on PfEMP1 trafficking when disrupted (yellow letters).

Expression and Homology of Genes Selected for Gene Knockout Screen in P. falciparum
The P. falciparum genome was scanned to generate a list that included known exported proteins, as well as those with a PEXEL motif (Hiller et al., 2004; Marti et al., 2004; Sargeant et al., 2006). Using these criteria we compiled a list of 83 candidate genes of which 46 had PEXEL motifs (shaded blue). Those not containing a PEXEL but known to be exported are shaded gray. Genes shaded green are not known to be exported or are present on the parasitophorous vacuole membrane (PVM) e.g. Exp-1 (PF11_0224). Shown in the first column is the gene number which can be found at http://www.plasmodb.org. The second column refers to a known name of the corresponding protein. The third column shows a transcription profile where red is an increased period of transcription and green a decreased period or no transcription. Gene transcription was obtained from the microarray data available on http://malaria.ucsf.edu/ (Bozdech et al., 2003). In the fourth column proteomic data is shown and + refers to peptides detected in ring (R), trophozoite (T) or schizont (S) asexual life cycle stages. Proteomic data is available on www.plasmodb.org. The fifth column refers to the presence (Yes) or absence (No) of a Plasmodium export element or PEXEL (Hiller et al., 2004; Marti et al., 2004; Sargeant et al., 2006). The sixth column refers to the subcellular localization of the protein in the P. falciparum-infected erythrocyte if known and if not known shown as ND. The seventh column shows the homology detected and in some cases putative function of the protein and where none was detected is shown as ND. The eighth column shows the conservation of genes within different Plasmodia spp.: Pf, P. falciparum; Pv, P. vivax; Pb, P. berghei; Pc, P. chabaudi; Py, P. yoelii. The ninth column refers to whether the gene can be genetically disrupted (Yes) or not (No).

Essentiality of Genes in P. falciparum
(A) Comparison of essentiality for genes judged by ability to genetically disrupt them. Comparison is shown for all genes (Overall), the exported and PEXEL containing proteins (Exported/PEXEL) and those not exported (non-PEXEL). Unsuccessful disruptions are in gray while successful ones are in green.
(B) Comparison of non-essential proteins to those not disrupted. The proteins are divided into hypothetical (blue), annotated (red) and those assigned to a metabolic pathway (white) (PlasmoDB, www.plasmodb.org).
(C) Essentiality of different gene groups. The essentiality of the genes was compared with respect to transcription profile, homologies, chromosomal position and allelic variability. The bars show essentiality (as determined by the percentage of unsuccessful gene knock-outs for each group) of exported (red) and non-exported gene products (yellow). n = number of attempted gene disruption for exported (red) and non-exported proteins (yellow) in each group.
(D) The essentiality of gene families as shown by the ability to generate a genetic disruption. n = number of attempted gene disruption/total members in gene family. PEXEL-containing families are shown in red and non-PEXEL families are depicted in yellow.

Microscopic Analysis of Mutants with Export Defects
(A) Localization of PfEMP1 and KAHRP in mutant P. falciparum-infected erythrocytes. The parasite lines shown are those with either no PfEMP1 or reduced levels on the surface of infected erythrocytes determined by FACS and trypsin analysis. The first panel depicts localization of PfEMP1 and the second panel shows localization of KAHRP. The first column of each panel shows a bright-field image, the second panel specific antibody (either anti-PfEMP1 or anti-KAHRP) and the third panel overlay of the previous two and a nuclear stain (DAPI).
(B) Localization pattern of three proteins which when deleted ablate surface exposure of PfEMP1. These proteins were detected with specific antibodies raised against the gene products of PFB0106c, MAL7P1.172 and PF14_0758 (see Figure 2C). The first panel shows a bright-field image, followed by a DAPI image, then the specific antibody (green), then antibodies against the Maurer's cleft resident protein SBP1 (red) and an overlay of the specific antibody with SBP1 localization. (C) Localization of PfEMP1 (first panel) and KAHRP (second panel) for parasite lines CS2ΔPF10_0381 and CS2ΔPFD1170c. Shown are a brightfield image, specific antibody (either anti-PfEMP1 or anti-KAHRP) and an overlay of the two with a nuclear stain (DAPI).
(D) Scanning electron microscopy of CS2ΔPF10_0381 and CS2ΔPFD1170c infected erythrocytes. The first panel shows parental CS2-infected erythrocytes with normal knobs compared to the two mutant lines in which knobs are absent (PFD1170c) or greatly reduced in size (PF10_0381). The scale bar represents 2 μm.

Function and Properties of Selected Proteins Identified by the P. falciparum Gene Knockout Screen
These proteins play a role in function or trafficking of the major virulence protein PfEMP1 or a putative role in determination of rigidity. The full description of the genes and proteins can be found at http://plasmodb.org/. Most proteins have a PEXEL, which is required for export to the infected erythrocyte. Changes in rigidity conferred on the host cell by each mutant P. falciparum line was compared to parent CS2 and uninfected red cells at increasing shear stress and are relative to CS2-infected erythrocytes. Those shown as a positive number have a significantly increased rigidity whilst those that are negative have a decrease in rigidity that is more than the CS2ΔPFA0110w (RESA) parasite line that has been shown previously to have an altered rigidity (Silva et al., 2005). The TIGR FAM01639 domain in MAL7P1.172 represents a conserved sequence of about 60 amino acids found in over 40 predicted proteins of P. falciparum. It is not found elsewhere, including closely related species such as P. yoelii. The PHIST proteins share a homologous domain unique to Plasmodium of 150 amino acids and have been divided into a number of subfamilies (a, b, and c) (Sargeant et al., 2006). The skeleton binding protein 1 is included as a reference protein that is involved in PfEMP1 trafficking (Maier et al., 2007; Cooke et al., 2006).