Another candidate gene deleted on chromosome 9 is the Maurer’s cleft-resident ring exported protein 1 (REX1). Its deletion results in alteration of Maurer’s clefts architecture [49] and a reduction of cytoadhesion to CD36 [13]. These data point to REX1 as a plausible candidate gene for the observed adhesion phenotype in D10. We assume that additional co-deleted genes may act together with REX1 resulting in the complete loss of adhesion. Although D10 is not known to possess any of the REX proteins on chromosome 9, our data indicate that transit of proteins in this parasite strain appears functional. Indeed, labelling of PfEMP1 in D10 seems identical to that observed with the cytoadherent FCR3CD36. REX1 mutation resulted in the co- deletion of a subtelometic region of 3D7 chromosome 2 including KAHRP [13]. In our D10 parasites this is not observed since our antibodies label KAHRP and AFM demonstrates the presence of knobs. Dixon et al. [13] observe that REX1 deletion leads to trypsin-resistant PfEMP1 and conclude that no PfEMP1 is exposed on the PRBC. In the absence of surface iodination experiments, however, it cannot be excluded that trypsin-resistant PfEMP1 is expressed on the surface. Further characterization of REX1 may unravel the function of this protein in the adhesion process. A large-scale loss-of-function study designed to characterize candidate genes involved in interactions with PfEMP1 or with a role in knob formation was performed by the Cowman laboratory [11]. Fifty genes were functionally analysed and a number of different adhesion phenotypes were linked to proteins required for loading of PfEMP1 to Maurer’s clefts and function on the PRBC surface. However, this study did not include genes deleted on chromosome 9. In addition, the surface exposure of PfEMP1 was only analysed by trypsin treatment, which releases a small trypsin- resistant fragment (mainly composed of the ATS domain) between 70 and 90 kDa. Trypsin-resistant PfEMP1 was interpreted as being not exposed on the surface of PRBC. Our data using surface iodination experiments, however, show that parasites can expose non-functional trypsin-resistant PfEMP1. Since PfEMP1 mole- References 1. Kraemer SM, Smith JD (2006) A family affair: var genes, PfEMP1 binding, and malaria disease. Curr Opin Microbiol 9: 374–380. 2. Rasti N, Wahlgren M, Chen Q (2004) Molecular aspects of malaria pathogenesis. FEMS Immunol Med Microbiol 41: 9–26. 3. Maier AG, Cooke BM, Cowman AF, Tilley L (2009) Malaria parasite proteins that remodel the host erythrocyte. Nat Rev Microbiol 7: 341–354. 4. Gardner MJ, Hall N, Fung E, White O, Berriman M, et al. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419: 498–511. 5. 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Waller KL, Nunomura W, Cooke BM, Mohandas N, Coppel RL (2002) Mapping the domains of the cytoadherence ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) that bind to the knob-associated histidine-rich protein (KAHRP). Mol Biochem Parasitol 119: 125–129. 10. Scherf A, Lopez-Rubio JJ, Riviere L (2008) Antigenic variation in Plasmodium falciparum. Annu Rev Microbiol 62: 445–470. 11. Maier AG, Rug M, O’Neill MT, Brown M, Chakravorty S, et al. (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum- infected human erythrocytes. Cell 134: 48–61. Adhesion Is Not Linked to clag9 Deletion cules interact with KAHRP at the knob protrusions [9], we cannot rule out that the slightly modified knobs structure observed in D10 (see Fig. 4), could contribute to the complete loss of adhesion in D10. The role of the 25 genes deleted in D10 in this process will require further analysis. To answer these questions, work is in progress aiming to complement the deleted genes from chromo- some 9 in D10 to establish which genes contribute to the cytoadherence phenotype. In conclusion, we have identified mutant parasites with non- functional PfEMP1 surface expression. We also provide solid experimental evidence that CLAG9 does not contribute in the binding to CD36. In addition, our work illustrates that the factors leading to functional surface expression of PfEMP1 are not well elucidated and that the analysis of the genes deleted in D10 may contribute to our understanding of parasite virulence. Supporting Information Table S1 Primers used to amplify the P. falciparum D10var1 gene, PfEMP1 M2 minimal domain, clag9 gene and clag9 knockout. 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