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. 2018 Jan 22;14(1):e1006828. doi: 10.1371/journal.ppat.1006828

Fig 2. MYR2 (TGGT1_270700) is essential for effector translocation.

Fig 2

(A) Schematic of TGGT1_270700 (MYR2) disruption. To disrupt the MYR2 locus, a CRISPR-Cas9 plasmid encoding a sgRNA against TGGT1_270700 was co-transfected with linearized pGRA1-HA-HPT plasmid into RHΔhpt parasites. Arrows indicate location of primers used to confirm integration of vector in the gene locus (and consequent disruption of the gene ORF) by PCR. (B) Disruptive integration of the HXGPRT vector in an exon of MYR2 was confirmed by PCR and sequencing. (C) Effect of MYR2 disruption and complementation on effector translocation. Wild-type (WT) and RHΔmyr2 tachyzoites were transiently transfected with a plasmid expressing Myc-tagged GRA24. For complementation, the Δmyr2 strain was co-transfected with GRA24-Myc and pGRA1:MYR2-3xHA plasmids. Transfected tachyzoites were allowed to infect HFFs for 16–24 hours. The infected monolayers were then fixed and GRA24 and MYR2 localization was assessed with anti-myc tag and anti-HA antibodies, respectively. Scale bar indicates 5 μm. White arrow indicates host nucleus lacking detectable GRA24 in the cell infected with RHΔmyr2 tachyzoites. (D) Quantitation of percentage of infected cells showing GRA24 localization in the host nucleus based on three independent experiments, each with analysis of 10 fields on at least three coverslips. Statistics were performed with one-way ANOVA and Tukey’s multiple comparison’s test. **** indicates P < 0.0001.