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ABSTRACT Toxoplasma gondii tachyzoites co-opt host cell functions through introduction of a large set of rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing three components, MYR1, MYR2, and MYR3. To determine whether other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and thereby inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM but that translocation can be rescued “in trans” by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17’s role in regulating GRA translocation is carried out on the host cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments of Toxoplasma tachyzoites collaborate to modulate the host-parasite interaction. IMPORTANCE When Toxoplasma infects a cell, it establishes a protective parasitophorous vacuole surrounding it. While this vacuole provides protection, it also serves as a barrier to the export of parasite effector proteins that impact and take control of the host cell. Our discovery here that the parasite rhoptry protein ROP17 is necessary for export of these effector proteins provides a distinct, novel function for ROP17 apart from its known role in protecting the vacuole. This will enable future research into ways in which we can prevent the export of effector proteins, thereby preventing Toxoplasma from productively infecting its animal and human hosts.

ABSTRACT CRISPR-Cas9 technologies have enabled genome engineering in an unprecedented array of species, accelerating biological studies in both model and nonmodel systems. However, Cas9 can be inherently toxic, which has limited its use in some organisms. We previously described the serendipitous discovery of a single guide RNA (sgRNA) that helped overcome Cas9 toxicity in the apicomplexan parasite Toxoplasma gondii, enabling the first genome-wide loss-of-function screens in any apicomplexan. Even in the presence of the buffering sgRNA, low-level Cas9 toxicity persists and results in frequent loss of Cas9 expression, which can affect the outcome of these screens. Similar Cas9-mediated toxicity has also been described in other organisms. We therefore sought to define the requirements for stable Cas9 expression, comparing different expression constructs and characterizing the role of the buffering sgRNA to understand the basis of Cas9 toxicity. We find that viral 2A peptides can substantially improve the selection and stability of Cas9 expression. We also demonstrate that the sgRNA has two functions: primarily facilitating integration of the Cas9-expression construct following initial genome targeting and secondarily improving long-term parasite fitness by alleviating Cas9 toxicity. We define a set of guidelines for the expression of Cas9 with improved stability and selection stringency, which are directly applicable to a variety of genetic approaches in diverse organisms. Our work also emphasizes the need for further characterizing the effects of Cas9 expression. IMPORTANCE Toxoplasma gondii is an intracellular parasite that causes life-threatening disease in immunocompromised patients and affects the developing fetus when contracted during pregnancy. Closely related species cause malaria and severe diarrhea, thereby constituting leading causes for childhood mortality. Despite their importance to global health, this family of parasites has remained enigmatic. Given its remarkable experimental tractability, T. gondii has emerged as a model also for the study of related parasites. Genetic approaches are important tools for studying the biology of organisms, including T. gondii. As such, the recent developments of CRISPR-Cas9-based techniques for genome editing have vastly expanded our ability to study the biology of numerous species. In some organisms, however, CRISPR-Cas9 has been difficult to implement due to its inherent toxicity. Our research characterizes the basis of the observed toxicity, using T. gondii as a model, allowing us to develop approaches to aid the use of CRISPR-Cas9 in diverse species.