cyst wall is synthesized that provides protection to the parasite during transmission to a new 46 host. Toxoplasma secretes proteins into the vacuole to build its replicative niche and previous 47 studies identified many of these proteins as phosphorylated. We investigate two secreted 48 proteins and show that phosphorylation plays an important role in their regulation. We also 49 observed widespread phosphorylation of secreted proteins when parasites convert from acute 50 to chronic stages, providing new insight into how the cyst wall may be dynamically regulated. 51During infection Toxoplasma secretes proteins that subvert host cell signalling and develops its 67 replicative niche within the PV (6, 7). The repertoire of secreted proteins is thought to include 68 up to 200 proteins (ToxoDB, LOPIT dataset) including 50 kinases/pseudokinases (8), although 69 the functions of many of these remain unknown. These proteins are secreted from the 70 rhoptries or dense granules and are called ROPs or GRAs, respectively. Secreted proteins 71 extensively modify the PV allowing selective passage of molecules and proteins across the PV 72 membrane. For example, GRA17 and GRA23 form pores in the PV membrane allowing the 73 passage of small molecules into the PV (9), while the MYR complex mediates the transport of 74 secreted proteins to the host cell (10, 11). Additionally, the parasite develops a complex set of 75 membrane structures within the PV including GRA7-lined invaginations (12) and a network of 76 tubules called the intravacuolar network (IVN) (13). The formation of this network is dependent 77 on GRA2 and the accessory protein GRA6, and is required for full virulence in mice (14,15). 78Both structures have been reported to play a role in scavenging nutrients from the host cell 79 with the uptake of host cell proteins (16), lipid droplets (17), and endolysosomal compartments 80 (18). 81Previous phosphoproteome analysis revealed that a large number of Toxoplasma secreted 82 proteins are phosphorylated after their release (19), raising the intriguing possibility that their 83 function is dynamically regulated. Indeed, the PV localised kinase WNG1 was shown to 84 contribute to formation of a functional IVN (20). Furthermore, it was recently shown that IVN 85 associated GRAs show dynamic localisation patterns as the parasite remodels the PV to form 86 the chronic stage cyst (21). Here we analyse two secreted, strand forming proteins that are 87 phosphorylated after secretion and show that phosphorylation regulates their localisation, 88 disrupting normal strand formation. Both proteins disperse in chronic stage cysts, so we 89