Phytophthora infestans PiGK4 is a membrane bound GPCR-PIPK that functions as a PI4P5-kinase and has a role in virulence

Signalling networks with heterotrimeric G-proteins and phospholipids as key players lie at the base of many cellular processes in eukaryotes. Oomycetes possess a family of novel proteins called GPCR-PIPKs (GKs) that are composed of a G-protein coupled receptor (GPCR) domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain. Based on this domain structure GKs are anticipated to link G-protein and phospholipid signalling but their functions and biochemical activities are currently unknown. Previously we analysed the function of one of the twelve GKs in the potato late blight pathogen Phytophthora infestans by gene silencing and overexpression and showed that PiGK4 is involved in spore development, sporangial cleavage, hyphal elongation and virulence (Hua, Meijer et al. 2013, Mol. Microbiology). Overexpression of subdomains of PiGK4 fused to a fluorescent protein further revealed that the GPCR domain targets PiGK4 to membranes surrounding certain cellular compartments. To determine the enzymatic activity of the PIPK domain in PiGK4 we make use of the temperature sensitive yeast mutant mss4ts that can be complemented with the full-length PiGK4 gene. Here we will present a more detailed analysis of the function of the various conserved motifs and domains in PiGK4 by complementation essays in yeast mss4ts using modified versions of PiGK4 generated by deleting and swapping domains.