Accumulation of phosphoinositides in distinct regions of the periarbuscular membrane

Phosphoinositides and phosphatidic acid are small anionic lipids that comprise a minor proportion of total membrane lipids in eukaryotic cells but influence a broad range of cellular processes including endomembrane trafficking, signaling, exocytosis and endocytosis. To investigate the spatial distribution of phosphoinositides during arbuscular mycorrhizal symbiosis, we generated fluorescent reporters of PI(4,5)P-2 and PI4P, as well as phosphatidic acid and diacylglycerol and used them to monitor lipid distribution on the cytoplasmic side of membrane bilayers in colonized cortical cells. The PI4P reporter accumulated strongly on the periarbuscular membrane (PAM) and transiently labeled Golgi bodies, while the PA reporter showed differential labeling of endomembranes and the PAM. Surprisingly, the PI(4,5)P-2 reporter accumulated in small, discrete regions of the PAM on the arbuscule trunks, frequently in two regions on opposing sides of the hypha. A mutant reporter with reduced PI(4,5)P-2 binding capacity did not show these accumulations. The PI(4,5)P-2-rich regions were detected at all phases of arbuscule development following branching, co-localized with membrane marker proteins potentially indicating high membrane bilayer content, and were associated with an alteration in morphology of the hypha. A possible analogy to the biotrophic interfacial membrane complex formed in rice infected with Magnaporthe orzyae is discussed.