iDePP: a genetically encoded system for the inducible depletion of PI(4,5)P 2 in Arabidopsis thaliana

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Doumane, Mehdi | Colin, Léia | Lebecq, Alexis | Fangain, Aurélie | Bareille, Joseph | Hamant, Olivier | Belkhadir, Youssef | Jaillais, Yvon | Caillaud, Marie-Cécile

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Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] is a low abundant lipid present at the plasma membrane of eukaryotic cells. Extensive studies in animal cells revealed the pleiotropic functions of PI(4,5)P 2 . In plant cells, PI(4,5)P 2 is involved in various cellular processes including the regulation of cell polarity and tip growth, clathrin-mediated endocytosis, polar auxin transport, actin dynamics or membrane-contact sites. To date, most studies investigating the role of PI(4,5)P 2 in plants have relied on mutants lacking enzymes responsible for PI(4,5)P 2 synthesis and degradation. However, such genetic perturbations only allow steady-state analysis of plants undergoing their life cycle in PI(4,5)P 2 deficient conditions and the corresponding mutants are likely to induce a range of non-causal (untargeted) effects driven by compensatory mechanisms. In addition, there are no small molecule inhibitors that are available in plants to specifically block the production of this lipid. Thus, there is currently no system to fine tune PI(4,5)P 2 content in plant cells. Here we report a genetically encoded and inducible synthetic system, iDePP (Inducible De pletion of P I(4,5)P 2 in P lants), that efficiently removes PI(4,5)P 2 from the plasma membrane in different organs of Arabidopsis thaliana , including root meristem, root hair and shoot apical meristem. We show that iDePP allows the inducible depletion of PI(4,5)P 2 in less than three hours. Using this strategy, we reveal that PI(4,5)P 2 is critical for cortical microtubule organization. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P 2 in given cellular or developmental responses but also to evaluate the importance of this lipid in protein localization. . Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a low abundant lipid present at the plasma membrane of eukaryotic cells. Extensive studies in animal cells revealed the pleiotropic functions of PI(4,5)P2. In plant cells, PI(4,5)P2 is involved in various cellular processes including the regulation of cell polarity and tip growth, clathrin-mediated endocytosis, polar auxin transport, actin dynamics or membrane-contact sites. To date, most studies investigating the role of PI(4,5)P2 in plants have relied on mutants lacking enzymes responsible for PI(4,5)P2 synthesis and degradation. However, such genetic perturbations only allow steady-state analysis of plants undergoing their life cycle in PI(4,5)P2 deficient conditions and the corresponding mutants are likely to induce a range of non-causal (untargeted) effects driven by compensatory mechanisms. In addition, there are no small molecule inhibitors that are available in plants to specifically block the production of this lipid. Thus, there is currently no system to fine tune PI(4,5)P2 content in plant cells. Here we report a genetically encoded and inducible synthetic system, iDePP (Inducible Depletion of PI(4,5)P2 in Plants), that efficiently removes PI(4,5)P2 from the plasma membrane in different organs of Arabidopsis thaliana, including root meristem, root hair and shoot apical meristem. We show that iDePP allows the inducible depletion of PI(4,5)P2 in less than three hours. Using this strategy, we reveal that PI(4,5)P2 is critical for cortical microtubule organization. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P2 in given cellular or developmental responses but also to evaluate the importance of this lipid in protein localization.

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