iDePP: a genetically encoded system for the inducible depletion of PI(4,5)P₂ in Arabidopsis thaliana

Abstract

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.Research Organism: A. thaliana surface charge (Hammond et al., 2012), a basic property of the plasma membrane, which recruits many 36 proteins through electrostatic interaction (Platre and Jaillais, 2017). 37 38In plant cells, less is known about PI(4,5)P2. Intriguingly, it seems that some functions of PI(4,5)P2 39 in animals are not conserved in plants. For example, plasma membrane surface charges relies on PI4P, 40 phosphatidylserine (PS) and phosphatidic acid (PA), but not PI(4,5)P2 (Simon et al., 2016a; Platre and 41 Jaillais, 2017). Yet, mutants in PI4P 5-kinases (PIP5K) suggest that PI(4,5)P2 production is essential 42 and has critical roles in development, immunity and reproduction (Heilmann, 2016; Noack and Jaillais, 43 2017a). However, i) the synthesis of each phosphoinositide species is tied to each other and to other 44 lipids, ii) PI(4,5)P2 has pleiotropic cellular functions and iii) PI(4,5)P2 interacts with many different 45 proteins. Thus, it is likely that steady-state depletion of PI(4,5)P2 triggers a range of indirect effects 46 involving several types cellular and developmental compensatory mechanisms. It is therefore essential 47

The Arabidopsis SAC9 Enzyme is enriched in a cortical population of early endosomes and restricts PI(4,5)P2 at the Plasma Membrane

“… A, Schematic representation of the mode of action of the PAO on the PI4P and PI(4,5)P 2 metabolism based on previously published work (Doumane et al 2020; Platre et al 2018). B, Confocal images of WT root epidermal cells expressing mCIT-TUBBYc PI(4,5)P 2 biosensor that underwent mock, or 30 μM PAO treatments for 30- or 120-min.…”

Coiled-coil and RPW8-type immune receptors function at the plasma membrane in a phospholipid dependent manner

“…The strong effect of PI4P depletion at the PM on the function and localization of AtRNLs and AtRPM1 and the specific interaction of their CC R /CC domain with anionic lipids (including PI4P) in vitro , suggest that PI4P plays a major role for their interaction with and function at the PM. However, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) fulfils similar important cellular functions, is specifically found at the plant PM, and is also required for the interaction of many proteins with the PM 47 , like the mammalian MLKL proteins 26,27 . Although, PI(4,5)P2 is most likely not required for plant PM electronegativity 30 .…”

“…2×35Sprom/pDONRP4-P1R 68 , MAP-mCHERRYnoSTOP in pDONR207 and SAC1in pDONR-P2R-P3 (or SAC1dead in pDONR-P2RP3) 30 were recombined using LR reaction into pH7m34GW 69 to generate 2×35Sprom::MAP-mCHERRY-SAC1 in pH7m34GW (or 2×35Sprom::MAP-mCHERRY-SAC1dead in pH7m34GW). 2×35Sprom in pDONRP4-P1R 68 , MAP-mCHERRYnoSTOP in pDONR207 and dOCRL in pDONR-P2R-P3 (or dOCRLdead in pDONR-P2RP3) 47 were recombined using LR reaction into pH7m34GW 69 to generate 2×35Sprom::MAP-mCHERRY-dOCRL in pH7m34GW (or 2×35Sprom::MAP-mCHERRY-dOCRLdead in pH7m34GW). Constructs were verified by sequencing and transformed into Agrobacterium tumefaciens strain GV3101 and used for transient expression in Nicotiana benthamiana .…”

“…5), prompted us to test whether PI(4,5)P2 is also required for AtRNL and AtCNL PM localization and cell death function. We co-expressed the PM-anchored wildtype PI(4,5)P2 5-phosphatase domain from the Drosophila OCRL protein (dOCRL WT ) that specifically depletes the PI(4,5)P2 pool at the plant PM 47 with AtADR1, AtADR1-L1, AtADR1-L2, AtRPM1 and AtRPS5. As a control we co-expressed a phosphatase dead mutant version of dOCRL (dOCRL dead ) that is catalytically inactive 47 .…”

“…We co-expressed the PM-anchored wildtype PI(4,5)P2 5-phosphatase domain from the Drosophila OCRL protein (dOCRL WT ) that specifically depletes the PI(4,5)P2 pool at the plant PM 47 with AtADR1, AtADR1-L1, AtADR1-L2, AtRPM1 and AtRPS5. As a control we co-expressed a phosphatase dead mutant version of dOCRL (dOCRL dead ) that is catalytically inactive 47 . Co-expression of neither dOCRL WT nor dOCRL dead had a visible effect on the (PM-) localization or protein expression of the tested AtRNLs and AtCNLs (Supplementary Figure S2c,d and S6).…”