An Inducible FLARE Construct Shows Apical Accumulation of PtdIns(4,5)P2 and at High Levels of Expression, Disrupts Tip Growth in Physcomitrium patensPublic
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Plant cell tip growth is a highly regulated process involving many membrane trafficking elements, including the exocyst, a protein complex that tethers secretory vesicles to the plasma membrane. The exocyst subunit Sec3 contains a PH domain that binds to PtdIns(4,5)P2 (PIP2), localizing the complex to the plasma membrane. Phylogenetic analysis of Sec3 revealed that the PH domain is highly conserved across kingdoms, including the model plant, the moss Physcomitrium patens. To visualize PIP2 in vivo, a GFP-based sensor (PIP2 -FLARE) was transformed into P. patens to generate a constitutively expressing PTHUBI line and an estradiol inducible PGX8 line. PIP2 is primarily located in the tips of protonemal cells in both FLARE-expressing lines. The PTHUBI line, showed a slow-growth phenotype, while uninduced PGX8 plants showed a lesser phenotype. Growth assays show that increased expression of the FLARE with estradiol exposure caused slowed growth similar to, if not more severe than that of the PTHUBI line. We conclude that optimal levels of PIP2 are essential for tip growth in P. patens protonemal cells, likely allowing for localization of the exocyst complex and other membrane proteins to mediate polarized membrane trafficking.
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