Calmodulin Domain Protein Kinase PiCDPK1 Regulates Pollen Tube Growth Polarity through Interaction with RhoGDI

The pollen-specific calcium-dependent protein kinase PiCDPK1 of Petunia inflata has previously been shown to regulate polarity in tip growth in pollen tubes. Here we report the identification of a Rho Guanine Dissociation Inhibitor (PiRhoGDI1) as a PiCDPK1 interacting protein. We demonstrate that PiRhoGDI1 and PiCDPK1 interact in a yeast 2-hybrid assay, as well as in an in vitro pull-down assay, and that PiRhoGDI1 is phosphorylated by PiCDPK1 in vitro. We further demonstrate the PiRhoGDI1 is capable of rescuing the loss of growth polarity phenotype caused by over-expressing PiCDPK1 in vivo using stable transgenic plants. We confirmed that PiRhoGDI1 interacts with a pollen-expressed ROP GTPase isoform consistent with the established role of RhoGDIs in negatively regulating GTPases through their membrane removal and locking them in an inactive cytosolic complex. ROP is a central regulator of polarity in tip growth, upstream of Ca²⁺, and PiCDPK1 over-expression has been previously reported to lead to dramatic elevation of cytosolic Ca²⁺ through a positive feedback loop. The discovery that PiCDPK1 impacts ROP regulation via PiRhoGDI1 suggests that PiCDPK1 acts as RhoGDI displacement factor and leads us to propose a model which we hypothesize regulates the rapid recycling of ROP GTPase at the pollen tube tip.